Neural Stem Cells Differentiated From iPS Cells Spontaneously Regain Pluripotency

Abstract

Differentiated somatic cells can be reprogrammed into pluripotent stem cells by transduction of exogenous reprogramming factors. After induced pluripotent stem (iPS) cells are established, exogenous genes are silenced. In the pluripotent state, retroviral genes integrated in the host genome are kept inactive through epigenetic transcriptional regulation. In this study, we tried to determine whether exogenous genes remain silenced or are reactivated upon loss of pluripotency or on differentiation using an in vitro system. We induced differentiation of iPS cells into neural stem cells (NSCs) in vitro; the NSCs appeared morphologically indistinguishable from brain-derived NSCs and stained positive for the NSC markers Nestin and Sox2. These iPS cellderived NSCs (iPS-NSCs) were also capable of differentiating into all three neural subtypes. Interestingly, iPS-NSCs spontaneously formed aggregates on long-term culture and showed reactivation of the Oct4-GFP marker, which was followed by the formation of embryonic stem cell-like colonies. The spontaneously reverted green fluorescent protein (GFP)-positive (iPS-NSC-GFP 1 ) cells expressed high levels of pluripotency markers (Oct4 and Nanog) and formed germline chimeras, indicating that iPS-NSC-GFP 1 cells had the same pluripotency as the original iPS cells. The reactivation of silenced exogenous genes was tightly correlated with the downregulation of DNA methyltransferases (Dnmts) during differentiation of iPS cells. This phenomenon was not observed in doxycycline-inducible iPS cells, where the reactivation of exogenous genes could be induced only by doxycycline treatment. These results indicate that pluripotency can be regained through reactivation of exogenous genes, which is associated with dynamic change of Dnmt levels during differentiation of iPS cells.

Distinct Enhancer Activity of Oct4 in Naive and Primed Mouse Pluripotency

“…Approximately two decades ago, we first determined the activity of the genomic fragment containing Oct4 using a β-galactosidase reporter and found that DE activity was ESC- and germ cell-specific and that PE was epiblast- and embryonic carcinoma cell (P19)-specific (Yeom et al., 1996). Oct4 -GFP transgenic mice have been generated and used for the detection of PGCs and PSCs and for monitoring reprogramming through nuclear transfer, cell fusion, and the transduction of reprogramming factors (Boiani et al., 2002, Choi et al., 2014, Do and Schöler, 2004). However, these existing transgenic systems cannot concurrently discriminate between the activity of DE and PE.…”

Cellular Reprogramming Using Protein and Cell-Penetrating Peptides

“…As a result, iPSC generation using retroviral transduction was developed; however, it presented some challenges, the most important of which was the integration of exogenous transgenes into the host genome after viral transduction [8]. Ultimately, iPSC research aims to develop regenerative and therapeutic applications to cure intractable human diseases; therefore, this issue must be resolved before clinical trials.…”

Novel Medical Management of Spinal Cord Injury

“…Injury, though, may restrict production of new neurons and oligodendrocytes by endogenous cells into the spinal cord. Some cell transplantation studies have demonstrated reduced differentiation of exogenous stem cells compared to the endogenous differentiation after grafting to a spinal cord [5]. Thus, the environment of the spinal cord has a restrictive or inhibitory action related to the differentiation of OPCs.…”