Single-Factor SOX2 Mediates Direct Neural Reprogramming of Human Mesenchymal Stem Cells via Transfection of In Vitro Transcribed mRNA

Abstract

Neural stem cells (NSCs) are a prominent cell source for understanding neural pathogenesis and for developing therapeutic applications to treat neurodegenerative disease because of their regenerative capacity and multipotency. Recently, a variety of cellular reprogramming technologies have been developed to facilitate in vitro generation of NSCs, called induced NSCs (iNSCs). However, the genetic safety aspects of established virus-based reprogramming methods have been considered, and non-integrating reprogramming methods have been developed. Reprogramming with in vitro transcribed (IVT) mRNA is one of the genetically safe reprogramming methods because exogenous mRNA temporally exists in the cell and is not integrated into the chromosome. Here, we successfully generated expandable iNSCs from human umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) via transfection with IVT mRNA encoding SOX2 (SOX2 mRNA) with properly optimized conditions. We confirmed that generated human UCB-MSC-derived iNSCs (UM-iNSCs) possess characteristics of NSCs, including multipotency and self-renewal capacity. Additionally, we transfected human dermal fibroblasts (HDFs) with SOX2 mRNA. Compared with human embryonic stem cell-derived NSCs, HDFs transfected with SOX2 mRNA exhibited neural reprogramming with similar morphologies and NSC-enriched mRNA levels, but they showed limited proliferation ability. Our results demonstrated that human UCB-MSCs can be used for direct reprogramming into NSCs through transfection with IVT mRNA encoding a single factor, which provides an integration-free reprogramming tool for future therapeutic application.

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).…”

“…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). Self-renewal capacity was also assessed through immunocytochemical staining for proliferation markers, such as Ki-67, in combination with key markers for NSCs [25,26,30,34,57,59,74]. 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].…”

“…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,…”