Abstract
Cell therapies represent a promising approach to slow down the progression of currently untreatable neurodegenerative diseases (e.g., Alzheimer's and Parkinson's disease or amyotrophic lateral sclerosis), as well as to support the reconstruction of functional neural circuits after spinal cord injuries. In such therapies, the grafted cells could either functionally integrate into the damaged tissue, partially replacing dead or damaged cells, modulate inflammatory reaction, reduce tissue damage, or support neuronal survival by secretion of cytokines, growth, and trophic factors. Comprehensive characterization of cells and their proliferative potential, differentiation status, and population purity before transplantation is crucial to preventing safety risks, e.g., a tumorous growth due to the proliferation of undifferentiated stem cells. We characterized changes in the proteome and secretome of human neural stem cells (NSCs) during their spontaneous (EGF/FGF2 withdrawal) differentiation and differentiation with trophic support by BDNF/GDNF supplementation. We used LC-MS/MS in SWATH-MS mode for global cellular proteome profiling and quantified almost three thousand cellular proteins. Our analysis identified substantial protein differences in the early stages of NSC differentiation with more than a third of all the proteins regulated (including known neuronal and NSC multipotency markers) and revealed that the BDNF/GDNF support affected more the later stages of the NSC differentiation. Among the pathways identified as activated during both spontaneous and BDNF/GDNF differentiation were the HIF-1 signaling pathway, Wnt signaling pathway, and VEGF signaling pathway. Our follow-up secretome analysis using Luminex multiplex immunoassay revealed significant changes in the secretion of VEGF and IL-6 during NSC differentiation. Our results further demonstrated an increased expression of neuropilin-1 as well as catenin β-1, both known to participate in the regulation of VEGF signaling, and showed that VEGF-A isoform 121 (VEGF121), in particular, induces proliferation and supports survival of differentiating cells.
Highlights
The neural stem cells (NSCs) are undifferentiated cells capable of reproducing themselves and giving rise to progenitors that may further differentiate into neuronal and glial lineages
Expression of neuropilin-1 is induced by VEGF165 in rat NSCs (Maurer et al, 2003), our results suggest that increased abundance of neuropilin-1 during both differentiations may not be connected to VEGF165 expression and secretion in H9 NSCs
NSCs derived from H9 embryonic stem cells (ESCs) are broadly used for both in vitro and in vivo studies related to central nervous system (CNS) pathologies, including autism spectrum disorders (Nguyen et al, 2018), brain ischemia (Green et al, 2018), glioblastoma (Balbous et al, 2014), neuroblastoma (Carr-Wilkinson et al, 2018), or Parkinson’s disease (Iacovitti et al, 2007)
Summary
The neural stem cells (NSCs) are undifferentiated cells capable of reproducing themselves and giving rise to progenitors that may further differentiate into neuronal and glial (astrocyte and oligodendrocyte) lineages. The transplanted cells are expected to influence local microenvironment, reduce cellular stress in situ, promote cell survival, modulate inflammation, enhance remyelination, and maintain neuronal circuits (de Gioia et al, 2020; Fischer et al, 2020; Ottoboni et al, 2020). Some of these effects are mediated by a direct cell-to-cell contact, others by release of simple metabolites. A significant part of the effects is expected to come from secreted proteins including cytokines, growth factors, and other proteins essential for cellular communication and signaling (Kupcova Skalnikova, 2013), leading to, e.g., antiinflammatory effects (Einstein et al, 2007) or inhibition of apoptosis (Petrenko et al, 2020)
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