Int. J. Dev. Biol. 61: 81 - 88 (2017)
doi: 10.1387/ijdb.160006sb
© UPV/EHU Press

Valproic acid assisted reprogramming of fibroblasts for generation of pluripotent stem cells in buffalo (Bubalus bubalis)

Puspendra S. Mahapatra, Renu Singh, Kuldeep Kumar, Nihar R. Sahoo, Pranjali Agarwal, Bhabesh Mili, Kinsuk Das, Mihir Sarkar, Subrat K. Bhanja, Bikash C. Das, Sujoy K. Dhara and Sadhan Bag*

Division of Physiology & Climatology. Indian Veterinary Research Institute, Bareilly, India

ABSTRACT Generation of pluripotent stem cells by reprogramming somatic cells of quality animals has numerous potential applications in agricultural and biomedical sciences. Unfortunately, till now, reprogramming of buffalo fetal fibroblast cells (bFFs) has been very ineffient despite intensive efforts. Here, we attempted to enhance reprogramming efficiency by using the HDAC inhibitor valproic acid (VPA) in bFFs transfected with pLentG-KOSM pseudo virus carrying mouse specific pluripotent genes. FACS analysis revealed that VPA treatment significantly increased (p < 0.05) GFP+ cells in comparison to VPA untreated control. Further, among different concentrations, 1.5 mM VPA was found to be optimal, increasing about 5 fold GFP+ cells and 2.5-fold GFP+ colonies with significantly (P < 0.05) larger size as compared to control. These colonies were further propagated and characterised. The colonies displayed embryonic stem cell (ESC)-like morphology, normal karyotype, and were positive for alkaline phosphatase staining as well as immune-positive for the ESC specific markers Oct4, Nanog, SSEA1, TRA-1-60 and TRA-1-81. The primary colonies revealed significantly higher (P < 0.05) expression of pluripotent genes than control, which declined gradually on subsequent passages. The reprogrammed cells readily formed embryoid bodies in vitro and cells of all three germ layers. These results indicated that VPA treatment of viral transducted cells can improve the generation of induced pluripotent stem cells and help their long term maintenance in buffalo.

Keywords:

valproic acid, buffalo fetal fibroblast, reprogramming, mouse defined factor

*Corresponding author e-mail: bag658@gmail.com