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1. Introduction
2. Materials and methods
3. Results
4. Discussion
5. Conclusions
References
Theriogenology
Figures and tables Volume 77, Issue 5, 15 March 2012, Pages 829839

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Cardiomyocytes rhythmically beating generated from goat

embryonic stem cell
S. Garga, 1, R. Duttaa, 1, D. Malakara, ,
, M.K. Jenaa, D. Kumara, S. Sahua, B. Prakashb
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https://doi.org/10.1016/j.theriogenology.2011.05.029
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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 ActivinA, 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.

Keywords
Blastocyst; Cardiomyocyte; Embryonic stem cell; Goat; Rhythmic beating
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2. Materials and methods
3. Results Corresponding author. Tel.: 91-9416741839
4. Discussion 1 The first two authors have contributed equally to the work.
5. Conclusions
References Copyright 2012 Elsevier Inc. All rights reserved.

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Article outline Show full outline

Abstract
Keywords
1. Introduction
2. Materials and methods
3. Results
4. Discussion
5. Conclusions
References

Figures and tables

Table1
Table-2
Table-3