Conversion of Human Fibroblasts to Stably Self-Renewing Neural Stem Cells with a Single Zinc-Finger Transcription Factor

Abstract

SummaryDirect conversion of somatic cells into neural stem cells (NSCs) by defined factors holds great promise for mechanistic studies, drug screening, and potential cell therapies for different neurodegenerative diseases. Here, we report that a single zinc-finger transcription factor, Zfp521, is sufficient for direct conversion of human fibroblasts into long-term self-renewable and multipotent NSCs. In vitro, Zfp521-induced NSCs maintained their characteristics in the absence of exogenous factor expression and exhibited morphological, molecular, developmental, and functional properties that were similar to control NSCs. In addition, the single-seeded induced NSCs were able to form NSC colonies with efficiency comparable with control NSCs and expressed NSC markers. The converted cells were capable of surviving, migrating, and attaining neural phenotypes after transplantation into neonatal mouse and adult rat brains, without forming tumors. Moreover, the Zfp521-induced NSCs predominantly expressed rostral genes. Our results suggest a facilitated approach for establishing human NSCs through Zfp521-driven conversion of fibroblasts.

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

“…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).…”

“…As a matter of fact, the various protocols for the generation of iNSCs give rise to distinct iNSC populations that slightly differ in self renewal capacity, marker expression, and regional identity, as well as in vitro and in vivo differentiation potential. 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]. 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].…”

“…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,…”

“…Moreover, several studies strongly suggest the presence of functional synapses, as indicated by their response to excitatory or inhibitory neurotransmitters, as well as of spontaneous postsynaptic currents, and the ability to (partially) abolish synaptic activity with specific blockers [11,25,28,38,42,48,57,59,75,80]. In addition to electrophysiological experiments, the ability to form synapses was also investigated through immunocytochemical approaches showing colocalization of pre-and postsynaptic markers like synapsin 1, vGLUT1, or PSD-95 ( Table 2 [13,14,25,28,30,32,36,38,48,57,73,75]). However, the functionality of iNSC-derived neurons shall be characterized in detail.…”