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

Mingzhi Xu; Gang Chen; Yanan Dong; Shensi Xiang; Miaomiao Xue; Yongxue Liu; Haijing Song; Haifeng Song; Yi Wang

doi:10.1186/s13287-022-03131-4

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

Stem Cell Res Ther. 2022 Sep 2;13(1):436. doi: 10.1186/s13287-022-03131-4.

Authors

Mingzhi Xu¹, Gang Chen¹, Yanan Dong¹, Shensi Xiang¹, Miaomiao Xue¹, Yongxue Liu², Haijing Song³, Haifeng Song⁴, Yi Wang⁵

Affiliations

¹ State Key Laboratory of Proteomics, National Center for Protein Sciences(Beijing), Beijing Proteome Research Center, Beijing Institute of Lifeomics, Beijing, 102206, China.
² Anti-Radiation Medical Laboratory, Beijing Institute of Radiation Medicine, Beijing, 100039, China.
³ Emergency Medicine, PLA Strategic Support Force Medical Center, Beijing, 100101, China. sys306@163.com.
⁴ State Key Laboratory of Proteomics, National Center for Protein Sciences(Beijing), Beijing Proteome Research Center, Beijing Institute of Lifeomics, Beijing, 102206, China. songhf@vip.163.com.
⁵ State Key Laboratory of Proteomics, National Center for Protein Sciences(Beijing), Beijing Proteome Research Center, Beijing Institute of Lifeomics, Beijing, 102206, China. yves.wangy@vip.163.com.

Abstract

Background: 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.

Methods: 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 iNSCs^TLX. Transcriptomic and biomarker profile of iNSCs^TLX were investigated. In particular, the positivity ratios of Sox2/Nestin and Musashi/Vimentin were used to gauge the homogeneity of the iNSCs^TLX 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.

Results: 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.

Keywords: Extracellular vesicles; Induced pluripotent stem cells; Long-term culture; Neural stem cells; TLX.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Cell Differentiation / physiology
Cells, Cultured
Extracellular Vesicles* / genetics
Induced Pluripotent Stem Cells* / metabolism
Neural Stem Cells*