Abstract
Restoring pluripotency using chemical compounds alone would be a major step forward in developing clinical-grade pluripotent stem cells, but this has not yet been reported in human cells. We previously demonstrated that VPA_AFS cells, human amniocytes cultivated with valproic acid (VPA) acquired functional pluripotency while remaining distinct from human embryonic stem cells (hESCs), questioning the relationship between the modulation of cell fate and molecular regulation of the pluripotency network. Here, we used single-cell analysis and functional assays to reveal that VPA treatment resulted in a homogeneous population of self-renewing non-transformed cells that fulfill the hallmarks of pluripotency, i.e., a short G1 phase, a dependence on glycolytic metabolism, expression of epigenetic modifications on histones 3 and 4, and reactivation of endogenous OCT4 and downstream targets at a lower level than that observed in hESCs. Mechanistic insights into the process of VPA-induced reprogramming revealed that it was dependent on OCT4 promoter activation, which was achieved independently of the PI3K (phosphatidylinositol 3-kinase)/AKT/mTOR (mammalian target of rapamycin) pathway or GSK3β inhibition but was concomitant with the presence of acetylated histones H3K9 and H3K56, which promote pluripotency. Our data identify, for the first time, the pluripotent transcriptional and molecular signature and metabolic status of human chemically induced pluripotent stem cells.