NOTCH Signaling Is Essential for Maturation, Self-Renewal, and Tri-Differentiation of In Vitro Derived Human Neural Stem Cells.

Although neural stem cells (NSCs) have potential applications in treating neurological disorders, much still needs to be understood about the differentiation biology for their successful clinical translation. In this study, we aimed at deriving NSCs from human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) and explored the role of Notch signaling in the differentiation process. The hUCB-MSCs were characterized as per guidelines of the International Society of Cellular Therapy. NSCs were successfully generated from hUCB-MSCs by using epidermal and fibroblast growth factors under serum-free conditions. The expression of NSC markers (Nestin and Musashi-1) in the neurospheres generated from hUCB-MSCs in the presence or absence of N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (DAPT; Notch inhibitor) was immuno-phenotypically characterized by using immunofluorescence. DAPT showed significant (*p < 0.05) downregulated expression of the NSC markers-Nestin and SOX2-at different time points (6 hours, 12 hours, 24 hours, 36 hours, and 5 days) post-treatment. In addition, Mushashi-1 (NSC marker) expression in NSCs was also inhibited after DAPT treatment, which signifies that the process is Notch dependent. These data were further correlated with formation of a reduced average number of neurospheres derived from hUCB-MSCs (2 colonies vs. 11 colonies/field of view) in the presence of DAPT compared with the control (without DAPT). The expression of Notch target genes in NSC cultures (Notch intracellular domain [NICD], HES1, and HES5) was also significantly downregulated after DAPT treatment. In the presence of DAPT, the markers for neuronal (MAP2, NEFH); and glial (GFAP, GLUL, and MBP) lineages were significantly downregulated as seen via immunofluorescence and quantitative polymerase chain reaction, indicating the role of Notch in the tri-differentiation mechanism of NSCs as well. In addition, Notch signaling inhibition induced higher cell death during the lineage commitment of NSCs as measured 3 days (16.9% vs. 8.9%) and 6 days (42.9% vs. 20.8%) postinduction. These results suggest that the efficient derivation of NSCs and their subsequent lineage commitment from hUCB-MSCs requires the Notch signaling pathway.