Stable expression of a truncated TLX variant drives differentiation of induced pluripotent stem cells into self-renewing neural stem cells for production of extracellular vesicles

Neural stem cells (NSCs)-derived extracellular vesicles (EVs) possess great potential in treating severe neurological and cerebrovascular diseases, as they carry the modulatory and regenerative ingredients of NSCs. Induced pluripotent stem cells (iPSCs)-derived NSCs culture represents a sustainable source of therapeutic EVs. However, there exist two major challenges in obtaining a scalable culture of NSCs for high-efficiency EVs production: (1) the heterogeneity of iPSC-derived NSCs culture impairs the production of high-quality EVs and (2) the intrinsic propensity of neuronal or astroglial differentiation of NSCs during prolonged culturing reduces the number of NSCs for preparing EVs. A NSCs strain that is amenable to stable self-renewal and proliferation is thus greatly needed for scalable and long-term culture.

iPSC-derived NSCs stably expressing TLX-TP is a promising cell line for scalable production of EVs, which should be further exploited for therapeutic development in neurological treatment.

Various constructs of the genes encoding the orphan nuclear receptor NR2E1 (TLX) were stably transfected in iPSCs, which were subsequently cultured in a variety of differentiation media for generation of iNSCsTLX. Transcriptomic and biomarker profile of iNSCsTLX were investigated. In particular, the positivity ratios of Sox2/Nestin and Musashi/Vimentin were used to gauge the homogeneity of the iNSCsTLX culture. The iNSCs expressing a truncated version of TLX (TLX-TP) was expanded for up to 45 passages, after which its neuronal differentiation potential and EV activity were evaluated.

Stable expression of TLX-TP could confer the iPSCs with rapid and self-driven differentiation into NSCs through stable passaging up to 225 days. The long-term culture of NSCs maintained the highly homogenous expression of NSC-specific biomarkers and potential of neuronal differentiation. EVs harvested from the TLX-expressing NSCs cultures exhibited anti-inflammatory and neuroprotective activities. Conclusions: iPSC-derived NSCs stably expressing TLX-TP is a promising cell line for scalable production of EVs, which should be further exploited for therapeutic development in neurological treatment.