Maintenance of horse embryonic stem cells in different conditions

Document Type: Full paper (Original article)

Authors

1 Department of Basic Sciences, Faculty of VeterinaryMedicine, and Embryonic and Stem Cell Biology and Biotechnology Research Group, Instituteof Biotechnology, Ferdowsi University of Mashhad, Mashhad, Iran

2 Goulburn Valley Equine Hospital, Shepparton, Victoria 3630, Australia

3 Monash Immunology and Stem Cell Laboratories (MISCL), Monash University, Wellington Rd, Clayton, Victoria 3800, Australia

Abstract

Embryonic stem cells (ESCs) are originally derived from the ICM of blastocysts and are characterized by
their ability to self-renew and their pluripotencies. Only a few reports have been published on ESC isolations
and line establishment in animals, even fewer in horses. However, it is still important to isolate equine ESCs
for animal biotechnology and therapeutic applications. In the present study, we tried to derive horse ESC
lines from the ICM of blastocysts fertilized in vivoand maintain their pluripotencies in different conditions.
The primary horse ESCs were able to self-renew when they were cultured in basic medium on γ-irradiated
MEFs. After 15 passages, immunohistochemistry of the putative horse ESCs showed that some cells in the
colonies were positive for Oct-4, SSEA-1, GCTM-2, TRA-1-60 and TRA-1-81. Moreover, to optimize the
culture conditions, these putative horse ESCs were cultured in basic medium supplemented with human
leukemia inhibitory factor (hLIF) only, human basic fibroblastic growth factor (hbFGF) only, or hbFGF plus
hLIF with or without heterologous (MEF) feeder cells. Based on our results, the heterologous feeder (MEF)
cells are necessary to maintain the undifferentiated state for horse ESCs, and ESC-like cell morphology of
horse ESCs were well maintained in the basic medium supplemented with or without hLIF. This result
suggested that hLIF was neither prerequisite nor negative for maintenance of horse ESCs; bFGF seemed to
be negative for maintenance of horse ECSs and the combination of hLIF and bFGF was unable to improve
the culture condition.

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