Human multipotent neural stem cells could effectively be used for the treatment of a variety of neurological disorders. However, a defining signature of neural stem cell lines that would be expandable, non-tumorigenic, and differentiate into desirable neuronal/glial phenotype after in vivo grafting is not yet defined. Employing a mass spectrometry approach, based on selected reaction monitoring, we tested a panel of well-described culture conditions, and measured levels of protein markers routinely used to probe neural differentiation, i.e. POU5F1 (OCT4), SOX2, NES, DCX, TUBB3, MAP2, S100B, GFAP, GALC, and OLIG1. Our multiplexed assay enabled us to simultaneously identify the presence of pluripotent, multipotent, and lineage-committed neural cells, thus representing a powerful tool to optimize novel and highly specific propagation and differentiation protocols. The multiplexing capacity of this method permits the addition of other newly identified cell type-specific markers to further increase the specificity and quantitative accuracy in detecting targeted cell populations. Such an expandable assay may gain the advantage over traditional antibody-based assays, and represents a method of choice for quality control of neural stem cell lines intended for clinical use.
- MeSH
- biologické markery MeSH
- buněčná diferenciace * MeSH
- buněčné linie MeSH
- buněčný rodokmen genetika MeSH
- hmotnostní spektrometrie MeSH
- imunohistochemie MeSH
- lidé MeSH
- nervové kmenové buňky cytologie metabolismus MeSH
- neuroglie MeSH
- neurony MeSH
- stanovení celkové genové exprese MeSH
- vývojová regulace genové exprese MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
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.
- Publikační typ
- časopisecké články MeSH
Cytokines, chemokines, and growth factors are key mediators of cell proliferation, migration, and immune response, and in tumor microenvironment, such factors contribute to regulation of tumor growth, immune cell recruitment, angiogenesis, and metastasis. In body fluids, levels of inflammatory mediators reflect the patient immune response to the disease and may predict the effects of targeted therapies. Significant improvements in cytokine detection techniques have been made during last 10 years leading to sensitive quantification of such potent molecules present in low pg/mL levels. Among the techniques, Luminex xMAP® multiplex assays allow for simultaneous quantification of up to 100 analytes with high sensitivity, broad dynamic range of quantification, high throughput, and minimal sample requirements. In this chapter we describe a detailed protocol for the application of xMAP assays using Luminex® 200™ analyzer with xPonent® acquisition software to quantify cytokines, chemokines, and growth factors secreted to blood serum and plasma of cancer patients. We also discuss how sample preparation, instrument settings, and standard curve fitting algorithms can influence validity of obtained results. Special attention is paid to data analysis using open source R statistical environment and we provide an example dataset of cytokine levels measured in serum and corresponding R script for standard curve fitting and concentration estimates.
- MeSH
- buněčná diferenciace fyziologie genetika MeSH
- chiméra genetika metabolismus MeSH
- embryonální kmenové buňky cytologie fyziologie MeSH
- indukované pluripotentní kmenové buňky * cytologie fyziologie ultrastruktura MeSH
- modely u zvířat MeSH
- myši transgenní MeSH
- přeprogramování buněk fyziologie genetika MeSH
- techniky buněčného přeprogramování metody MeSH
- techniky in vitro MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- práce podpořená grantem MeSH
- MeSH
- CRISPR-Cas systémy MeSH
- genetická terapie metody MeSH
- klinická studie jako téma MeSH
- lidé MeSH
- objevování léků MeSH
- pluripotentní kmenové buňky * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- práce podpořená grantem MeSH
- MeSH
- embryonální kmenové buňky MeSH
- indukované pluripotentní kmenové buňky * MeSH
- lidé MeSH
- přeprogramování buněk MeSH
- transkripční faktory MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- práce podpořená grantem MeSH
