Proteomic Characterization of Human Neural Stem Cells and Their Secretome During in vitro Differentiation

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.

Targeted mass spectrometry for monitoring of neural differentiation

“…We aimed to target a set of protein markers routinely used in NSC differentiation studies ( ), including ESC markers (homeobox protein NANOG, NANOG; OCT4), NSC markers (SOX2; NES; paired box protein Pax-6, PAX6; proliferation marker protein Ki-67, MKI67), neuronal markers (DCX, TUBB3, MAP2), astrocyte markers (GFAP, S100B), and oligodendrocyte markers (GALC, OLIG1). We also intended to test the ability to detect low-abundant proteins previously found in our differentiation experiments, namely vascular endothelial growth factor A (VEGF-A) ( Červenka et al, 2021 ) and growth-regulated alpha protein (CXCL1) (unpublished work, Institute of Animal Physiology and Genetics of The Czech Academy of Sciences).…”

“…6 A). Our recent data revealed that these neurotrophic factors affected the later stages of differentiation ( Červenka et al, 2021 ), so we employed our SRM assay to depict this effect after 4 weeks of differentiation. As a reference, pluripotent ESCs and mature astrocytes were processed for MS analysis ( Fig.…”

“…To address shortcomings of antibody-based screens, targeted MS analysis by SRM can be used not only to accurately verify protein abundance changes emerging from global transcriptome and proteome profiling (Červenka et al, 2021;Donega et al, 2019;Tyleckova et al, 2016;Yocum et al, 2008), but rather to identify markers that would provide a reliable read-out for the differentiation potential of NSCs. We had developed a quantitative high-throughput assay for neuroscience studies, and evaluated its capability Alternatively, it may suggest persistent NES and SOX2 expression in non-neuronal populations, e.g., differentiating glia.…”

“…Gibco Human Neural Stem Cells (H9-derived) generated from the NIH approved human ESCs (WA09; 46, XX) had been obtained from Life Technologies (catalogue number 510088, lot number 1402001, Thermo Fisher Scientific Inc.) and cultured as described previously ( Červenka et al, 2021 ) with modifications. Briefly, the H9-derived NSCs (condition NSC) were grown on 20 μg/ml poly-L-ornithine and 5 μg/ml laminin-coated plates (both from Sigma-Aldrich, St. Louis, MO, USA) in the NSC proliferation medium containing KnockOut Dulbecco's modified Eagle's medium (DMEM)/F-12, 2 mM GlutaMAX, 1% penicillin-streptomycin, 2% StemPro Neural Supplement, 20 ng/ml human recombinant FGF-2, and 20 ng/ml human recombinant EGF.…”

“…MS-based quantifications following enrichment strategies for capturing candidate markers of NSCs were performed ( Melo-Braga et al, 2014 ; Song et al, 2019 ; Tyleckova et al, 2016 ) using a conventional shotgun approach, where a subset of peptides was automatically and in part stochastically measured in the process of data-dependent precursor selection ( Aebersold and Mann, 2003 ). Recently, we applied the data-independent acquisition MS method that combined global feature detection with targeted data extraction to simultaneously quantify thousands of proteins in the course of NSC differentiation ( Červenka et al, 2021 ). This altogether helped to improve our understanding of the NSC differentiation and to identify potential protein markers of distinct steps in this process.…”