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.

Trichostatin A-Assisted Epigenomic Modulation Affects the Expression Profiles of Not Only Recombinant Human α1,2-Fucosyltransferase and α-Galactosidase A Enzymes But Also Galα1→3Gal Epitopes in Porcine Bi-Transgenic Adult Cutaneous Fibroblast Cells

“…Analogous methods of HDACiand/or DNMTi-dependent epigenetic transformation have been adapted to enhance capabilities of parental genomes to be epigenetically reprogrammed in the in vitro fertilization (IVF)-derived embryos [17][18][19][20]. Such approaches have also been used either to expedite the epigenomic reprogramming and molecular dedifferentiation of adult somatic cells or mesenchymal stem cells (MSCs) into induced pluripotent stem cells (iPSCs) [21][22][23][24][25] or to facilitate the molecular rejuvenation of adult MSCs [21,26,27]. Moreover, the efforts that are aimed at the epigenomic modulation dependent on HDACi and/or DNMTi approved by the U.S. Food and Drug Administration (FDA) or by European Medicines Agency (EMA) have been undertaken to accomplish clinical treatments (i.e., epigenetic therapies) in oncologic patients and medical patients afflicted by neurodegenerative diseases or psychiatric disorders [28][29][30][31].…”

Evaluation of Cytocompatibility as Assessed by Genomic Stability of Canine induced Pluripotent Stem Cells Propagated on Carbon Nanotube Substrates

“…After seeding, the colonies were attached and spread eventually in control and CNT scaffolds with compact roundness like morphology of pluripotent stem cells (Fig 1C). The compact round morphology of typical iPSC colonies were maintained on all the surfaces even after 72 hours of incubation (Fig 2D ) The pluripotency of iPSC is defined in terms that whether the cells have the capability to differentiate into three germ layers in vitro or in vivo (Mahapatra et al, 2016). ciPSC are unique in their ability to express pluripotency markers and higher ALP activity (Baird et al, 2015).…”

Epigenetic Features of Animal Biotechnologies

“…It was shown that even at low doses, the histone deacetylase inhibitor TSA was able to stabilize expression of pluripotent genes. Similarly, VPA was found to stabilize histone acetylation and the expression of pluripotent gene in bovine iPS cells (Mahapatra et al 2015). It was also shown that the use of such chemicals to increase reprogramming efficiency could also replace one or more of the key factors found to induce reprogramming (Ma et al 2017).…”