Cardiomyocytes rhythmically beating generated from goat embryonic stem cell S. Garg a,1 , R. Dutta a,1 , D. Malakar a, *, M.K. Jena a , D. Kumar a , S. Sahu a , B. Prakash b a Animal Biotechnology Center, National Dairy Research Institute, Karnal-132001, India b 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 goat embryonic stem cells produced from in vitro fertilized goat embryos. Goat blastocysts were produced in vitro by standard methods of in vitro maturation (IVM), in vitro fertilization (IVF) and in vitro culture (IVC) techniques. The ICMs isolated from IVF blastocysts were cultured on 10 l/ml mitomycin-C inactivated fetal fibroblast feeder layer with LIF. The putative ES colonies were characterized for extracellular markers like alkaline phosphatase, TRA-1-60, TRA-1-81, SSEA-1, SSEA-4 by immunocytochemistry and intracellular markers like Oct4, Sox2 and Nanog with reverse-transcription-PCR. The ES cells were successfully subcultured up to 22nd passage with feeder layer and LIF and up to 12th passage without feeder layer with LIF only. They exhibited normal karyotyping (20th passage) and maintained the expression of specific surface markers like alkaline 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. The embryoid bodies were analyzed with markers like Gata4, BMP4 and Nestin. The rhythmic beating of cardiomyocytes was observed after 30 d and the beating was still continuing even after 160 d of culturing. Similarly, 2nd and 3rd batches of EBs were also beating and the beating continues after 75 d and on. The beating cells were observed positive for cardiac specific markers like  Actinin, C-Troponin and -Myosin heavy chain. Histological studies also revealed morphology similar to cardiomyocytes. Prominent contractions typical of cardiac tissue have been maintained in the differentiated cells up to 160 d and still continuing beating at the rate of 30 beats/min. It could be concluded that ES cells generated from goat embryos were maintained undifferentiated up to 22nd passage on feeder layer and to 12th passage without feed layer using LIF and that the 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 cells derived from the inner cell mass of blastocyst stage embryos [1]. These have the capacity to self-renew as well as the ability to generate differentiated cells. They are unique in their capacity to self-renew indefinitely in culture, 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 Theriogenology 77 (2012) 829 – 839 www.theriojournal.com 0093-691X/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2011.05.029