Cardiomyocytes rhythmically beating generated from goatembryonic stem cell

S. Garga,1, R. Duttaa,1, D. Malakara,*, M.K. Jenaa, D. Kumara, S. Sahua, B. Prakashb

a Animal Biotechnology Center, National Dairy Research Institute, Karnal-132001, Indiab National Bureau of Animal Genetic Resources P.B. 129, GT Road By-Pass Karnal-132001, India

Received 30 January 2011; received in revised form 10 May 2011; accepted 13 May 2011

Abstract

The aim of present investigation was isolation, characterization and differentiation into cardiomyocytes of putative goatembryonic stem cells produced from in vitro fertilized goat embryos. Goat blastocysts were produced in vitro by standardmethods of in vitro maturation (IVM), in vitro fertilization (IVF) and in vitro culture (IVC) techniques. The ICMs isolatedfrom IVF blastocysts were cultured on 10 !l/ml mitomycin-C inactivated fetal fibroblast feeder layer with LIF. The putativeES colonies were characterized for extracellular markers like alkaline phosphatase, TRA-1-60, TRA-1-81, SSEA-1, SSEA-4by immunocytochemistry and intracellular markers like Oct4, Sox2 and Nanog with reverse-transcription-PCR. The ES cellswere successfully subcultured up to 22nd passage with feeder layer and LIF and up to 12th passage without feeder layer withLIF only. They exhibited normal karyotyping (20th passage) and maintained the expression of specific surface markers likealkaline phosphatase, SSEA-4, TRA-1-61, TRA-1-81 and intracellular markers Oct4, Sox2 and Nanog. The embryoid bodies(EBs) were generated from goat ES cells of 20th passage and were analyzed with markers like Gata4, BMP4 and Nestin.Differentiation was induced by medium containing 100 ng/ml Activin–A, 10 ng/ml FGF-2 and 100 ng/ml BMP-4. Theembryoid bodies were analyzed with markers like Gata4, BMP4 and Nestin. The rhythmic beating of cardiomyocytes wasobserved after 30 d and the beating was still continuing even after 160 d of culturing. Similarly, 2nd and 3rd batches of EBswere also beating and the beating continues after 75 d and on. The beating cells were observed positive for cardiac specificmarkers like " Actinin, C-Troponin and "-Myosin heavy chain. Histological studies also revealed morphology similar tocardiomyocytes. Prominent contractions typical of cardiac tissue have been maintained in the differentiated cells up to 160 dand still continuing beating at the rate of 30 beats/min. It could be concluded that ES cells generated from goat embryos weremaintained undifferentiated up to 22nd passage on feeder layer and to 12th passage without feed layer using LIF and thatthe differentiation protocol induced rhythmic beating cells.© 2012 Elsevier Inc. All rights reserved.

Keywords: Blastocyst; Cardiomyocyte; Embryonic stem cell; Goat; Rhythmic beating

1. Introduction

Embryonic stem (ES) cells are undifferentiated cellsderived from the inner cell mass of blastocyst stageembryos [1]. These have the capacity to self-renew aswell as the ability to generate differentiated cells. Theyare unique in their capacity to self-renew indefinitely inculture, maintaining a normal karyotyping, and remain-

1 The first two authors have contributed equally to the work.

* Corresponding author. Tel.: 91-9416741839.E-mail address: dhrubamalakar@gmail.com (D. Malakar).

Available online at www.sciencedirect.com

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0093-691X/$ – see front matter © 2012 Elsevier Inc. All rights reserved.doi:10.1016/j.theriogenology.2011.05.029

ing pluripotent, harboring the capacity to differentiateinto multiple cell types of the three germ layers. Theirdifferentiation potential has been demonstrated in vitrothrough the creation of embryonic bodies (EBs) [2,3],which are cell aggregates comprised of multiple celltypes.

Currently, differentiation of stem cells to particular celllineages is an area of intensive study. Most of the methodswhich are utilized for differentiation studies involve sev-eral steps: Production of EBs from suspension culture,exposure of EBs to agents designed to induce differenti-ation of a specific lineage, growth of EBs on tissue cultureplates coated with biological molecules such as gelatin orlaminin [4,5]. When the precise growth factor combina-tions that promote cell-type-specific differentiation are notknown, ES cells can be co-cultured with target tissues topromote differentiation [6].

Differentiation of ES cells can occur in two ways, byspontaneous or by directed differentiation [7,8]. WhenEBs are cultured in suspension without anti-differentiationagents, they spontaneously differentiate into multicel-lular lineage. Within 2–4 d of culture, visceral endo-derm [9] are formed from the ICM, thereby giving riseto simple EB [10]. Approximately on day 4, the differ-entiation of columnar epithelium with a basal laminaand formation of a central cavity occurs. At this stage,the EBs are called cystic embryoid bodies [11] and bearsimilarity to the egg-cylinder stage of mouse embryos[12]. Because their size, differentiation capacity, andgene expression profile resemble the early post implan-tation embryo, they are often employed as models ofdifferentiation and gene expression in early develop-ment. RNA in situ hybridization analyses have demon-strated that derivatives of all germ layers differentiateto EBs in some ways, recapitulating in vivo gene ex-pression patterns [10]. In directed differentiation, theES cells can be differentiated to form a specific type ofcell in response to a specific signal. The chemicalsignals used for inducing directed differentiation inmice include retinoic acid for producing neurons [13],BMP2 and FGF2 [14], nitric oxide [15], ascorbic acid[16] for producing cardiomyocyte, BMP4 ! bFGF forproducing trophectoderm cells, ascorbic acid for pro-ducing osteocytes [17], TGF-#3/ parathyroid hormonefor producing chondrocytes [18].

In contrast to mouse, a limited number of cells havebeen derived from human ES cells e.g., neuron-likecells by exposure to FGF [19] or trans-retinoic acid[20], cardiomyocytes by exposure to nitric oxide [15]or oxytocin [21], hematopoietic cells by exposure tocytokines and BMP-4 [22], and insulin-producing cells

by exposure to nicotinamide [23]. Synthesis of hemo-globin has been induced in bovine pluripotent stemcells by exposure to DMSO [24]. Skeletal muscle cellshave been formed by stimulating the ES cells withDMSO and retinoic acid [25].

At present, there is a vast need of generating ES cellsin domestic animals because they are immunologicallyand physiologically more similar to human than rodent.For these reasons they are a better model for studyinghuman pathology. Goat is an important livestock spe-cies contributing to milk, meat and wool production. EScells have 10–20 fold higher efficiency than somaticcell nucleus donor in somatic cell cloning than cumu-lous cells [26,27]. The present study was carried out toisolate and characterize embryonic stem cell-like cellsfrom in vitro produced goat embryos and to directlydifferentiate ES cells to cardiomyocytes.

2. Materials and methods

2.1. Chemicals

Except where otherwise stated, all chemicals andcell culture media were purchased from Sigma Chem-icals Co. (St. Louis, MO) and were of cell culture/embryo tested. The same batch of fetal calf serum(FCS, 054K8416) was used throughout the study. Theplastic wares used were purchased from Nunc (Nunc,Denmark) and filters were from Millipore (MilliporeIndia Pvt. Ltd. India).

2.2. In vitro production of goat embryos

Morulae, blastocysts and hatched blastocysts were pro-duced following in vitro maturation, fertilization, cultureand vitrification procedures, as described by Pawar et al,2009 [27]. Briefly, oocytes collected from slaughterhousewere matured in TCM 199 (HEPES modified), containing10 !g/ml luteinizing hormone (LH), 5 !g/ml follicle-stimulating hormone (FSH), 1 !g/ml oestradiol-17#, 50!g/ml sodium pyruvate, 3.5 !g/ml L-glutamine, 50 !g/mlgentamicin, 5.5 mg/ml glucose, 3 mg/ml Bovine SerumAlbumin (BSA) and 10% Fetal Calf Serum (FCS) [28].After in vitro fertilization with fresh semen, the morulae,blastocyst and hatched blastocysts were cultured with amedium containing TCM 199 (HEPES modification),30 !g/ml sodium pyruvate, 100 !g/ml L-glutamine, 50!g/ml gentamicin, 10 !l/ml essential amino acids, 5!l/ml non-essential amino acids (NEAA), 10 mg/ml BSA(Fraction-V), 10% FCS and 50 mM cysteamine for 5, 7and 8 days respectively.

830 S. Garg et al. / Theriogenology 77 (2012) 829–839

2.3. Preparation of feeder layer

Goat fetal fibroblast was used as feeder layer. Goatfetuses of 60 days old obtained from local slaughterhousewere dissected out from uteri and washed 4 to 5 times insterile Dulbecco’s phosphate buffer saline (DPBS). Skinexplants were taken and washed 8 to 9 times with sterileDPBS. Then the tissue pieces were cultured in separatefour well tissue culture dishes in Dulbecco’s modifiedeagles medium (DMEM) supplemented with 10% FCSand 50 !g/ml gentamicin in 5% CO2 in air at 37 °C. Afterproliferation and establishment of fibroblasts the explantswere removed. Monolayer fibroblasts were allowed togrow till confluence. For preparation of feeder layer, themonolayer was inactivated by treatment with 10 !g/mlmitomycin-C for 3 h and washed 5 times in DPBS.

2.4. Derivation of stem cells

Blastocysts and hatched blastocysts were used forisolation and culture of putative goat embryonic stemcells. The inner cell masses (ICMs) were isolatedfrom expanded and hatched blastocysts using me-chanical isolation. The zona pellucida of blastocystswas removed by treatment of 1% pronase in DPBS(w/v) until complete dissolving of zona pellucida.After the disappearance of zona pellucida, the blas-tocysts were immediately washed in DPBS to inhibitfurther action of pronase. The ICM cells were dis-sected out with the help of two sharp pipettes underzoom stereomicroscope (Olympus SZ 61, Japan).

In case of hatched blastocysts, the pronase treatmentwas not required and the ICM cells were easily isolatedmechanically as they were clearly visible under zoomstereomicroscope. The isolated ICMs were seeded on 10!g/ml mitomycin-C inactivated feeder layers in ES me-dium containing DMEM supplemented with 20% FCS,1000 IU/ml of mLIF 1% nonessential amino acids, 0.1mM #-mercaptoethanol, and 2 mM L-glutamine. Themedium was changed every 48 h interval and the forma-

tion of colony was observed routinely under invertedmicroscope (Nikon, Japan).

2.5. Subculture of putative embryonic stem cells

The primary colonies were obtained 4 to 5 daysafter seeding of ICMs. The colonies exhibiting typ-ical morphological features of ES cell-like cells weretaken for sub-culturing. The colonies were disaggre-gated mechanically under the zoom stereomicro-scope. Aggregates of 50 to 100 cells were individu-ally reseeded onto a new feeder layer in 4-well cellculture plates containing 0.5 ml ES cell culture me-dium containing DMEM, 5 !g/ml L-glutamine, 50!g/ml gentamycin, 0.007 !l/ml #-Mercaptoethanol,1 !l/ml NEAA, 20% FCS and 1000 IU/ml LIF.Similarly Es cells were subcultured in ES mediumwithout feeder layer using only LIF.

2.6. Characterization of stem cells

The characterization of the putative stem cells wascarried out at different passages by the followingmethods.

2.7. Alkaline phosphatase staining

For alkaline phosphatase staining, the medium wasremoved from the goat ES cell cultures and then washedtwice with DPBS. The cells were fixed in citrate-acetone-formaldehyde fixative solution for 1 min. After fixation,the cells were washed 3 times with DPBS for 1 min andincubated for 15 min at room temperature with the alka-line dye. The cells were rinsed again 2–3 times withDPBS and counterstained with neutral red stain for 1–2min. Finally, colonies were washed 5 times to remove theextra neutral red stain and the response of the cells toalkaline phosphatase staining was observed under a mi-croscope (Olympus). The putative goat ES-like cells werestained red in color.

Table1Primer sequences of pluripotency markers.

Gene product Primer sequence (5=-3=) Annealing Temp.(0C)/Cycle no.

Product size (bp) Accession no.

Beta actin 5=-CTCTTCCAGCCTTCCTTCCT-3= 58/35 88 NM_007393.35=-GGGCAGTGATCTCTTTCTGC-3=

Nanog 5=GCAGAAATACCTCAGTCTCCAGCA-3= 58/35 289 AY7864375=-ACTGTTCCAGCTCTGATTGCTCCA-3=

Sox2 5=-ACAGCATGTCCTATTCTCAGCAGGG-3= 60/35 231 NM_0011054635=-TCTGGTAGTGCTGGGACATGTGAA-3=

Oct-4 5=-AGTCCCAGGACATCAAAGCTCTTC-3= 58/35 156 NM_1745805=-AACGGCAGATAGTCGTTTGGCTGA-3=

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2.8. Immunofluorescence staining of putative goat EScells

The expression of intracellular marker Oct4 andsurface markers SSEA-1, SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81 were examined by immunofluo-rescence staining of colonies of putative goat EScells. To rule out the possibility that the immunoflu-orescence stains have not worked, pretested buffaloembryonic stem cells were used as control [29]. Theputative ES cell colonies were fixed in 4% Parafor-maldehyde in DPBS for 30 min, washed 3 times withDPBS and then permeabilized by treatment with0.1% Triton X-100 in DPBS for 30 min. After thor-ough washing with DPBS, putative goat ES cellcolonies were incubated with the blocking solution(4% normal goat serum) for 30 min, and then withthe primary antibody (Millipore, USA) at a dilutionof 1:10 to 1:20 for 1 h. After washing 3 times withDPBS, putative ES cell colonies were incubated with theappropriate FITC-labeled secondary antibody goat anti-mouse IgG or IgM, diluted 1:100 to 1:200 for 2 h. Theputative goat ES cell colonies were then examined undera fluorescence microscope (Nikon, Tokyo, Japan).

2.9. Reverse transcription and polymerase chainreaction (RT-PCR)

Total RNA was isolated from putative goat EScell colonies, embryoid bodies and differentiated em-

bryoid bodies using the “Cells-to-cDNA kit-II” (Am-bion, Austin, TX, USA) as per manufacturer’s in-structions. Briefly, the cells were washed with icecold PBS after which 20 –50 !l of cold cell lysisbuffer was added and the mixture was incubated at75 °C for 10 min in a thermal cycler (BIO-RAD,USA). The genomic DNA was degraded by incubat-ing the cell lysates in DNase-I at 37 °C for 30 minand the remaining activity of DNase-I was inacti-vated by heating at 75 °C for 5 min. cDNA wasprepared by taking 10 !l of the cell lysates usingrandom primers. The PCR cycle included denatur-ation at 94 °C for 2 min followed by repeated cyclesof denaturation at 94 °C for 30 sec, annealing at thetemperature as indicated in primer table (Tables 1, 2and 3) and for 30 sec, and extension at 72 °C for 45sec followed by a final extension at 72 °C for 10 minfor 35 cycles. A negative RT control reaction (i.e.,RT reaction but without MMLV enzyme) and a neg-ative PCR without template was also set up. # actinwas amplified as the housekeeping marker gene. ThePCR products were analyzed in 1% agarose gel elec-trophoresis.

2.10. Chromosomal integrity of ES cell-like cells

Karyotyping analysis was performed on ES cell-like cells at 5th, 10th and 20th passages. Briefly, thecell colonies were incubated in DMEM supple-

Table-2Primer sequences for embryoid body characterization.

Gene product Primers(5=-3=) Annealing Temp.(0C)/Cycle no.

Product size (bp) Accession no.

NESTIN 5=-AACAGCGACGGAGGTCTCTA -3= 60/35 267 NM_0066175=-GAGTCCACAGTGGTGCTTGA -3=

BMP-4 5=-CCACTCGCTCTACGTGGACT-3= 60/35 198 NM_0011102775=-GTGGGAACACAACAGGCTTT -3=

GATA-4 5=-CTCCTACTCCAGCCCCTACC-3= 60/35 285 NM_0011928775=-TTGATGCCGTTCATCTTGTG-3=

Table-3Primer sequences for cardiomyocytes characterization.

Gene product Primer(5=-3=) Annealing Temp.(0C)/Cycles No.

Product size(bp) Accession no.

"-actinin 5=-GGCCAGTGTTACCAGATGCT -3= 60/35 268 XM_001927272.25=-TTCAGCAGCAGGACCTTTCT -3=

Cardiac C-troponin 5=-CTTCAGAGCCTGCCTCATCT -3 60/35 212 NM_0003635=-CTGCCAGGATGTAGGGCTTA -3=

"-Myosin heavy chain 5=-GAACCTCACTCTTGCCAACC-3= 60/35 152 Z206565=-GGCTCTGGCAGCTCTGATAC-3=

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mented with 0.1 mg/ml colcemid for 4 h at 38.5 °C.The cells were then washed, trypsinized and resus-pended in hypotonic solution (75 mM KCl) for 30min at 38.5 °C. The cells were washed and then fixedin chilled fixative (3:1 methanol/glacial acetic acid)for 30 min at room temperature and centrifuged at200 " g for 8 min. The pellets were resuspended in 5 mlof ice-chilled fixative for another 10 min and then centri-fuged again. The metaphase spreads were prepared bydropping the cells onto ice cold glass slides. Chromo-somes were stained with 2% Giemsa for 6 min and ob-served under oil immersion 1000" using a compoundmicroscope (Nikon, Microphot-FXA, Japan).

2.11. Embryoid bodies formation and spontaneousdifferentiation

When goat ES cells are cultured in suspensionwithout the support of LIF and embryonic fibroblastfeeder layer, these form 3-dimensional aggregatescalled ‘embryoid bodies’ (EBs). For the preparationof EBs, gES cell colonies were removed and were

disaggregated into individual cell using 0.25% tryp-sin–EDTA for 5– 6 min at 38 °C. The cells werecultured for two days in hanging drops (20 !l of EScell culture medium without LIF) in which the cellswere dispersed and suspended from the lid of a Petridish. Third day onwards, the EBs were transferred tobacteriological dishes for further culture. These EBswere placed on tissue culture dishes for adhesion and growth.The cells were fed every alternate day by tilting the plate,allowing them to settle, and carefully replacing the medium.

2.12. Directed differentiation of goat ES cells

The EBs produced by the hanging drop methodwere placed in suspension for 3 additional days andwere then transferred to non coated 24 well plateculture dish and bacteriological dishes containingstimulating agent. After 2 days, the EBs had adheredand had started to grow. The stimulating agents ex-amined in the present study included 100 ng/ml Ac-tivin A and 100 ng/ml BMP4 for directing the dif-ferentiation to cardiac muscle cell-like cells.

Fig. 1. (A) Hatched goat blastocyst. (B) Isolated Inner cell mass from blastocyst for culture of ES cells. (C) Proliferation of ICM on feeder layerafter 10 days. (D) Subculture of ES cells at 20th passage for production of embryoid bodies.

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2.13. Histology, morphology and contractile natureof differentiated cells.

The differentiated cells were preserved in 10%buffered neutral formalin. Cardiac muscle cell-likecells were dehydrated and cleared in different gradesof acetone and benzene thereafter, impregnated inparaffin and embedded in paraffin wax. Sectionswere cut to 5 micron with the help of microtone andwere stained with Hematoxylin and Eosin as routineprocedure recommended by Luna (1968) [30]. Thebasic morphology and contractile nature of differen-tiating cells were checked in routine period.

3. Results

3.1. Production of IVF embryos and goat embryonicstem cells

In the present study, a total 60.48% cleavage,24.01% morulae, 11.35% blastocyst and 3.4%hatched blastocyst were obtained (Fig.1A). ICMs

(Fig.1B) were isolated from 80 blastocysts and 20hatched blastocysts and primary colonies were ob-tained at a rate of 66% from blastocysts and 90%from hatched blastocysts respectively. Morphologi-cally the putative stem cell primary colonies weredome shaped. After 7– 8 d culture compact cell col-onies were formed. Confluent monolayer of putativestem cells was formed after 8 –9 d of culturing (Fig1C). The ICMs were cultured on fetal fibroblastfeeder layers. Three ES cell cultures were subcul-tured upto passages 22nd and 2 were subculturedupto 20th passage on fetal fibroblast feeder layers(Fig.1D). Interestingly, 3 of the obtained ES cellcultures were maintained upto 12th passages withoutfeeder layer using LIF only.

3.2. Characterization of putative goat ES cells byimmunostaining

3.2.1. Surface markers: alkaline phosphataseAlkaline phosphatase expression test was employed

as a preliminary examination for state of pluripotency

Fig. 2. Characterization of putative goat ES cells. (A) Positive expression of alkaline phosphatase in ES cell colonies. (B) Positive expressionof alkaline phosphatase in ES cell colonies without feeder layer. (C) Bright field image of ES cells. (D) Negative expression of SSEA-1on ES cells. (E) Bright field image of ES cells (F) Negative expression of SSEA-3 on ES cells. (G) Bright field image of ES cells. (H) Positiveexpression of SSEA-4 on ES cells. (I) Bright field image of ES cells. (J) Positive expression of TRA-1-60 on ES cells. (K) Bright field image of ES cells.(L) Positive expression of TRA-1-81 on ES cells. (M) Bright field image of ES cells (N) Positive expression of Oct4 on ES cells.

834 S. Garg et al. / Theriogenology 77 (2012) 829–839

of ES cells. The putative goat ES cells produced in thepresent study were subjected to alkaline phosphatasestaining at every 5th passage. The ES cells colonies(35) were found strongly positive for alkaline phospha-tase stain (Fig. 2A, B). In contrast, the fibroblast feederlayer cells using LIF, which were used as a control,remained unstained (Fig. 2A).

3.2.2. Stage specific embryonic antigens (SSEA) andTumor rejection antigens (TRA)

The stage specific embryonic antigens (SSEAs)include a number of markers like SSEA-1, SSEA-3and SSEA-4 (Fig. 2C, D, E, F, G, H). The putativegoat ES cells were observed to be positive forSSEA-4 expression (Fig 2H) at different passageswhereas SSEA-1 and SSEA-3 expression were notobserved (Fig. 2C, D, E, F). Moreover, both tumorrejection antigen TRA-1-60 and TRA-1-81 werefound to be expressed strongly at different passagesof goat ES cell culture (Fig. 2I, J, K, L).

3.2.3. Intracellular marker Oct4Oct4 was strongly detected by immunofluorescence

staining in putative goat ES cells (Fig. 2M, N).

3.3. Characterization of goat ES cells by RT-PCRand karyotyping of ES cells

The colonies which were positive for alkaline phos-phatase were selected for Oct4, Sox2 and Nanog ex-pression study. The putative ES cells were found toexpress Oct4, Sox2 and Nanog by RT-PCR (Fig. 3A).Karyotyping of the goat ES cells revealed normal 60chromosomal organizations in the ES cells at 10th and20th passage during culture (Fig. 3B).

3.4. Formation of embryoid bodies and spontaneousdifferentiation of embryoid bodies

The embryoid bodies were harvested from thehanging drops on day 3 of suspension culture, someof them were observed to be in the form of compactmass (Fig. 4A). The embryoid bodies were culturedfor 10 d and turned to cystic type spontaneously (Fig.4B). When embryoid bodies were cultured in suspen-sion without anti-differentiation agents i.e., LIF andfeeder layers, they spontaneously differentiated intocells of different lineages including neuron-like andepithelial-like cells (Fig. 4C, D).

3.5. Directed differentiation to cardiomyocytes

Following induced differentiation with BMP4 andActivin-A the outgrowths at the periphery of the

embryoid bodies (Fig. 5) increased in size and as-sumed muscle cell-like morphology in culture.

3.6. Characterization of putative goat ES cells byimmunostaining

The identity of these muscle cell-like cells wasexamined by immunofluorescence staining for "-actinin and cardiac C-troponin (Fig. 6A, B) 15 d postculture. The earliest expressed cardiac muscle genewas " actinin. This gene was expressed since 10 dafter induction of differentiation. The muscle specificgene C-troponin and myosin heavy chain were found to beexpressed in differentiated cardiac muscles (Fig. 6C). His-tological section of the differentiated mass showed inter-calated disc indicating that the differentiated cells areindeed cardiomyocytes (Fig. 6D).

3.7. Rhythmic beating of cardiomyocytes generatedfrom goat ES cells

The embryoid bodies were analyzed with markerslike Gata-4, BMP4 and Nestin. The rhythmic beatingof cardiomyocytes was observed after 30 d of culturein differentiation medium containing BMP4 and Ac-tivin-A and the beating continued up to 160 d and

Fig. 3. (A) Expression of pluripotency markers in goat putative EScells (M)100bp ladder (1) #-actin 88bp. (2) Oct4 156bp (3) Sox2233bp (4) Nanog 287bp (5) -ve control (B) Analysis of chromosomeon 20th passage of goat ES cells was normal.

835S. Garg et al. / Theriogenology 77 (2012) 829–839

longer (Fig. 7A). Similarly, the 2nd and 3rd batchesof EBs were also beating and the beating continuedafter 45 days. The contractile nature of the beatingwas characteristic of cardiomyocytes. The beatingcardiac tissues were observed to be positive for car-diac-specific markers like " Actinin, C-Troponin and"-Myosin heavy chain (Fig. 7B).

4. Discussion

For the production of embryonic stem (ES) cells,inner cell mass (ICM) cells are obtained from blas-tocysts by different methods like immunosurgery,intact blastocyst culture, mechanical isolation or en-zymatic digestion [29]. In the present investigation,ICMs were mechanically isolated from in vitro fer-tilized blastocysts. These cells were grown and main-tained for long periods of time in culture throughself-renewal process. ES cells could be cultured in-definitely in their relatively undifferentiated, pluri-potent state, for which they must both express theintrinsic and extrinsic transcription factors.

Expression of alkaline phosphatase activity hasbeen reported in ES cells obtained from many spe-cies [28] and in goat [27]. In the present study all ES

cells prominently expressed alkaline phosphatase.The stage specific embryonic antigens (SSEAs) likeSSEA-1, SSEA-3 and SSEA-4 have been used as

Fig. 4. (A) Group Of Compact embryoid bodies (B) Development of cystic embryoid bodies after 15th day. (C) Epithelial like cells grow after20 days (D) Neuron like cells grow after 20 days of spontaneous differentiation.

Fig. 5. Proliferation of embryoid body in directed differentiation after10th day.

836 S. Garg et al. / Theriogenology 77 (2012) 829–839

surface or extracellular markers for monitoring thepluripotency of ES cells in many species [31]. Bo-vine ES cells strongly expressed of SSEA-3 andSSEA-4 [32]. Buffalo ES cells expressed SSEA-3only [33]. While goat embryos expressed SSEA-1and SSEA-4 proteins but not SSEA-3 [34]. However,

in the present study, goat ES cells strongly expressedonly SSEA-4 at different passages but not SSEA-1and SSEA-3. The expression of SSEA-4 was alsoconfirmed with previous studies in which this markerwas found to be expressed on goat ES cells. In thepresent investigation, TRA-1-60 and TRA-1-81 were

Fig. 7. (A) Rhythmic beating cells was observed after 30 days of induced differentiated embryoid bodies. (B) Expression of Cardiac Specific Markersin directed Differentiated Cells Lane M- Marker, Lane-1 RT Negative, Lane-# actin, Lane-3 Blank, Lane-C-Troponin & Lane-5 Heavy Chain " myosin.

Fig. 6. (A) Expression of "-Actinin of 20th day and Expression of "-Actinin after induction of differentiation on 30th day (B) Expression ofC-Troponin on induced differentiation cells on 20th day and expression of C-Troponin after induction of differentiation on 30th day. (C)Expression of Ectodermal, Mesodermall and Endodermal markers in embryoid bodies (1) Negative control (2) GATA- 4 285bp (3) BMP4 198bp(4) Nestinin 268bp (5) #-actin 88bp (M) 100bp ladder. (D) Histology of rhythmic beating cardiomyocyte like cells by Hematoxylin and Eosinafter induction of differentiation on 40th day and the stained tissues show intercalated discs.

837S. Garg et al. / Theriogenology 77 (2012) 829–839

expressed at different passages of goat ES cells. Theresults of the present study are in agreement withthose of previous studies in which the expression ofTRA-1-60 and TRA-1-81 has been reported in goatES cells [34].

Out of many intracellular transcription factors thatplay critical roles in maintaining stem cell self-re-newal, Oct4, Sox2, Nanog, are thought to be themost important. The expression of transcription-based markers Oct4 was detected by immunofluores-cence staining as well as RT-PCR. It was found thatthe goat ES cells expressed this marker strongly.These results are similar to those of earlier studies inwhich its expression was reported in ES cells in goat[27,35]. Expression of Sox2 marker was also de-tected by RT-PCR and it was found to be prominentin goat ES cell colonies. The expression of thistranscription factor was also detected in human [36],rhesus monkey [37], and porcine ES cells [38]. Ingoat, the expression of Nanog was detected at the 8-to 16-cell stage embryos and it reportedly increasesat the morula and blastocyst stages [34]. Prominentexpression of Nanog was also observed in the goatES cells in the current investigation. Similar expres-sion patterns of Nanog were found in mouse, humanand bovine oocytes and preimplantation embryos[39,40].

The ‘hanging drop’ method and ‘static suspensionculture’ are the two most common methods used forthe formation of embryoid bodies from ES cells [5].ES cells are subjected to culture in the hanging dropmethod which controlled the microenvironment moreprecisely. This method has, therefore, been used forthe formation of embryoid bodies across many spe-cies [41]. The same protocol resulted in efficientderivation of EBs in the present investigation. Posi-tive expression of Ectodermal, Mesodermal andEndodermal markers like GATA-4, BMP4 and Nes-tin were found in the obtained embryoid bodies.

Several earlier reports are available in mouse and hu-man being regarding directed differentiation of embryonicstem cells to cardiomyocytes [15,16,19,20]. BMP4 andActivin-A stimulants were chosen for induced differenti-ation of the ES cells to cardiomyocytes based on earlierreports [42–44]. In the present investigation, goat ES cellswere successfully differentiated to cardiomyocytes withBMP4 and Activin-A. Thus the current findings are inconjunction with previous reports of directed differentia-tion to cardiomyocytes as reported in different species[15,16,19,20].

5. Conclusions

In conclusion, goat ES cell-like cells could beisolated from in vitro produced embryos. The pluri-potency of goat ES cells was confirmed with theexpression of AP, Oct-4, SSEA-4, TRA-1-81, TRA-1-61, Sox2, Nanog expression, EBs formation and invitro differentiation. The goat ES cells were signif-icantly differentiated to rhythmically beating cardi-omyocytes. To our knowledge this is probably thefirst time rhythmic beating of cardiomyocytes wasobserved in the goat embryonic stem cells. Except inmouse, monkey and human being, this observationhas not previously been reported in other domesti-cated animals like cattle, buffaloes, sheep and pig.

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