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PROGRAMA DE DOCTORADO EN BIOQUÍMICA Y

BIOMEDICINA

DEPARTAMENTO DE BIOQUÍMICA Y BIOLOGÍA MOLECULAR.

FACULTAD DE CIENCIAS BIOLÓGICAS. UNIVERSIDAD DE VALENCIA

TESIS DOCTORAL / THESIS

REPROGRAMACIÓN DIRECTA DE CÉLULAS

SOMÁTICAS ADULTAS A CÉLULAS GERMINALES

MEIÓTICAS

DIRECT REPROGRAMMING OF HUMAN SOMATIC

CELLS TO MEIOTIC GERM-LIKE CELLS

Ana Mª Martínez Arroyo

Directores / Thesis advisors:

Dr. Carlos Antonio Simón Vallés

Dr. Jose Vicente Medrano Plaza

Prof. Dr. D. Carlos Antonio Simón Vallés, Catedrático de Pediatría,

Obstetricia y Ginecología de la Universidad de Valencia, España; Profesor

clínico adjunto, Departamento de Obstetricia y Ginecología de la

Universidad de Stanford, CA, EEUU; y Director Científico del Instituto

Valenciano de Infertilidad (IVI) e IVIOMICS

CERTIFICA:

Que el trabajo de investigación titulado: “REPROGRAMACIÓN

DIRECTA DE CÉLULAS SOMÁTICAS ADULTAS A CÉLULAS

GERMINALES MEIÓTICAS” ha sido realizado íntegramente por Dª Ana

Mª Martínez Arroyo bajo mi dirección. Dicha memoria está concluida

y reúne todos los requisitos para su presentación y defensa como

TESIS DOCTORAL ante un tribunal.

Y para que así conste a todos los efectos, firmo la presente

certificación en Valencia, a 10 de Junio de 2014

Fdo. Prof. Dr. D. Carlos Antonio Simón Vallés

Dr. D. Jose Vicente Medrano Plaza, investigador de la Fundación del

Instituto Valenciano de Infertilidad (FIVI) – Fundación INCLIVA

CERTIFICA:

Que el trabajo de investigación titulado: “REPROGRAMACIÓN

DIRECTA DE CÉLULAS SOMÁTICAS ADULTAS A CÉLULAS

GERMINALES MEIÓTICAS” ha sido realizado íntegramente por Dª Ana

Mª Martínez Arroyo bajo mi dirección. Dicha memoria está concluida

y reúne todos los requisitos para su presentación y defensa como

TESIS DOCTORAL ante un tribunal.

Y para que así conste a todos los efectos, firmo la presente

certificación en Valencia, a 10 de junio de 2014

Fdo. Dr. D. Jose Vicente Medrano Plaza

AGRADECIMIENTOS / ACKNOWLEDGMENTS

A Carlos, más que un director, un mentor. Gracias a ti he aprendido muchas,

muchas cosas. Sólo espero poder, alguna vez, devolverte en igual medida

todo lo que has hecho por mí, todo lo que me has dado. Gracias.

A Jose, mi otro director, por supuesto. Sin ti, este trabajo no habría sido

posible. Alegría, ilusión, ganas y trabajo duro han hecho posible esta tesis.

Continuar con tu trabajo y trabajar codo con codo han sido un privilegio y un

placer. Tus consejos y opiniones han sido imprescindibles para llegar donde

estamos hoy. Gracias por todo, Jose.

Al resto de gente de la Fundación. Felip, Paco, Irene, Toni, Sebas, Juanma,

Amparo, Aymara, Ali, Claudia, Patri... A los que estáis en Medicina, Horten,

Bea, Raúl, Sonia. A los que se acaban de incorporar con tanta la ilusión

Alessia, Nuria, María, Stefania, Hannes, Anna… junto a vosotros he

aprendido y he crecido como persona y como científica. Lo que me habéis

aportado es difícil de explicar y transmitir, no digamos de resumir después

de tantos años, en unas cuantas líneas. Pero no lo olvidaré. Gracias a todos.

Como no, a ti, Carmen. ¿Desde hace cuánto tiempo nos conocemos? Va a

hacer una eternidad… Y quién nos iba a decir que nuestros caminos

discurrirían paralelos… Han sido tantas cosas las que hemos vivido juntas:

alegrías, risas, malos ratos, problemas. Es curioso, pero ahora mismo,

escribiendo estas líneas, recuerdo los problemas como borrosos, lejanos;

mientras que las alegrías, y las risas me llegan a la mente vívidos con toda su

fuerza y color. Hemos compartido mucho. Y espero que la vida y los caminos

que escojamos nos permitan seguir compartiéndolo. Has sido (y eres) una

gran amiga. Gracias.

A Inma y a toda la gente del animalario, por su paciencia y habilidad, por su

comprensión y su cariño hacia los animales. Sin ellos, esto hubiese sido

mucho más difícil todavía.

Al Dr. Manel Esteller, por la magnífica oportunidad que me ofreció

permitiéndome estar en su laboratorio de Barcelona. Con vosotros aprendí

muchísimo, a nivel científico y personal. Gracias. A la gente de Barcelona. A

María, a Miguel, a Paula, a Fer, a Karol, a Paco… Reír y disfrutar mientras

trabajas no es ni sencillo, ni frecuente, ni habitual. Por eso, gracias.

A los compañeros de IVIOMICS, por vuestro apoyo y ayuda. Muchas veces

nos olvidamos del valor de una sonrisa de buenos días, pero vosotros me lo

recordáis. Gracias. A Eva y Mª Eugenia, antes también compañeras del CIPF,

gracias.

Por motivos sentimentales, a toda la gente que dejamos atrás en el CIPF. A

algunos de vosotros os he vuelto a encontrar. María, gracias por los cafés

secretos-exprés en la máquina. Ha sido una suerte poder contar contigo.

Gracias.

A mis amigas Esther y Mariam. Tantos años juntas, que ya no es necesario

decir nada más. Gracias por escucharme, entenderme y ayudarme. En

momentos difíciles pero también en los buenos ratos, las risas tontas y los

sueños soñados juntas. Desde que empezamos la universidad hasta el día de

hoy, sólo buenos recuerdos. Fiesta, risa y cariño desinteresado. ¿Qué más se

puede pedir? Gracias, chicas.

A todos los amigos de la carrera. Juntos recorrimos un camino que, a día de

hoy, me sigue pareciendo precioso e irrepetible. Sin vosotros no sería como

soy hoy. A Sara, a Carol, a Clara, a Mar. No me dejo a nadie, ¿verdad? Si es

así, perdonadme, pero a vosotros también, gracias.

¡Laura! ¡Que no me olvido de ti! ¡Y tampoco de ti, Aida! Qué bien nos lo

hemos pasado, ¿a que sí? Creo que con quien mejor he sabido compartir mi

amor y gusto por la ciencia ha sido con vosotras. Estaremos lejos las unas de

las otras, pero eso no quita para que cuando nos reencontramos, sea como

si nos hubiéramos despedido ayer. Gracias por todo, y por eso, también.

A mis chicos del pueblo. Nos vemos poco, pero esas largas charlas hasta las

tantas en el bar son insustituibles. Nuria, Pedro, Diana, Iñaki, Zaida, Mili,

Raúl… Gracias.

Mis principales compañeros en este viaje han sido, sin duda, mi familia. A

Miguel, mi primo. Sé que puedo contar contigo siempre y tú sabes que

puedes contar conmigo para lo que sea. Gracias por tu apoyo incondicional.

Y por tus mails. Gracias.

A mi hermana Olga, sin duda alguna, mi mejor amiga. Reímos, lloramos,

discutimos, peleamos, nos abrazamos y consolamos. No me dejes aunque te

enfades conmigo de vez en cuando. Y gracias.

A mis padres. Gracias. Gracias por ser mi apoyo, mi hogar, mi referente, mi

faro en estos años que no han sido precisamente fáciles. Gracias mamá por

ser mi confidente. Tú, con tu capacidad lectora de mentes desarrollada a

base de cariño y observación todos estos años, has sabido siempre qué era

aquello que me preocupaba y cómo conseguir que dejara de hacerlo.

Gracias, por dejar que siguiera mi camino, con mis propias equivocaciones, y

gracias por no permitir que me desviara de él. Gracias papá por tu confianza

en mí. Es reconfortante. Gracias a ambos por tantas y tantas cosas. Sólo

espero no decepcionaros, haberme convertido en la persona que querías

que fuera cuando de pequeña os imaginabais cómo sería de mayor. Os

quiero. Gracias.

Gracias.

El presente trabajo de tesis doctoral ha sido realizado en los laboratorios de

la Fundación del Instituto Valenciano de Infertilidad (FIVI) – Fundación

INCLIVA, Parque Científico de la UNIVERSIDAD DE VALENCIA (España) así

como en los laboratorios del Programa de Epigenética y Biología del Cáncer

(PEBC) de Barcelona (España), gracias a la ayuda de una beca predoctoral

del programa de Formación del Profesorado Universitario (FPU) concedida

por el Ministerio de Educación, Cultura y Deporte del Gobierno de España

(Ref. AP-2009-4522)

This work was performed at the research laboratories in the IVI Foundation

(FIVI), Institute of Health Research INCLIVA, and the University of Valencia,

Scientific Park (Valencia, Spain); and at research laboratories of the Cancer

Epigenetics and Biology Program (PEBC; Barcelona, Spain). Funding come

from a grant from the Formación del Profesorado Universitario (FPU) PhD

Program from the Ministry of Education, Culture and Sport from the Spanish

Government (# AP-2009-4522)

RESUMEN / SUMMARY

El desarrollo de la línea germinal comienza en la especie humana

durante el desarrollo embrionario con la aparición de las células germinales

primordiales (CGP) desde el epiblasto proximal en la semana 2 a 3 de

gestación. Las CGP migran a las crestas gonadales y cambian su perfil de

expresión genético y epigenético desde un patrón somático predefinido a

un patrón germinal. Durante el proceso de maduración germinal que

depende del sexo del embrión, la adquisición del patrón gamético permite

que células germinales puedan entrar en meiosis, un proceso crucial por el

cual los gametos reducen a la mitad la carga cromosómica para, tras la

fecundación, dar lugar a un nuevo individuo diploide.

Sin embargo, debido a las dificultades metodológicas, éticas, morales

y legales que implica su estudio durante el desarrollo embrionario humano

todo nuestro conocimiento acerca del desarrollo de la línea germinal en

mamíferos procede de la extrapolación del modelo murino.

Por lo tanto, la generación de modelos in vitro para la derivación de

células germinales humanas es de gran utilidad para estudiar tanto su

desarrollo, como su biología y comportamiento. Varios estudios indican que

tanto las células madre embrionarias humanas como las células madre

pluripotentes inducidas humanas son capaces de mostrar un fenotipo

similar al de una célula germinal tras su diferenciación llegando a mostrar

marcadores meióticos y postmeióticos. Es más, recientemente se ha

demostrado que células germinales inducidas in vitro a partir de hiPSC son

capaces de recolonizar el nicho de las células madre espermatogoniales en

ensayos de xenotransplante en ratones inmunodeprimidos.

Por otra parte, la reprogramación directa de un tipo celular

diferenciado a otro mediante la sobreexpresión de factores de transcripción

específicos, permite plantearse la hipótesis de la reprogramación directa de

células somáticas adultas hacia células germinales.

HIPÓTESIS Y OBJETIVOS:

La hipótesis de trabajo es investigar la conversión directa de células

somáticas adultas a células con fenotipo germinal inducidas in vitro

mediante sobreexpresión de un conjunto de genes implicados en las

distintas fases del desarrollo de las células germinales.

El objetivo principal es la obtención in vitro de células germinales

inducidas mediante sobreexpresión ectópica de un conjunto de genes

descritos como clave durante el desarrollo de la línea germinal.

En base a lo anteriormente descrito, los objetivos específicos son:

1. Identificación, aislamiento y clonación en vectores lentivirales de genes

humanos clave en el desarrollo de la línea germinal.

2. Optimización de las condiciones de cultivo para mejorar el

mantenimiento de las células somáticas adultas humanas reprogramadas.

3. Cribado de los factores de reprogramación para encontrar la

combinación más eficiente en términos de inducción y avance de la meiosis.

4. Caracterización a nivel transcriptómico y epigenético de las células

germinales inducidas in vitro.

5. Análisis de la progresión meiótica y ploidía de dichas las células

germinales inducidas in vitro.

6. Análisis de la funcionalidad las células germinales inducidas in vitro en

modelos de xenotranplante.

METODOLOGÍA Y DISEÑO EXPERIMENTAL:

Se han seleccionado y clonado en vectores lentivirales los siguientes

genes relacionados con diferentes fases del desarrollo de la línea germinal:-

Genes inductores de la formación de células germinales

1) PRDM1: Factor de transcripción encargado de silenciar el programa

somático en las CGP.

2) PRDM14: Interactúa con PRDM1 en su función y participa en la inducción

del proceso de reprogramación epigenética de la línea germinal in vivo.

3) NANOS3: Relacionado con la supervivencia y el mantenimiento en estado

quiescente de las células germinales mediante represión transduccional de

ARNs mensajeros.

4) LIN28A: Proteína de unión al ARN encargada de silenciar varios micro

ARNs y relacionada con la especificación de las CGP mediante la regulación

de PRDM1.

5) NANOG: Factor de transcripción relacionado con la pluripotencia y

proliferación de las CGP.

Genes necesarios para la maduración y supervivencia de las células

germinales:

6) DAZ2: Proteína de unión al ARN codificada en el cromosoma Y, participa

de forma esencial para que se desarrolle correctamente la

espermatogénesis. Su pérdida de función produce el Síndrome de sólo

células de Sertoli.

7) DAZL: Proteína de unión al ARN descrita como esencial para la correcta

maduración de las células germinales. Su pérdida de función produce

esterilidad en ambos sexos.

8) BOLL: Proteína de unión al ARN de la misma familia que DAZ2 y DAZL, su

pérdida de función produce fallos en la meiosis.

9) VASA: Proteína de unión al ARN implicada en la meiosis y maduración

germinal mediante su función como regulador de los ARNs asociados a

PIWI. Su pérdida de función produce esterilidad en ratones macho.

10) STRA8: Proteína implicada en la inducción de la meiosis en respuesta al

ácido retinoico.

Genes implicados en la meiosis:

11) DMC1: Recombinasa que se encarga de la reparación de roturas

programadas en el ADN durante la recombinación de los cromosomas

homólogos en la profase I de la meiosis.

12) SYCP3: Componente estructural esencial del complejo sinaptonémico

durante la profase I de la meiosis.

Una vez clonados, fueron transfectados en dos tipos celulares

humanos primarios XY: fibroblastos de prepucio humano (hFSK de sus siglas

en inglés) y células madre mesenquimales (hMSC, de sus siglas en inglés).

Los días 7, 14 y 21 post-transfección, los cultivos celulares fueron

analizados mediante su caracterización transcriptómica, e

inmunofenotipado. Además, en el día 14, se analizó su estatus epigenético

mediante secuenciación por bisulfito de dos genes improntados, su

progresión meiótica y ploidía. Igualmente, 14 días después de su

transfección las células germinales inducidas fueron introducidas en el

testículo del ratón para el ensayo de xenotransplante.

La metodología seguida se detalla a continuación:

- Caracterización transcriptómica mediante array de expresión y qRT-PCR,

análisis epigenético del estado de metilación por secuenciación por

bisulfito, inmunofenotipado por inmunofluorescencia.

- Determinación del estado meiótico mediante inmunofluorescencia para

SYCP3. Determinación de la haploidía mediante separación celular activada

por fluorescencia (FACS) basada en intensidad de yoduro de propidio

seguido de FISH para los cromosomas 18, X, Y y recomprobado con sondas

para los cromosomas 10, 12 y 3.

- Para el ensayo de xenotransplante, las células transfectadas se inyectaron

en el conducto eferente de ratones NUDE (Crl:NU-Foxn1nu) previamente

esterilizados con busulfán, según el protocolo descrito en la literatura

(Ogawa et al. 1997). Dos meses tras el xenotransplante, se analizaron los

testículos de los ratones para determinar su localización y progresión.

CONCLUSIONES:

La sobreexpresión ectópica de un conjunto de genes reguladores del

desarrollo de la línea germinal, en dos líneas celulares humanas masculinas

(hFSK y hMSC) induce la aparición de una población heterogénea de células

con fenotipo similar al germinal.

Estas células germinales inducidas muestran un perfil de expresión y

metilación compatible con el de células germinales en diferentes estadios

de maduración.

Algunas de estas células germinales inducidas se pueden

correlacionar con un estado similar al de espermatogonia dado que son

capaces de avanzar en la meiosis e incluso terminarla, dando lugar a células

haploides, tras ser expuestas a ácido retinoico.

La combinación más efectiva de factores para conseguir un fenotipo

meiótico se disminuyó de las 12 originales a 6 incluyendo PRDM1 y

PRDM14, proteínas que actúan como factores de transcripción; junto con

LIN28A, DAZL y VASA, que son proteínas de unión a ARN; y SYCP3, una

proteína estructural del complejo sinaptonémico.

Al ser xenotransplantadas, algunas de las células germinales

inducidas muestran características similares a las de células madre

espermatogoniales, al demostrar su capacidad para colonizar la lámina

basal de los túbulos seminíferos, probando su funcionalidad in vivo.

Por lo tanto, se propone que este estudio sea considerado como un

modelo piloto para el estudio in vitro del desarrollo de la línea germinal en

humanos.

LIST OF ABBREVIATIONS293-T - 293-T packaging cell line 5caC - 5-carboxylcytosine 5fC - 5-formylcytosine 5hmC - 5-hydroxymehylcytosine ABP – androgen binding protein AMH - anti-müllerian hormone AP - alkaline phosphatase BER - base excision repair

bFGF - basic fibroblast growth factor Bi-seq - bisulfite sequencing bp - base pairs BS - busulfan BSA - bovine serum albumin C - cytosine cDNA - copy deoxyribonucleic acid CGI - CpG Islands CpG – cytosine- guanine dinucleotides DAPI - 4',6-diamidino-2-phenylindole DAZ - deleted in azoospermia ddATP - 2',3'-dideoxyadenosine-5'-triphosphate ddCTP - 2',3'-dideoxycytidine-5'-triphosphate ddGTP - 2',3'-dideoxyguanosine-5'-triphosphate ddNTP - dideoxynucleotide ddTTP - 2',3'-dideoxythymidine-5'-triphosphate D-MEM/F12 - Dulbecco's modified Eagle's medium/ nutrient F-12 ham DMR - differentially demethylated region DNA: deoxyribonucleic acid E - embryonic day EB - embryoid body EGF - epidermal growth factor EmGFP - emerald green fluorescent protein endo-siRNA - small interfering endogenous RNA EP - elongated spermatid Epi – epiblast EpiSC – Epiblastic stem cells EpiLC - epiblast-like cell ESC - embryonic stem cell ExE - extraembryonic ectoderm FACS - fluorescence activated cell-sorting

FBS - fetal bovine serum FDR - false discovery rate FISH - fluorescent in situ hybridization FSH - follicle-stimulating hormone G - guanine GC-M - germ-cell media for induced germ-cell survival gDNA - genomic deoxyribonucleic acid GDNF - glial cell line-derived neurotrophic factor GFP - green fluorescent protein ɤH2AX - Phosphorylation form of H2A.X GnRH - gonadotropin-releasing hormone GO – gene ontology H1 – histone 1 H1LS - histone-1-like protein in spermatids H1T2 - histone 1 variant T2 H2A - histone 2A H2A.X – histone 2A variant X H2A/H4R3me2s - histone 2A and histone 4 arginine 3 symmetric dimethylation. H2B – histone 2B H2BK5ac – histone 2B lysine 5 acetylation H3.3 – histone 3 variant 3 H3K20me - histone 3 lysine 20 methylation H3K27ac - histone 3 lysine 27 acetylation H3K27me3 - histone 3 lysine 27 trimethylation H3K36me - histone 3 lysine 36 methylation H3K4ac - histone 3 lysine acetylation H3K4me - histone 3 lysine 4 methylation H3K6ac - histone 3 lysine 6 acetylation H3K6me3 - histone 3 lysine 6 trimethylation H3K79ac - histone 3 lysine 79 acetylation H3K79me3 - histone 3 lysine 79 trimethylation H3K9ac - histone 3 lysine 9 acetylation H3K9me - histone 3 lysine 9 methylation H4 – histone 4 H4K20me – histone 4 lysine 20 methylation

H4R3me2s - histone 4 arginine-3 symmetric dimethylation hESC - human embryonic stem cell hFSK - human foreskin fibroblast hiPSC - human induced pluripotent stem cell hMSC: human mesenchymal stem cell i12F – induced with twelve factors i6F – induced with six factors IAP - intracisternal A particle ICR - imprinting control regions ICSI - intra-cytoplasmic sperm injection IF - immunofluorescence iGC - induced germ cell-like cell iPSC - induced pluripotent stem cell KSR - knock-out serum replacement LH - luteinizing hormone LIF - leukaemia inhibitory factor LINE - long interspersed nuclear element ƛMAX Exc – maximum excitation wave length ƛMAX Em - maximum emission wave length MACS - magnetic activated cell-sorting MD - Müllerian paramesonephric duct mESC - mouse ESC miPSCs - murine induced pluripotent cells miRISC - miRNA-induced silencing complex miRNA - microRNA mRNA - messenger RNA MSCI - meiotic sex chromosome inactivation MSUC - meiotic silencing of unpaired chromatin

ORF – open reading frames OSKM - OCT4, SOX2, KFL4, and cMYC reprogramming factors (also known as Yamanaka’s factors) OSKMV- OCT4, SOX2, KFL4, cMYC, and VASA reprogramming factors PBS - phosphate-buffered serum PCA – principal component analysis PCR - polymerase chain reaction PFA - paraformaldehyde PGC - primordial germ cell PGCLC - PGC-like cell PI - propidium iodide piRNA - protein interacting RNA PMSF - phenylmethylsulfonyl fluoride PR - positive regulatory PSC - pluripotent stem cell p-value - probability value qRT-PCR – quantitative real-time PCR RA - retinoic acid RNA - ribonucleic acid rpm - rotations per minute RS - round spermatid SAC - spindle assembly checkpoint SCF – stem cell factor SEM – standard error of mean siRNA - small interfering RNA Spc I - primary spermatocyte Spc II - secondary spermatocyte Spg - spermatogonium SSC - spermatogonial stem cell T - thymidine U - uracil UTR - untranslated region WD - Wolffian mesonephric duct

LIST OF GENES All the genes listed here refer to human annotated genes, although in many cases the text

refers to the murine homologue when the gene names are the same. The species name is

indicated only when the gene refers to other animal models.

ACR – acrosin

AID – activation–induced cytidine

deaminase, coded by AICDA gene

AKT – v–akt murine thymoma viral

oncogene homolog 1

ALK3 – bone morphogenetic protein

receptor, type IA, coded by BMPR1A

gene

APOBEC – apolipoprotein B mRNA

editing enzyme, catalytic polypetides

Argonaute proteins – they are the

catalytic components of the RNA–

induced silencing complex (RISC). Eight

members of this family have been

identified in humans, being classified in

two subfamilies: PIWI (PIWIL1, PIWIL2,

PIWIL3, and PIWIL4), and eIF2C/AGO

(hAGO1, hAGO2, hAGO3, and hAGO4).

BAX – BCL2-associated X protein

BLIMP1 – PR domain containing 1, with

ZNF domain (also known as PRDM1), B–

lymphocyte induced maturation protein

1

BMP – bone morphogenetic proteins

BMP2 – bone morphogenetic protein 2

BMP4 – bone morphogenetic protein 4

BMP8b – bone morphogenetic protein

BMPRII – bone morphogenetic protein

receptor, type II (serine/threonine

kinase)

BOLL – boule-like RNA-binding protein

(also known as BOULE)

BOULE – boule-like RNA-binding protein

(also known as BOLL)

BUB – budding uninhibited by

benzimidazole

BUB1 – BUB1 mitotic checkpoint

serine/threonine kinase

BUB3 – BUB3 mitotic checkpoint protein

BUBRI – BUB1 mitotic checkpoint

serine/threonine kinase B, coded by

BUB1B gene

CD117 – tyrosine–protein kinase

receptor (also known as SCFR or c–KIT)

CD49f – integrin alpha 6, coded by ITGA6

gene (also known as 6-integrin)

cd9 – CD9 molecule

CD90 – Thy-1 cell surface antigen (also

known as Thy-1)

Cerberus-like – cerberus 1 homolog

(Xenopus laevis) in M. musculus. The

human homologue is called DAN domain

family member 5, BMP antagonist

c-KIT – tyrosine-protein kinase receptor

(also known as CD117 or SCFR)

c-Kit-ligand – KIT ligand, coded by KITLG

gene (also known as stem cell factor,

SCF)

cMYC – v-myc avian myelocytomatosis

viral oncogene homolog

CYP26B1 – cytochrome P450, family 26,

subfamily B, polypeptide 1

DAZ – deleted in azoospermia 1, coded

by DAZ1 gene

DAZL – deleted in azoospermia-like

DICER – dicer 1, ribonuclease type III

DKK – dickkopf WNT signaling pathway

inhibitor 3

DMC1 – dosage suppressor of MCK1

homologue

DND1 – DND microRNA-mediated

repression inhibitor 1

DNMT: DNA (cytosine-5-)-methyl-

transferase

DNMT1 – DNA (cytosine-5-)-

methyltransferase 1

DNMT3A – DNA (cytosine-5-)-

methyltransferase 3 alpha

DNMT3B – DNA (cytosine-5-)-

methyltransferase 3 beta

DNMT3L – DNA (cytosine-5-)-

methyltransferase 3-like

DRSOHA – drosha, ribonuclease type III

FOXH1 – forkhead box H1

FRAGILIS – interferon induced

transmembrane protein 3 (also known as

IFITM3). Only in mouse

GDNF – glial cell derived neurotrophic

factor

GFRA1 – GDNF family receptor alpha 1

GLP – euchromatic histone–lysine N–

methyltransferase 1, coded by EHMT1

gene (also known as KMT1D)

H19 – H19, imprinted maternally

expressed transcript (non-protein coding)

HILI – piwi-like RNA-mediated gene

silencing 2; homologue to Piwil2 in

mouse (also known as PIWIL2)

HIWI – piwi-like RNA-mediated gene

silencing 1; homologue to Piwi in mouse

(also known as PIWI, MIWI)

HIWI2 – PIWIL4 piwi-like RNA-mediated

gene silencing 4; homologue to Piwil4 in

mouse (also known as PIWIL4, MIWI2)

HOX – homeobox gene

HOXA1 – homeobox A1



HOXB1 – homeobox B1

HOXB2 – homeobox B2

HOXD1 – homeobox D1

HOXD9 – homeobox D9

IGFR2 – Fc fragment of IgG, low affinity

IIa, receptor (CD32)

KLF4 – Krüppel-like factor 4 (gut)

KMT1D – euchromatic histone–lysine N–

methyltransferase 1, coded by EHMT1

gene (also known as GLP)

Lefty – left right determination factor 1

in M. musculus. The human homologue is

called LEFTY1

Let7 microRNA family – The lethal-7 (let-

7) gene was first discovered in the

nematode as a key developmental

regulator. The mature form of let–

7 family members is highly conserved

across species. In human genome, there

are four clusters: let-7a-1/let-7f-1/let-

7d (at chromosome 9); miR-125b1/let-

7a-2/miR-100 (at chromosome 11); miR-

99a/let-7c/miR-125b-2 (at chromosome

21); let-7g/miR-135-1 (at chromosome 3)

LIN28A – lin-28 homolog A (C. elegans)

MAD – mitotic arrest deficient

MADI – MAD1 mitotic arrest deficient-

like 1 (yeast)

MADII – MAD1 mitotic arrest deficient-

like 2 (yeast)

MESP1 – mesoderm posterior 1 homolog

MEST – mesoderm specific transcript

(also known as PEG1)

Mili – piwi-like RNA-mediated gene

silencing 2 (also known as Piwil2) in M.

musculus

MIWI – piwi-like RNA-mediated gene


(also known as PIWI, HIWI)

Miwi – piwi-like RNA-mediated gene


musculus

MIWI2 – PIWIL4 piwi-like RNA-mediated


mouse (also known as PIWIL4, HIWI2)

Miwi2 – piwi-like RNA-mediated gene


musculus

MLH1 – mutL homolog 1

MLH3 – mutL homolog 3

MPS1 – TTK protein kinase, coded by TTK

gene

Mvh – mouse vasa homolog in M.

musculus

NANOG – Nanog homeobox

NANOS – coded by nos gene in

Drosophila

NANOS3 – nanos homolog 3 (Drosophila)

NODAL – nodal growth differentiation

factor

NOTCH1 – notch 1

OCT4 – POU class 5 homeobox 1, coded

by POU5F1 gene

PEG1 – mesoderm specific transcript

(also known as MEST)

PIWI – piwi-like RNA-mediated gene


(also known as HIWI, MIWI)

Piwil1 – piwi-like RNA-mediated gene

silencing 1 (also known as Miwi) in M.

musculus

PIWIL2 – piwi-like RNA-mediated gene

silencing 2; homologue to Piwil2 in

mouse (also known as HILI)


silencing 2 (also known as Mili) in M.

musculus

PIWIL4 – PIWIL4 piwi-like RNA-mediated


mouse (also known as HIWI2, MIWI2)


silencing 4 (also known as Miwi2) in M.

musculus

PLZF – zinc finger and BTB domain

containing 16, coded by ZBTB16 gene

PRDM1 – PR domain containing 1, with

ZNF domain (also known as BLIMP1)

PRDM14 – PR domain containing 14

PRMT1 – protein arginine

methyltransferase 1

PRMT5 – protein arginine

methyltransferase 5

PRM1 – protamine 1

pumilio – pumilio [Drosophila

melanogaster (fruit fly)]

RAD51 – RAD51 recombinase (also

known as RECA)

REC8 – REC8 meiotic recombination

protein

RECA – RAD51 recombinase (also known

as RAD51)

RPL19 – ribosomal protein L19

SAMD1 – sterile alpha motif domain

containing

SCF – stem cell factor, coded by KITLG

gene (also known as, c–KIT ligand)

SCFR – tyrosine–protein kinase receptor

(also known as c–Kit or CD117)

SMAD2 – SMAD family member 2

SMAD5 – SMAD family member 5

SMC1B – structural maintenance of

chromosomes 1B

SNAI1 – snail family zinc finger 1

SOX2 – SRY (sex determining region Y)–

box 2

SOX9 – SRY (sex determining region Y)–

box 9

SPO11 – SPO11 meiotic protein

covalently bound to DSB

SRY – sex determining region Y

SSEA1 – stage-specific embryonic antigen

1; coded by fucosyltransferase 4 (alpha

(1,3) fucosyltransferase, myeloid-specific)

FUT4 gene (also known as CD15)

STAG3 – stromal antigen 3

STELLA – developmental pluripotency

associated 3, coded by DPPA3 gene

STRA8 – stimulated by retinoic acid 8

SYCE1 – synaptonemal complex central

element protein 1

SYCE2 – synaptonemal complex central

element protein 2

SYCP1 – synaptonemal complex protein 1



TBX3 – T-box 3

TBX6 – T-box 6

TET – ten-eleven translocation

methylcytosine dioxygenase

TET1 – tet methylcytosine dioxygenase 1



TFAP2C – transcription factor AP–2

gamma (activating enhancer binding

protein 2 gamma; also known as AP2ɣ)

Thy-1 – Thy-1 cell surface antigen (also

known as CD90)

TNP1 – transition nuclear protein 1

TNP2 – transition nuclear protein 2

TR-KIT – truncated form of the tyrosine

kinase receptor c–kit

TUDOR – gene described in Drosophila,

tudor domain proteins function as

molecular adaptors during development,

most notably in the Piwi–interacting RNA

pathway, but also in other aspects of

RNA metabolism

Uhrf – ubiquitin-like, containing PHD and

RING finger domains 1 in M. musculus

WNT3 – wingless-type MMTV integration

site family, member 3

α6-integrin – integrin alpha 6, coded by

ITGA6 gene (also known as CD49f)

β1-integrin – integrin, beta 1 (fibronectin

receptor, beta polypeptide, antigen CD29

includes MDF2, MS

- 1 -

INDEX OF CONTENTS Page

1. INTRODUCTION..……………………………………………………………………….. 5

1.1 THE GERM LINE IN MAMMALS………………………………………………… 7

1.1.1 Germ line development.…………………………………………………… 7

1.1.1.1 Specification and migration.…………………………………………. 8

1.1.1.2 Sex determination, meiotic entry, and maturation……… 10

1.1.2 Control of gene expression during germ line development.. 13

1.1.2.1 BMP signaling………………………………………………………….…… 14

1.1.2.2 The role of PRDM1 and PRDM14 during specification… 15

1.1.2.3 Post-transcriptional regulation by RNA-binding proteins 17

1.1.2.4 Determinants of germ cell proliferation and survival……. 21

1.1.3 EPIGENETIC CHANGES DURING GAMETOGENESIS ……………. 22

1.1.3.1 Methylome reprogramming…………………………………………. 23

1.1.3.2 Chromatin reprogramming and meiotic control…………… 29

1.2. MALE GAMETOGENESIS………….………………………………………………. 32

1.2.1 The spermatogonial stem cell microenvironment ……………. 32

1.2.1.1 Spermatogonial stem cell culture…….………………………….. 34

1.2.1.2 Germ cell transplants.…….……………………………………………. 36

1.2.2 The stages of spermatogenesis….……………………………………… 36

1.2.2.1 The proliferative phase.……………………………………………….. 37

1.2.2.2 Meiosis………..………………………………………………………………. 38

1.2.2.3 Spermiogenesis……………………………………………………………. 40

1.3 IN VITRO GAMETE GENERATION.…..………………………………………… 41

1.3.1 Germ cell derivation from pluripotent stem cells……………… 45

1.3.3 Direct reprogramming………………………………………………………. 49

2. HYPOTHESIS………………………………………………………………………………. 51

- 2 -

3. OBJECTIVES……………………………………………………………………………….. 55

3.1 GENERAL OBJECTIVE…….…………………………………………………………. 57

3.2 SPECIFIC OBJECTIVES……………………………………………………………….. 57

4. EXPERIMENTAL DESIGN…………………………………………………………….. 59

5. METHODS………………………………………………………………………………….. 63

5.1 GENE SELECTION, ISOLATION, AND CLONING...……………………… 65

5.1.1 Gene selection………………………………………………………………….. 65

5.1.2 Gene isolation and cloning….……………………………………………. 66

5.1.2.1 Isolation…..…………………………………………………………………… 66

5.1.2.2 pENTRTMTOPO® vector cloning……………………………………… 69

5.1.2.3 Destination vector cloning…...………….………………………….. 70

5.2 REPROGRAMMING FACTOR OVEREXPRESSION AND CELL

CULTURE OPTIMIZATION..…………………………………………………………….. 71

5.2.1 Somatic primary cultures………………………………………………….. 71

5.2.2 Lentiviral transduction……………………………………………………… 71

5.2.3 Culture conditions……………………………………………………………. 73

5.3 CHARACTERIZATION OF GERM CELLS INDUCED IN VITRO.………… 74

5.3.1 Transcriptomic characterization……………………………………….. 74

5.3.1.1 Expression array…..………………………………………………………. 74

5.3.1.2 Quantitative real-time PCR..…………………………………………. 76

5.3.2 Epigenetic characterization by bisulfite sequencing.…………. 76

5.3.3 Meiotic Progression Analysis……………………………………………. 78

5.3.3.1 SYCP3 staining……..…..………………………………………………….. 78

5.3.3.2 Ploidy analysis: DNA-sorter and fluorescence in situ

hybridization..………………………………………………………………………….. 79

5.3.4 Immunofluorescence………………………………………………………… 80

- 3 -

5.4 XENOTRANSPLANT ASSAYS …………………………………………………….. 81

5.4.1 Mouse strain selection …………….……………………..………………. 81

5.4.2 Busulfan sterilization...…..………………………………………………… 82

5.4.3 Preparation of donor cells.…………..…………………………………… 83

5.4.4 Xenotransplantation…………………………………………………………. 84

5.4.5 Xenotransplanted tissue analysis…..……….………………………... 86

5.5 STATISTICAL EVALUATION OF THE RESULTS....…………………………. 88

6. RESULTS……………………………………………………………………………………. 89

6.1 HUMAN FORESKIN FIBROBLAST REPROGRAMMING USING

TWELVE GERM LINE FACTORS INDUCES THE FORMATION OF

PUTATIVE GERM CELL-LIKE CELLS.…………………………………………………. 91

6.1.1 Ectopic expression of transduced genes……………………………. 91

6.1.2 Reprogrammed cell morphological changes and the

establishment of cell culture conditions.…………………………………… 92

6.2 TWELVE FACTOR-REPROGRAMMED CELLS HAVE A GERM LINE-

LIKE TRANSCRIPTOMIC AND EPIGENETIC PROFILE…………………………. 94

6.2.1 Transcriptomic analysis by expression array and

quantitative real-time PCR........................................................... 94

6.2.2 H19 and PEG1 imprinting control region bisulfite

sequencing.................................................................................. 97

6.2.3 Phenotypic characterization of i12F clumps........................ 100

6.3 TWELVE FACTOR-REPROGRAMMED CELLS CAN PROGRESS

THROUGH MEIOSIS IN VITRO........................................................... 100

6.3.1 Meiotic spread analysis by SYCP3 staining........................... 100

6.3.2 Ploidy analysis by fluorescence in situ hybridization............ 103

- 4 -

6.4 GERM CELL-RELATED GENE SCREENING REVEALS OPTIMAL

COMBINATIONS THAT INDUCE MEIOTIC PROGRESSION AS THE

MAIN READ-OUT............................................................................. 108

6.4.1 Six factor-treated cells behave like twelve factor-induced

germ cell-like cells at the transcriptomic, epigenetic, and meiotic

levels………………………………………………………………………………………… 113

6.5 DIRECT GERM LINE REPROGRAMMING WITH TWELVE AND SIX

FACTORS CAN BE REPRODUCED IN HUMAN MESENCHYMAL CELLS

FROM BONE MARROW ORIGIN........................................................ 119

6.5.1 Transcriptomics of mesenchymal iGCs................................. 120

6.5.2 Mesenchymal-iGCs progress through meiosis and form

haploid cells................................................................................ 122

6.6 XENOTRANSPLANTATION DEMONSTRATES THAT iGCs ARE

ABLE TO COLONIZE THE GERMINAL EPITHELIUM OF SEMINIFEROUS

TUBULES.......................................................................................... 125

6.6.1 Flow cytometry results........................................................ 131

6.6.2 Immunohistological analysis............................................... 133

7. DISCUSSION………………………………………………………………………………. 137

8. CONCLUSIONS…………………………………………………………………………… 159

9. APPENDIX …………………………………………………………………………………. 163

10. REFERENCES ………….………………………………………………………………… 173

- 5 -

1. Introduction

Anemone, Daffodil, Plum

Sincere, respect, pure and true

1. Introduction

- 7 -

1.1 THE GERM LINE IN MAMMALS

In sexually reproducing organisms germ cells allow the life-cycle to continue

through generations. Specifically, in mammals, the germ line is clearly

separated from somatic lineages during early embryonic development. In

fact, according to the murine model, the germ line in mammals is formed at

gastrulation from a small number of founder cells called primordial germ

cells (PGCs) which escape their somatic fate to acquire a germinal role in

order to complete meiosis and form mature haploid gametes. Thus, these

are the only two cell types (i.e. oocytes and sperm cells) able to generate a

complete organism after fecundation.

1.1.1 GERM LINE DEVELOPMENT

The whole process of germ line development takes place sequentially

during early embryo development when PGCs change their gene expression

pattern and undergo an epigenetic reprogramming process in which they

proliferate and migrate from the posterior hindgut to the forming gonad.

Next, germ cells continue their development by differentiation, according

to the embryonic sex, into oocytes or sperm cells, a process that finishes at

puberty when they complete their maturation. The germ-line

developmental process involves global epigenetic reprogramming

mechanisms that `turn off’ the somatic program and `switch on’ the germ

line program to acquire pluripotent properties, `erasing’ the epigenetic

memory that would otherwise drive them towards a somatic fate. This

process, which also entails chromatin remodeling, is essential for the zygote

to establish totipotency and is widely regulated at both the genetic and

epigenetic levels.

Direct reprogramming of human somatic cells to meiotic germ-like cells

- 8 -

1.1.1.1 SPECIFICATION AND MIGRATION

In mice, PGCs can first be identified at embryonic day 6.25 (E6.25), when

some cells in the proximal epiblast start to express Prdm1 (Ohinata et al.,

2005) which is induced by the BMP4 and BMP8b secreted by

extraembryonic ectoderm (Fig.1.1A).

Prdm1 suppresses the somatic gene expression program, allowing the

expression of germ-cell specific markers STELLA (coded by the Dppa3 gene)

and Nanos3 as well as the pluripotency markers Sox2, Oct4, and Nanog

(Saga, 2008, Suzuki et al., 2008). Prdm1 expression is enhanced by LIN28A, a

microRNA-binding protein that binds the microRNA Let7 (West et al., 2009),

preventing binding to Prdm1 messenger RNA (mRNA). Prdm14 (which is

also activated by BMP) acts synergistically with Prdm1 by inducing the re-

acquisition of pluripotency and by initiating epigenetic reprogramming

(Kurimoto et al., 2008) and together with Nanog maintains pluripotency

networks (Ma et al., 2011, Mitsui et al., 2003). In mice at E7.5 alkaline

phosphatase (AP) positive PGCs are located in the posterior area of the

primitive ridge, in the base of the allantoides (Culty, 2009). They start

expressing Fragilis (also known as Ifitm3) and Stella (Saitou et al., 2002) and

maintain Oct4, Nanog, and AP expression. In humans, the specification

process occurs at two to three weeks of development (Ginsburg et al.,

1990, Mitsui et al., 2003).

1. Introduction

- 9 -

After specification (E8.5), PGCs migrate and proliferate through the

subjacent endoderm toward the gonadal ridges (Lawson and Hage, 1994,

Godin et al., 1990) located next to the primitive hindgut. They pass through

the mesoderm (E9.5) and start to bilaterally migrate towards the gonadal

ridges, contributing to gonadal formation (E10.5-11.5; Fig. 1.1B), which in

humans corresponds to gestation weeks four to five (Bendel-Stenzel et al.,

1998, Richardson and Lehmann, 2010). Their survival and migration are

maintained by the apoptotic suppression role of Nanos3 (Suzuki et al.,

2008). At the gonadal ridge PGCs proliferate and form gonocytes (E11.0-

11.5), characterized by their rounded morphology and low nucleus to

cytoplasm ratio (Donovan et al., 1986). Form this point, germ cells change

their genetic expression program and epigenetic profiles, starting to

express key genes for survival and maturation like DAZL and VASA

(Castrillon et al., 2000a, Noce et al., 2001, Tanaka et al., 2000). Other genes

from the DAZ (deleted in azoospermia) family, such as DAZ2 or BOULE (also

FIG. 1.1. Specification and migration in mouse PGCs. Modified from (Saitou et al., 2012) A) Specification and migration of the developmental embryo. PGC precursors at embryonic day 6.5(E6.5) and PGCs in embryos from E6.5 to E12.5 are shown in green, and the direction of their migration is indicated by green arrows; B) Detailed view of PGC migration at E9.5-E10.5. The direction of migration is indicated by green arrows; Al, allantoides; AVE, anterior visceral endoderm; DE, distal endoderm; DVE, distal visceral endoderm; EM, embryonic mesoderm; Epi, epiblast; ExE, extraembryonic ectoderm; ExM, extraembryonic mesoderm; Sm, somite; VE, visceral endoderm.

A B


- 10 -

known as BOLL) in humans, have also been demonstrated to be necessary

for the proper development of the germ line. In mouse, Daz is a master

regulator of spermatogenesis, while Dazl seems to be critical for proper

entry into meiosis (Ruggiu et al., 1997).

1.1.1.2 SEX DETERMINATION, MEIOTIC ENTRY, AND MATURATION

Germ cell sex determination depends on SRY gene expression, which is

codified by chromosome Y (Byskov, 1986). In males, SRY activates SOX9

expression which induces gonadal cells to differentiate into Sertoli cells. In

turn Sertoli cells induce the bi-potential gonad to differentiate by causing

the degeneration of Müllerian ducts as response to anti-müllerian hormone

(Sinclair et al., 1990, McLaren, 1988, Koopman et al., 1990, Burgoyne,

1988). At the same time, the Sertoli cells recruit cells from the

mesonephros and coelomic epithelium to form the testicular cords (Kanai

et al., 2005, Barsoum and Yao, 2006). In the absence of SRY and/or SOX9,

i.e. in females, the Müllerian ducts give rise to the female gonad while the

Wolf ducts degenerate (Fig. 1.2A).

After their sexual specification, male and female germ cells follow different

maturation patterns. During embryonic development, in the female gonad

germ cells enter meiosis and stop at prophase I, at E13.5 in mouse and

week 12 in humans, and only continue after activation of the hypothalamic-

pituitary axis at puberty (Monk, 1981, Monk and McLaren, 1981, Gondos et

al., 1986). However, in males the germ cells (now called gonocytes)

continue dividing by mitosis until they reach the pre-spermatogonia stage,

at which point mitosis stops and meiosis is not initiated until puberty

1. Introduction

- 11 -

FIG. 1.2. Sexual determination. A) Gonad sexual differentiation. In males, SRY expression

induces SOX9 which, via AMH, causes MD degeneration. In females, WD degenerates. B) PGC

sexual determination by niche induction. RA production induces entry into meiosis via STRA8

in females, with oocytes arresting in meiosis I. In males, Sertoli cells metabolize RA, preventing

entry into meiosis until puberty, when spermatogenesis initiates. MD, Müllerian

paramesonephric duct; WD, Wolffian mesonephric duct; AMH, anti-müllerian hormone; RA,

retinoic acid.

BIPOTENTIAL GENITAL RIDGES

MD

WD

Oviduct

Ovaries

Uterus

Testis

Vas deferens

Seminal vesicle

STRA8 expression

Meiosis initiation

Meiotic arrest(Prophase I)

Meioticprogression

Sertoli cells

RA degradation

Mitoticarrest

Spermatogenesis

PUBERTY

SRY/SOX9expression

AMH

RA PRODUCTION

♀

♂

CYP26B1

A B

(McLaren, 1984, Goto et al., 1999).

The induction of meiosis is similar in both sexes: retinoic acid (RA) secreted

by the mesonephros during PGC migration plays a critical role. In the ovary

RA induces meiosis by promoting Stra8 gene expression (Bowles et al.,

2006), whereas SRY induces RA degradation through Cytochrome P450

CYP26B1 expression in Sertoli cells (Fig. 1.2B). Thus, RA degradation

prevents gonocytes from entering meiosis until puberty when hormonal

changes cause CYP26B1 downregulation, preventing Sertoli cells from

metabolizing RA (Koubova et al., 2006).


- 12 -

Meiosis is a unique and crucial process in sexual reproduction. It comprises

a reductive cellular division in which four haploid cells are formed from one

diploid cell. The first meiotic division (meiosis I) is preceded by one round of

DNA replication and begins with a prophase divided into four stages

(leptotene, zygotene, pachytene, diplotene). Then, two cells are formed

with one copy of each pair of homologous chromosomes. At the second

meiotic division (meiosis II) there is no DNA duplication, and sister

chromatids separate to form four haploid cells (FIG. 1.3).

FIG. 1.3. Female and male meiosis. A) At oogenesis, meiotic divisions are asymmetric, with

three final polar bodies and one haploid oocyte. Meiosis only completes upon fecundation. B)

In spermatogenesis, from a diploid spermatogonium four haploid spermatocytes are

produced. These spermatocytes continue their maturation by a process called spermiogenesis.

C) Prophase I stages. The axial element of the synaptonemal complex SYCP3 throughout

prophase I is shown in red.

LEPTOTENE ZYGOTENE

PACHYTENE DIPLOTENE

Spermatogenesis

MITOSIS

MITOSIS

MEIOSIS I

MEIOSIS II

SPERMIOGENESIS

Oogenesis

PGC (2n)

Oogonium/ Spermatogonium

(2n)

Oocyte/ primary

spermatocyte

Oocyte/ Secondary

spermatocyte(n)

PROPHASE IA B C

Fecundation

1. Introduction

- 13 -

Meiosis allows genetic recombination to occur in order to create new

genetic variants which can be inherited by the next generation. This

happens when recombination events occur in the prophase of meiosis I.

Homologous chromosomes become paired, thus allowing exchange of

genetic material via non-sister homologous chromatids at recombination

spots or synapses (Fig.1. 3C). These loci are formed by the synaptonemal

complex comprising the highly phylogenetically and evolutionarily

conserved SYCP1, SYCP2, and SYCP3 proteins. Recombinases such as DMC1

repair the DNA double strand breaks formed during recombination events

(Yoshida et al., 1998); all these processes are highly controlled because

recombination mistakes would otherwise cause meiotic arrest (Pittman et

al., 1998).

As meiosis moves forward, germ cells maturate acquiring the rest of their

features. Should be noted that whilst oocytes finalize meiosis at

fecundation (Fig. 1.3A) spermatozoa continue their maturation after

finishing meiosis in a process called spermiogenesis. Their nuclei

condensate, they lose all their cytoplasm and acquire motility (Fig. 1.3B).

1.1.2 CONTROL OF GENE EXPRESSION DURING GERM LINE DEVELOPMENT

Germ line development is controlled by a unique three-step genetic

expression program: Firstly, somatic genetic program suppression is

required, then the acquisition of pluripotency, and finally reprogramming to

a unique and non-restrictive epigenome (Leitch and Smith, 2013, Saitou,

2009). During these early stages, changes are similar in female and male

germ cells. PGCs lose the vast majority of methylation marks and chromatin


- 14 -

modifications associated with somatic gene downregulation and

pluripotency factors activation. Hence germ cells are able to initiate zygote

formation and post-fecundation development. As gametogenesis goes

forward, pluripotency gene expression decreases and the germ cell-specific

expression program activates. This process is highly controlled at both the

genetic and epigenetic levels, where germ line specific RNA-regulating

proteins also have a specific role which is essential for completing meiosis

and giving rise to a new generation.

1.1.2.1 BMP SIGNALING

PGC specification occurs very early in embryo development; in mouse it is

initiated by a BMP4/8b signal from the extraembryonic ectoderm which

activates PRDM1 (Fig. 1.4). Anteroposterior axis signals from the anterior

visceral endoderm (AVE; i.e. LEFTY1 vs. NODAL, DKK vs. WNT3, or

CERBEROUS-LIKE vs. BMP) define a germ line fate to proximal epiblast. At

around E5.5 epiblast cells become competent to respond to BMP4 signaling

through NODAL and WNT3. At this moment, NODAL signaling, mediated by

SMAD2/FOXH1, specifies distal visceral endoderm cells to send anti-

posteriorization signals. Concomitantly, signals from the extraembryonic

ectoderm (ExE), including BMP8b, prevent differentiation of proximal to

distal visceral-endoderm, thus limiting anti-posteriorization events. At

about E6.0-E6.5, distal visceral-endoderm moves anteriorly to form anterior

visceral endoderm and the subpopulation of epiblast cells that receives the

highest amount of BMP4 signaling becomes specified to PGCs and starts to

express Prdm1 and Prdm14 (Saitou, 2009). BMP4 induces Prdm1 and

Prdm14 expression in a dose-specific manner through the Alk3 complex and

1. Introduction

- 15 -

BMP type II receptors (mainly BmprII) via SMAD1 and SMAD5 in the

epiblast. BMP2 produced by visceral endoderm, acts in a similar way on

PRDM1, but is not as efficient as BMP4. BMP8b controls anteroposterior

development, preventing inhibitory signals against germ line specification

from the AVE. Thus, three BMP signals act in a different but cooperative

way to ensure sufficient exposure to epiblast BMP-SMAD signaling to

specify a germ lineage fate (Saitou, 2009, Saitou et al., 2002).

1.1.2.2 THE ROLE OF PRDM1 AND PRDM14 DURING SPECIFICATION

The transcriptional repressor PRDM1 is considered to be the master

regulator of germ line specification in mice. Prdm1 expression starts at

E6.25 (Ohinata et al., 2005) in a group of 40 cells located in the posterior

epiblast by AVE agonist signaling. PRDM1 is essential for suppressing the

somatic program and creating the epigenetic status necessary for PGC-

related gene expression (Fig. 1.4). PRDM1 represses almost all of the

downregulated genes in PGCs and activates some of the upregulated ones

(Lehmann, 2012). It also associates with PRMT5, an arginine methyl

transferase, through its positive regulatory (PR) domain to control the

symmetrical dimethylation of H2A and H4 at arginine 3 (H2A/H4R3me2s)

which generates a signal to inhibit the somatic program. After E11.5 this

signal is lost as PGCs become epigenetically reprogrammed (Ancelin et al.,

2006).

After repression of the somatic-program PRDM1 together with PRDM14

promote epigenetic reprogramming so that a pluripotency status can be

reacquired (Fig. 1.4). Prdm14 is expressed in the morula and the inner cell


- 16 -

FIG. 1.4. Murine PGC specification pathway. Signaling by BMPs activates PRDM1 and PRDM14 expression through SMAD. In the absence of LIN28A, the mature form of microRNA Let7 (miR-let7) blocks PRDM1 translation. LIN28A binds these pri-miRNAs/ pre-miRNA hairpin loops, preventing their processing, hence enabling PRDM1 translation. PRDM1, through PRMT5/7 and TFAP2C, acts synergistically with PRDM14 by downregulating differentiation and upregulating specific germ line gene expression. Modified from (Saitou and Yamaji, 2010).

BMP4

SMAD1/5

PRDM1(BLIMP1)

PRDM14

Hoxclusters

Mesodermalgenes: Snai1, Mesp1, Tbx3, Tbx6

Cell cycleregulators

Dnmt3bDnmt3a Np95Glp

Sox2Dnd1KitStellaNanos3E-CadherinFragilis

miR-let7

PRDM1(BLIMP1)

differentiation

H2A/ H4me2s PGC

specification

LIN28A

PRMT5/7TFAP2C

Prdm1(Blimp1)

Prdm1(Blimp1)

(SMAD4)

PRMT5/7TFAP2C

mass cells, but it disappears during development and is expressed again in

PGCs only between E6.5 and E13.5-E14.5, unlike Prdm1 which can be

expressed in other cell types (Saitou and Yamaji, 2010). Initially, Prdm14

expression is independent of PRDM1, but its maintenance strictly depends

on PRDM1 (Kurimoto et al., 2008, Saitou and Yamaji, 2010).

Both PRDM1 and PRDM14 repress de novo DNA-methylases Dnmt3a and

Dnmt3b, as well as Uhrf, another component of the DNA-methylation

1. Introduction

- 17 -

maintenance-machinery. PRDM14 is essential for epigenetic

reprogramming due to its ability to reactivate Sox2 expression and repress

the histone-methyl-transferase Glp. This repression directly affects DNA-

methylation machinery by two mechanisms (dependent and independent)

to other chromatin modifications, mainly histone 3 lysine 9 dimethylation,

(3K9me2; Hackett et al., 2012).

Tcfap2C (or AP2ɣ) is another critical element which is downstream to

PRDM1 (Fig. 1.4); its expression starts at E7.5 after PGC specification and is

maintained during gonocyte migration until they reach the gonadal ridge

when its expression stops at E13.5. TCFAP2C prevents mesodermal

differentiation and enhances the germ cell program (Weber et al., 2010).

Because PGCs emerge in the context of mesodermal induction, TCFAP2C

repression is critical.

1.1.2.3 POST-TRANSCRIPTIONAL REGULATION BY RNA-BINDING PROTEINS

Germ line RNA-binding proteins prevent somatic differentiation and control

many cellular functions, such as germ cell specification, sexual identity,

proliferation, survival, migration, and regulation of transposable elements.

These proteins are associated in large RNA-protein complexes called P-

bodies (or nuage). The RNA-binding protein group, including VASA, NANOS,

PUMILIO, DND1, DAZL, PIWI, and TUDOR, is highly conserved and present in

different stages of germ line development (Lehmann, 2012).

LIN28A is a RNA-binding protein that acts as an upstream regulator of

PRDM1 (West et al., 2009) by inhibiting Let7 family microRNA maturation

(Fig. 1.4), whereas Let7 is a microRNA that binds to the 5’-untranslated


- 18 -

FIG.1.5. Gene expression changes during germ line development. A) Germ line related gene expression; B) male gametogenesis control by RNA-binding proteins; C) male gametogenesis control by piRNA.

E8.5 E10.5 E12.5 E15.5

Germ cell migration

Prophase I

E7.5

PGC specification

MEIOSISMeiosis arrest

(female)Sex determination

FRAGILIS

PRDM1

STELLA

c-KIT

OCT4

SYCP1

SYCP3

C-MOS, GDF9 (only oocytes)

OCT4

ACR, TNAP, TEKT1, PRM1 (only sperm)

Let7 family

DAZL

VASA

LIN28A

MILI

MIWI2 MIWIRepetitive piRNA Non-repetitive piRNA (only

sperm)

(onlysperm)

A

B

C

Mitosis arrest(male)

region (5’UTR) sequence of Prdm1 mRNA, blocking its translation and

preventing germ line development (West et al., 2009). By inhibiting Let7

maturation and thus activating Prdm1, LIN28A is involved in PGC

specification (Fig. 1.5B). Indeed, LIN28A, together with OCT4, SOX2, and

NANOG are sufficient to reprogram human fibroblasts into induced

pluripotent stem cells (iPSCs; Saitou and Yamaji, 2010), and seems to be

involved in the main core of the pluripotency network.

Genes from the DAZ family form another group of RNA-binding proteins

involved in germ line development (Ruggiu et al., 1997) which are

expressed in migrating PGCs until the spermatocyte I stage in males (Fig.

1.5B).

1. Introduction

- 19 -

In humans this family is composed of the autosomal genes BOULE and

DAZL, and the DAZ gene cluster, encoded by the Y chromosome. In mouse,

Daz acts as a master regulator of spermatogenesis, whilst Dazl is critical for

meiosis: in knock-out mouse models apoptosis increases at the beginning of

meiosis. (Ruggiu et al., 1997). Loss of BOULE gene function results in

azoospermia with primary defects at the meiotic transition(Kee et al.,

2006).

VASA is an important RNA-binding protein required for germ cell

maturation and their proper entry into meiosis. Similar to Daz family genes,

it is highly conserved in mammals and is located in perinuclear, electron-

dense structures known as germplasm which are enriched in RNA and other

RNA-binding proteins (Castrillon et al., 2000b). In humans and mice it is

codified by the DDX4 gene, a member of the DEAD-box family, and

translates an ATP-dependent RNA-helicase with several functions in germ

cell lineages, including proliferation, differentiation, and transposable

element silencing (Lehmann, 2012). It is expressed specifically by germ cells

(Fig. 1.5B) when they arrive to the genital ridge and its expression continues

until peri-meiotic stages (Lasko and Ashburner, 1988). Interestingly, mouse

knock-out models for mouse vasa homolog (Mvh) show male infertility, but

in Drosophila the phenotype is the opposite, causing female sterility

(Tanaka et al., 2000).

In contrast, Piwi, Tudor, and Argonaute form part of the PIWI interacting

RNA (piRNA) family and control transposable element expression. piRNAs

are small non-coding RNAs defined by their ability to bind Argonaute family


- 20 -

BOX 1. SMALL NON-CODING RNA BIOGENESIS Small non-coding RNAs are single stranded

RNAs that specifically bind to a target 3’UTR mRNA sequence, inhibiting their translation or inducing their degradation. There are three main types in mammals: microRNAs (miRNAs), small interfering endogenous RNAs (endo-siRNAs) and PIWI-interacting RNAs (Suh and Blelloch, 2011).

miRNAs are transcribed by RNA-

polymerase II and are recognized by the RNase

DROSHA, which cuts them into hairpin loops (Han

et al., 2009) which are recognized by DICER, a

cytoplasmic endonuclease that produces the

double stranded mature miRNA. siRNAs are

derived from large double-stranded RNAs and are

directly processed by DICER to produce multiple

copies (Fabian et al., 2010). miRNAs and siRNAs act

similarly: they bind to the miRNA-induced silencing

complex (miRISC) where proteins such as

Argonaute bind to mediate post-transcriptional

regulation of target mRNA by sequence

complementarity (Suh and Blelloch, 2011).

piRNAs do not require DICER for their

processing. They are generated from large single

strand precursors manly codified by intergenic

repetitive sequences (Aravin et al., 2008, Suh and

Blelloch, 2011). According to the ping-pong model,

Mili cuts

primary piRNAs to define a 5’end that will be

recognized by Miwi2. Then, Miwi2 cuts the other

precursor strand, generating a new 5’end to bind

Mili, leading to an amplification loop (Suh and

Blelloch, 2011). Most of the details of this model

are not well understood. The ping-pong cycle only

explains repetitive sequence-derived piRNA

biogenesis, while there is a second class of piRNA,

derived from intergenic sequences and associated

with late spermatogenesis that is not yet

understood.

Transposon transcripts

MILI

MIWI25’ 3’

5’3’

TRANSPOSON mRNA

MILI MIWI2

5’ 3’

SENSE PRIMARY

MILIMIWI2

5’ 3’5’3’

SENSEANTISENSE

TRANSCRIPT

Ping-pong cycle

DNA methylation

?

U

U

A

MILI MIWI23’ 5’

ANTISENSE SECONDARY

A

A

U

Slicerclaveage

Slicerclaveage

members (including Miwi/Piwil1, Miwi2/Piwil4, and Mili/Piwil2 in mouse;

Box 1; Suh and Blelloch, 2011, Aravin et al., 2008).

During spermatogenesis two types of piRNA are expressed (Fig. 1.5C). The

first, with a repetitive nature, are expressed before pachytene in prophase

I, while the second is non-repetitive and its expression is upregulated after

zygotene in prophase I. They both interact with Mili (the murine homolog

for HILI in humans) but the first type does so in conjunction with Miwi2 (the

1. Introduction

- 21 -

murine homolog for HIWI2 in humans) while the second type interacts with

Miwi (the murine homolog for HIWI in humans). Deletions in Mili and/or

Miwi2 lead to arrest in meiosis I whilst mice defective for Miwi suffer arrest

in meiosis II at the round spermatid stage (Kuramochi-Miyagawa et al.,

2010, Carmell et al., 2007). There are also some data indicating that VASA is

necessary for proper piRNA ping-pong amplifying-cycles because Mvh

knock-out mice have the same phenotype as Miwi, Mili, and Miwi2 knock-

out mice (Kuramochi-Miyagawa et al., 2010, Medrano et al., 2013).

piRNAs act to promote the de novo methylation of transposons and other

genomic repetitive sequences, thus promoting their silencing and, as a

consequence, genomic stability during meiosis (Suh and Blelloch, 2011,

Fabian et al., 2010, Aravin et al., 2008). This function is particularly relevant

in germ cell lineages in order to prevent somatic differentiation and to

protect the future generation’s genome throughout the cell cycle.

Nevertheless the mechanism by which piRNA acts on the DNA methylation

machinery remains unknown (Lehmann, 2012).

1.1.2.4 DETERMINANTS OF GERM CELL PROLIFERATION AND SURVIVAL

Specified PGCs start to express AP, STELLA (Dppa3) and to downregulate

Hox genes (i.e. Hoxa1, Hoxa2, Hoxa3, Hoxb1, Hoxb2, Hoxd1 and Hoxd9), as

well as other mesodermal-program genes (i.e. Snai1, Tbx3, Tbx6, Mesp1,

etc.; Kurimoto et al., 2008). Hence, PGC precursors that previously had a

somatic fate, lose their mesodermal features to recover their pluripotency

potential and activate transcriptional regulators which are specific for germ

line lineages (Fig. 1.5A). They upregulate genes required for germ line


- 22 -

specification, i.e. Prdm1, Dppa3, Ifitm3, Dnd1, and Mvh, and characteristic

of pluripotency, i.e. Sox2 and Nanog (Kurimoto et al., 2008, Saitou et al.,

2012).

During PGC migration and proliferation, the transmembrane receptor c-Kit

and its ligand stem cell factor (SCF) regulate their proliferation and survival,

probably via AKT/mTOR/BAX. At later stages (E9.5) OCT4 and NANOG are

also critical for PGC survival (Fig. 1.5A; Arnold and Robertson, 2009). PGCs

express relatively high levels of c-KIT (Donovan et al., 1986) which plays a

dual role in male fertility control: firstly, it is expressed and is functional in

postnatal spermatogonia, and secondly, TR-KIT, an intra-cellular protein

encoded by an internal promoter, accumulates during spermatogenesis. At

the beginning of meiosis, c-KIT expression ceases but TR-KIT maintains its

levels until postmeiotic stages. The SCF receptor (c-Kit-ligand) also plays a

very important role in regulating PGC proliferation, survival, and migration.

PGC differentiation into gonocytes induces marker changes: SSEA1 and

OCT4, together with c-KIT and AP activity, continues but reduces as

gonocyte β1- and α6-integrin expression levels increase; c-KIT expression

later returns in spermatogonia.

1.1.3 EPIGENETIC CHANGES DURING GAMETOGENESIS

Just after specification, PGCs have a stable epigenetic status, involving DNA

methylation and X chromosome inactivation in females, which establishes

an epigenetic barrier for the acquisition of totipotency. When PGCs enter

the gonadal ridges they undergo global epigenetic methylation mark

erasure (in two well-defined waves of demethylation) and chromatin is

1. Introduction

- 23 -

remodeled, a process that ends at around E13.5 reaching a basal (the most

naïve possible in mammals) epigenetic status. This is a different

reprogramming process to that which occurs during early embryo

development where imprinted genes and other loci stay methylated and

polycomb-based chromatin modifications remain. PGCs undergo such

profound reprogramming events because they must overcome multiple

epigenomic barriers, mainly due to the inductive mechanism of germ line

specification in somatic-fate cells instead of an inherited germplasm, as in

Drosophila (Hackett et al., 2012).

1.1.3.1 METHYLOME REPROGRAMMING

The methylome is defined as the set of covalent modifications (formed by

the addition of methyl groups to DNA at position 5 of the cytosine [C]

pyrimidine ring) in a certain cell at a given time (Box 2). DNA methylation

occurs mainly in the C located in Cytosine-Guanine (CpG) dinucleotides and

are usually grouped into CpG islands (CGIs). These CpG dinucleotides are

found in only approximately 1% of mammal genomes, generally at

promoter regions, 5’UTRs, and first exons, and are involved in

transcriptional regulatory functions (Portela and Esteller, 2010). An increase

of methylation is usually associated with transcriptional repression and CGIs

in the promoter regions of each particular cell lineage acquire a tissue-

specific methylation pattern throughout development, a process which is

usually associated with transcriptional repression and an increase in

methylation.; however, many important DNA methylation marks are also

located in repetitive genomic regions, i.e. transposable elements, which can

involve up to 45% of the genome. These sequences propagate by randomly


- 24 -

BOX 2. DNA METHYLATION, CONCEPTS, AND ASSOCIATED FUNCTIONS (Part one) 5mC, 5hmC, 5fC, 5caC: the methyl group

from 5mC undergoes TET protein-mediated oxidation, forming 5-hydroxymehylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). 5hmC is considered to be the signal that starts the active erasure of methylation marks.

CpG Islands (CGIs), are regions of more than 200 bp (300-3000 bp) with a C+G content of at least 55% and a statistically predicted CpG ratio of, at least, 0.6%. They are usually located in 5’UTR regions, promoters, and the first exon. Methylation is generally associated with transcriptional inactivation, unless these regions are located in gene bodies, where methylation acts as a transcriptional activator because it prevents the start of spurious transcription (Berdasco and Esteller, 2010).

Differentially Methylated Regions (DMRs),

are regions with a different methylation status

between tissues, cells, individuals, etc.; Imprinting

regions are genomic loci that represent a different

methylation status depending on the parental origin

of the chromosome (maternal or paternal);

imprinting control regions (ICRs) are formed when

imprinting

regions control more than one gene (Berdasco and Esteller, 2010)

Methods of studying the methylome: methylation-sensitive enzyme digestion, is based on the use of restriction enzymes which target sequences sensitive to methylation; methylated/ hydroxymethylated DNA immunoprecipitation (MeDIP/hMeDIP) is used where fragmented genomic DNA is enriched for 5mC or 5hmC by immunoprecipitation; in bisulfite sequencing (Bi-seq), genomic DNA is treated with sodium bisulfite salt to transform Cs, but not 5mC/5hmCs, to uracil (U); treated DNA is sequenced and compared to a reference sequence. If C is maintained, this specific locus was initially methylated and vice versa. This method allows for single nucleotide resolution and with sufficient deep reading it enables absolute quantification of methylation levels. It is the most widely used method, but it does not distinguish between 5mC and 5hmC, thus levels of 5hmC in the genome may be underestimated by this technique (Saitou et al., 2012).

inserting new copies of themselves, hence their methylation acts to

maintain chromosomal integrity (Berdasco and Esteller, 2010). Throughout

the mammalian life cycle there are two epigenetic reprogramming events

that involve massive DNA demethylation: The first in the zygote, just after

fertilization, in order to erase the epigenetic signature inherited from

gametes (except imprinting marks) and to reacquire totipotency; the

second during PGC development, to restore the developmental potency

and to erase imprinting marks. This erasure of parental imprinting marks is

essential for the proper development of the next generation, establishing

new marks according to the somatic sex of the embryo.

1. Introduction

- 25 -

BOX 2. DNA METHYLATION, CONCEPTS, AND ASSOCIATED FUNCTIONS (Part two) DNMT (DNA-5-cytosine-methyl-

transferase) enzyme family catalyzes the transfer

of methyl groups from S-adenosyl-methyonine to

DNA. In mammals there are five DNMT family

members: DNMT1, DNMT2, DNMT3a, DNMT3b,

and DNMT3L, but only DNMT1, 3a, and 3b have

methyltransferase activity. DNMT1 is considered

to be the maintenance methylase because it

copies the methylation mark from the template

strand during DNA replication. DNMT3a and 3b

are de novo methylases and are considered to be

responsible for establishing methylation patterns

during development. DNMT3L recruits DNMT3a

and 3b when non-methylated H3K4 nucleosomes

are recognized and is required for maintaining maternal imprinting marks (Saitou et al., 2012).

TET (ten-eleven translocation) proteins, including TET1, TET2, and TET3, catalyse the oxidation of 5-methycytosine to 5-hydroxymehylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC).

AID (activation-induced deaminase) and APOBEC (apolipoprotein B mRNA editing enzyme, catalytic polypeptides), are cytidine deaminases that transform C to U; BER (base excision repair) is involved in the DNA repair pathway, and eliminates miss-paired bases by cutting the 5’end of the phosphodiester link of the abasic site which is then completed by a DNA polymerase (Saitou et al., 2012).

- Methylation mark erasure

Classically, it was considered that this second reprogramming process

begins at PGC specification and gonadal entry at E10.5, with demethylation

occurring in imprinting regions and other differentially demethylated

regions (DMR) at E11.0-E12.5 (Seisenberger et al., 2013).

However, new results indicate that the reprogramming process initiates

much earlier than previously supposed, at E8.0-E8.5. According to the most

recent studies, PGC demethylation takes place in two phases (Fig. 1.6A):

the first being an early phase, during migration, where some specific

regions are maintained actively methylated; and the second after gonadal

ridge colonization, affecting sequences with epigenetic memory (e.g.

imprinting regions, X chromosome CGIs, and CGIs in germ cell-related

promoters), although, there are other regions such as intracisternal A

particles (IAPs) that are not affected by this epigenetic erasure.


- 26 -

FIG. 1.6. Epigenetic changes during gametogenesis. A) Methylome reprogramming; B) Chromatin changes.

E8.5 E10.5 E12.5 E15.5

Germ cell migration

Prophase I

E7.5Mitosis arrest

(male)PGC

specification

MEIOSISMeiosis arrest

(female)Sex determination

sperm

oocyte

5mC

5hmC

active

passive

% genome methylation

FASE 1 FASE 2

TET1

TET2

DNMT1

DNMT3A

DNMT3B

H3K9me2

H3K9ac

H2A/H4 R3 me2s

H3K27me3

H4ac

H3K4me3

H3K9ac

H3H3.3

ɣH2A.X

MSCI

A

B

In fact, another recent study demonstrated that PGC demethylation,

including erasure of imprinting marks, seems to involve conversion of 5-

methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) via TET

methylcytosine dioxygenase 1 and 2 (TET1/ TET2; Seisenberger et al., 2012,

Hackett et al., 2013). Between E9.5 and E10.5, as 5mC is lost, PGCs

asynchronously gain 5hmC which is then progressively lost at a rate specific

to each locus. This fact agrees with the increase in TET1 and TET2

expression observed between E10.5-E11.5, suggesting that there is a strict

temporal order of 5mC erasure. Additionally, methylation may be passively

lost, meaning that 5mC, as well as its derivatives (5hmC, 5fC, and 5caC; Fig.

1.7A; Box 2) would be lost over rounds of cellular divisions giving rise to a

hypomethylated status at E13.5 (Seisenberger et al., 2012).

1. Introduction

- 27 -

At this moment, downregulation of the de novo demethylases Dnmt3a and

Dnmt3b, and Uhrf1 cofactor, the methylation maintenance system in PGCs,

prevents remethylation. This, together with the progressive loss of 5hmC

widely enhances the epigenetic erasure associated with cell division.

Furthermore, demethylation depends on the AID/APOBEC system (Popp et

al., 2010) to convert 5mC into thymidine (T). This system, followed by the

base excision repair (BER) system (Hackett et al., 2012), may act as an

auxiliary active demethylation mechanism in PGCs (Hackett et al., 2013).

In conclusion, global demethylation in PGCs is consistent with a passive

erasure mechanism, which may be supplemented by active methylation

maintenance at some specific loci (Fig. 1.7B), which disappears after they

arrive to the gonads (Seisenberger et al., 2012, Seisenberger et al., 2013).

Genomic regions that escape from demethylation (i.e. long interspersed

nuclear elements, LINEs; or IAPs) retain 5mC marks, suggesting the

existence of specific systems to prevent both 5hmC conversion and direct

5mC erasure (Seki et al., 2005).

Thus while 5mC loss in zygotes depends on the conversion of 5mC in to

5hmC by TET3 in the paternal genome or the passive direct depletion of

5mC in the maternal genome, both mechanisms (TET1/2 and passive

demethylation) cooperate in PGCs (Hackett et al., 2013). However, the vast

majority of these studies were all based on methylome studies after

bisulfite conversion which does not allow 5mC and 5hmC to be

distinguished, meaning that the role of 5hmC plays in PGC development

may remain underestimated.


- 28 -

- Acquisition of the gametic methylation pattern:

After PGC demethylation, the genome must remethylate de novo in order

to reach the sex-specific methylation profile of mature gametes. Again, our

current knowledge of this process is extrapolated from imprinting regions

and retrotransposons. De novo methylation in male PGCs occurs several

days after the end of erasure, between E14.5-E16.5, and continues until the

spermatogonial stages, while in female PGCs, the methylation pattern is

established in the growing oocyte after birth. This de novo methylation

results in the mature methylation pattern characteristic of gametes, with

FIG. 1.7. Epigenetic erasure mechanisms and their intermediates. A) The process of 5mC oxidation by TET and the possible cellular mechanisms involved. B) Differences between active and passive demethylation processes (replication-independent and dependent, respectively).

T

C

hmU

TET1/2

TET1/2

TET1/2

?

AID/ APOBEC

AID/ APOBEC

BER

passive

APassive demethylation

(replication-dependent)Active demethylation

(replication-independent)

B

1. Introduction

- 29 -

their respective somatic sex imprinting marks (Seisenberger et al., 2012)

and requires the activity of DNMT3a, DNMT3b, and DNMT3L to establish

these de novo marks. For some maternal imprinting marks, DNMT3L is

recruited to non-methylated histone 3 lysine 4 (H3K4) tails to allow

DNMT3a and DNMT3b to bind these sequences. Therefore, the mechanism

by which imprinting marks are reacquired is different from the transposable

elements mechanism mediated by piRNAs. At the end of the process, in

males, spermatozoa are highly methylated (around 80%) whist in females,

oocytes are about 30% methylated (Seisenberger et al., 2012).

1.1.3.2 CHROMATIN REPROGRAMMING AND MEIOTIC CONTROL.

Chromatin structure changes which control the transcriptional status of

DNA (Box 3), are initiated directly by PRDM1 by binding through its PR

domain with PRMT5, an arginine methyltransferase, to increase

H2A/H4R3me2s levels. This mark is involved in the somatic-to-germ-cell

fate transition (Ancelin et al., 2006) and it initiates chromatin

rearrangements (Fig. 1.6B).

These changes are similar in both sexes and occur in concert with the

suppression of the somatic program. From day E7.5 repressive

modifications are detected in PGCs: specifically, there is a gradual loss of

H3K9me2 and an increase in histone 3 lysine 27 trimethylation (H3K27me3)

between E8.5-E9.0 to E12.5. This epigenetic transition coincides with loss of

lysine methyltransferase Glp (Seki et al., 2005) expression. Additionally,

unlike H3K9me2, global H3K9me3 levels are maintained in migrating PGCs

(Hackett et al., 2012). Nevertheless, only the X chromosome from female


- 30 -

BOX 3. CHROMATIN STRUCTURE AND HISTONE MODIFICATIONS Chromatin structure controls the

transcriptional status of DNA. Nucleosomes, formed by the histone core H2A, H2B, H3, and H4 and the linker histone H1, are the structures responsible for the extent of chromatin packing. All the histones can undergo post-translational modifications associated with gene expression; the different combinations of all these marks determine a permissive or non-permissive transcriptional status.

In general, acetylation, methylation, phosphorylation, and ubiquitination are related with transcriptional activation, whilst methylation, sumoylation, and proline isomerization are associated to repression. Compacted heterochromatin is transcriptionally inactive, and has low levels of acetylation, especially on H3K9, and high levels of H3K9me2/me3, H3K27me, and

H4K20me, which are all marks which are

considered to be transcriptional repressors. In

euchromatin there are high levels of acetylation

and it is enriched in H3K9me3/ac, H3K4me3/ac,

H3K36me3/ac, and H3K79me/ac marks.

Transcriptionally active genes are, in turn,

enriched in H3K4me3, H3K27ac, H2BK5ac, and

H4K20me1 marks at promoter regions, and

H3K79me1 and H4K20me1 on gene bodies. The

different levels of histone modifications can be

predictive of the gene expression status, but it

depends on the context. For example, H3K36me

and H3K9me have a positive effect on coding

regions but a negative one on promoter regions

(Kouzarides, 2007).

PGCs show an extended decline in H3K27me3 levels which is directly

related to the beginning of X chromosome reactivation, hence there is an

established chromatin status which characterizes pluripotency (Hackett et

al., 2012).

In the second step, methylome erasure starts the chromatin remodelling

process at around E11.5 when PGCs colonize the gonadal ridges. The main

nuclear architecture reorganizations detected at this time imply that there

are histone modification changes which allow exchange of these histone

variants. This includes transient reorganization of H1, H3K27me3, and

H3K9me3, and stable reorganization of histone 3 lysine 9 acetylation

(H3K9ac) and H2A/H4R3me2s (Hackett et al., 2012). Hence, when PGCs are

colonizing the genital ridges, methylation marks on histone 3 lysine 4

(H3K4me) and H3K9ac increase, associating these two marks with a

transcriptionally permissive state (Seki et al., 2005).

1. Introduction

- 31 -

Proper epigenetic reprogramming of germ cells is crucial for proper

progression through meiosis, but there are also some histone modifications

that occur during meiosis. H3K4 mono-, di- and trimethylation levels, as

well as H3K9me2, are globally changed during prophase I; the relevance of

these epigenetic modifications remains unknown, but many of the factors

involved in histone methylation are essential, especially for male germ cell

development (Kota and Feil, 2010).

During meiosis, recombination events occur preferentially at specific

regions called recombination hotspots. They are sequence-specific and

need a concrete chromatin configuration: starting recombination sites are

enriched in H3K4me3 and H3K9ac (Kota and Feil, 2010). It is thought that

epigenetic erasure is necessary to promote the formation of chiasmata at

these hotspots during meiosis because they enable PRDM9, a lysine

methyltransferase, to recognize H3K4me3, a signal for recombination to

take place (Hackett et al., 2012).

Other chromosome regions that do not recombine during meiosis are

transcriptionally silenced via a mechanism called meiotic silencing of

unpaired chromatin (MSUC) which prevents non-homologous

recombination. In mammals, the best studied example is the condensation

and inactivation-silencing of the sexual chromosomes during male meiosis,

which can be distinguished as sexual bodies. This process is called meiotic

sex chromosome inactivation (MSCI) and involves H3 to H3.3 exchange, as

well as the accumulation of phosphorylated H2A.X (Kelly and Aramayo,

2007, Kota and Feil, 2010).


- 32 -

This extensive and organized epigenetic reprogramming is required for the

subsequent acquisition of totipotency in mature gametes, to rearrange

imprinting marks and to produce gametes with the suitable epigenome to

enable the proper development of a new individual (Seki et al., 2005).

1.2. MALE GAMETOGENESIS

Gamete maturation is different depending on the sex of the embryo. In

females, germ cells go into meiotic arrest until puberty when hormonal

cycles start. Hormonal signaling stimulates meiosis to progress, but from all

the follicles that are stimulated in each cycle, only one will ovulate

producing a metaphase II oocyte that will finalize meiosis, which can only

occur after fertilization (McLaren, 1995). In males, germ cells instead

remain in mitotic arrest, and when puberty arrives, germ cells re-start their

cellular divisions and differentiate continuously in order to start meiosis

and give rise to mature spermatozoa.

1.2.1 THE SPERMATOGONIAL STEM CELL MICROENVIRONMENT.

Spermatogenesis occurs in the lumen of the seminiferous tubules,

mediated by and surrounded by Sertoli cells that act as germ-cell support

cells. These cells are located from the basal lamina of the tubule to the

lumen and their function is to nurture and to support developing germ cells

(Fig. 1.8). Immature Sertoli cells are present from birth and proliferate until

puberty when they start to differentiate into mature Sertoli cells. They are

joined together by tight junctions and form the blood-testis barrier that

divides the seminiferous tubule into two compartments: the basal (pre-

meiotic) and the adluminal compartments (Dufour et al., 2005). Leydig cells,

1. Introduction

- 33 -

together with macrophages, lymphocytes, blood vessels, etc. comprise the

interstitial space located between these seminiferous tubules (Lehmann,

2012).

The basal compartment controls and delimitates spermatogonial stem cell

(SSC) differentiation. SSCs are the cell subpopulation that lead to male germ

cell propagation throughout the whole adult life (Kanatsu-Shinohara et al.,

2008) so they are considered the germ line stem cells in the male germ line

(Kyurkchiev et al., 2012). In humans, SSCs are present at the basal

membrane of seminiferous tubules (Fig. 1.8) from the moment of birth in

prepuberal tissue; while in mice the gonocyte-to-spermatogonia transition

takes place 1-4 days postpartum (de Rooij, 2009). Once sexual maturity is

FIG. 1.8. Stages of spermatogenesis. SSCs, spermatogonial stem cells; Spg, spermatogonium; Spc I, primary spermatocyte; Spc II, secondary spermatocyte; RS, round spermatid; EP,

elongated spermatid.

SSC

Spg

Spg

Spc I

Spc II

RS

ES

MEIOSIS I

MEIOSIS II

SPERMIOGENESIS


- 34 -

reached spermatogonia start to asymmetrically divide to give rise to

mitotically active spermatogonia, which can advance into spermatogenesis,

whilst also self-renewing to maintain adult testicular stem cells (Kolasa et

al., 2012, Waheeb and Hofmann, 2011, Phillips et al., 2010, Hermann et al.,

2010).

The SSC niche is the microenvironment which regulates stem cell fate by

maintaining the balance between self-renewal and differentiation and is

maintained by Sertoli cells and the basal lamina of the seminiferous tubule

to which they are bound. Sertoli cells produce large amounts of the growth

factors required for the maintenance of the SSC subpopulation. Among

them, the production of glial cell line-derived neurotrophic factor (GDNF) is

particularly important, because it promotes SSC self-renewal in vitro and in

vivo to initiate an intracellular response when bound to its receptor, GFRA1,

which is present in spermatogonia. Other relevant factors involved in

maintaining the cells in an undifferentiated state are basic fibroblast growth

factor (bFGF) and epidermal growth factor (EGF), which are also secreted by

Sertoli cells (Simon et al., 2010, Kanatsu-Shinohara et al., 2008, Dores et al.,

2012).

1.2.1.1 SPERMATOGONIAL STEM CELL CULTURE

There are different techniques that enable the identification and

purification of SSCs (Goossens et al., 2013, Wyns et al., 2010), including

several membrane markers which are characteristic of spermatogonia.

Among them there are 6-integrin (CD49f) and 1-integrin, two laminin

receptors (Conrad et al., 2008), CD9, a basal membrane cell attachment

1. Introduction

- 35 -

marker (Kanatsu-Shinohara et al., 2004), Thy-1 (CD90), a immunoglobulin-

protein family member which is overexpressed in neonatal mouse and

primate testis, including in humans (Kubota et al., 2003, Ryu et al., 2004,

Hermann et al., 2010), PLZF (also known as ZBTB16) which is SSC-specific

(Buaas et al., 2004), GFRA1, a GDNF receptor (Hofmann et al., 2005, von

Schonfeldt et al., 2004), and NOTCH1 (von Schonfeldt et al., 2004). Use of

such markers enables cellular subpopulations to be identified and

separated by fluorescence activated cell-sorting (FACS) or magnetic

activated cell-sorting (MACS).

SSC culture is challenging, in particular, blocking differentiation,

maintenance of long-term survival, and promotion of proliferation (Geens

et al., 2008). This is accomplished by culturing them in conditioned media

supplemented with GDNF, EGF, bFGF, and leukemia inhibitory factor (LIF;

Kanatsu-Shinohara et al., 2003, Guan et al., 2009), or SCF (Oatley and

Brinster, 2006), to simulate the natural physiological niche and the signals

sent by Sertoli and Leydig cells. Serum-free and feeder cell monolayer co-

culture systems have also been developed, demonstrating that SSCs can

proliferate under these conditions (Kanatsu-Shinohara et al., 2004, Kanatsu-

Shinohara et al., 2005). Co-culture with somatic cells, in particular with

Sertoli cells, seems to be necessary to differentiate SSC towards male

haploid gametes. This technique has already demonstrated promising

results in humans, where SSCs isolated from human biopsies and co-

cultured with Sertoli cells, have shown expression of markers for meiotic

progression and haploid cell formation (Riboldi et al., 2012).


- 36 -

1.2.1.2 GERM CELL TRANSPLANTS

Transplantation of SSCs into mice is an assay which defines the functional

activity of this cellular subpopulation (Geens et al., 2008, Brinster and

Avarbock, 1994, Brinster and Zimmermann, 1994). In the murine model,

donor SSCs are injected into the seminiferous tubules of sterilized mice,

either through deferent ducts, rete testis, or the seminiferous tubules

themselves (Ogawa et al., 1997), and colonize the niche, in some cases even

regenerating active spermatogenesis (Avarbock et al., 1996) to produce

healthy and fertile offspring (Wu et al., 2012, Schlatt et al., 2003). Similar

results have been produced with SSC xenotransplants from other animal

models, including primates, by transplanting not only purified SSCs, but also

a pool of gamete cells at different stages of spermatogenesis (Schlatt et al.,

2002, Hermann et al., 2007, Hermann et al., 2012, Wu et al., 2012).

Although there are evolutionary differences between species, these can be

overcome so that complete spermatogenesis can be obtained from rat-to-

mouse or human-to-non-human primate tubule transplantation. However,

human germ cell transplantation into mouse tubules has been shown to be

limited to the formation of pro-spermatogonia, spermatogonia, or very

early meiotic derivatives (Nagano et al., 2008).

1.2.2 THE STAGES OF SPERMATOGENESIS

Spermatogenesis is the process in which diploid spermatogonia terminally

differentiate into mature, mobile, and haploid spermatozoa (Fig. 1.8). It can

be divided in three stages: the proliferative phase, meiosis, and

spermiogenesis (McLaren, 1984, Hess and Renato de Franca, 2008).

1. Introduction

- 37 -

1.2.2.1 THE PROLIFERATIVE PHASE

Spermatogonia attached to the basal lamina divide by mitosis. A small

subpopulation constitutes the SSC pool while the rest are their progeny. In

prepuberal children, the germ cell population is exclusively constituted by

SSCs, whereas in an adult testis only 1% of spermatogonia are SSCs (de

Rooij, 2009). Hence, maturating spermatogonia divide by mitosis following

waves of maturation from puberty onwards which continues throughout

adult life under hormonal control.

The hypothalamus secretes gonadotropin-releasing hormone (GnRH) which

acts on the pituitary gland, triggering the release of luteinizing hormone

(LH) and follicle-stimulating hormone (FSH). LH causes Leydig cells to

secrete testosterone which stimulates Sertoli cells to promote and regulate

spermatogenesis, and limits GnRH and LH production at the pituitary level

in a negative feedback loop. FSH, together with testosterone, induces

Sertoli cells to transport nutrients to the inner part of the tubule to activate

spermatogenesis, as well as androgen binding protein (ABP) synthesis; ABP

is necessary for maintaining testosterone levels in the seminiferous

epithelium. Sertoli cells also produce inhibin in response to FSH, which

inhibits FSH and GnRH production at the pituitary level (de Kretser et al.,

1998, Phillips et al., 2010).

From this point, spermatogonia are able to differentiate into primary

spermatocytes, diploid cells with 23 pairs of homologous chromosomes,

each one with two sister chromatids (2n, 2c) ready to start meiosis.


- 38 -

1.2.2.2 MEIOSIS

Primary spermatocytes break through the blood-testis barrier to the

adluminal compartment where they initiate meiosis. Meiosis consists of

two cellular divisions (meiosis I/ meiosis II) with similar stages to mitosis

(prophase, metaphase, anaphase, and telophase) but these are preceded

by only one DNA replication. At meiosis I, primary spermatocytes duplicate

their DNA content (2n, 4c) before prophase I, which is divided into five well-

differentiated stages (Handel and Schimenti, 2010, Medrano et al., 2013):

1) Leptotene: homologous chromosomes are aligned but not yet paired.

Each pair of sister chromatids, bound by cohesins, start to form a

chromosomal scaffold called the synaptonemal complex, formed by

cohesins REC8, STAG3, and SMC1B, and axial elements SYCP3 and SYCP2.

2) Zygotene: topoisomerase SPO11 mediates programmed double-strand

DNA breaks that allow recombination repair machinery to recognize them.

Phosphorylation of H2A.X to form ɤH2A.X is necessary in order to recruit

the recombinase-related RECA, DMC1, and RAD51 proteins, among others,

to repair breaks by homologous recombination. At this stage, chromosome

pairing is completed and axial elements become lateral elements in the

synaptonemal complex along the strands of the DNA which are being

zipped.

3) Pachytene: chromosomal synapsis is completed by the structural

function of SYCP1 and the central elements SYCE1 and SYCE2. Bivalents

(crossovers) appear at the specific recombination sites between

1. Introduction

- 39 -

homologous chromosomes. MLH1 and MLH3 mediate DNA break mismatch

repair by homologous recombination.

4) Diplotene: the synaptonemal complex disappears, the chromosomes

undergo desynapsis and the bivalents start to condensate. At this stage the

recombination sites can be observed by the formation of chiasmata (the

point where two homologous non-sister chromatids exchange genetic

material).

5) Diakinesis: chromosomes complete condensation, but the sister

chromatids remain bound by cohesins.

The process goes through metaphase I, anaphase I, and telophase I, when

primary spermatocytes (spermatocyte I) divide into two secondary

spermatocytes (spermatocyte II). Spermatocyte II cells are haploid

(homologous chromosomes are segregated) with 23 chromosomes, each

one with two sister chromatids (1n, 2c). Meiosis II is a cellular division

similar to mitosis but without a previous DNA replication. At the end,

spermatocyte II cells divide into two haploid spermatids with 23

chromosomes, each one with one sister chromatid (1n, 1c).

Meiosis has several checkpoints, most of them also present in mitosis,

which are concentrated along the different stages of prophase I to avoid

synapsis or recombination mistakes (e.g. the pachytene checkpoint). One of

the most important is the spindle assembly checkpoint (SAC), at anaphase I.

This SAC is formed by MADI and MADII proteins; the BUB proteins BUBI,

BUB3, and BUBRI; and MPSI. It is a mechanism that detects unattached

tubules or the loss of tension in the kinetochores of chromosomes during


- 40 -

metaphase in order to ensure correct chromosome segregation (Medrano

et al., 2013, Handel and Schimenti, 2010).

1.2.2.3 SPERMIOGENESIS

Following meiosis II, male germ cells undergo morphological differentiation

from spermatid to mature spermatozoa. This process is called

spermiogenesis and is characterized by: i) nuclear condensation, in which

histones are exchanged for protamines (Gaucher et al., 2010); ii) acrosome

formation from the Golgi apparatus; iii) flagellum creation from the

centrosome (Handel and Schimenti, 2010, Phillips et al., 2010) $(Fig. 1.8).

Throughout this process, spermatids change from a round to an elongated

morphology, as the chromatin compacts; when spermiogenesis ends

spermatozoa are released into the lumen of the seminiferous tubule.

Histone-to-protamine exchange is one of the most studied chromatin

remodelling processes in which nucleosome histones are substituted by

small basic proteins called transition nuclear proteins 1/2 (TNP1/TNP2)

$(Okada et al., 2007). Moreover, the vast majority of described histone H1,

H2A, and H2B variants are present in the testis, and in vitro studies

demonstrate that these nucleosomes bind to the DNA in a less stable way.

The presence of histone variants would explain why TNPs only help

protamine exchange: they are not essential for this process but they help to

avoid aberrant packaging events (Gaucher et al., 2010). This process is also

helped by covalent histone modifications, such as H4 lysine acetylation or

histone ubiquitination, in order to facilitate their degradation, and allowing

1. Introduction

- 41 -

BOX 4. BIVALENT DOMAINS Bivalent domains are chromatin

domains with histone modification marks from both transcriptional activators and repressors. For example, in pluripotent cells, H3K4me3 and H3K27me3 co-exist even though the H3K4me is associated with transcriptional activation and the H3K27me mark is associated with transcriptional repression. The result is a low basal expression level of transcription factors that are controlled by these domains.

As pluripotent cells differentiate bivalent domains

tend to maintain only one of these marks but never

both. Thanks to this importance of the ‘stemness-

maintenance’ mechanism, pluripotent cells can

activate gene transcription during development in a

controlled and quick manner, an ability that is lost

during differentiation (Portela and Esteller, 2010,

Kouzarides, 2007).

the chromatin to become more accessible (Hazzouri et al., 2000, Awe and

Renkawitz-Pohl, 2010).

In a second step, TNPs are exchanged by protamines, which are essential

for postmeiotic development in mammals. Besides compacting the

spermatozoon genome, histone exchange by protamines helps to protect

the genome against mutagen-induced damage (Rathke et al., 2010).

However, certain regions of the genome retain their histones. These regions

include the loci of key developmental genes, imprinted genes, microRNA,

and homeotic genes. These histones are enriched in H3K4me3 and

H3K27me3 chromatin modifications with the opposite ‘meanings’ (Box 4)

but which co-exist in bivalent domains (Farthing et al., 2008).

1.3 IN VITRO GAMETE GENERATION

According to the World Health Organization (WHO), infertility affects up to

14% of reproductive-aged couples. This percentage is increasing in

developed countries mainly due to a delay in the age of maternity and an

increase in harmful habits.


- 42 -

In fact, according to the 2005 American National Survey on Family Growth,

there was a 20% increase in couples with fertility problems between 1995

and 2002 (Stephen and Chandra, 2006). Gamete donation may be a

solution when these infertility problems are caused by poor gamete quality

or an absence of gametes. Considering only the male factor, sperm counts

in more than 100 worldwide studies demonstrated a progressive decline in

the quality of sperm from between 1930-1940 to 2012 (Sharpe, 2012).

Moreover, standardized and prospective studies with men aged between

18-25 years from seven different European countries confirm that, although

the mean of spermatozoa concentration is between 45-65 million/mL, the

proportion of men with less than 20 million/mL is close to 20% (Jorgensen

et al., 2006). Furthermore, low gamete counts are usually associated with

poor gamete quality, i.e. low mobility and/or abnormal sperm morphology.

Hence, a higher number of young men can be classified as sub-fertile

(Sharpe, 2012)

Despite the variety of results from different tracking reports, low gamete

quality seems to be one of the most important causes of infertility. In 2000,

32% of assisted reproduction cycles carried out in 49 countries involved

egg-donation (Adamson et al., 2006), and the US Department of Health and

Human Services established that, in 2004, 12.5% of these cases were

accounted for in the USA alone (Wright et al., 2007). Moreover, the Sixth

Report on the European Results of Assisted Reproductive Techniques,

published by the European Society of Human Reproduction and Embryology

(ESHRE) in 2006 as a part of the European IVF-Monitoring Program

(Andersen et al., 2006) indicated that the proportion of cycles with

1. Introduction

- 43 -

ovodonation increased to 22.4% in 2002. Although the clinical results were

excellent (recording a 34.9% clinical pregnancy rate) gamete donation is

associated with a number of ethical, legal, and personal difficulties, and so,

there is a growing interest in the study of germ line development (Medrano

et al., 2013).

Most of our knowledge about germ line development in mammals comes

from extrapolation from the murine model. However, due to moral, ethical,

and legal difficulties regarding the use of tissues from early human

embryological stages (from gestational week 2-3 where most of the key

events take place) still very little is known about this process in humans

(Marques-Mari et al., 2009). In fact, although pre-implantation embryo

development, including gastrulation, is similar between mice and humans,

there are some relevant differences. In mouse, cells from the proximal

epiblast remain in direct contact with ExE from E5.0 to E6.5, and ExE-cell

BMP4 signaling causes the germ cell fate to be adopted. In humans, before

gastrulation at around day 8 of development, some pluripotent cells from

the epiblast form the amnioblast, resulting in the formation of a new cavity

between the amnioblast and epiblast: the amniotic cavity. Therefore from

this point amnioblastic cells are in direct contact with cytotrophoblastic

cells; in the second week of development most epiblastic cells are already

separated from the trophoblastic layer by the amnioblastic layer and the

amniotic cavity (Fig. 1.9).

To date, there is no information about what part of the epiblast acquires

the germinal fate or where the induction signal comes from. At the end of

week three PGCs are detectable for the first time by their AP activity at the


- 44 -

FIG.1.9. Human post-implantation embryo development. At day 8, the human embryo consists of syncytiotrophoblast (not shown), cytotrophoblast (CT, purple), epiblast (Epi, blue), and hypoblast (Hypo, yellow). In contrast to the mouse embryo, these epiblast cells have an almost formed amnioblast (Am), and the amniotic cavity appears between the Epi and Am. At day 12, before gastrulation starts, epiblast cells are not in direct contact with CT. En: Extraembryonic Endoderm; SpM: Extraembryonic Splanchnopleuric mesoderm; SoM: Extraembryonic Somatopleuric mesoderm. From (Saitou and Yamaji, 2010)

wall of the yolk sac, at the base of allantoides, and the future umbilical cord

(Saitou and Yamaji, 2010).

Given that studying the cell fate specification mechanisms in human

embryos is so complicated, to better specify these mechanisms the

knowledge derived of mouse models must be supplemented information

obtained from other animal models with post-implantation embryo

development closer to humans (e.g. rabbit, pig, etc.) $(Saitou and Yamaji,

2010). Human embryonic stem cells (hESCs) and human induced

pluripotent stem cells (hiPSCs) have also been used to demonstrate that a

germ-cell-like state can be induced in vitro (Clark et al., 2004, Kee et al.,

2006, Park et al., 2009, Panula et al., 2011, Medrano et al., 2011, Bucay et

al., 2009 , Tilgner et al., 2008, Durruthy Durruthy et al., 2014). As both

1. Introduction

- 45 -

embryonic stem cells (ESCs) and iPSCs (in general, pluripotent stem cells,

PSC) are capable of differentiating not only into the three germinal layers

but also into germ cells, they can also be used as a model to understand the

genetic and epigenetic basis of germ cell specification and development in

humans, hence the recent increasing interest in in vitro gamete derivation

research (Medrano et al., 2013).

1.3.1 GERM CELL DERIVATION FROM PLURIPOTENT STEM CELLS

In 2003 Schöler’s group first demonstrated that mouse ESCs (mESCs)

expressing green fluorescent protein (GFP) under the control of a distal

Oct4 promoter spontaneously differentiated towards germ-line cell lineages

(Hubner et al., 2003). After differentiation, these GFP-positive cells were

selected and were maintained in culture, spontaneously differentiating,

until some structures similar to follicles appeared in which oocyte-like cells

(OLCs) were observed. OLCs were parthenogenetically activated and

formed pseudo-blastocysts. This breakthrough study opened up the

possibility of obtaining germ cells in vitro for study. In the same year, and

following a similar approach, another group reported the differentiation of

sperm-like cells (SLCs) from mESCs. In this study, they used the Mvh

promoter associated with GFP as a reporter; they differentiated tri-

dimensional mESC aggregates using BMP4 and engrafted GFP-positive cells

into testis. These cells participated in spermatogenesis in vivo, although the

authors did not specify if any offspring were obtained (Toyooka et al.,

2003).


- 46 -

One year later, Daley’s group proved the apparent functionality of in vitro

derived gametes: mESCs differentiated to embryoid bodies (EBs)

spontaneously formed SSEA1+ and OCT4+, putative germ cells. These cells

were isolated and cultured with RA to induce their entry into meiosis, and

they showed an Igfr2r and H19 imprinting pattern which correlated with

spermatozoa. Most importantly, these cells were able to form blastocysts

after injection into oocytes by intra-cytoplasmic sperm injection (ICSI)

(Geijsen et al., 2004).

Similar results from hESCs were later obtained by Reijo-Pera’s group by

spontaneous EB differentiation, which is a reference model for germ cell in

vitro differentiation. They established that undifferentiated hESCs already

express some germ-cell-related markers, such as c-KIT and DAZL, but not

other later markers such as VASA or SYCP3 and proposed that hESCs consist

of a heterogeneous population in which some cells are predisposed

towards germ-cell-fate differentiation (Clark et al., 2004). Many other

studies have demonstrated the formation of germ cells in vitro using

different strategies. The main approaches were based on purification of

spontaneously-derived germ-line cells, and gonadal tissue co-cultures (Park

et al., 2009 ), as well as cell culture media growth factor supplementation

(Kee et al., 2006).

- Meiotic progression and the role of RNA-binding proteins

Despite these findings, proper meiotic progression of germ cell derived

cells, either from mESCs or hESCs was still a challenge. Studies

demonstrated aneuploidies in mESC-derived oocytes because, despite

1. Introduction

- 47 -

detecting SYCP3 expression, other elements also required for meiotic

progression, such as SYCP1, SYCP2, REC8, STAG3, or SMC1-b, were not

expressed (Novak et al., 2006). Other groups achieved complete meiosis in

purified spontaneously-differentiated in vitro-derived germ cells. They

cultured these cells with a mixture of growth factors in the absence of

genetic manipulation (Eguizabal et al., 2011). However, following this work,

other authors described how ectopic expression of genes belonging to the

DAZ family (DAZ2, DAZL, BOULE) enables a proper meiotic progression in

both hESCs and hiPSCs (Panula et al., 2011, Kee et al., 2009). These papers

describe how overexpression of a pool of RNA-binding proteins with a

highly conserved structure can result in proper meiotic progression in the

absence of a gonadal niche. They also demonstrated the crucial role those

factors play in transcriptional regulation throughout germ line

development. Following this idea, ectopic expression of VASA, another

RNA-binding protein which is highly conserved in mammals, has shown a

similar effect on the induction of meiosis (Medrano et al., 2011).

In fact, the same group went further to explore, for the first time, the role

of one of these RNA-binding proteins on the in vivo differentiation of

human-derived germ cells. They derived these cells using an integration-

free hiPSC line via RNA-based reprogramming using only Yamanaka’s

factors (OCT4, SOX2, KFL4, and cMYC; OSKM) or by combining them with

VASA RNA (OSKMV). They transplanted undifferentiated hiPSCs into the

inner part of the seminiferous tubules of sterilized immunodeficient mice to

test their in vivo differentiation capacity. OSKMV-hiPSCs showed the

greatest capacity to differentiate in vivo into SYCP3-expressing germ-cell-


- 48 -

like, whereas only OSKM-hiPSCs that remained outside the seminiferous

tubules proliferated and formed small tumours similar to the teratomas

formed in ESC characterization assays. These results, apart from being the

first ones reporting the in vivo differentiation of human-derived germ cells,

highlight the role of VASA, an RNA-binding protein, in proper meiotic entry

and the control of the pluripotency-state (Durruthy Durruthy et al., 2014).

- Production of living offspring from in vitro-derived germ cells

Despite these promising results demonstrating the ability of ESCs and iPSCs

to generate germ cells in vitro, and this first approximation of in vivo

functionality of in vitro derived germ cells, there are few groups that have

been able to obtain living offspring from these induced germ cells. The first

such report used SSC lines derived from mESCs. Reporter genes under the

control of Stra8 and Prmt1 were used to purify SSCs and after purification

the culture media was supplemented with RA to induce meiosis. In vitro

sperm-like cells appeared and when they transplanted these cells into a

host-mouse testis they formed haploid sperm with limited motility;

functionality was demonstrated by producing living offspring after ICSI.

However, the pups showed phenotypic alterations such as growth

retardation and aberrant imprinting patterns that led to premature death

(Nayernia et al., 2006).

Saitou’s group produced another strategy for obtaining in vitro germ cells

capable of producing fertile offspring: they differentiated mESCs and

murine induced pluripotent cells (miPSCs) into epiblast-like cells (EpiLCs),

representing a cellular state very close to epiblast cells before gastrulation

1. Introduction

- 49 -

but different from epiblastic stem cells (EpiSCs). They generated PGC-like

cells (PGCLCs) which expressed β3-integrin and SSEA1 from these EpiLCs,

and differentiated these by adding BMP4 to the culture media. The PGCLCs

obtained by this technique showed imprinting patterns according to their

somatic sex and when transplanted into host mice, they went through both

complete spermatogenesis (Hayashi et al., 2011) and oogenesis and

produced fertile offspring after ICSI (Hayashi et al., 2012). Moreover, this

group recently showed how a similar PGCLC-phenotype could also be

obtained only by overexpressing Prdm1, Prdm14, and TFAP2C genes which

act downstream of the BMP4 signaling pathway, although in this case in the

absence of BMP4 as an inductor. Importantly, these PGCLCs are able to

complete spermatogenesis when injected into host mice leading to the

production of fertile offspring by ICSI (Nakaki et al., 2013).

1.3.3 DIRECT REPROGRAMMING

One of the main applications of ESCs and iPSCs is to direct their

differentiation towards almost any cell type. However, the advent of iPSC

reprogramming technologies represented a paradigm shift in our

understanding of how transcription factors can affect the phenotypic

plasticity of differentiated somatic cells to reprogram them. Hence, by

deepening in our understanding of transcription factors, it has been shown

that cellular identity can be directly changed following a methodology

similar to that used to reprogram iPSCs. To date, several cell types have

been obtained by direct reprogramming of differentiated somatic cells,

without first reverting to pluripotent state, including direct conversion of

fibroblasts to cardiomyocytes (Ieda et al., 2010), blood precursors (Szabo et


- 50 -

al., 2010), neurons (Kim et al., 2011, Vierbuchen et al., 2010), and even

Sertoli cells (Buganim et al., 2012), exocrine pancreas cells to insulin

producing β-cells (Zhou et al., 2008), or astrocytes to neurons (Corti et al.,

2012). However, there are no reports showing that germ cell like cells have

been directly obtained from somatic cells. Even so, in the light of previous

studies, it is known that differentiated cells maintain a high level of

phenotypic plasticity and cell fates can be changed with proper

manipulation. This has many potential applications, from pharmacological/

toxicological screening to illness modelling (Ladewig et al., 2013).

Nonetheless, there are many challenges in the reprogramming field. The

role of the epigenome remains undefined, and the question of whether it

functions equally-well when reprogrammed also remains. Research into

post-transcriptional and pre-translational control, involving miRNAs and

RNA-binding proteins, is also gaining momentum. Given the evidence

currently available regarding the possibility of deriving germ cells in vitro, it

seems that direct reprogramming or induction of germ cell formation from

adult somatic cells may be a feasible strategy that would create a more

realistic in vitro model of human germ line development allowing the role

of cells which are extremely difficult to study in vivo to be explored in vitro.

- 51 -

2. Hypothesis

Hydrangea, Pansy, Forsythia

Pride, thoughtful, hope

2. Hypothesis

- 53 -

Germ cell-like cells have been derived in vitro from pluripotent stem cells,

both ESCs and iPSCs. Following a similar approach to iPSC reprogramming,

adult cell types have been directly induced to convert into other

differentiated cell types.

Based on these findings the working hypothesis of this doctoral thesis is

that human somatic cells can be directly reprogrammed in vitro into

induced germ-like cells using a pool of key germ cell-related factors.

- 55 -

3. Objectives

Azalea, Daffodil, Edelweiss

Patience, respect, courage, and power

3. Objectives

- 57 -

3.1 GENERAL OBJECTIVE

To obtain induced germ cell-like cells (iGCs) in vitro from human somatic

cells by genetic overexpression of key germ cell-related factors.

3.2 SPECIFIC OBJECTIVES

1. To identify, isolate, and clone into lentiviral vectors the key human

germ cell developmental genes.

2. To optimize culture conditions in order to maintain putative derived

iGCs in vitro.

3. To screen the cocktail of reprogramming factors to identify the most

efficient combination of genes for inducing a meiotic phenotype.

4. To characterize the iGCs obtained at the transcriptomic and epigenetic

level.

5. To analyze the meiotic progression and ploidy of these iGCs.

6. To test the in vivo functionality of these iGCs by their ability to colonize

the spermatogenic niche in busulfan-sterilized mice.

- 59 -

4. Experimental design

Morning Glory, Peony, Camellia

Wilful promises, bravery, reliance and modesty

4. Experimental design

- 61 -

Based on the literature, together with previous reports from our group,

twelve germ cell-related genes were selected and cloned into lentiviral

vectors and overexpressed in two commercially available human male (XY)

somatic primary cell types: human foreskin fibroblasts (hFSKs) and human

mesenchymal stem cells (hMSCs).

Reprogrammed cells were analyzed on days 7, 14, and 21 after transduction

so as to establish a time-course pattern during the process of direct

reprogramming towards a germ-cell-like fate. Transcriptomic

characterization by expression arrays and real-time polymerase chain

reaction (qRT-PCR), as well as phenotyping by immunofluorescence were

performed at all of these time points. Moreover, epigenetic

characterization by bisulfite sequencing, meiotic progression, ploidy assays,

and xenotransplantation assays were performed on day 14 after

transduction (Fig. 4.1).

FIG. 4.1 Experimental design. Human somatic cells were transduced with germ cell-related genes and cultured under a specific germ cell medium. The reprogrammed cells where analyzed on days 7, 14 and 21 after transduction. A thorough explanation is included in the main text. FISH: fluorescent in situ hybridization; IF: immunofluorescence; KSR, Knock-out serum replacement

PRDM1PRDM14NANOS3

LIN28NANOG

DAZ2DAZLVASA

BOULESTRA8

DMC1SYCP3

MeiosisGerm cell determination Migration Genital ridge

DAY 21

- Lentiviraltranfection

- RNA -RNA, gDNA, (clusters & total population)- Expression array- Bisulfite sequencing-Meiotic progression analysis-FISH analysis (clusters & total population by DNA-FACS)-IF of germ cell markers-Xenotransplant

-RNA (clusters & total population)-IF of germ cell markers

DAY 0 DAY 7 DAY 14

Human somatic cell

Germ cell-like

cells

iGCLC clusters

GERM CELL MEDIUM:

+GDNF

+bFGF

+SCF

+LIF

+RA

+ KSR

Germ cell factor

overexpression

Lentiviral

transfection

- 63 -

5. Methods

Yellow poppy, Pink Rose, Verbena

Success, trust and confidence, cooperation

5. Methods

- 65 -

5.1. GENE SELECTION, ISOLATION, AND CLONING

5.1.1 GENE SELECTION

According to current knowledge and our group’s previous results, 12 genes

were chosen for ectopic overexpression in human somatic cells. These

genes are all involved in different stages of in vivo and in vitro germ cell

differentiation and development and are listed below:

- Genes related with germ-cell induction

PRDM1 (BLIMP1): A transcription factor involved in PGC specification

and somatic-program silencing (Kurimoto et al., 2008, Saitou, 2009,

Vincent et al., 2005).

PRDM14: Interacts with PRDM1 and has an important role in inducing

epigenetic reprogramming during in vivo PGC development (Kurimoto et

al., 2008).

NANOS3: Related to germ cell survival and quiescent state maintenance

by repressing mRNA translation (Suzuki et al., 2008).

LIN28: An RNA-binding protein which is involved in microRNA silencing

for PGC specification by regulating PRDM1 (West et al., 2009).

NANOG: A transcription factor involved in PGC pluripotency and

proliferation (Mitsui et al., 2003).

- Genes related with PGC survival and maturation

DAZ2: Is encoded by the Y chromosome and is an RNA-binding protein

essential for proper spermatogenesis. In vivo, DAZ2 deficiency causes

Sertoli-only syndrome (Farthing et al., 2008).


- 66 -

DAZL: Is an RNA-binding protein involved in germ cell maturation. Loss

of its function causes sterility in both sexes (Gill et al., 2011, Medrano et

al., 2011, Ruggiu et al., 1997, Kee et al., 2009).

BOLL: Is an RNA-binding protein, from the same family as DAZ2 and

DAZL. Loss of its function causes meiotic errors (Kee et al., 2009).

VASA: An RNA-binding protein involved in germ cell maturation and

meiosis via its piRNA-regulating function. Loss of function is a cause of

male sterility in mice and female sterility in Drosophila (Castrillon et al.,

2000b, Fujiwara et al., 1994, Medrano et al., 2011, Tanaka et al., 2000).

STRA8: Is involved in inducing meiosis by RA signaling (Guan et al., 2009,

Bowles et al., 2006).

- Genes involved in meiosis

DMC1: Is a DNA-recombinase that acts during homologous chromosome

recombination at prophase I of meiosis by repairing double strand

breaks (Pittman et al., 1998, Yoshida et al., 1998).

SYCP3: Is a structural component of the synaptonemal complex during

prophase I of meiosis (Reynolds et al., 2007, Costa et al., 2005).

5.1.2 GENE ISOLATION AND CLONING

5.1.2.1 ISOLATION

The open reading frame (ORF) sequences of the different genes of interest

(GenBank accession numbers are shown in Table 5.1) were isolated from

human testis copy DNA (cDNA) which was obtained by reverse transcription

of human adult testis total RNA (Clontech). These isolated polymerase chain

5. Methods

- 67 -

reaction (PCR) products were cloned into the pLenti7.3-V5 lentiviral

backbone (Invitrogen) using the Gateway system (Invitrogen). Primers were

designed using the Primer3 tool (http://frodo.wi.mit.edu/primer3/) in order

to specifically hybridize on the start and stop transcription sites of each

mRNA (Fig. 5.1).

Retrotranscription was performed as follows: RNA was quantified using a

NanoDrop spectrophotometer (ND-1000 Spectrophotometer, Thermo

Scientific) and about 500 ng of total RNA from each sample was converted

into cDNA with oligo-dTs for reverse transcription using the Advantage® RT-

for-PCR kit (Clontech) following the manufacturer’s instructions.

FIG. 5.1 General strategy for gene isolation and PCR amplification. TSS, transcription start

site; CDS, coding DNA sequence.

5’ 3’PROMOTOR

DNA

EXON 1 EXON 2 EXON 3INTRON 2INTRON 1

TSS

5’ 3’ Primarytranscript

EXON 1 EXON 2 EXON 3INTRON 2INTRON 1

atg

aug

aug(start codon)

5’ 3’MaturemRNA

AAAAAAACA

P

uga, uaa, uag(stop codon)

ORF

CDS

PROTEIN

TRANSCRIPTION

SPLICING

TRANSLATION

Fw Rv

Clonedsequence


- 68 -

cDNA amplification was performed by PCR using the thermostable proof-

reading polymerase Pfx50-DNA (#12355012, Invitrogen). The primer

sequences used to isolate the ORFs are shown in Table 5.1.

TABLE 5.1. GenBank accession numbers and primer sequences for cloned genes; according

to the Gateway technology manufacturer’s instructions 5’ forward primers all include a CACC

sequence.

GENE GenBank accession no.

Primer sequence 5’3’ Length (bp)

PRDM1 NM_182907.2 F: CACCATGGAAAAGATCTATTCCA 2080

R: TTAAGGATCCATTGGTTCAACTG

PRDM14 NM_024504.3 F: CACCATGGCTCTACCCCG 1720

R: CTAGTAGTCTTCATGAAACTTCA

LIN28 NM_024674.4 F: CACCATGGGCTCCGTGT 634

R: TCAATTCTGTGCCTCCGGGA

NANOS3 NM_001098622.2 F: CACCATGGGGACCTTTGACC 583

R: CTAGGTGGAGATGGAGGGAGAG

NANOG NM_024865.2 F: CACCATGGGTGTGGATCCAG 922

R: TCACACGTGTTCAGGTTGCATG

DAZ2 NM_020363.3 F: CACCATGTCTGCTGCAAATCCT 1692

R: CTCTTCTCTGACTATTTA

DAZL NM_001351.3 F: CACCATGGCTACTGCAAATCC 892

R: TCAAACAGATTTAAGCATTGCC

BOULE NM_197970.2 F: CACCATGGAAACCGAGTCC 892

R: TTAATAATGAATGCTCCACACTG

VASA NM_024415.2 F: CACCATGGGAGATGAAGATTG 2179

R: TTGGCTTTAATGCCATGACTC

STRA8 NM_182489.1 F: CACCATGGGGAAGATTGATGT 997

R: TTACAAATGTTCATCGTCAAAGG

SYCP3 NM_001177949.1 F: CACCATGGTGTCCTCCGGA 715

R: TCAGAATAACATGGATTGAAGAG

DMC1 NM_007068.2 F: CACCATGGAGGAGGATCAAGT 1027

R: CTACTCCTTGGCATCCCCAAT

5. Methods

- 69 -

Briefly, the PCR conditions were the following: Pfx50-DNA polymerase 10x

Buffer with MgSO4 (1x); dNTP mix (200 μM); primers (1.0 µM), template

DNA at less than 0.5 μg/50 μL, and Pfu-DNA polymerase (2-3 U/μL) at a

concentration of less than 1.25 U/50μL. In general the PCR programs were

as shown in Table 5.2. The PCR product was isolated using a QIAquick gel

extraction kit (#28704, Qiagen).

TABLE 5.2. PCR cycles for Pfx50-DNA polymerase extension.

Step Temperature Time Number of Cycles

Initial Denaturation 95° C 1-2 minutes 1 cycle

Denaturation 95° C 0.5-1 minute 25-35 cycles

Annealing 42–65° C 30 seconds

Extension 72–74° C 2-4 minutes

Final Extension 72–74° C 5 minutes 1 cycle

Soak 4° C Indefinite 1 cycle

5.1.2.2 pENTRTMTOPO® VECTOR CLONING

Briefly, the Gateway system (Invitrogen) is a two-step cloning system in

which the isolated PCR product is first cloned into a pENTRTM/D-TOPO®

system entry vector (#K2400-20, Live Technologies) and is then sub-cloned

into an expression vector with a lentiviral backbone. The PCR products are

directionally cloned with the pENTRTM/D-TOPO® system by adding four

specific bases to the forward primer (CACC) and the covalently bound

Topoisomerase I from Vaccinia virus allows these PCR products to be cloned

in the right direction. The pENTRTM/D-TOPO® map is shown in Appendix 1.

Cloning reactions were performed following the manufacturer’s

instructions. E. coli Transforming One Shot TOP10® (Invitrogen, Carlsbad,


- 70 -

CA, #C4040-10, Life Technologies) were transformed by heat shock and the

transformed clones were selected by conferred kanamycin resistance.

Transformed clones were analyzed using restriction enzymes to ensure

proper cloning of the insert. The plasmids were isolated using the PureLink®

HQ Mini Plasmid Purification Kit (#K2100-01, Life Technologies). Moreover,

pENTRTM/D-TOPO® has attL1/attL2 sequences which enables specific site

recombination with the destination/ expression vectors (pLENTI7.3/V5-

DEST).

5.1.2.3 DESTINATION VECTOR CLONING

The destination pLenti7.3/V5-DEST vector (#V534-06, Life Technologies)

includes the attR1/attR2 sites which are complementary to the attL1/attL2

sequences; this facilitates the proper insertion of the gene of interest from

the pENTRTM/D-TOPO® into the target. This plasmid has a V5-epitope tag at

the 3’ end of the insertion site, so the resulting recombinant protein has a

V5-fusion tag at the C-terminal end, and in addition, it also contains the

emerald green fluorescent protein (EmGFP) under control of the SV40

promoter as a reporter (Appendix 1). The recombination reactions were

performed according to the manufacturer’s instructions. Transformed

clones were selected by conferral of ampicillin resistance and plasmids

were purified as previously described. Recombinant plasmids were

sequenced using primers provided by the manufacturer:

forward primer 5′–CGCAAATGGGCGGTAGGCGTG–3′ (CMV);

reverse primer 5′–ACCGAGGAGAGGGTTAGGGAT–3 (V5 C-term).

5. Methods

- 71 -

Finally, the clones were amplified and purified with a QIAGEN Plasmid Maxi

Prep kit (#12965, QIAGEN).

5.2 REPROGRAMMING FACTOR OVEREXPRESSION AND CELL CULTURE

OPTIMIZATION

5.2.1 SOMATIC PRIMARY CULTURES

The following cell lines were cultured in vitro:

- Human foreskin fibroblasts (hFSKs; 46, XY) from the American Type Culture

Collection (Manassas, VA; Ref. ATTC #CRL-2429).

- Human mesenchymal cells (hMSCs) from bone marrow (46, XY; PT-2501,

Lonza).

- Patient-derived primary Sertoli cells (Riboldi et al., 2012)

- The 293-T packaging cell line (293-T) from the American Type Culture

Collection (Manassas, VA; Ref. ATCC # CRL-11268).

5.2.2 LENTIVIRAL TRANSDUCTION

Once the lentiviral expression constructs containing the inserts were

obtained they were ectopically overexpressed following a conventional

protocol for lentiviral transduction. Briefly, three plasmids were co-

transfected onto 293-T cells to produce lentiviral supernatants:

- Plasmid 1, containing the HIV-gag polymerase (retro-transcriptase) and

accessory proteins Tat and Rev (pCMVdeltaR8.91, derived from

pCMVR8.931).


- 72 -

- Plasmid 2, containing the envelope glycoprotein VSV-G (pMD.G) for

pseudotyped lentiviral particle production.

- Plasmid 3, destination/expression lentiviral vector pLenti7.3/V5-DEST with

the cloned gene.

These plasmids were obtained using a QIAGEN Plasmid Maxi Prep kit

(#12965, QIAGEN). Their respective maps are shown and described in

Appendix 1.

High-titre lentiviral supernatants were generated in the 293-T packaging

cell line by transitory co-transfection with the three plasmids at 0.5 µg/mL

with Fugene HD (Roche Diagnostics, Indianapolis, IN; Ref. n. 4709705001) at

5 µg/mL in OPTI-Mem low protein cell culture medium (Invitrogen; Ref. n.

31985-047). Co-transfection media were incubated for 6-8h at 37˚ C in a

humidified atmosphere with 5% CO2. This medium was then replaced with

D-MEM (Sigma, Ref. n. D6421) supplemented with 10% fetal bovine serum

(FBS; Life Technologies) without antibiotics. Viral supernatants were

collected and filtered after 72 h.

Infection was performed by adding the lentiviral supernatant to hFSK and

hMSC primary cultures for 8-12 h at 37˚ C in a humidified atmosphere with

5% CO2 in the presence of 10 µg/mL of polybrene (Sigma-Aldrich, St Louis,

MO; Ref. n. TR1003G) when they were at 60% confluence. After infection

these lentiviral supernatants were removed and replaced with conventional

culture media. Seventy-two hours after transduction the cells were checked

for EmGFP fluorescence as a lentiviral vector expression reporter.

5. Methods

- 73 -

5.2.3 CULTURE CONDITIONS

All cells were cultured at 37° C in an incubator with a thermostat, with

saturated humidity and 5% flow-regulated CO2. Cell manipulation was

carried out in laminar flow hood under sterile conditions. The culture media

were as follows:

- Conventional culture media: Dulbecco's modified Eagle's medium/

nutrient F-12 ham (D-MEM/F12; GIBCO, Invitrogen) supplemented with

10% FBS and 2 mM Glutamine, 0.1 mM MEM non-essential amino acids

(both Life Technologies), and 1% penicillin/streptomycin (Life Technologies)

- Induced germ-cell survival/germ-cell media (GC-M): DMEM/F12 with 20%

knock-out serum replacement (KSR; GIBCO, Invitrogen), 2 mM glutamine

(Life Technologies), and 0.1 mM non-essential amino acids (Labclinics, PAA),

supplemented with 1000 U/ml human recombinant LIF (Sigma), 20 ng/mL

human recombinant GDNF (Peprotech), 5 µM Forskolin (Sigma), 10 ng/ml

bFGF (Invitrogen), 0.1 mM 2-mercaptoethanol (Millipore), 5 ng/mL SCF

(Peprotech), and 2 µM RA (Sigma).

- Co-culture with Sertoli cells: twenty-four hours before seeding the

reprogrammed cells, Sertoli cells were plated on a collagen matrix (#C6745,

Sigma) at a density of 104 cells/cm2 and cultured under standard conditions.

3-days post-transduction, reprogrammed cells were harvested and seeded

on this Sertoli cell monolayer. The co-culture was maintained with GC-M

until day 14 when they were harvested for further analysis.


- 74 -

5.3 CHARACTERIZATION OF GERM CELLS INDUCED IN VITRO

5.3.1 TRANSCRIPTOMIC CHARACTERIZATION

Total RNA was prepared using the RNeasy Mini Kit (Qiagen) following the

manufacturer’s instructions. RNA quantification and purity were

determined using a NanoDrop spectrophotometer. The integrity of the RNA

samples (RNA quality control procedure) was assessed with a 2100

Bioanalyzer (Agilent Technologies, Madrid, Spain) by running an aliquot of

the RNA samples in the RNA 6000 Nano LabChip (Agilent Technologies,

Madrid, Spain). The Bioanalyzer-2100 software determines a parameter

indicative of RNA integrity called an RNA integrity number (RIN), which is

calculated using ribosomal RNA ratios; values higher than 7 confirm high

quality RNA. Transcriptomic characterization was performed either by an

expression array or qRT-PCR.

5.3.1.1 EXPRESSION ARRAY

All samples were hybridized onto the Whole Human Genome Oligo

Microarray (Agilent Technologies) that encompasses more than 44,000

human DNA probes. The sample preparation and hybridization protocols

were adapted from the Agilent Technical Manual. Briefly:

1) Microarray hybridization: First-strand cDNA was transcribed from 100 ng

of total RNA using the T7-Oligo(dT) promoter primer. Samples were

transcribed in vitro and Cyanine-3 (Cy-3) labelled (Quick- AMP labelling kit,

Agilent Technologies). Fragmented copy RNA (cRNA) samples (1.65 µg)

were hybridized onto chips by incubating them for 16 h at 65° C with

5. Methods

- 75 -

constant rotation. The microarrays were then washed in two one-min steps

in two washing buffers (Agilent Technologies, Madrid, Spain). Hybridized

microarrays were scanned in an Axon 4100A scanner (Molecular Devices,

Sunnyvale, CA, USA), and data were extracted with the GenePix Pro 6.0

software (Molecular Devices, Sunnyvale, CA, USA).

2) Data processing and analysis: The GenePix Pro 6.0 software was used for

array image analysis and to calculate the spot intensity measurements,

which are considered to be raw data. Spot intensities (medians) without

background subtraction were transformed to the log2 scale using R

software (R Development Core Team, 2004) and libraries from the

Bioconductor database (www.bioconductor.org). Before quantile

normalization, the data were initially represented on a box plot so that the

distribution could be analyzed and any abnormal microarray data could be

subtracted. Replicates by gene symbol were merged and the data were

filtered in order to delete unknown sequences or probes without a gene

description. The web-based Babelomics tool

(http://babelomics.bioinfo.cipf.es) was used on the resulting data matrix to

merge the replicates from each probe by mean and for principal

component analysis (PCA). Next, samples treated with i12F/i6F versus

MOCK controls were statistically analyzed using the Rank Products module

in MeV software for statistical analysis (www.tm4.org). A false discovery

rate (FDR) correction of less than 5% was used. The DAVID web-based tool

(http://david.abcc.ncifcrf.gov) was used to predict the biological processes

affected. Microarray results were validated by performing qPCR for two of

the most interesting upregulated genes.


- 76 -

5.3.1.2 QUANTITATIVE REAL-TIME PCR

RNA quantification was measured using a NanoDrop spectrophotometer.

Next, 500 ng of total RNA was converted into cDNA with oligo-dTs using the

Advantage® RT-for-PCR kit (Clontech) following the manufacturer´s

instructions.

Subsequently, 2 µl of cDNA were qRT-PCR amplified with SYBR®Green

(Invitrogen). Transcription levels were determined by using a LightCycler

480 (Roche) in triplicate reactions and normalized to the average of the

housekeeping gene RPL19 using the 2-ΔΔCt method. The primers used to

detect gene expression are described in Appendix 2 and were designed with

the Primer3 tool (http://frodo.wi.mit.edu/primer3/).

5.3.2 EPIGENETIC CHARACTERIZATION BY BISULFITE SEQUENCING

Epigenetic characterization was performed by bisulfite sequencing (bi-seq),

a technique which is based on bisulfite-converted genomic DNA (gDNA).

Treating gDNA, previously extracted with the QIAamp DNA MiniTM kit

(Qiagen), with bisulfite converts C into U residues. Hence, bisulfite

treatment changes the genomic sequence depending on the methylation

status of individual Cs; by comparing the post-treated sequence to a

reference sequence, the initial C methylation status can therefore be

determined (Fig. 5.2).

Genomic DNA was extracted with the QIAamp DNA Mini kit (Qiagen) and

100 ng of gDNA was processed using the EpiTect Bisulfite Kit (Qiagen)

according to the manufacturer’s instructions. 1 µL of bisulfite-treated gDNA

5. Methods

- 77 -

was PCR-amplified in the presence of 5 mM MgCl2, 0.2 mM dNTPs, 10 pmol

of each primer, and 1 U Platinum Taq polymerase (Invitrogen). PCR

amplifications for H19 and PEG1/MEST1 were initiated at 94˚ C for 3 min

followed by 45 cycles of 94˚ C for 30 sec; 58˚ C for 30 sec; and 72˚ C for 30

sec. Primer sequences are included in Appendix 2. The resulting PCR

products were gel-extracted using a QIAquick gel extraction kitTM (Qiagen)

and cloned into the pCR®2.1-TOPO® VECTOR using the TOPO-TA cloning®

kit (Ref. 45-0641, Invitrogen). At least 20 clones were sequenced for

analysis, and CpG methylation analysis was performed using BiQ Analyzer

software (Max Planck Institut Informatik).

FIG. 5.2 gDNA bisulfite conversion. A) Biochemical bisulfite conversion reaction; B) General methylation-status discrimination strategy; C) Interpretation of results after bisulfite treatment. Ref. Seq., Reference sequence used to compare with the Bs-converted seq., the sequenced bisulfite-converted DNA.

Ref. Seq.

Bs-converted seq.

Non-methylated

Methylated

A

B C


- 78 -

5.3.3 MEIOTIC PROGRESSION ANALYSIS

In order to assess the meiotic status and progression of treated cells, two

different assays were performed. SYCP3 staining on meiotic spreads allows

meiotic cells to be identified and to subsequently determine how advanced

through meiosis they are, while ploidy analysis by fluorescence in situ

hybridization (FISH) enables us to determine if reprogrammed cells have

completed meiosis, forming haploid cells.

5.3.3.1 SYCP3 STAINING

Meiotic spreads were performed as previously described (Kee et al., 2006,

Panula et al., 2011, Medrano et al., 2011). Briefly, cells were collected and

resuspended in hypoextraction buffer (30 mM Tris, 50 mM sucrose, 17 mM

citric acid, 0.5 mM dithiothreitol [DTT], 0.5 mM phenylmethylsulfonyl

fluoride [PMSF] all from Sigma Aldrich, and 5 mM

ethylenediaminetetraacetic acid [EDTA] from Invitrogen, at pH 8.2-8.4). The

solution was incubated for 30 min at room temperature and then put onto

slides which had previously been fixed with 1% paraformaldehyde (PFA).

Slides with the cellular solution were incubated overnight in a humid

chamber at room temperature. Then, the cells were permeabilized for 2

min with 0.04% Photoflo (KODAK) followed by incubation with blocking

solution for 60 min with 4% donkey serum in 1 % bovine serum albumin

(BSA) and 0.1% Triton X100 in phosphate-buffered serum (PBS). Human

anti-CREST antibody (Fisher Scientific) was used at a 1:1,000 dilution and

incubated overnight at 37˚ C in a humid chamber. Rabbit polyclonal anti-

SYCP3 primary antibody (Novus Biologicals) was used at a 1:100 dilution

5. Methods

- 79 -

and incubated for 3 h at 37˚ C in a humid chamber. The slides were washed

with blocking solution and the secondary donkey anti-human 4',6-

diamidino-2-phenylindole (DAPI)-conjugated antibody (Jackson

Immunoresearch) was applied at a 1:100 dilution and incubated overnight

at 37˚ C in a humid chamber. The following day the slides were washed as

described above and incubated with a donkey anti-rabbit

tetramethylrhodamine (TRITC)-conjugated antibody (Abcam) at a 1:200

dilution for 1 h at 37˚ C in a humid chamber. Prolong Gold anti-fade reagent

(Life Technologies, ref. P36935) was applied. Finally, the cells were counted

under a fluorescence microscope. SYCP1 co-staining with SYCP3 was also

performed following a similar protocol.

5.3.3.2 PLOIDY ANALYSIS: DNA-SORTER AND FLUORESCENCE IN SITU

HYBRIDIZATION

Ploidy analysis was performed following different strategies: i) in the total

population; ii) in the putative haploid population, which had previously

been purified by a DNA-content cell sorter; iii) in manually selected clumps.

- DNA-content fluorescent-activated cell-sorting (FACS): cells were

harvested into a single cell suspension and then fixed with 70% ethanol

over night at - 20° C. The cells were then washed and incubated in a

solution containing 0.2 mg/ml RNase A and 0.02 mg/ml propidium iodide

(PI, Invitrogen) in Hank’s balance salt solution (HBSS, Invitrogen) for 30 min

at 37° C. Cells were sorted according to their DNA content by a MoFlo®

(Modular Flow Cytometer; Beckman Coulter, Dako, Denmark,

http://www.dako.com) jet-in-air high-speed sorter.


- 80 -

- Fluorescence in situ hibridization (FISH): Cells were harvested into a single

cell suspension (total population or manually selected clumps) or sorted cell

populations were collected and post-fixed with Carnoy’s fixative (1:3 acetic

acid:methanol) for 5 min and then air dried. The slides were dehydrated in

a sequence of 70%, 80%, and then 100% ethanol and air dried. FISH probes

for chromosomes 18 (D18Z1,Kreatech, KI-20018-B) , and X and Y (SE-X

[DXZ1]/SE-Y [DYZ3], Kreatech, KBI-20030) were denatured on slides at 73° C

for 5 min and then hybridized at 37° C overnight. The slides were then

washed with 50% formamide, 2X saline sodium citrate at 43° C for 5 min

each. ProLong Gold anti-fade reagent with DAPI was applied to the slides

and the cells were counted under a fluorescence microscope.

5.3.4 IMMUNOFLUORESCENCE

Cultured cells were fixed with 4% PFA for 15 min at room temperature and

then washed twice with PBS. They were then permeabilized with 1%

TritonX100 in PBS for 15 min at room temperature, blocked with 0.5%

Tween20, 1% BSA, and 4% secondary-antibody host-serum. Primary

antibodies were incubated over night at 4˚ C, while secondary antibodies

were incubated for 2-3 h at room temperature in the dark. A detailed

description of the antibodies used is included in Table 5.3. ProLong Gold

anti-fade reagent with DAPI was applied to the slides and the cells were

counted under a fluorescence microscope.

For 5hmC immunostaining, a chromatin-opening step was performed

before permeabilizing: cells were incubated with 4M HCl for 15 min and

neutralized with 100 mM Tris-HCl at pH 8.5 for 30 min at room

5. Methods

- 81 -

temperature. Blocking was performed overnight at 4˚ C and primary

antibodies were incubated for 4 h at room temperature.

Table 5.3 Primary antibodies used.

ANTIGEN PRIMARY ANTIBODY REF COMPANY

CREST Human anti-centromere protein 15-234 Fisher

SYCP3 SCP-3 Purified rabbit antisera NB 300-232 Nobus

SYCP1 Goat anti SCP-1 sc-23600 Santa Cruz

5hmC Rabbit anti-5-hydroxymethylcytosine

39791 Active Motif

Alkaline Phosphatase (AP)

Mouse monoclonal anti-TRA2-54

MAB4354 Millipore

PLZF Mouse monoclonal anti-PLZF MAB2944 R&D

HIWI Rabbit anti-HIWI 710208 Novex-life technologies

ACRO Goat anti-acrosin sc-46284 Santa Cruz

Human VASA (hVASA)

Goat anti-VASA AF-2030 R&D

NuMA Rabbit anti-Human NuMA Ab36999 ABCAM

V5-epitope Mouse monoclonal anti-V5 r960-25 Invitrogen

5.4 XENOTRANSPLANT ASSAYS

In order to test the in vivo functionality of the germ cells induced in vitro, a

xenotransplant assay was performed (Fig. 5.3). Briefly, transduced cells

were injected into the efferent duct of previously busulfan-sterilized nude

mice to test if they were able to recolonize the SSC niche.

5.4.1 MOUSE STRAIN SELECTION

For this assay 4-6-week-old nude (Crl:NU-Foxn1nu) male mice were chosen

This strain is appropriate for these xenotransplant assays because they are

immunodeficient and so no immuno-cross-reactivity is expected with the


- 82 -

FIG. 5.3 General overview of xenotransplant assay. GFP; green fluorescent protein, hFSK; human foreskin fibroblast, IP; intraperitoneal.

STERILIZATION (Busulfan, IP)

4 weeks 8 weeks

GC-M

GERM CELL TRANSPLANTATION

TESTIS ANALYSIS

2 weeks

hFSKTRANSDUCTION

GFP+-transducedcells GFP+/CellVue+

CellVue® Claret FarRed CellLinker (Sigma) for membranelabeling

human transplanted cells.

This animal model assay was approved by the ethical committee (Comité de

Ética y Bienestar Animal, Universidad de Valencia, Ref, A1332863413145).

Since all these animals are immunodeficient, all the procedures and animal

manipulations were performed under sterile conditions.

5.4.2 BUSULFAN STERILIZATIONSTERILISATION

Busulfan is a DNA-alkylating agent (Fig. 5.4) that can eliminate germ cells

and breaks cell-to-cell junctions between Sertoli cells. These effects allow

the colonization of transplanted cells in the basal lamina from seminiferous

tubules. The mice were sterilized by an intraperitoneal (IP) injection of

busulfan (30-35 mg/kg) dissolved in dimethyl sulfoxide (DMSO) and after 4-

6 weeks when endogenous spermatogenesis is suppressed, the mice were

xenotransplanted.

5. Methods

- 83 -

Mice were kept under sterile conditions in a specific pathogen free area in

the Animal Facility at the Faculty of Pharmacy (University of Valencia) until

the xenotransplant surgery was carried out.

5.4.3 PREPARATION OF DONOR CELLS

hFSK cells were harvested 14 days post-transfection and prepared for

xenotransplant. Cell harvesting was synchronized to correspond with week

4 after busulfan sterilization of the recipient mice.

The EmGFP reporter gene, under the control of a constitutive promotor

contained in our lentiviral backbones, was used to track cells after

transplantation. This backbone also incorporates a short V5-epitope at the

C-terminal of the recombinant protein that can act as a marker of cells that

have been properly transduced. Even so, another non-viral marker was

chosen: the CellVue® Claret far red fluorescent from a cell linker Kit (Sigma-

Aldrich, ref. MIDCLARET/ 24849-1). This far red fluorescent molecule (ƛMAX

Exc = 655 nm; ƛMAX Em = 675 nm) binds to the aliphatic tails of phospholipids

and inserts into cellular lipid bilayers (Fig. 5.5) allowing cell tracking over

cell divisions.

Harvested cells were incubated with CellVue® Claret far red according to

the manufacturer’s instructions: briefly, 107cells/mL were incubated at a

FIG. 5.4 The chemical structure of Busulfan.


- 84 -

final concentration of 2 µM CellVue® Claret Far Red for 4 min at room

temperature in the dark. The cells were washed twice with 10% BSA in PBS

and then washed with GC-M.

Finally, the cells were concentrated to 1-3x105 cells/µL in GC-M and kept on

ice. Just before xenotransplant 10% (v/v) trypan blue dye and 1 mg/mL

DNase I were added to ensure that the injected cells could be properly

visualized while being injected into the seminiferous tubules and to prevent

the formation of cellular aggregations.

5.4.4 XENOTRANSPLANTATION

Four weeks after busulfan sterilization (approximately 2.5-3-month-old

mice) xenotransplant surgery was performed (Fig. 5.6). Mice were

anesthetized using 5% isofluorane and the abdominal area was prepared by

applying povidone-iodine. The recipient testes were exteriorized through a

0.75 cm mid-ventral abdominal incision.

FIG. 5.5 CellVue® Claret Far Red Fluorescent Cell Linker. A. Fluorescence absorption and emission spectra. B. Membrane-labeling insertions.

A B

5. Methods

- 85 -

FIG. 5.6 Mice xenotransplant. A. Surgery set-up overview; B. Mouse preparation and testis manipulation; C. Seminiferous tubules at the start of donor cell injection; D, Appearance of the xenografted testis after injection. 1: Inhalational anaesthesia; 2: Dissecting scope; 3: micromanipulator; 4: FemtoJet microinjector; 5: Injection pipette; 6: pre-cut “V” paper pad; 7: exteriorised testicle; 8: sponge; 9: tip of injection pipette inserted into the efferent duct.

1

2

3

4

5

6

8

9

A

B

C D

7


- 86 -

The first testis to be injected (right) was pulled out through the incision

using the testis fat pad and placed on the pre-cut ‘V’ paper pads to secure it

in position. The membrane connecting the testis to the epididymis was

gently pulled apart and fat cleared to expose the efferent duct. Using

sponges, the testis was positioned upright to align a long straight stretch of

efferent duct with the injection pipette (bevelled micropipettes with a 75

µm inner diameter). The pipette was filled with 8-10 µl of cell preparation

and, with the help of a micromanipulation unit (Leica), was inserted into the

efferent duct so that its tip was positioned in the rete testis.

Cell injections were performed with a FemtoJet microinjector (Eppendorf).

After the injection was completed on the first side, the sponges were

removed and the testis was returned to the abdominal cavity. The same

steps were repeated on the other testis, and finally the incision was

sutured. All these steps were performed under a dissecting scope with a flat

base and tilting binocular eyepiece tube (Nikon).

5.4.5 XENOTRANSPLANTED TISSUE ANALYSIS

Two months post-surgery the mice were sacrificed by cervical dislocation

and the testes were removed and analyzed for the formation of colonies of

iGCs by flow cytometry and confocal immunofluorescence using

cryosections.

- Flow cytometry protocol: Half of each xenotransplanted testis was

mechanically minced and enzymatically disaggregated (2 mg/mL

collagenase IA [Worthington, ref. LS004176] was added for 15 min at 37˚ C

and then centrifuged at 200 rpm, followed by two decantation steps and

5. Methods

- 87 -

trypsinization using 0.25% trypsin [Life technologies] for 10 min at 37˚ C).

Then, 10 mg/mL DNase I (Sigma-Aldrich, ref. D4513) was added to the

single cell suspension and immediately followed by lysis buffer to eliminate

erythrocytes (dilution 1:10 from red blood cell Lysis Buffer 10X, BioLegend,

ref. 420301). The single-cell suspension was filtered (with a 50 µm diameter

filter from Partec CellTrics®, ref. 04-004-2327) and then counted.

Conventional cell suspension immunofluorescence was then performed:

Briefly, cells were fixed with 1% PFA for 15 min at room temperature,

washed, and then resuspended in Perm/Wash Buffer 1X (dilution 1:10 from

BD Perm/WASH™ Buffer; BD, ref. 554723). Primary antibodies (Table 5.3)

were incubated at a 1:100 dilution for 1-2 h on ice and following washing,

the secondary antibodies were incubated consecutively for 1 h on ice in the

dark. Cells were analyzed with a 4-laser LSRFortessa BECTON DIKINSON

flow cytometer for antibody expression and the presence of the CellVue®

Claret Far Red signal.

- Tissue cryosections: The other part of each xenotransplanted testis was

cryopreserved in O.C.T.TM compound (Tissue Tek, ref. 4583); 8 µm

cryosections were made using a Leica CM1950 Cryostat, and conventional

immunofluorescence was carried out: Briefly, tissue sections were fixed

with 4% PFA for 10 min at room temperature. After washing, the slides

were permeabilized with 1% TritonX100 in PBS for 20 min at room

temperature, and blocked with 4% donkey serum (Sigma-Aldrich, ref

D9663), 3% BSA, and 0.1% Tween20. Primary antibodies were incubated at

a 1:200 dilution overnight at 4˚ C and consecutive secondary antibodies

were incubated for 1-2 h at room temperature in the dark. Finally the slides


- 88 -

were mounted with Prolong Gold anti-fade reagent with DAPI. Sections

were analyzed by confocal microscopy (Olympus FV1000), for the presence

of different primary antibodies and for the CellVue® Claret Far Red signal.

NuMA+/VASA+/CellVue+ triple positive cells located on the basal layer of

tubules were counted for 15 serial cryosections per transplanted testis

leaving a distance of 80 µm between them. The average number of triple

positive cells found by two independent researchers was divided by the

number of injected cells in each testis and multiplied by 105 to obtain the

colonization efficiency per 105 injected cells.

5.5 STATISTICAL EVALUATION OF THE RESULTS

For each experiment at least three independent samples (n = 3) were used.

The different values are represented as the mean plus or minus (±) the

standard error of mean (SEM).

Statistical significance was calculated with the SPSS statistical package (SPSS

Inc.). Statistical analysis of the RT-qPCR results as well as meiosis and FISH

counts was performed with one-way ANOVA and t-student pair-wise

comparisons. Statistical significance was accepted for P-values less than

0.05. The only exception was data from the expression arrays: an

explanation is included for these experiments in their respective results

sections.

- 89 -

6. Results

Four-leaf clover, Daisy, Iris

Lucky, faith, good news

6. Results

- 91 -

6.1 HUMAN FORESKIN FIBROBLAST REPROGRAMMING USING TWELVE

GERM LINE FACTORS INDUCES THE FORMATION OF PUTATIVE GERM CELL-

LIKE CELLS

6.1.1 ECTOPIC EXPRESSION OF TRANSDUCED GENES

As previously explained, based on the literature, a cocktail of twelve

candidate genes was chosen because of their role in mammalian germ line

development: PRDM1 (Vincent et al., 2005), PRDM14 (Kurimoto et al.,

2008), LIN28A (West et al., 2009), NANOG (Mitsui et al., 2003), NANOS3

(Suzuki et al., 2008), DAZ2, BOULE, DAZL (Ruggiu et al., 1997), VASA (also

known as DDX4) $(Fujiwara et al., 1994), STRA8 (Bowles et al., 2006), DMC1

(Yoshida et al., 1998), and SYCP3 (Costa et al., 2005). These genes were

isolated by PCR and their ORFs were cloned into a lentiviral expression

backbone containing a CMV promoter and tagged with a GFP reporter. The

lentiviral vector also included a V5 epitope at the C-terminal end of the

cloned protein (Appendix 1). The lentiviral particles carrying the transgenes

of interest were used in a mixture of equal proportions of the twelve

factors (i12F) to transduce a human somatic primary culture of foreskin

fibroblasts (hFSKs; 46, XY). The empty lentiviral backbone (MOCK) was also

included as an independent negative control condition. To check the

lentiviral infection efficiency GFP fluorescence emission was used as a

reporter (Fig. 6.1A-B). As shown by qRT-PCR, transduced hFSK primary

cultures overexpressed all the transgenes, although at different levels (Fig.

6.1C). PRDM1 and NANOG transgene expression levels were constantly

lower than the others because the infection efficiency was not as high as

that of the other lentiviral particles. In the case of PRDM1, the lower rate of


- 92 -

overexpression (compared with the levels reached by the other factors) is

explained by the relatively high basal levels of PRDM1 in hFSK cells. Even

though levels of ectopic NANOG overexpression were not as high as the

other factors, there was a 60 fold-change compared with MOCK controls

meaning that their levels were sufficient enough to obtain the expected

phenotype.

6.1.2 REPROGRAMMED CELL MORPHOLOGICAL CHANGES S AND THE

ESTABLISHMENT OF CELL CULTURE CONDITIONS

Initially, human primary cultures were transduced with the previously

mentioned twelve factors and were cultured in standard medium consisting

of DMEM/F12 with 20% of FBS. A morphological change was observed from

day 7 post-transduction, when some small clumps of compacted, small,

FIG. 6.1 Green fluorescence protein (GFP) and reprogramming factor expression in i12F-treated hFSKs. A) Bright-field microscopy; B) Dark-field microscopy; C) qRT-PCR transgene overexpression. Values are indicated as the fold-change vs. the MOCK-treated negative controls.

PR

DM

1; 2

3.61

5573

5

PR

DM

14; 4

162.

79

LIN

28; 2

057.

49

NA

NO

S3; 1

1692

.80

NA

NO

G; 6

3.41

DA

ZL; 8

39.4

7

VA

SA; 2

2558

8.40

STR

A8;

154

2315

.69

BO

ULE

; 676

59.2

7

DA

Z2; 4

3883

7.31

SCP

3; 8

2570

.19

DM

C1;

279

7.65

0

20

40

60

80

100

120

140

160

180

200

i12FPRDM1

PRDM14

LIN28

NANOG

NANOS3

DAZL

DAZ2

VASA

STRA8

BOULE

DMC1

SYCP3

C

B

A

6. Results

- 93 -

round cells began to appear. After few days, these clumps disappeared, an

indication that perhaps the culture conditions were not optimal for their

survival (Fig. 6.2A).

In order to improve the survival of these cell clumps, and based on previous

reports on the derivation of germ cells from testicular biopsies, a specific

cell culture media (Germ Cell Media; GC-M) was designed with the purpose

of promoting putative germ cell survival. This GC-M consisted of

DMEM/F12 supplemented with 20% KSR, 20 ng/mL GDNF, 10 ng/mL bFGF,

1000 U/mL LIF, 5 ng/mL SCF, and 2 μM RA (Conrad et al., 2008, Kossack et

FIG. 6.2 A) Morphological change were only maintained under GC-M culture conditions. B) i12F-treated cell clusters were alkaline phosphatase positive. Scale bar represents 10μm.

5 Days 16 Days13 Days 20 Days8 Days

i12

F St

and

ard

-Mi1

2F

GC

-MM

ock

Bright Field Alkaline Phosphatase MERGEDDAPI

28

Day

s5

0 D

ays

B

A


- 94 -

al., 2009, Golestaneh et al., 2009). Under these cell culture conditions an

increase in spontaneous cell clump formation was observed (Fig. 6.2A).

Moreover, these cell clumps could be maintained in culture for up to 50

days and were also positive for AP (Fig 6.2B). These results suggested that

these clumps had a putative induced germ cell like cell (iGC) identity that

could only be maintained in GC-M supplemented with growth factors.

6.2 TWELVE FACTOR-REPROGRAMMED CELLS HAVE A GERM LINE-LIKE

TRANSCRIPTOMIC AND EPIGENETIC PROFILE

6.2.1 TRANSCRIPTOMIC ANALYSIS BY EXPRESSION ARRAY AND

QUANTITATIVE REAL-TIME PCR

Expression arrays were performed using RNA samples to determine the

global expression profile of i12F-transduced cultures. Treated cultures from

different independent experiments clearly clustered in a defined group (Fig.

6.3A) and from these 21 genes that were significantly differentially

upregulated compared to MOCK controls were identified, indicating that

the gene expression profile of the transduced culture cells had switched.

Gene ontology (GO) analysis of these genes indicated that they were

implicated in germ cell-related processes (Fig. 6.3B-C) such as meiosis (P <

0.001), spermatogenesis (P < 0.001) or germ cell development (P < 0.001),

among others, with a predicted activation of translation regulation activity

(fold enrichment) of 63.95 (P < 0.001).

6. Results

- 95 -

FIG. 6.3. A) Hierarchical clustering of significantly upregulated genes in i12F-transduced samples vs. MOCK controls. B) Percentages of fold-enrichment in GO terms. C) Details of the enrichment in GO terms related to ‘germ line development’. (*) sexual reproduction (1%), multicellular organism reproduction (1%), reproductive processes in a multicellular organism (1%), reproductive developmental process (2%), gamete generation (1%).

Germ line development

46%

Epithelial cell differentiation 13%

Fluid transport 7%

Regulation of neuronal synaptic plasticity 6%

Defense response 4%

Myeloid leukocytedifferentiation 3%

Cell motion 3%

Negative regulation of celldevelopment 3%

Chemotaxis 3%Inflamatory response 3%

Leukocyte differentiation 2%

Cellular hormone metabolic process 2%

Meiotic cell cycle 3%

Meiosis 3%

M phase of meiotic cell cycle 3%Others related to Reproductive

developmental process (*)

Male meiosis I 18%

Spermatogenesis 2%

Male gamete generation 2%

Ovulation cycle process 4%

Female gonad development 4%

Ovulation cycle 4%

Ovarian follicle development 6%

Development of primary female sexual characteristics 4%

Female sex differentiation 4%

Positive regulation of translational initiation 33%

Regulation of cellproliferation 1%

Rhythmic process 3%

A

B

C


- 96 -

Following this, expression analysis by qRT-PCR was performed on days 7,

14, and 21 resulting in a significant increase in expression of several germ

line specific markers when compared with MOCK controls. Indeed,

spermatogonial markers such as PIWIL2 (HILI) and the GDNF receptor type

1α (GFRA1) , as well as meiotic and post-meiotic markers including Acrosin

(ACR), Transition Protein 2 (TNP2), and Protamin 1 (PRM1), were among the

germ line markers that were upregulated after i12F-transduction indicating

the presence of a heterogeneous germ cell-like population at different

developmental stages within the reprogrammed cultures.

Moreover, the expression of the markers HILI, TNP2, PRM1 and ACR, but

not GFRA1, was relatively enriched in the manually isolated iGC clumps

when compared with the total population, indicating the likelihood that

more developmentally advanced germ cell-like cells were localized within

the iGC clumps (Fig 6.4A-E). It is worth noting that germ line-related marker

expression changed with the time, showing a temporal germ line-like

expression pattern with an increase in the expression of later-stage markers

concurrently with a decrease of the earliest ones. For example, GFRA1

expression decreased over time in culture (Fig. 6.4A) while TNP2 was

overexpressed not only in the total population but especially in the

manually selected clumps (Fig. 6.4C). Hence, these germ line marker

expression temporal dynamics indicate that the i12F-reprogrammed cells

were a heterogeneous germ cell-like cell population that recapitulate the in

vivo germ cell developmental program in vitro.

6. Results

- 97 -

6.2.2 H19 AND PEG1 IMPRINTING CONTROL REGION BISULFITE

SEQUENCING

To determine whether i12F-treated cells were able to recapitulate the in

vivo germ line cell developmental epigenetic reprogramming in vitro, the

FIG. 6.4. Germ line marker expression. A) qRT-PCR expression analysis of the germ line markers GFRA1; B) PIWIL2; C) TNP2; D) PRM1; E) ACR on days 7, 14 and 21 post-transduction; F) Representative scheme of the in vivo expression of these different markers. Values are indicated as fold change vs. MOCK controls mean ± SEM; (*) = significant vs. MOCK controls; (^) = significant vs. total population. (*/^ = P-value < 0.05; **/^^ = P-value < 0.001).

A B

C D

E

SSC

Spg

Spg

Spc I

Spc II

RS

ES

GFRA1

PIWIL2

TNP2

PRM1ACR

F

05

101520253035

MOCK i12F i12F CLUMPS

PIWIL2

0

10

20

30

40

50


TNP2

01020304050607080


PRM1

***

*^

*

*

*

^

^^**

01234567


GFRA1

010203040506070


ACR

D7

D14

D21


- 98 -

methylation status of the parentally imprinted H19 and PEG1/MEST loci

promoters were analyzed by bi-seq. As expected for a somatic cell type,

MOCK controls showed approximately 50% CpG methylation for both loci.

However, in 14-day post-12F-transduction cultures methylation was

decreased in the paternally imprinted (and thus sperm-methylated) H19

locus to approximately 21% CpG methylation in the whole cell cultures and

35.15% in manually selected clumps (Fig. 6.5A) compared to the MOCK

control. On the other hand the maternally imprinted (and thus sperm de-

methylated) PEG1/MEST locus showed only a small increase (54.7%) in CpG

methylation compared with the MOCK cells which was enhanced to 65.4%

in the manually selected clumps (Fig. 6.5B).

The bi-seq analysis is based on bisulfite modification of genomic DNA. The

main handicap of this method is that it cannot distinguish between 5mC

and 5hmC so the percentages of methylation analyzed may be biased: some

of the 5mCs detected may in fact correspond to 5hmCs. 5hmC has been

described as a pre-erasure marker for CpGs that are about to be actively de-

methylated by the TET family demethylases (Tahiliani et al., 2009).

Interestingly, a relative 5hmC enrichment was observed in the iGC clumps

two weeks post-transduction (Fig. 6.5E) which correlates with enriched

TET1 and TET2 (but not TET3) expression (Fig. 6.5D), as well as the lack of

significant DNMT1, DNMT3A, or DNMT3B expression changes in these

clumps compared to controls (Fig. 6.5F).

6. Results

- 99 -

FIG. 6.5. Methylation status of two imprinted genes, H19 (A) and PEG1/MEST (B) by bi-seq analysis; C) Percentages of methylation. D) qRT-PCR for TET and DNMT DNA-methylation modifications (F); E) 5hmC immunofluorescence for i12F-hFSK treated cells. Data is presented as the normalised mean fold change ± the SEM. (*) represents significant differences (P < 0.05) compared to controls; (+) represents significant differences (P < 0.05) between the i12F cells alone and the isolated i12F-clumps.

0

100

200

300

400


TET1 TET2 TET3

0

20

40

60

80

100


0

5

10

15

20

25


MO

CK C

ontr

oli1

2F

H19

20.97% methylated

50.49% methylated

i12F

CLU

MPS

35.13% methylated

i12F

1N

30.74% methylated

PEG

1/M

EST

MO

CK C

ontr

oli1

2F

54.7% methylated

55.67% methylated

i12F

CLU

MPS

i12F

1N

65.4% methylated

49.03% methylated

A B

0

20

40

60

80

100

MOCK i12F i12F CLUMPS i12F 1N SORTED

% of Methylation

H19 PEG1

C

hmC MERGED

MO

CKD

14

DAPI

D14

D21

i12F

D

0.00

0.50

1.00

1.50

2.00


DNMT1 DNMT3A DNMT3B

E F

*+

*+

*+


- 100 -

FIG. 6.6 Immunostaining analyses of reprogrammed fibroblasts at 14 and 21 days post-transduction for the germ line markers alkaline phosphatase (AP) $(A), PLZF (B), HIWI (C), and ACR (D). The periphery of iGC clumps in i12F/16F conditions is highlighted by dashed lines. Scale bar represents 10µm. Larger images are provided on the CD-version accompanying this thesis.

C

A B

D

6.2.3 PHENOTYPIC CHARACTERIZATION OF i12F CLUMPS

Since our previous morphological, transcriptomic, and methylation results

suggested that i12F clumps are a differentiated cell subpopulation,

phenotypic characterization was carried out to better discriminate the

differences between these putative subpopulations. Further

immunostaining analysis allowed the expression of AP, PLZF, HIWI, and

ACROSIN to be detected at the protein level at different time points (Fig.

6.6). Interestingly, these markers were also relatively enriched, and were

mainly localized in the iGC clumps, supporting previous observations and

indicating relative germ cell enrichment in the clumps.

6.3 TWELVE FACTOR-REPROGRAMMED CELLS CAN PROGRESS THROUGH

MEIOSIS IN VITRO

6.3.1 MEIOTIC SPREAD ANALYSIS BY SYCP3 STAINING

Since i12F-reprogrammed cells showed post-meiotic marker expression

their prophase I meiotic progression status was analyzed by examining the

6. Results

- 101 -

localization and distribution of one of the axial elements of the

chromosomal scaffold of the synaptonemal complex: SYCP3 (Kee et al.,

2009, Medrano et al., 2011, Panula et al., 2011). Synaptonemal complexes

comprise the axial elements SYCP2 and SYCP3, among other proteins, and

their formation starts at leptotene during prophase I. At this time SYCP3

acquires a punctate conformation which changes to a more elongated one

as chromosome pairing is completed through prophase I during zygotene,

pachytene, and up to diplotene; at diplotene the synaptonemal complex

disappears, the chromosomes undergo desynapsis and the bivalents

condensate (Fig. 6.7A).

This analysis was performed on day 14 post-transduction because

represented the peak appearance of iGC clusters. Approximately 8.69% of

the reprogrammed hFSK cells positively stained for SYCP3 (Fig. 6.7B)

however, around 7.07% of the SYCP3 positive nuclei showed a staining

pattern considered aberrant because they represented random protein

aggregations (Fig. 6.7F). Thus, approximately 1.62% of reprogrammed cells

showed a correct positive SYCP3 staining pattern, suggesting that these

cells were involved in prophase I of meiosis. Approximately 35.8% of

meiotic nuclei (0.58% of the total population) had an elongated SYCP3

staining pattern corresponding to late zygotene and pachytene stages of

prophase I, whereas the other 64.2% (1.04% of the total population)

showed a punctate staining pattern corresponding to leptotene (Fig. 6.7C-

D). Completion of synapsis is mediated by SYCP1, a central element with a

structural function in the synaptonemal complex, which binds to

chromosomes in the late stages of prophase I.


- 102 -

FIG. 6.7 Meiotic progression of hFSKs 14 days post-transduction. A) Illustrative pictures of the SYCP3 staining pattern; B) Percentage of aberrant/dotted/elongated staining pattern found in each condition in hFSK cells; C) Representative images of SYCP3 staining pattern overlaid and merged with transduced-cell images; D) Percentage of dotted/elongated staining pattern found in each hFSK cell condition; E) Representative SYCP1 (green) and SYCP3 (red) co-immunostaining; F) Representative SYCP3 aberrant conformations. Scale bar represents 10μm. Data is presented as the mean ± the SEM. (*) represents significant differences (P < 0.05) compared to the control.

0 1.040

0.58

0

7.07

0

2

4

6

8

10

12

MOCK i12F

Leptotene Zygotene/Pachytene Aberrant

0

1.04

0

0.58

0

0.5

1

1.5

2

MOCK i12F

Leptotene Zygotene/Pachytene

LATERAL ELEMENTS SYCP2/SYCP3

CENTRAL ELEMENTS SYCP1

Point of recombination

Chromatin

Hey J (2004) What's So Hot about Recombination Hotspots? PLoS Biol 2(6): e190.

A B

C D

E

F

CREST MERGEDSYCP3

MO

CK

Pu

ncta

teE

lon

gate

di12F

Elo

ng

ate

d a

berr

ati

on

s

CREST MERGEDSYCP3

Pu

cta

tea

be

rra

tio

ns

CREST MERGEDSYCP3

Pu

ncta

teE

lon

gate

d

i12F

SYCP1

*

Leptotene Zygotene Pachytene

6. Results

- 103 -

Since SYCP1 may act as a better chromosomal synapsis marker than SYCP3,

co-immunostaining was performed in order to assess whether SYCP3 and

SYCP1 co-localized. In the i12F-treated hFSK cells SYCP3 and SYCP1 only co-

localized in the more advanced stages of prophase I (Fig. 6.7E).

With the aim of improving the culture media towards obtaining better

meiotic efficiency rates, a different strategy was tested: in order to mimic in

vivo spermatogenesis, three-day post-transduction i12F-hFSKs were co-

cultured with a Sertoli cell feeder monolayer in GC-M. These Sertoli cells

were obtained from human patients with obstructive azoospermia who

underwent testicular biopsies as a part of their clinical infertility treatment.

Primary Sertoli cell cultures were transduced with a constitutively

expressed red fluorescent protein (RFP) prior to the initiation of the co-

culture (Riboldi et al., 2012). However, no significant differences at the

morphological level (Fig. 6.8A) or in meiotic progression (Fig. 6.8B-C) were

detected 14 days post-transduction compared to the non-co-cultured

condition.

6.3.2 PLOIDY ANALYSIS BY FLUORESCENCE IN SITU HYBRIDIZATION

In order to asses if i12F-treated cells were able to complete meiosis a ploidy

analysis was performed. Since we expected a very low number of cells to

finalize meiosis the candidate haploid cell population was enriched

according to their DNA content.


- 104 -

For this purpose, i12F-reprogrammed cells were treated with PI and the

putative haploid (1N) population was sorted by FACS according to their DNA

content. FISH with probes for the centromeres of chromosomes 18, X, and Y

was then performed on the total cell population, the manually selected

clumps, and the FACS sorted (putative haploid) population (Fig 6.9B). Using

this method, between 1 and 2% of putative haploid cells from i12F-

transduced cell cultures were isolated, which correlates with the

percentage of meiotic cells found by SYCP3 staining (Fig 6.9D). The

percentage of haploid cells in the unsorted i12F-transduced hFSK was

FIG. 6.8 A) Representative pictures of transduced cells (labelled with GFP) co-cultured with primary Sertoli cells (labelled with red fluorescent protein; RFP). Asterisk indicates green iGC clumps surrounded by red Sertoli cells. Scale bar represents 10μm; B) Percentage of aberrant/dotted/elongated staining pattern found in each condition in hFSK cells; C) Percentage of dotted/elongated staining pattern found in each condition in hFSK cells; Data is presented as the mean ± the SEM. (*) represents significant differences (P < 0.05) compared to the control. CC, co-culture with Sertoli cells.

0 0

7.07

1.70

0

2

4

6

8

10

12

MOCK MOCK CC i12F i12F CC

Aberrant Zygotene/Pachytene Leptotene

0 1.040

0.58

0

7.07

0

2

4

6

8

10

12

MOCK i12F


0 0

1.04 0.90

0 0

0.58 0.10

0

0.5

1

1.5

2


Zygotene/Pachytene Leptotene

0

1.04

0

0.58

0

0.5

1

1.5

2

MOCK i12F


A

B C

6. Results

- 105 -

1.57%, whereas there was a haploid cell enrichment of up to 4% in the

sorted cell population (Fig 6.9D). Because the iGC clumps showed higher

expression of post-meiotic markers, FISH analysis was performed in order

to elucidate where these haploid cells were located, showing that there

were 2.56% within the iGC clumps compared to 1.57% in the total cell

population (Fig. 6.9D).

These results were validated by repeating the FISH analysis with

centromeric probes for chromosomes 10 (aqua), 12 (green), and 3 (red) in

the same nuclei whilst concomitantly determining their ploidy (Fig 6.9C),

demonstrating haploidy for at least five of the 23 chromosome pairs.

Since the putative haploid cell population within the i12F-treted cells may

have been enriched by this DNA-content FACS protocol, the DNA-

methylation status for the H19 and PEG1/MEST loci was also analyzed in

this putative haploid population by bi-seq. Interestingly, the 1N sorted

population showed similar patterns to the manually selected clumps, with a

CpG methylation reduction at the H19 locus (30.74% and 35.13%

methylation respectively in manually selected clumps vs. 50.49%

methylation compared to the somatic cell control). There were no

significant CpG methylation changes at the PEG1/MEST locus (49.03%)

compared to the somatic control (55.67%) although there was a slight

decrease compared to CpG methylation in manually selected clumps

(65.4%; Fig. 6.6A-C).

All these results (i.e., germ cell marker expression by qRT-PCR and immuno-

phenotyping, meiotic progression by FISH, and methylation levels by bi-seq)


- 106 -

FIG. 6.9 Ploidy analyses in hFSKs 14 days post-transduction. A) Illustrative FACS plots of the cell cycle in cells stained with propidium iodide (PI); B) Representative FISH results for probes against chromosomes 18 (aqua), X (green), and Y (red) in putative 1N sorted cells and (C) re-hybridization with probes for chromosomes 10 (aqua), 12 (green), and 3 (red). Scale bar represents 10μm. (D) Percentage of haploid cells found in each treatment based on the analysis of these three probes in hFSK cells. Data is presented as the mean ± SEM. (*) represents significant differences (P < 0.05) compared to the MOCK and MOCK 1N sorted controls.

i12F

Chr 18 Chr Y MERGEDChr X

MO

CK

1N

so

rte

d

i12

F 1

N

sort

ed

0.00 0.00

4.00

1.57

2.56

0.00

1.00

2.00

3.00

4.00

5.00

6.00

MOCK 1N sorted

MOCK TOTAL

i12F 1N sorted

i12F TOTAL i12F CLUMPS

A B

D

C

*

*

support the concept that a more developmentally advanced reprogrammed

cell population resided in these clumps. Differences in the percentages of

haploid cells and methylation levels between manually selected clumps and

DNA-content sorted cells can be explained by the experimental subjectivity

of the manual clump selection.

6. Results

- 107 -

Finally, a combination of SYCP3 staining and FISH was used to identify the

intermediate meiotic stages of prophase I from leptotene to pachytene by

using both SYCP3 staining and ploidy analysis by FISH with centromeric

probes against chromosomes 18, X, and Y on the same nuclei (Fig. 6.10).

Before prophase I, diploid cells duplicate their chromosomes (2n, 4c),

showing normal diploidy by FISH. At leptotene homologous chromosomes

start to condensate, so centromeric probes for somatic and sexual

chromosomes are represented by only one mark; they show a diploid (18,

18, X, Y) karyotype. At this stage, SYCP3 starts to become expressed with a

punctate conformation along the chromosomes.

As meiosis advances, chromosome pairing is completed and SYCP3 changes

its lateral synaptonemal complex position to an axial one, acquiring an

elongated conformation. Chromosome pairing means that the centromeric

probes used show different mark combinations as they progress from a

diploid to haploid status (from 18, 18, X, Y; 18, X, Y; 18, 18, X-Y; to 18, X-Y as

an overlaying mark), together with the elongated SYCP3 conformation

which is characteristic of late-zygotene and pachytene. Diplotene is a very

fast stage, during which the synaptonemal complex disappears.

At meiosis II, the resulting cells are haploid but have two sister chromatids

(1n, 2c), which is represented by the 18, X-Y chromosomal probe

conformation in the absence of SYCP3 staining. When meiosis is completely

finished and the sister chromatids are segregated, haploid cells (1n, 1c) are

formed, resulting in a chromosomal probe conformation of either 18, X or

18, Y. All these possible combinations were found using both these


- 108 -

techniques consecutively on the same nuclei (Fig. 6.10). Hence, the

detection of both SYCP3 staining and ploidy analysis for chromosomes 18,

X, and Y made it possible to identify iGCs in different stages of meiotic

progression.

6.4 GERM CELL-RELATED GENE SCREENING REVEALS OPTIMAL

COMBINATIONS THAT INDUCE MEIOTIC PROGRESSION AS THE MAIN

READ-OUT

With the i12F-reprogramming treatment we obtained a heterogeneous

population with spermatogonial stem cell-characteristics at several levels

(transcriptomic, epigenetic, and phenotypic) with the ability to initiate and

progress through meiosis, acquiring an iGC status. The next objective was to

find the minimal optimal combination of reprogramming factors which

FIG. 6.10. SYCP3 and FISH detection on the same nuclei. The expected and observed staining correlates with the different stages of meiosis. SYCP3 (red), chromosomes 18 (blue probe), X (green probe), and Y (red probe). Scale bar represents 10μm.

PROPHASE I (SPERMATOCYTE I)SPERMATOGONIA SPERMATOCYTE II SPERMATID

LEPTOTENE PACHYTENE

EXP

ECTE

DO

BSE

RV

ED

1818

Y X

ZYGOTENE

6. Results

- 109 -

would still produce similar results. To do this, different combinations within

the pool of 12 factors were screened using the progression of meiotic

spreads for each condition, as assessed by SYCP3 staining to measure the

efficiency of germ cell reprogramming.

Interestingly, several combinations of the twelve factors were able to

induce meiosis, but most of these combinations gave rise to SYCP3 staining

pattern aberrations (Fig. 6.11A). Some combinations were chosen as the

most efficient compared to the i12F-reprogramming treatment, as

measured by the percentages of elongated zygotene/pachytene SYCP3

staining (Fig. 6.11B). In all these cases (highlighted with arrows in Fig. 6.11)

the percentages of zygotene/pachytene were higher than those obtained

with the i12F-treated cells (1.1; 0.7; 0.6; 0.7; and 0.7% in the total

population respectively vs. 0.58% in the i12F treated cells). Among these

combinations, members of the DAZL/VASA genes were always present.

Interestingly the best results using the latest stages in meiotic progression

were obtained when different combinations of PRDM1/PRDM14/LIN28A

were present. None of these combinations alone were able to induce

endogenous expression of SYCP3 indicating that overexpression of this gene

was mandatory for reprogramming towards an iGC phenotype.


- 110 -

FIG. 6.11 Meiotic progression of different combinations of reprogramming factors 14 days post-transduction. A) Percentage of aberrant/punctate/elongated staining pattern and B) detailed percentage of punctate/elongated staining pattern found in each condition in hFSK cells 14 days post-transfection. Data are presented as mean; C) overexpressed factors included in each condition.

33,7

1110,4 14

11,613,55

15,3

10,4

3,1

8,412,1

8,2 7,1 3,94,3

6,7 6,3

46,5 5,2 4,2 7,07

0

5

10

15

20


0,55

2,92,1

0,8 0,81,2 1,2

1,6

0,5

2,1

1,20,8 0,7

3,1

1,11,8 1,6

1 0,91,4

2,4

1,040,53

0,3

0,5

0,4 0,5

1,10,7 0,1

0

0,1

0,10,2 0,3

0,2

0,3

0,6 0,7

0,2 0,70,2

0

0,58

0

0,5

1

1,5

2

2,5

3

3,5Zygotene/Pachytene Leptotene

A

B

C iSYCP3 iSYCP3

i DAZL+VASA+SYCP3

ii DAZL+STRA8+SYCP3

iii DAZL+BOULE+SYCP3

iv VASA+STRA8+SYCP3

v DAZL+VASA+STRA8+SYCP3 A

vi DAZL+VASA+BOULE+SYCP3 B

vii DAZL+STRA8+BOULE+SYCP3

viii VASA+STRA8+BOULE+SYCP3

ix DAZL+VASA+STRA8+BOULE+SYCP3

x DAZL+VASA+STRA8+BOULE+DAZ2+SYCP3

xi DAZL+VASA+STRA8+BOULE+SYCP3+DMC1

xi DAZL+VASA+STRA8+BOULE+DAZ2+SYCP3+DMC1

xiii PRDM1+PRDM14+LIN28+DAZL+SYCP3

xiv PRDM1+PRDM14+LIN28+VASA+SYP3

xv PRDM1+PRDM14+LIN28+DAZL+VASA+SYCP3 C

xvi PRDM1+PRDM14+LIN28+DAZL+VASA+STRA8+SYCP3 D

xvii PRDM14+LIN28+NANOS3+DAZL+SYCP3

xviii PRDM14+LIN28+NANOS3+VASA+SYCP3 E

xix PRDM14+LIN28+NANOS3+DAZL+STRA8+SYCP3

xx PRDM14+LIN28+NANOS3+DAZL+VASA+STRA8+SYCP3

i12F i12F

6. Results

- 111 -

In order to determine the most effective combination, their ability to

complete meiosis was interrogated by FISH for chromosomes 18, X, and Y,

following the same strategy as with the i12F-treated cells. Their phenotypic

characteristics and morphological changes were also analyzed by further

immunostaining analysis to detect the expression of AP, PLZF, HIWI, and

ACROSIN at the protein level on days 14 and 21 post-transduction.

The percentages of haploid cells within the total population and the

putative haploid cell-sorted population indicated that although all these

combinations were able to complete meiosis, resulting in the production of

haploid cells, there were some combinations which were more effective

than others compared to the i12F-reprogramming treatment. According to

the extent of meiotic progression, the conditions A, B, and C showed better

results than i12F-treated cells (Fig. 6.12A). However, when analyzing the

percentages of haploid cells in the putative 1N sorted population, in the

manually selected clumps as well as the total population, the conditions B

and C seemed to present higher rates of meiosis completion (Fig. 6.12B).

In conclusion, condition C, with an average of 1.88% meiotic cells (30.85%

of them showing an elongated SYCP3 pattern; Fig. 6.12A), 12.15% haploid

cells within the sorted putative 1N cell population, and 5.86% haploid cell

enrichment in iGC clumps (Fig. 6.12B) appeared to be the combination with

the most consistent results. The relationship between meiotic progression

and haploid cell formation, together with immunostaining for germ line-

related markers and morphological changes (Fig. 6.12C) points towards

condition C as the most efficient combination which includes the fewest

germ cell-related factors.


- 112 -

FIG. 6.12. A) Detail of the percentage of meiotic progression and B) ploidy analysis of hFSK cells with the five main combinations 14 days post-transduction; C) Immunostaining analysis of reprogrammed fibroblasts at 14 and 21 days post-transduction for the germ line markers AP, PLZF, HIWI, and ACR. The periphery of iGC clumps in i12F/16F conditions are highlighted by dashed lines. Scale bar represents 10µm. A larger image is provided on the CD-version of this thesis. Data are presented as mean ± SEM.

PLZF MERGED

iSC

P3

AB

CD

E

DAPI

D14 D21

PLZF MERGEDDAPI

HIWI MERGEDDAPI HIWI MERGEDDAPI

iSC

P3

AB

CD

E

D14 D21

ACRO MERGED

iSC

P3

AB

CD

E

DAPID14

ACRO MERGEDDAPID21

AP MERGED

iSC

P3

AB

CD

E

DAPID14

AP MERGEDDAPID21

0.55

1.43 1.23 1.230.83 0.93 1.04

0.53

1.33

1.030.65

0.93 0.65 0.58

0.00

0.50

1.00

1.50

2.00

2.50

3.00

3.50

iSYCP3 A B C D E i12F


0.00

2.00

4.00

6.00

8.00

10.00

12.00

14.00

16.00

MO

CK 1

N

MO

CK T

OTA

L

iSYC

P3 1

N

iSYC

P3 T

OTA

L

A 1N

A TO

TAL

A CL

UM

PS

B 1N

B TO

TAL

B CL

UM

PS

C 1N

C TO

TAL

C CL

UM

PS

D 1N

D TO

TAL

D CL

UM

PS

E 1N

E TO

TAL

E CL

UM

PS

i12F

1N

i12F

TO

TAL

i12F

CLU

MPS

A DAZL+VASA+STRA8+SYCP3

B DAZL+VASA+BOULE+SYCP3

C PRDM1+PRDM14+LIN28+DAZL+VASA+SYCP3

D PRDM1+PRDM14+LIN28+DAZL+VASA+STRA8+

SYCP3

E PRDM14+LIN28+NANOS3+VASA+SYCP3

A

B

c

6. Results

- 113 -

6.4.1 SIX FACTOR-TREATED CELLS BEHAVE LIKE TWELVE FACTOR-INDUCED

GERM CELL LIKE CELLS AT THE TRANSCRIPTOMIC, EPIGENETIC, AND

MEIOTIC LEVELS

Because the most effective treatment was the combined ectopic expression

of PRDM1, PRDM14, LIN28, DAZL, VASA, and SYCP3 (referred to above as

“combination C”, from hereon in termed “i6F”), the next objective was to

assess their transcriptomic, epigenetic, and phenotypic status following the

same strategies as with the i12F-reprogrammed cells. Expression arrays

showed that i6F reprogrammed cultures clustered into a well-defined

group, and resulted in a list of 21 significantly up-regulated genes compared

with MOCK control cells, indicating that a significant switch in their

expression program had occurred (Fig 6.13A).

Hierarchical clustering of statistically significant differentially expressed

genes clustered i12F- and i6F-reprogrammed cultures as well as MOCK

controls separately (Fig. 6.13B). A different condition (iSYCP3) according to

the results extrapolated from the screening assay was added as a second

negative control to exclude any collateral effects of SYCP3 overexpression

which also clustered into another group (Fig. 6.13B). Similarly, PCA of these

statistically significant genes clustered the i12F-and i6F-reprogrammed cells

into defined groups which were different from those of the MOCK controls.

(Fig. 6.13C).


- 114 -

Moreover, similar to i12F-treated cultures, cell transduction with the i6F

pool and subsequent culture in GC-M induced the formation of iGC clumps

and the qRT-PCR results showed that these were relatively enriched for the

expression of PIWIL2, TNP2, and PRM1 on day 21 of culture, but not for

GFRA1 or ACR. Interestingly, ACR was maximally expressed in the clumps on

day 14 instead of day 21 (Fig. 6.14).

FIG. 6.13. A) Hierarchical cluster representation of significantly upregulated genes in i6F samples vs. MOCK controls; B) Hierarchical clustering of the differentially expressed genes when MOCK, iSYCP3, i12F, and i6F samples were compared; C) Principal component analysis (PCA) of statistically significant differentially expressed genes.

BA

C

MOCKi12Fi6F

6. Results

- 115 -

DNA methylation-level analysis by bisulfite sequencing of i6F-

reprogrammed cells showed a decrease in methylation in the promotor

region of the paternally imprinted gene H19 (33.21%), but a

hypermethylated status for the maternally imprinted gene PEG1/MEST

FIG. 6.14. Germ line marker expression. A) qRT-PCR expression analysis of the germ line markers GFRA1; B) PIWIL2; C) TNP2; D) PRM1; E) ACR on days 7, 14, and 21 post-transduction; F) Representative scheme for the in vivo expression of the different markers. Values are indicated as fold change vs. MOCK controls; (*) = significant vs. MOCK controls; (^) = significant vs. total population. (*/^ = P-value < 0.05; **/^^ = P-value < 0.001).

0

10

20

30

40

50

60

MOCK iSCP3 i6F i6F CLUMPS

ACR

D7

D14

D21

iSYCP3

0

10

20

30

40

50

60


TNP2

iSYCP3

0

5

10

15

20


PIWIL2

iSYCP3

0

2

4

6

8

10

12


GFRA1

iSYCP3

A B

C D

E

SSC

Spg

Spg

Spc I

Spc II

RS

ES

GFRA1

PIWIL2

TNP2

PRM1ACR

F

*

^

+

^^

**

*

** + ^

*

**++

05

10152025303540


PRM1

iSYCP3


- 116 -

(93.49%), indicating that i6F treated cells may have acquired an imprinting

pattern corresponding to that of mature oocytes (Fig. 6. 15A-C). This

hypomethylation trend was also reproduced in the pattern obtained for

H19 methylation within the manually selected clumps (36.75%) and the

putative haploid population sorted by FACS according to their DNA-content

(17.89%; Fig. 6.15A, C) Interestingly, PEG1/MEST methylation levels within

manually selected clumps (59.56%) and putative haploid population

(52.85%) maintain similar levels to somatic controls (55.57%) while total

population is highly hypermethylated (93.49%; Fig. 6.15B-C). Also, i6F-

transduced clumps were enriched in 5hmC (Fig. 6.15E) which is consistent

with both the expression of TET1, TET2, and TET3 (Fig. 6.15D) and also with

non-significant changes observed in DNMT expression levels when

compared to controls (Fig. 6.15F).

Therefore i6F-treated cells showed an epigenetic and transcriptomic profile

similar to a spermatogonial stem cell and were able to initiate meiosis and

even form haploid cells, albeit as a heterogeneous population (Fig. 6.16A-

D). Co-localization of SYCP1 with SYCP3 was also checked as previously

described, confirming synaptic recombination (Fig. 6.16C). Interestingly, the

iSYCP3 control showed the highest percentage of aberrant SYCP3

conformation (Fig. 6.16B) probably because of the lack of the proper

genetic background; moreover, the percentage of haploid cells resulting

from the i6F-treatment was statistically significant compared to the iSYCP3

control (Fig. 6.16F).

6. Results

- 117 -

FIG. 6.15. Methylation status of two imprinted genes, H19 (A) and PEG1/MEST (B) by bi-seq analysis; C) Percentages of methylation. D) qRT-PCR for DNA-methylation TET or DNMT modifications (F); E) 5hmC immunofluorescence for i6F-hFSK treated cells. Data is presented as the normalized fold-change mean ± SEM. (*) represents significant differences (P < 0.05) compared to the controls; (+) represents significant differences (P < 0.05) between i6F whole cultures and i6F clumps.

0

20

40

60

80

100

MOCK i6F i6F CLUMPS i6F 1N SORTED

% of Methylation

H19 PEG1

hmC MERGED

iSYC

P3

Cont

rol

D14

DAPI

10X

20X

10X

D21

i6F

0.00

1.00

2.00

3.00

iSYCP3 i6F i6F CLUMPS

DNMT1 DNMT3A DNMT3B

0

20

40

60

80


TET1 TET2 TET3

0

5

10

15

20

25


TET1 TET2 TET3

A B

C D

E F

i6F

CLU

MPS

36.75% methylated

i6F

1N

17.89% methylated

MO

CK C

ont

rol

H19

50.49% methylated

i6F

33.21% methylated

PEG

1/M

EST

MO

CK C

ont

rol

55.67% methylated

i6F

93.49% methylated

i6F

CLU

MPS

59.56% methylated

i6F

1N

52.85% methylated

*+

*+

*+


- 118 -

FIG. 6.16. Meiotic progression and ploidy analysis of hFSKs 14 days post-transduction. A) Illustrative pictures of the SYCP3 staining pattern in transduced cells. SYCP3 (red); CREST (blue); B, D) Percentage of punctate/elongated staining pattern. Data is presented as the mean ± SEM. (*) represents significant differences (P < 0.05) between leptotene nuclei and controls. (+) represents significant differences (P < 0.05) between zygotene/pachytene nuclei

00

33.70

10.280

10

20

30

40

50

60

MOCK iSYCP3 i6F


00.55

1.23

0

0.53

0.65

0

0.5

1

1.5

2

2.5

MOCK iSYCP3 i6F


0.00 0.003.69

0.67

12.15

1.07

5.86

0.00

2.00

4.00

6.00

8.00

10.00

12.00

14.00

MOCK 1N sorted

MOCK TOTAL

iSYCP3 1N iSYCP3 TOTAL

i6F 1N sorted

i6F TOTAL i6F CLUMPS

* + +

Elo

nga

tedi6

FCREST MERGEDSYCP3

MO

CK

Pu

ncta

te

CREST MERGEDSYCP3

Pu

nc

tate

Elo

ng

ate

d

i6F

SYCP1

Chr 18 Chr Y MERGEDChr X

Co

ntr

ol

i6F

A

B

DC

E

F

**

6. Results

- 119 -

and controls. C) SYCP1 (green) and SYCP3 (red) co-localization. E) Representative FISH results for probes against chromosomes 18 (aqua), X (green), and Y (red) in putative 1n-sorted cells; F) Percentage of haploid cells found in each treatment based on the analysis of these three probes. Data are presented as the mean ± SEM. (*) represents significant differences (P < 0.05) compared to MOCK 1N-sorted controls. (+) represents significant differences (P < 0.05) compared to iSYCP3 1N-sorted controls. Scale bar represents 10μm.

As shown by IF analysis (Fig. 6.12C), the detection of characteristic markers

for pre- and post-meiotic spermatogenic stages (i.e. AP, PLZF, HIWI, and

ACR) confirmed that these markers were relatively enriched and were

mainly localized in the iGC clumps (Fig. 6.12C). As expected, the iSYCP3

negative control did not show any morphological changes or expressed any

germ line markers at the transcriptomic (Fig. 6.14A-E) or protein level (Fig.

6.12C).

6.5 DIRECT GERM LINE REPROGRAMMING WITH TWELVE AND SIX

FACTORS CAN BE REPRODUCED IN HUMAN MESENCHYMAL CELLS FROM

BONE MARROW ORIGIN

In order to determine the reproducibility of our findings, the same

experimental design using the overexpression of both i12F and i6F gene

sets was applied to a different somatic cell type. Commercial primary

human male mesenchymal cells (hMSCs, 46, XY) from bone marrow origin

were chosen for these experiments because previous reports indicated that

these cells can spontaneously express germ line-related genes (Johnson et

al., 2005). These hMSCs were transduced with the i12F or i6F gene cocktails

and cultured in the same GC-M as for hFSKs; MOCK and iSYCP3 conditions

were included as negative controls. Following the same hFSK experimental

set up, the transcriptomic and phenotypic status of treated cells was


- 120 -

FIG. 6.17. Morphological changes in i12F- and i6F-treated hMSCs 14 days after transduction. Brightfield and darkfield images showing GFP fluorescence; Scale bar represents 10μm.

hMSC i12F

Bright Field GFP Bright Field GFP

hMSC i6FA B

interrogated, as well as their capacity to reach, enter, and progress through

meiosis and to form haploid cells. In contrast with previous observations in

hFSK reprogrammed cells, where most of the cells kept their fibroblastic

morphology and some iGC clumps appeared, hMSCs were much more

sensitive to the reprogramming process. During the first week of lentiviral

transduction most of the cells de-attached from the culture plates and the

only ones that remained attached had switched their morphology to form

iGC clumps (Fig. 6.17).

6.5.1 TRANSCRIPTOMICS OF MESENCHYMAL iGCs

Fourteen days post-transduction, i12F- and i6F-treated hMSC cultures were

analyzed by qRT-PCR to check germ line marker expression (Fig. 6.18A-E).

They were enriched in pre- and post-meiotic markers which changed over

time, following a germ line-like compatible pattern. Moreover, both i12F

and i6F-transduced-hMSCs expressed AP, PLZF, PIWIL2/HIWI, and ACR germ

line markers at the protein level at 14 days post-transduction as shown by

immunostaining (Fig. 6.18G), indicating that, similar to hFSK-iGCs, these

reprogrammed cell cultures contained different subpopulations at different

stages of germ line maturation.

6. Results

- 121 -

FIG. 6.18. Germ line marker expression. A) qRT-PCR expression analysis of the germ line markers GFRA1; B) PIWIL2; C) TNP2; D) PRM1; E) ACR; in the different reprogramming conditions on days 7, 14, and 21 post-transduction. Values are indicated as mean fold change ± SEM vs. MOCK controls; (*) = significant vs. MOCK controls; (^) = significant vs. total population. (*/^ = P-value < 0.05; **/^^^ = P-value < 0.001). (+) represents significant differences (^ = P < 0.05) with iSYCP3 condition; F) Representative scheme of the in vivo expression of the different markers; G) Immunofluorescence of i12F and i6F-hMSC treated cells for different germ line markers. Larger images are provided on the CD-version.

G

0

20

40

60

80

100

120

140

MOCK iSCP3 i12F i6F

PIWIL2

** ++

* +

** ++

+

A B

C D

E

SSC

Spg

Spg

Spc I

Spc II

RS

ES

GFRA1

PIWIL2

TNP2

PRM1ACR

F

0

2

4

6

8

10

MOCK iSCP3 i12F i6F

ACR

D7

D14

D21

*+

iSYCP3

0

50

100

150

200

MOCK iSCP3 i12F i6F

PRM1

** ++

iSYCP3

0

50

100

150

200

MOCK iSCP3 i12F i6F

TNP2

*++** ++

iSYCP3

0

10

20

30

40

MOCK iSCP3 i12F i6F

GFRA1

iSYCP30

20

40

60

80

100

120

140

MOCK iSCP3 i12F i6F

PIWIL2

** ++

* +

** ++

+

A B

C D

E

SSC

Spg

Spg

Spc I

Spc II

RS

ES

GFRA1

PIWIL2

TNP2

PRM1ACR

F

0

2

4

6

8

10

MOCK iSCP3 i12F i6F

ACR

D7

D14

D21

*+

iSYCP3

0

50

100

150

200

MOCK iSCP3 i12F i6F

PRM1

** ++

iSYCP3

0

50

100

150

200

MOCK iSCP3 i12F i6F

TNP2

*++** ++

iSYCP3

0

10

20

30

40

MOCK iSCP3 i12F i6F

GFRA1

iSYCP3


- 122 -

Interestingly, the time-expression pattern was different between hMSC-

and hFSK-iGCs. Later germ cell-markers (i.e. TNP2, PRM1, and ACR) were

maximally expressed on day 21 in both i12F- and i6F-hFSK-iGCs (Fig. 6.5C-E;

6.14C-E) whilst these same markers reached maximum expression levels on

days 7 and 14 in hMSC-iGCs in both i12F and i6F conditions (Fig. 6.18C-E).

Moreover, manually selected clumps of i12F- and i6F-transduced hFSKs

presented a similar time-expression pattern to hMSC-iGCs. They showed

higher expression levels of the later germ cell markers PRM1 and ACR on

day 14 post-transduction instead of on day 21 (Fig. 6.5D-E; 6.14D-E)

suggesting that hFSK-iGCs-clusters may be closer to hMSC-iGCs in terms of

the timing of transcriptomic reprogramming because the reprogrammed

population is enriched in these clusters.

6.5.2 MESENCHYMAL iGCs PROGRESS THROUGH MEIOSIS AND FORM

HAPLOID CELLS

When hMSCs were subjected to reprogramming, approximately 10.3% and

18.99% of mesenchymal transduced cells showed a positive SYCP3 staining

profile for the i12F and i6F conditions respectively (Fig. 6.19A). Similar to

the results with hFSKs, around 7.99% of i12F- and 16.97% of i6F-transduced

SYCP3 positive nuclei showed an aberrant staining pattern representing

protein aggregations (Fig. 6.19B). Thus, only 2.31% of i12F- and 2.02% of

i6F-transduced hMSCs showed a correct positive SYCP3 staining pattern.

The percentage of meiotic cells with an elongated SYCP3 pattern in the i12F

cultures was 31.6% (0.73% of the total population), whereas 68.39% of cells

(1.58% of the total population) showed a punctate staining pattern.

6. Results

- 123 -

FIG. 6. 19 Meiotic progression and ploidy analysis 14 days post-transduction in reprogrammed hMSCs. A) Illustrative pictures of the SYCP3 staining pattern in transduced cells. SCP3 staining pattern (red) is co-localized with centromeres stained with CREST (blue); B) Percentage of aberrant/punctate/elongated staining pattern, and C) detailed percentage of punctate/elongated staining pattern found in each condition in hFSK cells. Data is

Elo

ngate

di6F

hMSCC

ontr

ol

Dott

ed

Elo

ngate

dCREST MergedSYCP3

Dott

ed

i12F

Chr 18 Chr Y MergedChr X

Co

ntr

ol

i12

Fi6

F

0

5

10

15

20

25

30

MOCK 1N

sorted

MOCK TOTAL

iSCP3 1N sorted

iSCP3 TOTAL

i12F 1N sorted

i12F TOTAL

i6F 1N sorted

i6F TOTAL

0

0.981.58

0.9

0

0.49

0.73

1.1

0

0.5

1

1.5

2

2.5

3

MOCK iSYCP3 i12F i6F


0

53.05

7.9917.0

0

10

20

30

40

50

60

MOCK iSYCP3 i12F i6F


**++

**++

**++

**++

*+

A B

C

D

E


- 124 -

presented as the mean ± SEM. (*) represents significant differences (P < 0.05) between leptotene nuclei and controls. (+) represents significant differences (P < 0.05) between zygotene/pachytene nuclei and controls; C) Illustrative FACS plots of the cell cycle in cells stained with PI; D) Representative FISH results for probes against chromosomes 18 (aqua), X (green), and Y (red) in putative 1N sorted cells; E) Percentage of haploid cells found in each treatment based on the analysis of these three probes in hFSK cells. Data is presented as the mean ± SEM. (*) represents significant differences (P < 0.05) compared with the MOCK 1N sorted controls. (+) represents a significant difference (P < 0.05) compared with the iSCP3 1n sorted controls. Scale bar represents 10μm.

In contrast, the percentage of meiotic cells with an elongated SYCP3

pattern in the i6F mesenchymal cultures was 56.93% (1.15% of the total

population), and 43.07% of cells (0.87% of the total population) showed a

punctate staining pattern (Fig. 6.19C). Additionally, only around 1% within

the unsorted populations in the i12F reprogrammed hMSCs were haploid,

whereas the percentage rose to 1.11% in the i6F condition. Furthermore,

we observed an enrichment of 17.87% in i12F and 20% in i6F cultures of

haploid cells within the putative 1N sorted cell population (Fig. 6.19D-E).

In order to improve hMSC culture conditions in terms of survival and

meiotic progression efficiency, the same co-culture system strategy as for

i12F-hFSK cultures, using RFP-Sertoli cells as feeders, was attempted.

However, no significant differences were observed in the morphology of

i12F-hMSC treated cells (Fig. 20A) or in the percentages of the meiotic cells,

in any of the analyzed stages for SYCP3: leptotene, zygotene/pachytene,

and aberrant conformations (Fig. 20B-C).

6. Results

- 125 -

FIG. 6.20. A) Representative pictures of i12F-hMSC transduced cells (labeled with GFP) co-cultured with Sertoli primary cells (labeled with RFP). Asterisks indicate green iGC clumps surrounded by red Sertoli cells. Scale bar represents 10μm; B) Percentage of aberrant/punctate/elongated staining pattern, and C) detailed percentage of punctate/elongated staining pattern found in each condition in hFSK cells. Data is presented as the mean ± SEM.

A

B

0 0

1.47

2.4

0 0

0.67

0.2

0

0.5

1

1.5

2

2.5

3


Zygotene/Pachytene Leptotene

0

1.04

0

0.58

0

0.5

1

1.5

2

MOCK i12F


0 0

7.35 6.8

0

2

4

6

8

10

12



0 1.040

0.58

0

7.07

0

2

4

6

8

10

12

MOCK i12F

Leptotene Zygotene/Pachytene Aberrant C

6.6 XENOTRANSPLANTATION DEMONSTRATES THAT iGCs ARE ABLE TO

COLONIZE THE GERMINAL EPITHELIUM OF SEMINIFEROUS TUBULES

Based on the previously described results indicating spontaneous meiotic

induction and a suitable germ-cell like expression profile, we hypothesized

that part of the reprogrammed iGCs would have a spermatogonia-like

phenotype. Nevertheless, this is an in vitro model that needs a functional

proof of concept to demonstrate its in vivo functionality. A key feature of

germ cells is that they can contribute to the spermatogenesis of sterilized

mice. As iGCs show spermatogonia-like features, they are also expected to

be able to colonize the SSC physiological niche, i.e. the basal lamina of the

seminiferous ducts (Brinster and Zimmermann, 1994). Therefore, a


- 126 -

xenograft assay was performed with the iGCs to test their functionality in

vivo. This assay requires the use of immunosuppressed mice, such as the

nude mouse strain, which must be chemically sterilized using busulfan prior

to transplant (Ogawa et al., 1997) so that xenotransplanted cells are

presented with an empty niche without the presence of competing cells

such as the host’s own spermatogonial cells (see Fig. 5.3 in Methods for a

general overview of the procedure).

The busulfan dose-response effect on mouse spermatogenesis 12 weeks

post-injection, the length of the complete experimental design, is shown in

(Fig. 6.21). Different busulfan doses were tested in terms of survival and

sterilization 12 weeks post-injection in this mouse strain (Fig. 6.21A).

FIG. 6.21. The effects of busulfan dosage. A) dosage-mortality curve; B) histological effects of busulfan 12 weeks post-injection. Asterisks indicate seminiferous tubules with no evidence of spermatogenesis.

020406080

100

25 30 35 40 45

% m

ort

alit

y

Dosage Busulfan (mg/Kg)

Dosage-mortality curve

0 30 35 40

Dosage busulfan (mg/Kg)

10X

20X **

*

* *

*

* **

*

A

B

(mg/Kg)

6. Results

- 127 -

Busulfan showed a sterilizing effect on spermatogenesis, although 12 weeks

after intraperitoneal injection (IP) the mice started to recover some

spermatogenic capacity (Fig. 6.21B). As expected, the highest levels of

spermatogenesis-recovery correlated with the lowest levels of busulfan

dosage and vice versa: the highest levels of busulfan caused the highest

rates of mortality (Fig. 6.21A.

Therefore, based on the literature (Wang et al., 2010) and our preliminary

data, a range of 30-35 mg/Kg of busulfan was used to chemically sterilize

the animals. All the male mice used were sterilized by an IP injection of 30-

35mg/kg busulfan dissolved in DMSO at 4-6 weeks of age prior to

xenotransplant (details shown in Table 6.1). Because this mouse strain is

immunodeficient, no immuno-cross-reactivity due to the human origin of

the transplanted iGCs was expected after the xenotransplantation. After

four weeks, when endogenous spermatogenesis is suppressed, the mice

were ready for xenotransplant.

For this experiment, hFSKs were used as primary somatic cells and were

transfected with MOCK, iSYCP3, i12F, and i6F lentiviral supernatants.

Transduced hFSK cells were cultured in GC-M and harvested 14 days post-

infection, so as to correspond with the fourth week after the IP busulfan

dose. Cells were harvested and incubated with CellVue® Claret Far Red

Fluorescent Cell Linker Kit to verify the origin of the xenotransplanted cells

in future analyses using two methods: V5-peptide (included in the lentiviral

backbone; Appendix 1) immunoreactivity; and fluorescent emission (at ƛMAX

Exc = 655 nm; ƛMAX Em= 675 nm) from CellVue® Claret Far Red Fluorescent Cell

Linker (henceforth CellVue).


- 128 -

Table 6.1 Detailed data on the mice used.

MICE PER CONDITION

Busulfan dosage (mg/g)

Final body weight (g)

Median testis weight (mg)

SD testis weight (mg)

MOCK 1 40 31.8 33.2 0.50

2 35 33.2 25.9 3.74

3 30 37.2 * *

4 30 37.7 82.2 10.18

5 30 38.6 13.05 12.51 Number of animals (N)

5

iSYCP3 1 40 31.8 34.2 0.50

2 30 35.8 * *

3 30 -- -- --

4 30 34.4 97.85 23.40


4

i12F 1 40 40.8 43.6 10.11

2 40 -- -- --

3 35 36.1 31.35 0.21

4 35 38.2 69.8 12.44

5 35 37.3 51.75 7.42


5

i6F 1 40 40.8 57.9 10.11

2 40 -- -- --

3 35 32.8 29.9 2.30

4 35 35.5 36.5 0.00

5 35 32.9 28.85 0.85


5

CONTROL 1 30 35.3 32.9 2.75


2

-- death after surgery * data not-collected

6. Results

- 129 -

Once the cell solution was prepared, the xenotransplantation surgery was

performed (details are provided in table 6.2) so that the in vitro-derived

iGCs could be injected into the inner part (up to 70%) of the seminiferous

tubules.

Table 6.2 Number of injected testes.

CONDITION Number of animals (N)

Total testes injected (n)

Negative controls

MOCK 5 5 5

iSYCP3 4 4 4

i12F 5 6 4

i6F 5 6 4

CONTROL 2 0 2

Two months after xenotransplantation, mice were sacrificed and the testes

were removed to analyze whether the injected iGCs were able to colonize

the SSC niche. The weight of the testes was similar between the different

busulfan dosages (Fig. 6.22A-B) in MOCK, iSYCP3, i12F, and i6F treatments

(Fig. 6.22C-D).

Moreover, the relative normalized ratio of testis weight vs. their respective

body weight compared to control non-sterilized mice was analyzed. In the

case of mice transplanted with the negative control samples (i.e., MOCK,

iSYCP3) the relative weight of the testes were lower than the non-sterilized

controls, suggesting that the transplanted cells were not able to colonize

the empty tubules. On the contrary, the ratios of the samples from mice

transplanted with i12F- and i6F-iGCs were higher than the negative controls

and, in i6F-iGCs transplanted samples, higher than the non-sterilized


- 130 -

FIG. 6. 22. Relationship between testicular weight (A) and normalized testicular weight (B) with busulfan dosage. Relationship between testicular weight (C) and normalized testicular weight (D) with busulfan dosage disaggregated by conditions.

R² = 0,0346

0

20

40

60

80

100

120

140

29 31 33 35 37 39 41

Test

icu

lar

we

igh

t(m

g)

BS-dosage (mg/Kg)

Testicular weight vs. BS-dosage

0

20

40

60

80

100

120

140

29 34 39 44

Test

icu

lar

we

igh

t(m

g)

BS-dosage (mg/Kg)

Testicular weight vs. BS-dosage

MOCK

iSYCP3

i12F

i6F

CONTROL

R² = 0,0457

0

0,5

1

1,5

2

2,5

3

3,5

29 31 33 35 37 39 41Test

icu

lar

we

igh

t/ b

od

yw

eig

ht

(mg

/g)

BS-dosage (mg/Kg)

Normalized testicular weight vs. BS-dosage

0

0,5

1

1,5

2

2,5

3

3,5

29 34 39 44Test

icu

lar

we

igh

t/ b

od

y w

eig

ht

(mg

/g)

BS-dosage (mg/Kg)

Normalized testicular weight vs. BS-dosage

MOCK

iSYCP3

i12F

i6F

CONTROL

A B

C D

controls (Fig. 6.23A). This ratio, together with the GFP emission detected

inside the seminiferous tubules immediately before their analysis (Fig.

6.23B) indicated iGC-colonization for both i12F- and i6F-iGCs.

Xenotransplanted testes were divided into two parts: one was used for

immuno-phenotyping by flow cytometry to confirm the presence of the

xenotransplanted iGCs, and the other was immunohistologically analyzed to

assess the localization of colonizing xenotransplanted iGCs.

6. Results

- 131 -

FIG. 6.23. Average of normalized testis weight/body weight (mg/g) ratio compared to control samples (A); Illustrative picture of how i12F treatment shows GFP expression inside the tubules (B) White arrows indicate the highest intensity spots for GFP emission, which correlates with GFP emission from iGCs attached to the inner part of the tubules.

0

0,2

0,4

0,6

0,8

1

1,2

1,4

1,6

Controls MOCK iSYCP3 i12F i6F

Normalized ratio testis weight/body weight (mg/g)

A B

6.6.1 FLOW CYTOMETY RESULTS

The presence of cells of human origin in the xenotransplanted testes was

analyzed by flow cytometry using three different markers: an antibody

against human VASA which is overexpressed in both the i12F- and i6F-iGCs;

an antibody against V5-peptide in the C-terminal of the transgenes, which is

included in the lentiviral backbone (Appendix 1); and CellVue far red

emission resulting from iGC linkage before transplantation. Two well-

differentiated populations of cells double-positive for vasa and CellVue

were observed only in the transplanted i12F- and i6F-iGCs (Fig. 6.24D-E).

Therefore, based on the presence of cells positive for V5, VASA, and CellVue

within the disaggregated tubules in some of the i6F- (5 out of 6) and i12F-

iGCs (4 out of 6) transplanted testis, we concluded that human-origin cells

were located inside the testis two months after transplantation.


- 132 -

FIG. 6. 24. Illustrative flow cytometry histogram plots for the presence of V5 (FITC-A), VASA (PE-Texas Red-A), and CellVue (APC-A) positive cells n the transplanted testes in the different conditions: MOCK (B), iSYCP3 (C), i12F (D), and i6F (E) transduced hFSKs, and in a busulfan-treated testis as a negative control (A). Cells positive for CellVue are indicated in red in the VASA-positive histogram and Vasa positive cells are indicated in pink in the CellVue-positive histogram.

MO

CKCO

NTR

OL

i12F

i6F

iSYC

P3A

B

C

D

E

0

5

10

15

20

25

CONTROL MOCK + iSYCP3 + i12F + i6F +

% VASA positive cells

0

10

20

30


% CellVue positive cells

0

5

10

15

20

25


% V5 positive cellsF G H

6. Results

- 133 -

Interestingly, a few cells positive for the analyzed markers were also

observed in MOCK and iSYCP3 transplanted testes (Fig. 6.24B-C), indicating

that some human-origin cells were also still present in these tissues. In

these cases, CellVue-positive cells were not positive for VASA. Since CellVue

is a membrane marker, these CellVue positive cells may have been Sertoli

cells which had phagocytized non-attached transplanted human cells.

Nevertheless, this technique only allows cells of human-origin to be

detected in the testis, so the next step was to detect the presence of these

cells in the inner part of the seminiferous tubules.

6.6.2 IMMUNOHISTOLOGICAL ANALYSIS

Frozen sections were stained for VASA and NuMA antigen detection

analysis, whilst also searching for co-localization of these markers with a

CellVue signal. Even though many tubules which had spontaneously

recovered spermatogenesis were observed in almost every analyzed testis,

triple positive cells were detected in the basal layer of some germ cell-

depleted tubules only in i6F- and i12F-iGC transplanted testis, indicating

both their human origin and germ cell identity (Fig. 6.25C).

Interestingly, VASA was detected in the nucleus, co-localizing with NuMA,

which correlates with previous reports indicating a nuclear rather than

cytoplasmic VASA location for human germ cells located in the 16-week old

human fetal testis (Anderson et al., 2007); this may indicate the possible

immature state of these transplanted colonizing cells.


- 134 -

FIG. 6. 25. Percentage of tubules containing NuMA+/VASA+ cells (A) and efficiency of

colonization per 105 injected cells (B) in both i12F and i6F transplanted testis. Data is presented as the mean ± SEM. Illustrative pictures showing NuMA+/VASA/CellVue+ triple positive cells in the basal layer of germ cell depleted seminiferous tubules in both i12F and i6F transplanted testis (C). Illustrative pictures of the mouse and human testis tissue as negative and positive controls, respectively, for the specificity of the NuMA antibody to mark the nucleus of human cells (D). Scale bar represents 10μm.

0

0.5

1

1.5

2

2.5

3

i12F i6F

% Tubules with NuMA+/VASA+ cells

0

0.1

0.2

0.3

0.4

i12F i6F

Efficiency of colony formation per 10e5 injected cells

A B

C

DAPI NuMA

Mo

use t

esti

s

MERGED

Hu

man

feta

l

testi

s

D

DAPI NuMAVASA CELLVUE MERGED

MO

CK

iSY

CP

3i1

2F

i6F

6. Results

- 135 -

Although there was a high variability in the number of triple positive cells

observed in each transplanted testis, tubule cross-sections indicated an

average of around 1.45% triple positive cells located in the basal layer of

seminiferous tubules for i12F transplanted testis, and 2.1% in i6F

transplanted testes (Fig. 6.25A) with efficiencies of around 0.2 colonizing

cells per 105 injected cells for i12F- and 0.3 colonizing cells per 105 injected

cells for i6F-iGCs (Fig. 6.25B). These percentages showed no statistically

significant differences between i12F- and i6F-iGCs, suggesting that both

combinations of factors had a similar capacity to induce cells to colonize the

SSC niche, demonstrating behavior typical of SSCs.

- 137 -

7. Discussion

White Chrysanthemum, Violet, Lavender

Truth, honesty, faithful

7. Discussion

- 139 -

To date several studies have shown that human germ cells can be

spontaneously derived in vitro either from hESCs (Clark et al., 2004, Kee et

al., 2009) or hiPSCs (Eguizabal et al., 2011, Medrano et al., 2011, Panula et

al., 2011, Park et al., 2009). Many groups have also been inspired by

successes in reprogramming terminally differentiated cells into a

pluripotent state using defined factors (Takahashi and Yamanaka, 2006),

thus increasing research aiming to directly reprogram adult cell types into

other terminally differentiated lineages, bypassing the undifferentiated

state. Examples in this field include the direct conversion of fibroblasts into

neurons (Kim et al., 2011, Vierbuchen et al., 2010), blood precursors (Szabo

et al., 2010), cardiomyocytes and even Sertoli cells (Buganim et al., 2012).

Based on this background, the possibility of directly reprogramming human

somatic cells into germ cell-like cells was investigated and the findings are

presented in this thesis. Following a similar strategy to iPSC reprogramming,

human primary cells were transduced in order to overexpress different

germ-line related factors. Germ cells are characterized by a set of specific

features, the most important of which are their ability to reduce their

chromosomal charge during meiosis and to erase epigenetic modifications

so as to subsequently re-establish their sex-specific epigenetic profile

during germ line epigenetic reprogramming. The results presented

demonstrate that the overexpression of these germ cell related genes

induced the formation of a heterogeneous cell population with

spermatogonial features which is able to progress through and complete

meiosis, giving rise to a very small fraction of haploid cells. Moreover, some

of these cells show spermatogonial stem cell-like characteristics, and are


- 140 -

able to colonize murine seminiferous ducts in human-to-mouse

xenotransplant experiments

- The transcriptomic and epigenomic characteristics of the in vitro human

induced germ cell-like cell model

When two non-related types of human primary somatic cell cultures were

transduced with a combination of 12 germ-line related factors a

morphological change that resembled a mesenchymal-to-epithelial

transition was observed which had a similar morphology to that previously

described in some reports focused on deriving SSCs (Conrad et al., 2008).

Interestingly, this morphology was only maintained when a specifically

designed culture media was used, i.e. GC-M (Fig. 6.2) supplemented with

different factors (i.e. GDNF, bFGF, LIF, SCF, and RA) which are thought to

promote putative germ cell survival (Guan et al., 2009, Kanatsu-Shinohara

et al., 2003, Oatley and Brinster, 2006, Kanatsu-Shinohara et al., 2007,

Kanatsu-Shinohara et al., 2005). Additionally, similar to its use for the

derivation and maintenance of hESCs (Galan and Simon, 2012) and hiPSCs

(Takahashi and Yamanaka, 2006, Park et al., 2009 ) GC-M was also

supplemented with 20% KSR. The use of KSR has recently been described

for culturing both mouse (Aoshima et al., 2013) and human origin (Riboldi

et al., 2012) male germ cells, and moreover, it has also been successfully

used for in vitro testicular organ culture without cytokines (Sato et al.,

2011). Together this work shows how the use of KSR promotes the

maintenance of stem cell status and putative SSC survival, supporting its

use and the results obtained with the specifically designed GC-M.

7. Discussion

- 141 -

Subsequent expression analysis of these treated cultures showed a

significant increase in the expression of several pre- and post-meiotic

markers compared with untreated controls (Fig. 6.4; 6.14; 6.18A-E),

suggesting the presence of a heterogeneous germ cell-like population at

different maturation stages. Even though both types of primary cultures

responded in a similar way, maximum expression of the post-meiotic

markers TNP2, PRM1 and ACR in hMSCs was reached during the first week

post-transduction (Fig. 6.18C-E) and not during the third week as occurred

with hFSK cells (Fig. 6.4C-E), indicating a difference in the timing of the

reprogramming process between these two somatic cell types.

Since one of the key features of germ cells is the epigenetic reprogramming

that they undergo during in vivo development, the methylation levels of the

imprinted loci promoters widely used in previous reports is a good indicator

of the epigenetic status of the in vitro derived germ cells (Eguizabal et al.,

2011, Medrano et al., 2011, Panula et al., 2011, Park et al., 2009). Thus, the

methylation status of the DMRs, of the paternally methylated H19 and of

the maternally methylated PEG1/MEST loci was determined in the

reprogrammed cells (Fig. 6.5A-C; 6.15A-C). As expected, approximately

50% of CpGs were methylated in MOCK controls in somatic lineages,

however only the paternally methylated H19 gene showed a hypo-

methylated status in both i12F and i6F-treated cultures, which was

reconfirmed by analyzing manually selected clumps and putative 1N sorted

cells (Fig. 6.5A,C; 6.15A,C). However, with the exception of the hyper-

methylation observed in the i6F total population, no substantial changes


- 142 -

were observed in the methylation pattern of the maternally methylated

PEG1/MEST gene (Fig. 6.6B-C; 6.15B-C).

All the methylation assays performed here are based on the bisulfite-

conversion technique that cannot distinguish between 5mC and 5hmC.

Immunofluorescence results showed that iGC clumps were enriched in

5hmC (Fig. 6.5E; 6.15E) and gene expression results showed increased

expression of the TET1, TET2, and TET3 enzymes (Fig. 6.5D; 6.15D) involved

in the oxidation pathway of 5mC to 5hmC. Given these results, several

explanations could be considered. Firstly, it is possible that the methylation

status of these imprinted genes was biased, and so was overestimated, and

that the hyper-methylation found in PEG1/MEST actually correlates with

5hmC enrichment in 5hmC before methylation erasure. Secondly, the clear

upregulation in the expression of the TET-mediated demethylation pathway

members TET1, TET2, and TET3 in reprogrammed cells, and especially in the

case of the i12F and i6F clumps, together with any substantial differences in

DNA methyl-transferase DNMT1, DNMT3A and DNMT3B expression (Fig.

6.5F; 6.15F), must be taken in consideration.

Taking all these results together, it seems that the TET-mediated de-

methylation pathway is active in both i12F and i6F reprogrammed cells,

inducing a decrease in the methylation pattern of the paternally methylated

DMRs such as H19, but failing in the case of maternally methylated

PEG1/MEST. This situation could reflect the fact that most of the

reprogrammed cells in these cultures were probably at an early maturation

stage, when methylation erasure is just starting rather than at a stage when

a sex-specific imprinting pattern is being re-acquired, although it may

7. Discussion

- 143 -

indicate a failure in the reacquisition of the methylation pattern in these

cells. Consequently, these observations implicate that reprogrammed cells

are unable to correctly recapitulate epigenetic reprogramming due to their

inability to properly erase their methylation marks and the lack of

expression of the de novo DNA methyltransferases DNMT3A and DNMT3B

implicated in the reacquisition of the sex-specific methylation marks in

germ cells in vivo. Therefore further research will be required to find key

factors that can confer this ability to the iGCs generated in this model.

- In vitro induced germ cell-like cells show characteristic features of

spermatogonia-like cells at different maturation stages

In contrast to somatic tissues the germ line is composed of cells which

transmit genetic information to the offspring, and so they are characterized

by a set of specific features, among which the most important is their ability

to reduce their chromosomal charge during meiosis. When the meiotic

status of the i12F and i6F-iGCs was studied, a subpopulation of these i12F

and i6F-iGCs was found to be involved in different stages of Prophase I after

exposure to RA (Fig. 6.6; 6.16A-D; 6.19A-C). Indeed, most of these meiotic

cells were able to complete meiosis and form haploid cells (Fig. 6.9; 6.16E-F;

6.19D-E). According to these results, only 1-2% of haploid cells were

obtained from reprogrammed cultures. However, after cell sorting for the

putative 1N population within these reprogrammed cell cultures, the purity

was increased up to 20% in the case of the i6F-hMSC-iGCs (Fig. 6.19E). The

higher rate of haploid cell formation in hMSC-iGCs may be explained by the

different differentiation status of the starting hFSK and hMSC cell cultures

(Kassem, 2004, Solter, 2006, Takahashi and Yamanaka, 2006). Overall, these


- 144 -

data suggest that reprogrammed cell cultures are constituted by a

heterogeneous population of germ cells in different stages of maturation

and some of these iGCs may correlate with a spermatogonia-like stage

because they are capable of going through meiosis after exposure to RA.

Based on the meiotic progression (Fig. 6.6; 6.16A-D; 6.19A-C) and the

relative enrichment of post-meiotic marker expression in the iGC clumps

(Fig. 6.6; 6.12C; 6.18C), the localization of haploid cells within the i12F- and

i6F-reprogrammed cultures was interrogated. For this purpose, FISH

analysis was performed on the manually selected iGC clumps, showing that

haploid cells were relatively enriched compared to the total cell population.

However, in contrast to the significant enrichment of haploid cells that was

found when the putative 1N sorted cell populations were analyzed, this

enrichment was not-significant compared to the percentage of haploid cells

within the total population of reprogrammed cells (Fig. 6.9D; 6.16F; 6.19F).

Similarly, results from gDNA bi-seq assays indicated that manually selected

clumps behave similarly (but not exactly) to the putatively iGC-enriched 1N

sorted cells (Fig. 6.5A-C; 6.15A-C). This can be explained by contamination

with cells from outside the iGC clumps at the time the clumps were

manually collected and therefore it is impossible to specifically pinpoint

whether these haploid cells are located within iGC clumps or if they are

homogeneously distributed throughout reprogrammed cell cultures.

Finally, since iGCs appeared to have a SSC-like phenotype, their ability to

colonize the spermatogenic niche in vivo was tested. During male germ cell

maturation only the SSCs, which are spermatogonial precursors, are able to

colonize and repopulate the SSC niche (i.e. the seminiferous tubules) when

7. Discussion

- 145 -

transplanted into the germ cell-depleted spermatogenic niche of chemically

sterilized mice (Geens et al., 2008, Brinster and Avarbock, 1994, Brinster

and Zimmermann, 1994). Although the efficiency was very poor, i12F- and

i6F-iGCs were able to colonize this in vivo murine niche in a human-to-mice

xenotransplant (Fig. 6.25A-C); this low colonization efficiency might be

explained by the heterogeneous nature of the transplanted cells, which

were at different stages of maturation due to low reprogramming

efficiency. However, because of the limitation in cell numbers, the

xenotransplantation assay was performed with cells from whole i12F- and

i6F-iGC cultures; similar to previous publications (Geens et al., 2008,

Goossens et al., 2013, Hermann et al., 2007, Hermann et al., 2012) it can be

hypothesized that SSC purification prior to xenotransplant might have

improved colonization efficiency, and so research is this area should be

continued.. In addition, future research should also address if i12F- and i6F-

iGC xenotransplantation into the spermatogenic niche can help them to

acquire the correct epigenetic status, a step which they are unable to

overcome in in vitro culture conditions.

It should be noted that mouse-to-mouse transplantation assays not only

evaluate the colonization abilities of transplanted cells but also their

repopulation capacity, as measured by the formation of spermatozoa and

thus the recovery of fertility. However, in the case of human-to-mouse

xenotransplantation human SSCs are able to colonize, but not repopulate,

the seminiferous tubules due to inter-species barriers (Schlatt et al., 2002,

Hermann et al., 2007, Hermann et al., 2012). In fact, one recent paper

attempts to analyze the functionality of human PSC-derived germ cells in


- 146 -

vivo in mice (Durruthy Durruthy et al., 2014). Similar to the findings in the

iGC model presented here, the authors describe seminiferous tubule

colonization but no repopulation. In summary, these results indicate that

some iGCs also behave like SSC-like cells demonstrating an ability to

colonize mouse seminiferous ducts.

- Proposed model for germ line reprogramming and induction of meiotic

progression

Screening the 12 germ cell-related factor pool showed that the combination

which was the most effective at giving rise to meiotic-like germ cells

included the two transcription factors PRDM1 and PRDM14 together with

the RNA-binding proteins LIN28A, DAZL, VASA, and the structural protein

from the synaptonemal complex SYCP3 (Fig. 6.11; 6.12).

Among these factors, PRDM1, PRDM14, and LIN28A are implicated in the

core network that regulates escape from the somatic cell fate during germ

line specification in E7.5 mouse embryos (Saitou et al., 2002). Classically

Prdm1 is considered to be the only germ cell specification master regulator,

although recently the role of Prdm14 has also been shown to be more

important (Kurimoto et al., 2008). In mouse, overexpression of the Prdm1,

Prdm14, and Tfap2C genes that act downstream of BMP4, together

initiating germ cell specification pathway signaling, triggers EpiSCs to form

germ cells capable of producing live offspring (Nakaki et al., 2013); in this

same work, the authors also showed that Prdm14 is a prerequisite to

obtaining this phenotype, which is mainly due to its role in switching on the

germ line program.

7. Discussion

- 147 -

Moreover, DAZL and VASA have also been described as essential for germ

cell survival and meiotic progression in vivo (Tanaka et al., 2000, Ruggiu et

al., 1997, Gill et al., 2011, Castrillon et al., 2000b) and in vitro (Medrano et

al., 2011, Kee et al., 2009, Panula et al., 2011). Furthermore, the essential

role of VASA in priming hiPSCs to form germ cells in vivo has also recently

been shown by transplanting OSKMV-reprogrammed iPSCs into the testis of

chemically sterilized mice (Durruthy Durruthy et al., 2014).

It was, however, surprising to find that SYCP3 is essential for the initiation

of meiosis in the iGCs (Fig. 6.11). Perhaps this can be explained because

none of the factors used in this study are implicated in activating

synaptonemal complex proteins. Even though there was no endogenous

activation of SYCP3 in response to overexpression of the i12F- and i6F-

reprogramming pools, these factors were essential for correct

synaptonemal complex assembly because most cells subjected to SYCP3

overexpression (alone or in combination with factors other than the ones

included in the i6F- or i12F- cocktail) formed a high percentage of aberrant

synaptonemal patterns.

The RNA-binding protein LIN28A mediates Let-7 miRNA repression which in

turn upregulates the reprogramming transcription factor PRDM1 and

prevents PRDM1 mRNA degradation. The core transcription factors PRDM1

and PRDM14 bind to their specific genomic targets to switch on the germ

line transcription program, in so generating a pool of germ line-related

mRNA transcripts. Hence, based on the literature and on the results

presented here, the meiotic phenotype which is observed might be

explained by the regulatory functions that the RNA-binding proteins DAZL


- 148 -

and VASA have on this transcript pool. These regulatory functions are also

likely to control entry of germ-like reprogrammed cells into meiosis and the

orchestration of meiotic prophase I (likely in conjunction with other

downstream translated proteins) by organizing the structural protein SYCP3

in the synaptonemal complex, thus promoting organization of the proper

chromosome conformation required for a meiotic-like phenotype (Fig. 7.1).

- Germ line development and the pluripotency barrier

Germ line development is associated with pluripotency maintenance: only

germ cells, classically considered as unipotent, can recover pluripotency by

FIG. 7.1. Proposed model for the germ line reprogramming and induction of meiotic

progression. The transcription factors PRDM1 and PRDM14 reinforced by LIN28A enhance

the expression of germ cell-related transcripts (1). The RNA-binding proteins VASA and DAZL

act over this pool of transcripts (2) allowing the structural protein SCYP3 to mediate the

proper chromosome conformation required for a meiotic-like phenotype (3).

7. Discussion

- 149 -

fertilization; hence, germ cells express pluripotency factors in order to

control a non-restrictive epigenome. In other words, there is a pluripotency

cycle, which is naïve in early epiblast and latent in germ cells, which is

maintained only by a pluripotency network common to these cells.

Therefore the moment of PGC specification in mammals is critical. Firstly,

since PGC precursors belong to cell population with a somatic fate, they

must ‘escape’ from that fate and acquire a gametic fate. Secondly, these

processes are crucial for future oocytes and sperm to be able to generate a

complete embryo; interestingly, pluripotency markers seem to be lost after

sexual determination, although SSCs still express some of them (Leitch and

Smith, 2013).

It is worth noting that all of our knowledge about germ cell specification is

based on developmental mouse models. According to this data, BMP signal

induction is triggered by the extra-embryonic ectoderm (Ohinata et al.,

2005). The exposure of epiblast cells to this signal specifies them to acquire

a germ cell fate which means that they can overcome the epigenetic

barriers to reach a naïve pluripotent state. However, although embryonic

pre-implantation development, including gastrulation, shares some

characteristics between mouse and humans there are differences that must

be considered (Fig. 7.2). For example, at week two of human pregnancy,

(the equivalent of murine E5.5-E6.5) most of the epiblastic cells are

separated from trophoblast by the amniotic cavity, meaning that there is no

structure equivalent to me murine ExE (Chuva de Sousa Lopes and

Mummer, 2009); while in mouse embryos, proximal epiblastic cells remain

in direct contact with the ExE, from which they receive the BMP signaling


- 150 -

FIG. 7.2. Comparison of human and mouse embryonic development. A) Human blastocyst at day 13. 1. Secondary yolk sac; 2. Exocoelomic cyst; 3. Amniotic cavity; 4. Extra-embryonic coelom; 5. Epiblast; 6. Connecting stalk; 7. Hypoblast; 8. Primary villi; 9. Trophoblastic lacunae; 10. Extraembryonic somatic mesoderm; 11. Extraembryonic splanchnic mesoderm; B) Midline section of the discoid embryo at day 16. 1. Ectoderm; 2. Primitive pit; 3. Endoderm; 4. Neurenteric canal; 5. Notochord; 6. Intra-embryonic mesoderm; 7. Amniotic cavity; 8. Yolk sac; 9. Allantois; 10. Cloacal membrane; 11. Prechordal plate; C) Human embryo (without extra-embryonic tissues) at day 18. 1. Ectoderm; 2. Mesoderm; 3. Notochord; 4. Endoderm; 5. Prechordal plate; 6. Primitive node; 7. Allantois; 8. Primitive streak; 9. Caudal mesoderm; 10. Amnion; 11. Yolk sac cavity; D) Mouse egg cylinder at E6.0; E) Mouse egg cylinder at E6.5. Onset of gastrulation; F) Mouse embryo at E7.5. Full length primitive streak. Images modified from www.embryo.chronolab.com/ and www.mun.ca/biology/desmid/brian/BIOL3530.

Mouse egg cylinder E6.0 Mouse egg cylinder E6.5 Mouse embryo E7.5

Human blastocyst day 13 Human embryo day 16 (midline section)

Human embryo day 18A CB

D FE

(Saitou and Yamaji, 2010). The way these morphological differences affect

to human PGC specification and, concomitantly, the cycle of pluripotency,

are still not understood in great detail and therefore more in depth studies

should be performed (Saitou and Yamaji, 2010) since the most critical step

in germ cell development seems to be the epigenetic barriers and the

underlying reprogramming processes which are initiated during these

periods of early development (Ng and Surani, 2011).

7. Discussion

- 151 -

As previously mentioned, germ like-cell derivation from PSCs has already

been described (Clark et al., 2004, Kee et al., 2009, Eguizabal et al., 2011,

Medrano et al., 2011, Panula et al., 2011, Park et al., 2009) however, PGCs

isolated from mice just after their specification can also give rise to another

type of PSC line, embryonic germ cells (Ng and Surani, 2011), demonstrating

the reversibility and elasticity of the pluripotent cycle. Interestingly, there

are other differences between human and mouse related to the core of

pluripotency and how it is involved in the derivation of ESC lines. Mouse

ESCs are derived from the inner cell mass of pre-implantation embryos.

However, EpiSCs are another type of PSC which can be derived from post-

implantation epiblastic cells and which are able to differentiate into several

cell lineages and form teratomas (although they do not contribute to

chimera formation as efficiently as ESCs). Under specific culture conditions

ESCs show features similar to pre-implantation epiblastic cells which can

then be induced into expressing post-implantation-like characteristics by

changing the culture conditions, thus becoming EpiSC-like; moreover,

EpiSCs can be derived in vitro into PGCs, demonstrating the relevance of a

primed fate towards pluripotency. Interestingly, hESC are derived in a

similar way from human blastocysts, but there are studies that suggest that

hESCs are closer to mouse EpiSCs than to mESCs (Tesar et al., 2007, Sato et

al., 2011).

This plasticity related to pluripotency networks in PSCs, the strong

correlation between germ line specification and pluripotency maintenance

(Aoshima et al., 2013), and paradoxically, the fact that in vivo PGCs must

overcome epigenetic pluripotency barriers may explain why, to date,


- 152 -

gamete derivation in vitro has only been described from a PSC origin.

In the model of direct conversion of human somatic cells into iGCs

described in this thesis, initiation of this pluripotency cycle may be induced

and maintained by the earlier markers (PRDM1, PRDM14, and LIN28A)

included in the i12F- and i6F-reprogramming cocktail. These three genes

are described to act in an ‘initiating’ PGC specification network (Fig. 1.4)

that leads PGCs into a naïve pluripotency state (Nakaki et al., 2013,

Kurimoto et al., 2008). The overexpression of these early markers seems to

be necessary for treated cells to achieve a ‘near-to-pluripontency’ state.

Since iGCs are directly reprogrammed from somatic cells, a proper

pluripotency state is not reached, as demonstrated by experiments in which

i12F-iGCs do not express the classical pluripotency markers (i.e. OCT4,

SOX2) and cannot form teratomas when directly injected in the testes of

SCID male mice (data not shown).

In fact, it has been reported that genetic manipulation of PRDM1

expression in hESCs can drive them to differentiate towards a germ cell fate

(Lin et al., 2014); indeed, some studies suggest that PRDM14 operates

together with NANOG, another factor also included in the i12F cocktail (Fig.

6.1), to maintain pluripotency networks (Ma et al., 2011, Mitsui et al.,

2003). Moreover, LIN28A in conjunction with other reprogramming factors

(i.e. OCT4, SOX2 and NANOG) is sufficient to reprogram human fibroblasts

into hiPSCs, suggesting it has an important role in the main core of

pluripotency factors (Saitou and Yamaji, 2010).

7. Discussion

- 153 -

According to the literature (Nakaki et al., 2013) this near-pluripotent state

would allow iGCs to start the epigenetic reprogramming process, and turn

off expression of the somatic program without reaching full naïve

pluripotency levels. This may explain why the efficiency of this direct

conversion model (in terms of gene expression, methylation

reprogramming and haploid cell formation) is lower than that achieved by

germ cell derivation from PSCs.

As mentioned above, overexpression of the RNA-binding proteins DAZL and

VASA in combination with other early genes might orchestrate the

formation of a pool of mRNA transcripts in this near-pluripotent state in

which PRDM1 and PRDM14 transcription factors would promote the

expression of the germ cell-related program. It has been proposed that

RNA-binding proteins involved in germ line development may act as

chaperones which mediate correct protein folding to enable proper target-

RNA interaction with accessory proteins (Mohr et al., 2002), as well as

acting as post-transcriptional meiosis-related protein regulators. In fact,

recent results suggest that DAZL may be an upstream transcriptional

regulator of Sycp3 and Mvh in mouse (Reynolds et al., 2007, Reynolds et al.,

2005). It has also been proposed that VASA interacts with Nanos2 mRNA,

thus maintaining mitotic arrest in spermatogonia (Becalska and Gavis,

2009). In this scenario DAZL and VASA overexpression may control entry

into meiosis in vitro, as previously described (Medrano et al., 2011,

Durruthy Durruthy et al., 2014) allowing SYCP3 to find an appropriate

background in which is can attain the correct conformation.


- 154 -

Therefore, studying the overexpression of these reprogramming factors in a

different system, e.g. in human PSCs, may be very useful to better

understand how they interact in a proper pluripotent state. Moreover,

whole genome expression or whole methylome studies (e.g. methylation

arrays or bisulfite deep sequencing techniques) may also help to elucidate

how these reprogramming factors (alone or in combinations) act in a

complete genome context and these experiments may even help us to

design a more accurate human germ cell development model.

- Future perspectives and concluding remarks

Several groups have reported in vitro human germ cell derivation starting

from a pluripotent state (Clark et al., 2004, Kee et al., 2009, Eguizabal et al.,

2011, Medrano et al., 2011, Panula et al., 2011, Park et al., 2009, Durruthy

Durruthy et al., 2014). But, to date, this is the first study that has

interrogated the possibility of directly converting human somatic cells into

cells with germ cell-like features. From this point of view, the results

presented here must be considered as a pilot study from which an in vitro

model can be created to study and gain insights into human germ cell

development. However, a lot of work is still required to improve this model

to the point at which it would serve as an effective technology for the

derivation of clinical-grade gametes in vitro.

Future work will aim to improve the efficiency of this system, for instance

by the possible inclusion of other candidate genes to control human germ

line development. Because some of the genes used in both reprogramming

i12F- and i6F- cocktails are expressed at different moments of mammalian

7. Discussion

- 155 -

germ line development, it is possible that some factors, especially the

earlier markers PRDM1, PRDM14, and LIN28A, must be silenced during the

later germ cell maturation stages such as in the onset of meiosis. In fact,

some work has already highlighted that pluripotency core silencing is

required for proper gamete development not only during germ cell

migration (Seisenberger et al., 2012) but also for entry into meiosis

(Anderson et al., 2007). Hence, sequential expression of factors is a

potential strategy which could be used to better mimic these physiological

processes. Therefore an inducible model for the activation of each of the

transduced genes could be used to increase the effectiveness of this iGC

model. Moreover, changing the reprogramming strategy from a constitutive

to an inducible system (i.e., doxycycline-inducible lentiviral vectors) would

be very useful, also helping to avoid variability in the results and

standardizing the induction response of treated cells.

Indeed, as mentioned above, some groups have reported how genetic

manipulation of the expression of some RNA-binding proteins such as the

DAZ gene family can drive human and mouse pluripotent stem cell germ

line differentiation and haploid cell formation (Panula et al., 2011, Kee et

al., 2009, Medrano et al., 2011, Yu et al., 2009). The importance of

translational control as a germ cell proteome regulating mechanism is also

noteworthy: transcriptional upregulation of individual RNAs is as relevant as

an increase in the translation efficiency. Recent studies have shown that,

there is a higher translational efficiency from the quiescent-gonocyte to

mitotic-spermatogonium stages in neonatal mice due to a higher ribosomal

binding rate, including to both germ line specific, housekeeping, and cell


- 156 -

cycle gene transcripts, rather than an increase in the abundance of

transcripts (Chappell et al., 2013), thus highlighting the importance of

mRNA translational control during germ line development. Hence, more

research into the role of RNA-binding proteins in germ cell differentiation

should be undertaken, not only including DAZ-family members, but also

PIWI proteins.

It would also be interesting to focus on the methylation regulating

pathways because the reprogramming process in germ cell development is

even more extensive than in early embryos, and achieving the proper

methylation status is one of the main challenges for this model. As

previously mentioned, it would also be interesting to overexpress the

factors used in this model in human PSCs to test their efficiency in forming

haploid cells because their expression and epigenetic profile is much closer

to that of PGCs than somatic cells.

Additionally, it would also be of great interest to apply both the i12F- and

i6F- reprogramming combinations to a female human somatic cell line.

Results from this approach may help to elucidate the role of the niche in

the sexual differentiation of gametes and how these genes interplay in a

female genomic context, focusing on X-chromosome silencing/reactivation

and the acquisition of the imprinting pattern. As shown here, some of the

iGCs show some SSC-like characteristics; these could therefore be of great

help for improving our ability to identify, culture, and mature SSCs in vitro,

all of which are strategies proposed for preserving and recovering fertility in

adulthood for oncogenic prepuberal children (Goossens et al., 2013, Baert

et al., 2013). From a more basic perspective, iGCs could also act as a base to

7. Discussion

- 157 -

study human spermatogenesis at a molecular level which could be

compared with other mammalian murine, rabbit or pig models, especially

given that the latter two exhibit peri-implantation embryo development

which is closer to human development (Saitou and Yamaji, 2012).

Based on these results, this model is still very far away from obtaining

functional gametes in vitro. Until a method for producing proper

reprogramming events is reported this technology only serves to model

germ cell development and perhaps also pathological situations. Because

iGCs are able to go through and to complete meiosis they could also be

used to study the causes of meiotic failure from a personalized medicine

point of view. From a future perspective, this model of direct

reprogramming human somatic cells into meiotic germ cells could be used

to help to discover therapies to treat patient-derived fertility problems.

With this objective, non-viral integration strategies for reprogramming (i.e.,

RNA-reprogramming) should also be tested.

In conclusion, to date this is the first study that describes the direct

conversion of human somatic cells into meiotic-like cells in vitro. Therefore

these results should be considered as proof of concept that iGCs could be

generated to produce a robust in vitro model for studying human germ cell

development. Hence, more work must be performed in order to improve

this in vitro iGC model in both terms of its efficiency and its efficacy. In the

same sense, further research is required to fully implement this model’s

potential applications at a translational and clinical level.

- 159 -

8. Conclusions

Bluebell, White poppy, Sweet Pea

Grateful, rejoice, goodbye

8. Conclusions

- 161 -

Therefore, the final conclusions of this thesis project are:

1. By reprogramming two different human primary somatic cells (hFSKs,

and hMSCs) with key gene regulators a heterogeneous iGC population

at different stages of maturation was obtained.

2. iGCs show an transcriptomic expression and methylation profile that

correlate with that of germ cells at different maturation stages

3. Some of these iGCs may correlate with a spermatogonia-like stage since

they are able to go through and complete meiosis after exposure to

retinoic acid.

4. The most effective combination of factors used to achieve direct

reprogramming to a meiotic phenotype included the transcription

factors PRDM1, PRDM14, the RNA-binding proteins LIN28A, DAZL, and

VASA, and SYCP3, a structural protein which is part of the

synaptonemal complex.

5. Moreover, some of these iGCs demonstrated their in vivo functionality

by their ability to colonize seminiferous ducts in a murine xenograft

model.

6. This work must be considered as a pilot study in which iGCs were

created in vitro using direct reprogramming to gain insights into human

germ cell development.

- 163 -

9. Appendix

Hanakotoba is the Japanese language of flowers

9. Appendix

- 165 -

APPENDIX 1. PLASIMD MAPS

- ENTRY VECTOR


- 166 -

- EXPRESSION VECTOR (plasmid 3 for third generation lentiviral

transduction)

9. Appendix

- 167 -

- PLASMIDS 1 AND 2 for third generation lentiviral transduction:

PLASMID 1 (GAG-pol; Tat, Rev)

PLASMID 2 (VSV-G)


- 168 -

- PLASMID FOR Bi-seq sub-cloning

9. Appendix

- 169 -

APPENDIX 2. PRIMERS SEQUENCES

GENE Primer sequences for detect ectopic expression Length (bp)

PRDM1 F: GCCAAGTTCACCCAGTTTGT 183

R: GATTCGGGTCAGATCTTCCA

PRDM14 F: TCTCTACGATCTGCCCTGGT 231

R: CTCAGCCCCTCAGGTAACAG

LIN28A F: TGCACCAGAGTAAGCTGCAC 189

R: CTCCTTTTGATCTGGCTTC

NANOS3 F: GGGAAAGAGGGTCCTGAAAC 221

R: AGCACGTGGGACTGGTAGAT

NANOG F: GATTTGTGGGCCTGAAGAAA 155

R: CAGTCTGGACACTGGCTGAA

DAZ2 F: GAGATTGGAAGCTGCTTTGG 212

R: TGCACATGACGAGCACATAA

DAZL F: CATCCTCCTCCACCACAGTT 202

R: AACTGTGGTGGAGGAGGATG

BOULE F:CTAATCCTGTGTCACCTGTGC 205

R:ACGAAACCATACCCTTTGGA

VASA F:ATGGATGATGGACCTTCTCG 248

R:CCTCTGTTCCGTGTTGGATT

STRA8 F:AATCCCCATGACAGAGCAAC 227

R:TTATCCAGGGTTTGCTCCAG

SYCP3 F: AGCCGTCTGTGGAAGATCAG 197

R: AACTCCAACTCCTTCCAGCA

DMC1 F: AATCAAATGACTGCCGATCC 166

R: CAGGCATCTCAGGACTGTCA

RPL19 F: ACCTGAAGGTGAAGGGGAAT 140

R: GCGTGCTTCCTTGGTCTTAG


- 170 -

GENE Primer sequences for germ cell markers Length (bp)

GFRA1 F: GCAAGGAGACCAACTTCAGC 190

R: TCCTCCAGCAGATGATTTCC

PIWIL2 F: GTTAATGGTGATCGGGATGG

R: ATGCATGCCATTTATCAGCA

120

TNP2 F: CCAACACTAGTCCACCACCA 197

R: GTTGGATTTCCATCCTGAGC

PRM1 F: CCGCCAGAGACAAAGAAGTC 200

R: GGATGGTGGCATTTTCAAGA

ACRO F: ATTCTGCTGGTCTTGGCAGT 192

R: TGTGTGGTACCTGTGGCTGT

DNMT1 F: TACCTGGACGACCCTGACCTC 103

R: CGTTGGCATCAAAGATGGACA

DNMT3A F: TATTGATGAGCGCACAAGAGAGC 111

R: GGGTGTTCCAGGGTAACATTGAG

DNMT3B F: GGCAAGTTCTCCGAGGTCTCTG 188

R: TGGTACATGGCTTTTCGATAGGA

TET1 F: GCTATACACAGAGCTCACAG 139

R: GCCAAAAGAGAATGAAGCTCC

TET2 F: CTTTCCTCCCTGGAGAACAGCTC 146

R: TGCTGGGACTGCTGCATGACT

TET3 F: ATTCTGCTGGTCTTGGCAGT 93

R: TGTGTGGTACCTGTGGCTGT

RPL19 F: GTTCCTGGAGCATGTACTTC 199

R: CTTCCTCTTTGGGATTGTCC

ICR Primer sequences for bisulfite sequencing Length (bp)

hH19 F: TGTATAGTATATGGGTATTTTTGGAGGTTT 231

R: TCCTATAAATATCCTATTCCCAAATAACC

hPEG1/MEST1 F: TCGTTGTTGGTTAGTTTTGTACGGTTGC 290

R: ATAAACTCCTCCCCCACAATAAAAA

9. Appendix

- 171 -

APPENDIX 3. DERIVED PUBLICATIONS AND SCIENTIFIC COMMUNICATIONS Articles - Martínez-Arroyo, AM.*; Medrano, JV*; Remohí J.; Simón, C. Germ line development: lessons learnt from pluripotent stem cells. Current opinion in genetics and development. REVIEW. In press. * Equally contibuting authors. - Medrano, JV*; Martínez-Arroyo, AM*; Quiñonero, A; Marqués-Marí, AI; Pellicer, A; Remohí, J; Simón C. Direct Conversion of Human Somatic Cells to Meiotic Germ-like Cells by Genetic Reprogramming. ORIGINAL ARTICLE. Submitted. * Equally contibuting authors - Medrano, JV; Martínez-Arroyo, AM; Sukhwani, M; Noguera, I; Quiñonero, A; Martínez-Jabaloyas, JM; Pellicer, A; Remohí, J; Orwig, KE; Simón, C. Germ Cell Transplantation Into Mouse Testes Procedure. Fertility and Sterility. ORIGINAL ARTICLE. Submitted. Book chapters - Medrano, Jose V.; Martínez-Arroyo, Ana M.; Simón, Carlos; Reijo

Pera, Renee A. Germ Cell Differentiation from Pluripotent Cells. At: Stem Cells in Human Reproduction: Basic Science & Therapeutic Potential, Third Edition. Cap. 4. Cambridge University Press

Scientific communications: - Martínez-Arroyo, Ana M.; Medrano, Jose V.; Quiñonero, Alicia;

Marqués-Marín, Ana I.; Simón C. Direct reprogramming of human somatic cells to meiotic germ cells. Conjoint Meeting of the International Federation of Fertility Societies and the American Society for Reproductive Medicine (IFFS/ASRM). Boston, MA, USA. (October 12-17, 2013) ORAL PRESENTATION.

- Medrano, Jose V.; Martínez-Arroyo, Ana M., Quiñonero, Alicia; Marqués-Marí, Ana I.; Simón C. Direct conversion of human somatic cells to meiotic germ cells by genetic reprogramming. Gordon Reserch


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Conference on Germinal Stem Cell Biology. Hong-Kong, China. (July 12-16 2012) POSTER

- Martínez-Arroyo, Ana M.; Medrano, Jose V.; Simón, Carlos. Direct reprogramming of human fibroblast to meiotic germ cell-like cells by defined factors. Epigenetics and Stem Cells conference. Cambridge, UK. (October 16-17 2012). ORAL PRESENTATION

- Medrano, Jose V.; Marqués-Marí, Ana I.; Martínez-Arroyo Ana M.; Simón, Carlos. Direct conversion of human adult somatic cells to meiotic germ cells by genetic manipulation. International Society for Stem Cell Research 10th Annual Meeting. Yokohama, Japan. (June, 13-16 2012). POSTER

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