SOX2 and SOX2-MYC Reprogramming Process of Fibroblasts to the Neural Stem Cells Compromised by Senescence

Abstract

Tumorigenic potential of induced pluripotent stem cells (iPSCs) infiltrating population of induced neural stem cells (iNSCs) generated from iPSCs may limit their medical applications. To overcome such a difficulty, direct reprogramming of adult somatic cells into iNSCs was proposed. The aim of this study was the systematic comparison of induced neural cells (iNc) obtained with different methods—direct reprogramming of human adult fibroblasts with either SOX2 (SiNSc-like) or SOX2 and c-MYC (SMiNSc-like) and induced pluripotent stem cells differentiation to ebiNSc—in terms of gene expression profile, differentiation potential as well as proliferation properties. Immunocytochemistry and real-time PCR analyses were used to evaluate gene expression profile and differentiation potential of various iNc types. Bromodeoxyuridine (BrdU) incorporation and senescence-associated beta-galactosidase (SA-β-gal) assays were used to estimate proliferation potential. All three types of iNc were capable of neuronal differentiation; however, astrocytic differentiation was possible only in case of ebiNSc. Contrary to ebiNSc generation, the direct reprogramming was rarely a propitious process, despite 100% transduction efficiency. The potency of direct iNSCs-like cells generation was lower as compared to iNSCs obtained by iPSCs differentiation, and only slightly improved when c-MYC was added. Directly reprogrammed iNSCs-like cells were lacking the ability to differentiate into astrocytic cells and characterized by poor efficiency of neuronal cells formation. Such features indicated that these cells could not be fully reprogrammed, as confirmed mainly with senescence detection. Importantly, SiNSc-like and SMiNSc-like cells were unable to achieve the long-term survival and became senescent, which limits their possible therapeutic applicability. Our results suggest that iNSCs-like cells, generated in the direct reprogramming attempts, were either not fully reprogrammed or reprogrammed only into neuronal progenitors, mainly because of the inaccuracies of currently available protocols.

IDH1R132H in Neural Stem Cells: Differentiation Impaired by Increased Apoptosis

“…The ebiNSc, a human induced neural stem cell line obtained from induced pluripotent stem cells following embryoid bodies generation and induction of neural differentiation, and ebiNSc IDH1R132H , a human cell line with an induced expression of mutated IDH1 R132H gene, were gifts received from Celther Polska Ltd. The ebiNSc were obtained as described previously [ 14 ]. The ebiNSc IDH1WT and ebiNSc empty were generated, analogously to ebiNSc IDH1R132H , via the transduction with the respective vector (as described below).…”

ProNGF Is a Cell-Type-Specific Mitogen for Adult Hippocampal and for Induced Neural Stem Cells

“…We derived iNSCs cultures by infecting MEF with the p‐Sox‐2 retrovirus . One limitation of current iNSCs reprogramming protocols is that they produce mainly late Nestin + /DCX + progenitors, with restricted propagation potential (≤8–9 passages ; this article). To explore the possibility of overcoming this limitation, we chronically exposed mouse iNSCs to 0.4 nM proNGF‐KR from passage 0 to passage 6 (before the culture undergoes spontaneous differentiation despite the presence of mitogenic stimulus in the medium).…”

“…iNSCs technology is a powerful tool for studying neural development and neurological disorders, both in vitro and in animal model . One main obstacle for an efficient use of iNSCs is their limited expansion potential, being composed mainly of late multipotent and late neural precursors that soon reach senescence , while earlier progenitors are poorly represented in the culture. In this view, our results on the mitogenic effect of proNGF‐KR on these latter cells are important, as this allows expanding this population of cells prior to their differentiation into neurons.…”

Perfluorooctane sulfonate (PFOS) impairs the proliferation of C17.2 neural stem cells via the downregulation of GSK‐3β/β‐catenin signaling

“…The pivotal role of the Wnt/β‐catenin pathway in NSC biology is related to various β‐catenin target genes. For example, C‐myc, which is a well‐known β‐catenin target, is a master regulator of stem cell pluripotency (Winiecka‐Klimek et al , ). Cyclin D1 is a key regulator of cell cycle entrance and stem cell expansion, and has been recently reported to promote the expansion of basal neural progenitor cells (Lange et al , ).…”