ROCK inhibitor Y-27632 enhances the survivability of dissociated buffalo (Bubalus bubalis) embryonic stem cell-like cells

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Contents Vol 25(2)

doi:10.1071/RD11315

Vertebrate Reproductive Science & Technology

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ROCK inhibitor Y-27632 enhances the survivability of dissociated buffalo (Bubalus bubalis) embryonic stem cell-like cells

Ruchi Sharma ^A, Aman George ^A, Manmohan S. Chauhan ^A, Suresh Singla ^A, Radhey S. Manik ^A and Prabhat Palta ^A ^B

^A Embryo Biotechnology Lab, Animal Biotechnology Centre, National Dairy Research Institute, Karnal132001, Haryana, India.
^B Corresponding author. Email: prabhatpalta@yahoo.com

Reproduction, Fertility and Development 25(2) 446-455 http://dx.doi.org/10.1071/RD11315
Submitted: 19 December 2011 Accepted: 5 April 2012 Published: 3 May 2012





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Abstract

This study investigated the effects of supplementation of culture medium with 10 μM Y-27632, a specific inhibitor of Rho kinase activity, for 6 days on self-renewal of buffalo embryonic stem (ES) cell-like cells at Passage 50–80. Y-27632 increased mean colony area (P < 0.05) although it did not improve their survival. It decreased OCT4 expression (P < 0.05), increased NANOG expression (P < 0.05), but had no effect on SOX2 expression. It also increased expression of anti-apoptotic gene BCL-2 (P < 0.05) and decreased that of pro-apoptotic genes BAX and BID (P < 0.05). It increased plating efficiency of single-cell suspensions of ES cells (P < 0.05). Following vitrification, the presence of Y-27632 in the vitrification solution or thawing medium or both did not improve ES cell colony survival. However, following seeding of clumps of ES cells transfected with pAcGFP1N1 carrying green fluorescent protein (GFP), Y-27632 increased colony formation rate (P < 0.01). ES cell colonies that formed in all Y-27632-supplemented groups were confirmed for expression of pluripotency markers alkaline phosphatase, SSEA-4 and TRA-1–60, and for their ability to generate embryoid bodies containing cells that expressed markers of ectoderm, mesoderm and endoderm. In conclusion, Y-27632 improves survival of buffalo ES cells under unfavourable conditions such as enzymatic dissociation to single cells or antibiotic-assisted selection after transfection, without compromising their pluripotency.

Additional keywords: apoptosis, cryopreservation, pluripotency, transfection.

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