5-Aza-2′-Deoxycytidine and Valproic Acid in Combination with CHIR99021 and A83-01 Induce Pluripotency Genes Expression in Human Adult Somatic Cells

Abstract

A generation of induced pluripotent stem cells (iPSC) by ectopic expression of OCT4, SOX2, KLF4, and c-MYC has established promising opportunities for stem cell research, drug discovery, and disease modeling. While this forced genetic expression represents an advantage, there will always be an issue with genomic instability and transient pluripotency genes reactivation that might preclude their clinical application. During the reprogramming process, a somatic cell must undergo several epigenetic modifications to induce groups of genes capable of reactivating the endogenous pluripotency core. Here, looking to increase the reprograming efficiency in somatic cells, we evaluated the effect of epigenetic molecules 5-aza-2′-deoxycytidine (5AZ) and valproic acid (VPA) and two small molecules reported as reprogramming enhancers, CHIR99021 and A83-01, on the expression of pluripotency genes and the methylation profile of the OCT4 promoter in a human dermal fibroblasts cell strain. The addition of this cocktail to culture medium increased the expression of OCT4, SOX2, and KLF4 expression by 2.1-fold, 8.5-fold, and 2-fold, respectively, with respect to controls; concomitantly, a reduction in methylated CpG sites in OCT4 promoter region was observed. The epigenetic cocktail also induced the expression of the metastasis-associated gene S100A4. However, the epigenetic cocktail did not induce the morphological changes characteristic of the reprogramming process. In summary, 5AZ, VPA, CHIR99021, and A83-01 induced the expression of OCT4 and SOX2, two critical genes for iPSC. Future studies will allow us to precise the mechanisms by which these compounds exert their reprogramming effects.

Revealing the pathogenesis of gastric intestinal metaplasia based on the mucosoid air-liquid interface

“…The basal medium widely utilized for culturing mammalian cells is Advanced DMEM/F-12 (ADF) medium [22]. To maintain cell stemness, Wnt3a, R-spondin1, and A83-01 were employed [23,24]. Tissue culture buffer was provided by 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid [25].…”

Application of Small Molecules in the Central Nervous System Direct Neuronal Reprogramming

“…Some small molecules can regulate the efficiency of reprogramming by modulating epigenetics ( Aguirre-Vázquez et al, 2021 ). These epigenetic modifications are heritable differences that include “Tags” such as DNA methylation and diverse histone modifications that can affect DNA accessibility and chromatin structure ( Ebrahimi et al, 2019 ).…”