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Germ cells production from established goat

embryonic stem cells and iPS cells

Dhruba Malakar*, Hruda Nanda Malik and Dinesh Kumar

Animal Biotechnology Centre, National Dairy Research Institute, Karnal-132001, India

*Corresponding author. Tel:+91-9416741839; fax: +91-184 2250042

E-mail address: dhrubamalakar@gmail.com

Abstract:

Objective/Background:

World trend of fertility rate was declined drastically. In vitro derivation of both germ

cells from embryonic stem cell (ESC) and induced pluripotent stem cell (iPSC) can be an

alternative technique. Here we are presenting how ESC and iPSC are differentiating into

germ cells like cells using goat as an animal model with this objective. The aim of the present

study was to produce in vitro germ cells like cells from IVF ES cells, parthenogenetically

produced ES cells and iPSC.

Material & Methods: Embryonic stem cells were generated by IVF and parthenogenetically

activated blastocyst. Oocytes were collected from the ovaries of slaughtered goats and

matured in TCM-199 supplemented with FSH, LH and β-estradiol as maturation factors.

Matured oocytes were in vitro fertilized with capacitated goat spermatozoa and cultured in

RVCL medium. For parthenogenesis, matured oocytes were activated with 5 µM Ca

ionophore for 5 min and 2 mM 6-DMAP for 4 h and cultured in RVCL medium for

blastocyst. Inner cell masses were isolated from blastocysts and cultured for ES cells on 10

µg/ml mitomycin-C treated feeder layer. The iPS cells were produced using transcription

factors of Nanog, Oct4, Sox2 and Lin28 with lentiviruses in goat adult fibroblasts cells.

Selected iPS cells were characterized using different methods. Characterization of ES cells

and iPS cells was carried with Alkaline phosphatase staining, intracellular markers of Oct4,

Nanog, sox2 and surface markers of SSEA-1, SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81.

The embryoid bodies of goat ES cells were spontaneous differentiated to neuron, fibroblast,

muscle cells and directed differentiation to rhythmic beating of cardiomyocytes.

Cardiomyocyte was characterized with α-actinin, C-Troponin and Gata4 genes expressed in

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RT-PCR and immunohistochemistry. The present study was experimentally established goat

ES cells and further subcultured to 22 passages and cryopreserved these cells. Directed

differentiation of ES cells into germ cell like cells: Goat ES cell colonies were cultured for

embryoid bodies. These embryoid bodies were cultured in ES cell medium supplemented

with retinoic acid and BMP-4. The differentiated ES cells for germ cell like cells were

characterized with germ cell marker genes like VASA, STELLA and PUM1 immunostaining

and Western Blotting of differentiated ES cells of these genes were expressed in the present

study.

Results:

The VASA, STELLA and PUM1 germ cell specific marker genes were expressed in

germ cells of directed differentiated ES cells and genes were 891bp, 365bp and 822bp

respectively. Immunostaining germ cell like cells of differentiated goat ES cells with VASA

primary antibody was already shown positive sign. VASA, STELLA and PUM1 germ cell

specific marker proteins were also identified in Western blotting as these proteins expressed

in the differentiated germ cells of directed differentiating embryonic stem cells. Here we

mentioned that within one month, we will able to complete our rest of the research work.

Conclusion:

VASA, STELLA and PUM1 germ cell markers genes were expressed in directed

differentiating ES cells. The proteins of these genes have already expressed in

immunohistochemistry and western blotting in the differentiated ES cells and iPS cells.

Characterization of germ cells from IVF, parthenogenetic ES cells and iPS cells were

obtained optimistic result.

Key words: Cardiomyocyte, Embryonic stem cell, Germ cell, Goat, iPSC, VASA

Introduction:

The ability to generate functional haploid germ cells acts as a yard stick to measure

reproductive performance of mammals. One of the most common causes of male infertility is

abnormal germ cell differentiation leading to azoospermia or oligospermia. In females, the

most common cause of infertility involves ovulatory dysfunction.

World trend of fertility rate declined gradually is shown in the figure below.

Elucidating the molecular mechanisms involved in establishing the oocyte reserve and

formation of spermatogonial stem cells is challenging because these events are completed

before birth. In vitro derivation of both germ cells and matured functional gametes from

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embryonic stem cell (Esc) and induced pluripotent stem cell (iPSc) can be an alternative

technique to meet upto these challenges.

Here we are presenting how embryonic stem cells and iPSc are differentiating into

germ cells like cells using goat as an animal model. Male germ cells derived from ESCs and

IPSc can be engrafted in host testes to produce viable sperm which helps in understanding of

molecular mechanism of spermatogenesis and possibly provide new treatments for male

infertility. A limitless supply of eggs derived from ESC and iPSc can have a radical impact

on medicine. An ability to efficiently develop ESC and iPSc derived oocytes for nuclear

transfer studies would be a significant advance and may provide a limitless source of oocytes.

If these oocytes provide all the cues necessary to allow reprogramming of donor nuclei and

successfully develop to the blastocyst stage, patient-specific ESC and iPSc lines resulting

from nuclear transfer could be created, helping circumvent the major obstacle of donor

oocyte availability in the construction of patient-specific ESC lines by nuclear transfer.

Establishment of iPS cell line from adult goat fibroblast cells as a model for production

of germ cells. Embryonic stem (ES) cells derived from inner cell mass of mammalian

blastocysts grow rapidly and infinitely having the ability to differentiate into all types of cells

(Evans and Kaufman, 1981; Martin, 1981). These properties of ES cells are maintained by

symmetrical self-renewal, producing two identical stem cell daughters upon cell division

(Burdon et al., 2002). The generation of pluripotent cells from differentiated adult cells has

vast therapeutic implications, particularly in the context of in vitro disease modelling,

pharmaceutical screening, and cellular replacement therapies. In addition, the ability to revert

somatic cells to an embryonic state provides a unique tool to dissect the molecular events that

permit the conversion of one cell type to another.

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Thus, the direct generation of pluripotent cells without the use of embryonic material

has been deemed a more suitable approach that lends itself well to mechanistic analysis and

has fewer ethical implications. The direct reprogramming of somatic cells to pluripotency

was accomplished in 2006, when Takahashi and Yamanaka converted adult mouse fibroblasts

to iPSCs through ectopic expression of a selected group of transcription factors. Subsequent

reports optimized this technique, demonstrating that iPSCs were indeed highly similar to

ESCs when tested across a rigorous set of assays (Maherali et al., 2007; Okita et al., 2007;

Wernig et al., 2007). In 2007, direct reprogramming was achieved in human cells (Takahashi

et al., 2007b; Yu et al., 2007), providing an invaluable contribution to the field of

regenerative medicine.

Materials and Methods:

In vitro production of goat embryos

Blastocysts were produced following in vitro maturation, fertilization, culture and

vitrification procedures, as described by Pawar et al, (2009). Briefly, oocytes collected from

slaughterhouse were matured in TCM 199 (HEPES modified), containing 10 µg/ml

luteinizing hormone (LH), 5 µg/ml follicle stimulating hormone (FSH), 1 µg/ml oestradiol-

17β, 50 µg/ml sodium pyruvate, 3.5 µg/ml L-glutamine, 50 µg/ml gentamicin, 5.5 mg/ml

glucose, 3 mg/ml Bovine Serum Albumin (BSA) and 10% FCS (Malakar and Majumdar,

2005).

After in vitro fertilization with fresh semen, the blastocyst and hatched blastocysts

were cultured with a medium containing TCM 199 (HEPES modification), 30 µg/ml sodium

pyruvate, 100 µg/ml L-glutamine, 50 µg/ml gentamicin, 10 µl/ml essential amino acids, 5

µl/ml non-essential amino acids (NEAA), 10 mg/ml BSA (Fraction-V), 10% FCS and 50 mM

cysteamine for 8 days.

Parthenogenetic activated goat embryos:

Parthenogenetic embryos were produced with different methods such as electrical

stimulus, the use of chemical agents such as Ca2+ ionophore, ethanol, strontium chloride,

phorbol ester, thimerosal and phospholipase zeta (Ross et al., 2008) have been successfully

used to activate bovine parthenotes. The present study was conducted by chemical activation

of oocytes with the aid of a Ca ionophore and 6-DMAP. In vitro matured oocytes were

activated with 5 µM Ca ionophore for 5 min and 2 mM 6-DMAP for 4 h. The putative

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zygotes were cultured in the 3 different media, i.e. RVCL, mSOF and EDM (Jena et al.,

2012). The ICM of blastocyst and hatched blastocyst were isolated and culture for

parthenogenetic embryonic stem cell production and further characterized with different

methods as given below.

Production and characterization of embryonic stem cell from IVF and parthenogenetic

activated goat embryos:

Blastocysts and hatched blastocysts were used for isolation and culture of putative

goat embryonic stem cells. The inner cell masses (ICMs) were isolated from expanded and

hatched blastocysts using mechanical isolation. The isolated ICMs were seeded on 10 µg/ml

mitomycin-C inactivated feeder layers in ES medium containing DMEM supplemented with

20% FCS, 1000 IU/ml of mLIF 1% nonessential amino acids, 0.1 mM β-mercaptoethanol,

and 2 mM L-glutamine. The medium was changed every 48 h interval and the formation of

colony was observed routinely under inverted microscope and further subculture of the ES

cells.

Culture of goat fibroblast cells for iPS cells:

Cell culturing: Goat adult fibroblast was grown in Dulbecco’s Modified Eagal

Medium (DMEM) containing 10% fetal bovine serum and 0.5% penicillin and streptomycin.

The fibroblast cells were used after 3-4 passages for gene transfection. The 293T cells was

maintained in DMEM containing10% FBS, mM L glutamine, 0.1 mM nonessential amino

acids, 1 mM sodium pyruvate and 0.5% penicillin and streptomycin. Goat induced

pluripotent stem cells was generated and maintained in primate embryonic stem cells medium

(ESCM) supplemented with 0.4 ng/ml basic fibroblast growth factor (bFGF). The goat fetal

skin feeders and feeder free culture with the conditioned medium was carried out.

Preparation of expression constructs: Total RNA was isolated from goat embryonic

stem cells by Trizol method. cDNA will be prepared by RT-PCR. DNA of Oct4, Nanog,

Sox2 and Lin28 was amplified using specific primers and cloning of these genes was done in

a suitable cloning vector. Clones were transfected and amplified in the E.coli Top10

competence cells. The open reading frame of goat Oct4, Nanog, Sox2 and Lin28 will be

amplified and subcloned into a plentiV4/5 DEST vector.

Induction of pluripotency in goat adult fibroblast cells:

Plat-E cells were seeded at 8 X 106 cells per 100 mm dish day before transduction. On

the next day, pMXs-based retroviral vectors was introduced into Plat-E cells using Fugene 6

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transfection reagent (Roche) according to the manufacturer’s recommendations. Twenty-

seven microlitres of Fugene 6 transfection reagent was diluted in 300 ml DMEM and

incubated for 5 min at room temperature. Nine micrograms of plasmid DNA was added to the

mixture, which will be incubated for another 15 min at room temperature. After incubation,

the DNA/Fugene 6 mixture was added drop by drop onto Plat-E cells and incubated overnight

at 37oC with 5% CO2 in air. The virus containing supernatants was used to infect the goat

fibroblast cells. Virus-containing supernatants derived from Plat-E cultures was filtered

through a 0.45 mm cellulose acetate filter and supplemented with 4 mg/ml polybrene after 24

hrs.

Equal amount of supernatants containing each of four retroviruses was mixed in equal

amount and transferred to the fibroblast dish. Target cells were incubated in the

virus/polybrene-containing supernatants for 4 hrs to overnight. Two or three rounds of

overnight infection will be performed and cultured on feeder layer and matrigel coated plate.

Next day the medium was replaced with Primate ES cell medium supplemented with 4 ng/ml

bFGF.

The medium was changed every other day and observed the colonies. The goat iPS

cells was cultured on goat fetal skin feeders and matrigel coated plate in standard ES media

(DMEM supplemented with 15% FBS, Nonessential amino acids, L-glutamine, penicillin

streptomycin, b-mercaptoethanol and with 1000 U/ml LIF). The iPS cells were again

subcultured as per the colony formation.

Characterization of stem cells with different methods.

The characterization of the putative stem cells was carried out at different passages by

the following methods.

Alkaline phosphatase staining, the expression of intracellular marker Oct4 and surface

markers SSEA-1, SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81 were examined by

immunofluorescence staining of colonies of putative goat ES cells. Reverse transcription and

polymerase chain reaction (RT-PCR), chromosomal integrity of ES cell-like cells, embryoid

bodies formation and spontaneous differentiation to neurone cells, fibroblast cells, muscle

cells and directed differentiation to cardiomyocyte of goat ES cells. The differentiation of ES

cells to cardiomyocyte was characterized with α-actinin, C- Troponin and Gata4 genes

expressed in RT-PCR and immunohistochemistry.

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Directed differentiation of embryonic stem cell into putative primordial germ cell:

Goat putative ES cell colonies was cultured in suspension or in hanging drop method

with ES cell medium in absence of leukemia inhibitor factor to form embryoid bodies. Then

these embryoid bodies was cultured in conditioned medium which consists of ES cell media

supplemented with retinoic acid and BMP-4.

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Characterization of differentiated embryonic stem cell:

Amplification of germ cell specific gene:

Total RNA was isolated from differentiated ES colonies by using RNeasy Mini Kit

(Qiagen). Then total RNA was converted into cDNA by using RevertAidTM First Strand

cDNA Synthesis Kit (Fermentas). The cDNA was amplified by STELLA, VASA and PUM1

gene specific primer. Primers used in PCR reaction were VASA-F: (5’- CTG GTG GCA

TTT TTG GTT CT -3’) and VASA-R: (5’- GCT GTT CCT TTG ATG GCA TT -3’),

STELLA- F (5’- AAC CCA ACC TGG ACC CTA GA -3’) and STELLA- R (5’- TGG AAT

CTT CGC ACT CTT GA -3’), PUM1-F (5’- AGA ATG GGA TTG ACG CAG AC -3’) and

PUM1-R (5’- AGT AAG CAG CAG GAG CCA AG -3’). These germ cell specific genes

were amplified at initial denaturation 94ºC for 5 min, then again denatured at 94ºC for 30 sec,

annealling at 58ºC for 30 sec, extension at 72ºC for 30 sec for 40 cycles and final extension at

72ºC for 5 min. A total of 25µl of PCR reaction mixture was taken which contains PCR

master mix 1X 12.5µl, cDNA 5µl, and forward primer and reverse primer 1µl (10 pmol) each

and Nuclease free water 5.5 µl. The amplified product was resolved on agarose gel and

observed the band under UV transilluminator.

Immunostaining of differentiated putative ES cells

Differentiated goat ES cells were immunostained with VASA primary antibody.

These cells were fixed in 4% paraformaldehyde in DPBS for 30 min, washed 3 times with

DPBS and then permeabilized by treatment with 0.1% Triton X-100 in DPBS for 30 min.

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After thorough washing with DPBS, the differentiated goat ES cells were incubated with the

blocking solution (4% normal goat serum) for 30 min, and then with the VASA (primary

antibody at a dilution of 1:200) for 1 h. In the respective controls, the addition of the primary

antibody was omitted. After washing 3 times with DPBS, above cells were incubated with the

FITC-labeled secondary antibody (goat anti-mouse IgG or IgM, diluted 1:2000) for 2 h. The

differentiated goat ES cells were then examined under a fluorescence microscope (Diaphot,

Nikon, Tokyo, Japan.)

Results:

Amplification of germ cell specific gene:

The result obtained by resolving PCR product on 1.5% agarose gel containing

ethidium bromide (0.5ug/ml) are shown in Fig.1. The cDNA amplified by PCR using VASA,

STELLA and PUM1 gene specific primers yielded product of 891 bp, 365 bp and 822 bp

respectively, visible in lane 1, 2 and 3 (Fig. 1).

Fig1. Amplification of germ cell specific genes: L: Ladder (100 bp); 1: Vasa (891 bp); 2:

Stella (365 bp); 3: PUM1 (822bp).

Immunostaining of differentiated goat ES cells

The result obtained by immunostaining of differentiated goat embryonic stem cell to

putative germ cells with VASA primary antibody was shown positive result in figure 2 and

similarly negative control without VASA primary antibody was shown in figure 3.

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Western blotting of directed differentiating embryonic stem cells:

Vasa, Stella and PUM1 proteins were identified in Western blotting as these proteins

expressed in the differentiated germ cells.

Conclusion:

Vasa, Stella and PUM1 germ cell markers genes were expressed in directed

differentiating ES cells and product size in RT-PCR were 891bp, 365bp and 822bp

respectively. The proteins of these genes have already expressed in immunohistochemistry

and western blotting in the differentiated embryonic stem cells and iPS cells in the present

study. The present study for characterization of germ cells from IVF, parthenogenetic ES

cells were obtained optimistic result. Within one month we will able to complete our rest of

the research work. Germ cells derived from embryonic stem cell and iPS cells have vast

social implications and have the ability to transform the existing assisted reproductive

technologies into a new horizon. Continuous production of germ cell and functional gametes

from embryonic stem cells and iPS cells for practical application is not too distant future.

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