title: Valproic acid assisted reprogramming of fibroblasts for generation of pluripotent stem cells in buffalo (Bubalus bubalis).
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.
authors.affiliation: Division of Physiology and Climatology. Indian Veterinary Research Institute, Bareilly, India.
authors.forename: Puspendra S
authors.initials: PS
authors.lastname: Mahapatra
authors.forename: Renu
authors.initials: R
authors.lastname: Singh
authors.forename: Kuldeep
authors.initials: K
authors.lastname: Kumar
authors.forename: Nihar R
authors.initials: NR
authors.lastname: Sahoo
authors.forename: Pranjali
authors.initials: P
authors.lastname: Agarwal
authors.forename: Bhabesh
authors.initials: B
authors.lastname: Mili
authors.forename: Kinsuk
authors.initials: K
authors.lastname: Das
authors.forename: Mihir
authors.initials: M
authors.lastname: Sarkar
authors.forename: Subrat K
authors.initials: SK
authors.lastname: Bhanja
authors.forename: Bikash C
authors.initials: BC
authors.lastname: Das
authors.forename: Sujoy K
authors.initials: SK
authors.lastname: Dhara
authors.forename: Sadhan
authors.initials: S
authors.lastname: Bag
mesh_terms: D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D002020:Buffaloes; D065150:Cellular Reprogramming / Q000187:drug effects*; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D057026:Induced Pluripotent Stem Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D014635:Valproic Acid / Q000494:pharmacology*
chemical_list: D014635:Valproic Acid; D000469:Alkaline Phosphatase