Induced pluripoten (iPS) cells: an emerging technology...

Induced pluripoten (iPS) cells: an emerging technology platformMaria J. Barrero

Centre de Medicina Regenerativa de Barcelona

Our body is composed by more than 200 different cell types…

…among them some are considered stem cells

Stem cell:

‐

can self‐renew

‐

potenciality: differentiate into other cell types.

Types of stem cells:

‐

Embryonic stem cells

‐

Adult stem cells

5‐7 day embryoSingle cellembryo

6 week embryo

Embryonic Embryonic Adult Adult

TotipotentTotipotent OligopotentOligopotent

Embryonic Stem (ES) cellsPluripotent

Embryonic germ (EG) cellsPluripotent

Cord blood Stem cellsMultipotent

Adultstem cellsMultipotent

• Self‐renewal• Pluripotent

5‐7 days

ICM

Embryonic vs. Adult Stem Cells

•Ethical issues/iPS

•Known how to isolate them

•Easy to expand in vitro

•Give rise to many tissues

•Difficult to control differentiation

•First clinical trials just started

•Difficult to identify and isolate

•Difficult to expand in vitro

•Give rise to limited tissues

•Easier to control differentiation

•Currently used to treat deseases

Embryonic Adult

Other sources of embryonic stem cells

Somatic cell REPROGRAMMING

Embryonic stem cell programmSkin cell programm

In vitro manipulation

1956 1981 1988 1992 1996

1998 2002 2004

First bone

marrow

transplant

mouseESisolated

HSC from

mouse

isolated

SC in human

brain

identified

human ESisolated

first animalcreated by

nuclear

transfer

pancreatic

cells from mouse ES

cure diabetic

mice

dopaminergic

neurons from

human ES

Stem cell time line

2006 2007

mouse iPS human iPS

Skin cells culture

iPS cells= induced pluripotent stem cells

Reprogramming using defined factors

Reprogramming

Oct4Sox2Klf‐4c‐myc

• Self‐renewal• Pluripotent

Induced pluripoten stem (iPS) cells.

Generation of Induced pluripoten stem (iPS)cells.

1. Goal: therapeutic, basic research, screenings.

2. Choice of somatic cell.

3. Choice of reprogramming factors.

4. Delivery of reprogramming factors.

5. Selection and expansion of reprogrammed cells

6. Characterization of iPS cells

7. Set up of drug screening.

Generation of Induced pluripoten stem (iPS)cells.

Seeding

somatic

cells

Delivery

of

factors

Splitand

change to

ES media

appearance

of first

colonies

1‐2 days 2‐5 days 7‐30 days

expansion and

characterization

of colonies

3 months

Choice of somatic cellChoice of somatic cell

Amniotic fluid

Endometrium

Fetal lung fibroblasts

Umbilical vein endothelial cells

Cord Blood

Menstrual blood

Neural adult stem cells

Astrocytes

Keratinocytes

Adult dermal fibroblast

B‐lymphocytes

Reprogramming efficiency

Disease type Disease name Genetic cause N. of lines Cell type Control line Phenotype Drug test

Neurological Parkinson's disease Polygenic 23 Dopaminergic neurons hiPSC No obvious defect ND

Polygenic (with

LRRK2

mutation)4 Dopaminergic neurons hiPSC Neuronal death with chemicals Yes

Amyotrophic lateral sclerosis Polygenic 3 Motor neurons hESC ND ND

Spinal muscular atrophy Monogenic 2 Motor neurons hiPSC Loss of neuron formation, loss of SMN

gene expression

Yes

Familial dysautonomia Monogenic 2 Neural crest cells hiPSC, hESC Loss of neural crest cells Yes

RETT syndrome Monogenic 4 Neurons hiPSC Loss of synapses, reduced spine density,

smaller soma size

Yes

Huntington's disease Monogenic 2 ND hiPSC, hESC ND ND

Friedreich ataxia Monogenic 6+ ND hESC Changes GAA‐TTC repeat ND

Blood Fanconi anaemia Monogenic 19 Blood cell hiPSC, hESC Corrected loss of FANCA

function ND

Fragile X syndrome Monogenic 11 ND hiPSC, hESC Loss of FMR1 expression ND

Cardiac and

vascular

Long QT 1 syndrome Monogenic 6 Cardiomyocytes hiPSC Increased cardiomyocyte depolarization Yes

Long QT 2 syndrome Monogenic Not reported Cardiomyocytes hiPSC Increased cardiomyocyte depolarization Yes

LEOPARD syndrome Monogenic 6 Cardiomyocytes hiPSC, hESC Increased cardiomyocyte size, decreased

MAPK signaling

ND

Timothy syndrome Monogenic 16 Cardiomyocytes hiPSC Increased cardiomyocyte depolarization Yes

Hutchinson Gilford Progeria Monogenic 4 Smooth muscle cells,

mesenchymal stem cells

hiPSC, hESC Smooth muscle and mes‐enchymal cell

apoptosis

ND

Monogenic 6 Smooth muscle cells hiPSC Smooth muscle cell nuclear morphology

and ageing phenotype

ND

Duchenne muscular dystrophy Monogenic 2 ND hiPSC, hESC ND ND

Pancreatic Type 1 diabetes Polygenic 4 Insulin‐

and glucagon‐

producing cells

hESC ND ND

Hepatic A1‐antitrypsin deficiency Monogenic 19 Hepatocytes hiPSC Loss of A1‐antitrypsin expression Yes

Others Prader‐Willi syndrome Monogenic 4 Neurons hiPSC, hESC Imprint disorder ND

Angelman and Prader‐

Willi

syndrome

Monogenic 13 Neurons hiPSC, hESC Loss of paternal UBE3A expression ND

Down syndrome Monogenic 2 ND hiPSC, hESC ND ND

Choice of Reprogramming Factors.

Sox2

c‐mycKlf4

Oct4 Nanog Lin28

1. Proteins

2. miRNAs

3. ChemicalsVPA

UTF Sall4

BIX‐01294

5′‐azacytidine SAHA TSA

miR302/367

mir‐200c /302 s /369 s

miR‐302/372

Virus: lentivirus,Retrovirus, adenovirus

DNA: transfection

Protein: transfection,PTD

RNA: transfection

Advantage Disadvantage

Delivery methods

CheapEasy to produceefficient

integrative

CheapEasy to producenon integrative

Inefficient

Integrative

Expensiveineficient

Non integrative

CheapEasy to producenon integrative

ineficient

iPS cell time line

2006 2007 2008 2009

2010 2011

mouse iPS OSKM Retrovirus

human iPS OSKM retrovirus

iPS without myc

iPS from NSC with two factors

iPS with two factors+ small molecules

Non integrative iPS (adenovirus)

Non integrative iPS (excisable lenti)

Desease specific iPS cells

Non integrative iPS (episomal)

Non integrative iPS (proteins)

Non integrative iPS (RNA)

iPS using miRNAs

iPS cells have a high rate of mutations

Selection and expansion of coloniesSelection and expansion of colonies

Seeding

somatic

cells

Delivery

of

factors

Splitand

change to

ES media

appearance

of first

colonies

1‐2 days 2‐5 days 7‐30 days

expansion and

characterization

of colonies

3 months

ES/iPS cell culture conditions

matrigelHES media: Serum replacement

FGFFibroblast conditioned

Passage: Enzymatic

Feeder:

Human irradiated

fibroblast

HES media: Serum replacementFGF

Passage: Mechanical

matrigelDefined media (mTeSR)Passage: Enzymatic

Selection and expansion of coloniesSelection and expansion of colonies

Partially Reprogrammed Cells

•Selfrenewal•Downregulation of cell

specific expression programs•Expression of transgenes

Fibroblast

Oct4Sox2Klf4c‐Myc

Fully Reprogrammed Cells

•Pluripotent•Activation of pluripotency

genes.•Silencing of transgenes•Bivalent modifications

Stress/Senescence

Chromatin

Tumor Teratoma

iPS

Characterization of iPS lines coloniesCharacterization of iPS lines colonies


1. Molecular characterization:• Silencing of transgenes

expression of transgenes

0

0.2

0.4

0.6

0.8

1

1.2KE

RATO

S

keratos 4F

shRD

#1 P4

shRD

#2 p4

shRD

#3 p4

shRD

#4 p4

shRD

#5 p4

shM1#1 p4

shM1#2 p

shM1 #3

P4

shM2#1 p4

shM2 #2

P4

shM2#3 p4

shM2 #4

P4

ES4 p1

4

Trans‐Oct4Trans‐Sox2Trans‐Klf4Trans‐Myc

Levels to

GAPD

H


1. Molecular characterization:• Induction of pluripotency factors

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

keratos

ES4 p1

4

shRD

#1 P4

shRD

#2 p4

shRD

#3 p4

shRD

#4 p4

shRD

#5 p4

shM1#1 p4

shM1#2 p4

shM1 #3

P4

shM2#1 p4

shM2 #2

P4

shM2#3 p4

shM2 #4

P4

KLF4cMYCOct4SOX2NanogCRIPTORex1

expression of endogenous genes

Levels to

GAPD

H


1. Molecular characterization:• Induction of pluripotency factors


2. Functional characterization:• In vitro differentiation: formation of embryoid bodies

FGFSerum replacement

Fetal bovine serumAscorbic acidActivine A

Characterization of iPS linesCharacterization of iPS lines

2. Functional characterization:• In vivo differentiation: teratoma formation

Characterization of mouse iPS linesCharacterization of mouse iPS lines

2. Functional characterization:• Germline contribution in chimaeras

"We can differentiate the disease‐specific iPS cells into certain lineages, and we can

screen for a drug that can alleviate the phenotype of the disease"

Advantage:Unlimited source of human cell types

Challenges:

Drug companies require large and consistent numbers of differentiated cells at high

purity to be able to use them in their discovery and toxicity screening systems.→

Improvement of differentiation protocols

Easy readout of phenotypes. (reporter genes, colorimetric assays...)

Drug screeningsDrug screenings

Drug screeningsDrug screenings

La ley españolaLa ley española

Para generar o trabajar con "human

ES/iPS cell lines":

1. Obtener el informe favorable del Comitè

d’Ètica d’Investigació

Clínica (CEIC):

•Valorar els projectes de recerca (PR) i protocols d’assaigs clínics (AC) i les seves modificacions, en relació

amb els objectius definits, l’evidència científica i la justificació

dels

riscs i molèsties previsibles per als participants en funció

dels potencials beneficis.

•

Avaluar la preparació

dels investigadors per al projecte o l’assaig proposat, així

com

també

la idoneïtat de les instal∙lacions i equipaments disponibles en el centre on es pretén

dur a terme el PR o AC

2. Con el informe favorable del comité

ético solicitar la aprobación al Departamento de

Salud. (Banco Nacional de Líneas Celulares o a la Comisión de Garantías de la donación y

utilización de células y tejidos humanos)

3. El investigador principal se compromete a informar a la comisión de los progresos del

proyecto y a proporcionar al Banco Nacional de Líneas Celulares las líneas obtenidas.

La ley españolaLa ley española

Es obligatorio registrar las "human

ES/iPS cell lines" generadas en el Banco Nacional de

Líneas Celulares

Copia de la autorización de derivación de la línea celular, junto con informe del Comité

Ético.

Copia de cualquier publicación científica relacionada con la derivación y/o caracterización de

la línea.C. V. del investigador principal

Descripción de las características morfológicas de la línea en cultivoControles microbiológicos realizados (micoplasma)Marcadores pluripotenciaCapacidad de diferenciación in vitro (embryoid bodies)Capacidad de diferenciación in vivo (teratoma)Consistencia celular tras 6 pases de congelación y descongelaciónPase en el momento del registro

ComitComitèè

dd’È’Èticatica

dd’’InvestigaciInvestigacióó

ClClíínica nica

Banco Nacional de LBanco Nacional de Lííneas Celularesneas Celulares

Induced pluripoten (iPS) cells: an emerging technology...

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