Adult stem cells age during long-term in vitro culture, and neural stem cells (NSCs), which can self-renew and differentiate into neurons and glial cells, also display reduced differentiation potential after repeated passaging. However, the mechanistic details underlying this process remain unclear. In this study, we found that long-term in vitro culture of NSCs resulted in aging-related upregulation of inflammatory-and endoplasmic reticulum (ER) stress-related genes, including the proinflammatory cytokines interleukin (IL)1β and IL6, the senescence-associated enzyme matrix metallopeptidase 13 (MMP13), and the ER stress-responsive transcription factor activating transcription factor 4 (ATF4) and C/EBP homologous protein (CHOP). However, the cyclic and transient induction of four reprogramming factors (POU domain, class 5, transcription factor 1, also known as octamer-binding transcription factor 4; SRY [sex determining region Y]-box 2; Kruppel-like factor 4; and myelocytomatosis oncogene; collectively referred to as OSKM) can inhibit NSC aging, as indicated by the decreased expression of the inflammatory and ER stress-related genes. We used ROSA-4F NSCs, which express OSKM from only one allele, to minimize the potential for full reprogramming or tumor formation during NSC rejuvenation. We expect that this novel rejuvenation method will enhance the potential of NSCs as a clinical approach to the treatment of neurological diseases.
Human-induced pluripotent stem cells (hiPSCs) have facilitated studies on organ development and differentiation into specific lineages in in vitro systems. Although numerous studies have focused on cellular differentiation into neural lineage using hPSCs, most studies have initially evaluated embryoid body (EB) formation, eventually yielding terminally differentiated neurons with limited proliferation potential. This study aimed to establish human primitive neural stem cells (pNSCs) from exogenefree hiPSCs without EB formation. To derive pNSCs, we optimized N2B27 neural differentiation medium through supplementation of two inhibitors, CHIR99021 (GSK-3 inhibitor) and PD0325901 (MEK inhibitor), and growth factors including basic fibroblast growth factor (bFGF) and human leukemia inhibitory factor (hLIF). Consequently, pNSCs were efficiently derived and cultured over a long term. pNSCs displayed differentiation potential into neurons, astrocytes, and oligodendrocytes.These early NSC types potentially promote the clinical application of hiPSCs to cure human neurological disorders. K E Y W O R D S differentiation, human-induced pluripotent stem cells, neural lineage, primitive neural stem cells
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