Quantitative Microplate Assay for Studying Mesenchymal Stromal Cell-Induced Neuropoiesis

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

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.

MeSH terms

  • 2',3'-Cyclic Nucleotide 3'-Phosphodiesterase / metabolism
  • Animals
  • Biological Assay* / methods
  • Biomarkers / metabolism
  • Bone Morphogenetic Protein 4 / metabolism
  • Cell Proliferation
  • Cell Shape
  • Cells, Cultured
  • Cerebral Cortex / embryology
  • Cerebral Cortex / metabolism*
  • Coculture Techniques
  • Culture Media, Conditioned / metabolism
  • Extracellular Matrix / metabolism
  • Fibroblast Growth Factor 2 / metabolism
  • Gene Expression Regulation
  • Heparan Sulfate Proteoglycans / metabolism
  • Humans
  • Immunohistochemistry
  • Intermediate Filament Proteins / metabolism
  • Mesenchymal Stem Cells / metabolism*
  • Miniaturization
  • Nerve Tissue Proteins / metabolism
  • Nestin
  • Neural Stem Cells / metabolism*
  • Neurogenesis* / genetics
  • Paracrine Communication*
  • Rats
  • Reverse Transcriptase Polymerase Chain Reaction
  • Time Factors
  • Transfection

Substances

  • BMP4 protein, human
  • Biomarkers
  • Bone Morphogenetic Protein 4
  • Culture Media, Conditioned
  • Heparan Sulfate Proteoglycans
  • Intermediate Filament Proteins
  • NES protein, human
  • Nerve Tissue Proteins
  • Nes protein, rat
  • Nestin
  • Fibroblast Growth Factor 2
  • 2',3'-Cyclic Nucleotide 3'-Phosphodiesterase
  • Cnp protein, rat