title: Quantitative microplate assay for studying mesenchymal stromal cell-induced neuropoiesis.
abstract: Transplanting mesenchymal stromal cells (MSCs) or their derivatives in a neurodegenerative environment is believed to be beneficial because of the trophic support, migratory guidance, and neurogenic stimuli they provide. There is a growing need for in vitro models of mesenchymal-neural cell interactions to enable identification of mediators of the MSC activity and quantitative assessment of neuropoietic potency of MSC preparations. Here, we characterize a microplate-format coculture system in which primary embryonic rat cortex cells are directly cocultured with human MSCs on cell-derived extracellular matrix (ECM) in the absence of exogenous growth factors. In this system, expression levels of the rat neural stem/early progenitor marker nestin, as well as neuronal and astrocytic markers, directly depended on MSC dose, whereas an oligodendrogenic marker exhibited a biphasic MSC-dose response, as measured using species-specific quantitative reverse transcription-polymerase chain reaction in total cell lysates and confirmed using immunostaining. Both neural cell proliferation and differentiation contributed to the MSC-mediated neuropoiesis. ECM's heparan sulfate proteoglycans were essential for the growth of the nestin-positive cell population. Neutralization studies showed that MSC-derived fibroblast growth factor 2 was a major and diffusible inducer of rat nestin, whereas MSC-derived bone morphogenetic proteins (BMPs), particularly, BMP4, were astrogenesis mediators, predominantly acting in a coculture setting. This system enables analysis of multifactorial MSC-neural cell interactions and can be used for elucidating the neuropoietic potency of MSCs and their derivative preparations.
authors: SanBio, Inc., Mountain View, CA, USA. irina.aizman@san-bio.com
authors: Irina
authors: I
authors: Aizman
authors: Michael
authors: M
authors: McGrogan
authors: Casey C
authors: CC
authors: Case
mesh_terms: D063309:2',3'-Cyclic Nucleotide 3'-Phosphodiesterase / Q000378:metabolism; D000818:Animals; D001681:Biological Assay / Q000379:methods; D015415:Biomarkers / Q000378:metabolism; D055415:Bone Morphogenetic Protein 4 / Q000378:metabolism; D049109:Cell Proliferation; D048430:Cell Shape; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000196:embryology / Q000378:metabolism*; D018920:Coculture Techniques; D017077:Culture Media, Conditioned / Q000378:metabolism; D005109:Extracellular Matrix / Q000378:metabolism; D016222:Fibroblast Growth Factor 2 / Q000378:metabolism; D005786:Gene Expression Regulation; D019812:Heparan Sulfate Proteoglycans / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D007381:Intermediate Filament Proteins / Q000378:metabolism; D059630:Mesenchymal Stem Cells / Q000378:metabolism*; D008904:Miniaturization; D009419:Nerve Tissue Proteins / Q000378:metabolism; D064231:Nestin; D058953:Neural Stem Cells / Q000378:metabolism*; D055495:Neurogenesis / Q000235:genetics; D019899:Paracrine Communication; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D014162:Transfection
chemical_list: C525809:BMP4 protein, human; D015415:Biomarkers; D055415:Bone Morphogenetic Protein 4; D017077:Culture Media, Conditioned; D019812:Heparan Sulfate Proteoglycans; D007381:Intermediate Filament Proteins; C577484:NES protein, human; D009419:Nerve Tissue Proteins; C577486:Nes protein, rat; D064231:Nestin; D016222:Fibroblast Growth Factor 2; D063309:2',3'-Cyclic Nucleotide 3'-Phosphodiesterase; C571339:Cnp protein, rat