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Stem Cells Transl Med. 2013 Mar;2(3):223-32. doi: 10.5966/sctm.2012-0119. Epub 2013 Feb 19.

Quantitative microplate assay for studying mesenchymal stromal cell-induced neuropoiesis.

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  • 1SanBio, Inc., Mountain View, CA, USA. irina.aizman@san-bio.com

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.

PMID:
23430693
PMCID:
PMC3659769
DOI:
10.5966/sctm.2012-0119
[PubMed - indexed for MEDLINE]
Free PMC Article
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