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

Abstract

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.

Characterization of Neurodevelopmental Abnormalities in iPSC-Derived Striatal Cultures from Patients with Huntington’s Disease

“…4D). This data is corelates with recent findings of the role of Notch in self-renewal of NPCs and subsequent differentiation to glial and neuronal fate [43]. There was significant increase observed in JHD 71 and JHD 180 ( p < 0.05) in TuJ1-expressing neuronal population (Fig.…”

“…The receptors are triggered by cell-cell contact and a subsequent cascade of signals dictates the cellular fate, including but not limited to neurogenesis [39–41]. Recent findings suggest that Notch signaling plays vital role in subsequent differentiation of NPCs to neuronal or glial fate and inhibition of this pathway also impedes the differentiation [43].…”

Integrated Analysis of Whole Exome Sequencing and Copy Number Evaluation in Parkinson’s Disease

“…The p.Q1257X variant, identified in PD-64 falls in the (LNR) repeat involved in receptor regulation, while the splice variant (c.2865 + 2T > C) affects an exon that falls within Calcium-binding EFG-like domain. It is well established that Notch signalling is an evolutionarily conserved pathway involved in a wide variety of developmental processes, including adult homeostasis, stem cell maintenance, cell proliferation and apoptosis 62,64 .…”

Molecular Mechanisms of Transdifferentiation of Adipose-Derived Stem Cells into Neural Cells: Current Status and Perspectives

“…Previous reports have indicated that Notch signaling occurs in proliferating hADSCs and is downregulated when cells are transdifferentiated to a neuronal phenotype [ 119 ]. On the other hand, Notch was found to be required for the expansion and self-renewal of NSCs in vitro and in vivo [ 121 ], and this signaling pathway is also a key regulator of stem cell lineage commitment and differentiation [ 121 ]. Notch receptor activation induces expression of the specific target genes hairy and enhancer of split 3 (HES3) and sonic hedgehog (Shh) through rapid activation of cytoplasmic signals, including Akt and STAT3, and promotes NSC survival [ 122 ].…”