Induction of Neural Progenitor-Like Cells from Human Fibroblasts via a Genetic Material-Free Approach

Abstract

BackgroundA number of studies generated induced neural progenitor cells (iNPCs) from human fibroblasts by viral delivering defined transcription factors. However, the potential risks associated with gene delivery systems have limited their clinical use. We propose it would be safer to induce neural progenitor-like cells from human adult fibroblasts via a direct non-genetic alternative approach.Methodology/Principal FindingsHere, we have reported that seven rounds of TAT-SOX2 protein transduction in a defined chemical cocktail under a 3D sphere culture gradually morphed fibroblasts into neuroepithelial-like colonies. We were able to expand these cells for up to 20 passages. These cells could give rise to cells that expressed neurons and glia cell markers both in vitro and in vivo.Conclusions/SignificanceThese results show that our approach is beneficial for the genetic material-free generation of iNPCs from human fibroblasts where small chemical molecules can provide a valuable, viable strategy to boost and improve induction in a 3D sphere culture.

Take the shortcut – direct conversion of somatic cells into induced neural stem cells and their biomedical applications

“…Two studies reported that lentivirus-mediated expression of OCT4 is sufficient to obtain iNSCs from adult human fibroblasts, neonatal cord, and peripheral blood cells [29,30]. Besides Oct4, also Sox2 has been reported to be sufficient for direct conversion of somatic cells into iNSCs [32][33][34]. Dual SMAD inhibition is known to efficiently and rapidly convert pluripotent cells into neural cells [31], potentially indicating that reprogrammed cells might have passed a transient pluripotent stage.…”

“…A key question to assess the outcome of direct conversion strategies is to which extent directly converted iNSCs recapitulate these characteristics [11,71]. In order to demonstrate selfrenewal potential and clonal growth ability, iNSCs were either cultivated as primary and secondary neurospheres [33,34], analyzed in colony formation assays [28,33,36,39,42,48,72], and/or passaged several times [11,13,14,26,30,32,33,38,57,59,60,73]. On the other hand, all iNSC populations have been reported to express pan-neural markers, to be at least bipotential, and to show self-renewal and clonal growth ( Table 2).…”

“…While Kim et al [11] reported passage of their mouse iNSCs for 3-5 times only, more recent studies demonstrated maintenance of NSC characteristics for more than 30 passages [13,14,28,34,36,38,58,73]. The neural expansion media were supplemented with either LIF and small molecules like CHIR99021, SB431542, purmorphamine, A83-01, and/or ascorbic acid [25,26,48], or basic fibroblast growth factor (FGF2) and epidermal growth factor (EGF) [14,30,32,34,36,38,[57][58][59][60]62], or even a combination of them [33,35,41]. Among the various publications reporting iNSC generation, there is extensive accordance in terms of expression of bona fide neural stem cell markers, such as SOX1, SOX2, PAX6, NESTIN, CD133, and BLBP.…”

“…Other iNSC populations gave only rise to neurons and astrocytes [11,26,35,78]. Regarding neuronal subtypes, iNSCs were shown to have the potential to differentiate into the main neuronal subtypes, including glutamatergic [28,30,32,36,48,57,59,60,73,77], GABAergic [11,13,28,30,32,36,38,48,57,59,60,77], dopaminergic [14,25,28,30,33,34,36,48,57,73], serotonergic [25,28], and cholinergic neurons [14,25,28,34,36,48,60,73]. Regarding neuronal subtypes, iNSCs were shown to have the potential to differentiate into the main neuronal subtypes, including glutamatergic [28,30,…”

Direct reprogramming and biomaterials for controlling cell fate

“…Actually, the result showed a quite low transduction efficiency, but a high viability (~100%) after transfection. Similarly, 3D sphere induced neural progenitor like cells from fibroblast were reported [106, 107]. Although the repeated transduction is necessary due to the low transduction efficiency, it can be free form the potential risk of viral vector such as gene disruption, instability, and tumor formation [108].…”