Induced pluripotent stem cell generation using mRNAs: the effect of valproic acid, 5-azacytidine and ascorbic acid

Abstract

In the bourgeoning fields of tissue engineering and regenerative medicine, induced pluripotent stem cells (iPSCs) technology with gene therapy are promising candidates for alternative stem cell source and cell transplantation. In this study, small molecules as anti-oxidant; ascorbic acid (ASA), histone deacetylase inhibitors (HDACi); Valproic acid (VPA), and DNA methyltransferase inhibitors (DNMTi); 5-Azacytidine (5-AzaC) were examined during the generation of murine iPSCs using mRNA of Yamanaka factors from mouse embryonic fibroblasts (MEFs). These modulators were selected based on their well-known effect on the epigenetic status and chromatin modification during early reprogramming. iPSC generation was performed by using synthesized mRNAs of Yamanaka factors Oct4, Sox2, c-Myc, and Klf4 (OSCK) as a standard reprogramming strategy. Both morphological changes and the expression level of the pluripotency markers were examined. 5-AzaC with 1 µM concentration has a slightly toxic effect on the cells, affecting its proliferation and growing efficiency. In contrast, the use of VPA or ASA led to a two-fold increase in the number of iPSC colonies. The iPSCs cultured with the addition of VPA or ASA showed a high expression of the tested pluripotency markers, with a significant increase, more than that of the cells cultured with the addition of 5-AzaC. These findings shed light on the role of ASA, VPA, and 5-AzaC during murine iPSCs generation using a mRNA reprogramming strategy.