title: Comparison of the neuropoietic activity of gene-modified versus parental mesenchymal stromal cells and the identification of soluble and extracellular matrix-related neuropoietic mediators.
abstract: BACKGROUND
  Transplanting mesenchymal stromal cells (MSCs) or their derivatives into a neurodegenerative environment is believed to be beneficial because of the trophic support, migratory guidance, immunosuppression, and neurogenic stimuli they provide. SB623, a cell therapy for the treatment of chronic stroke, currently in a clinical trial, is derived from bone marrow MSCs by using transient transfection with a vector encoding the human Notch1 intracellular domain. This creates a new phenotype, which is effective in experimental stroke, exhibits immunosuppressive and angiogenic activity equal or superior to parental MSCs in vitro, and produces extracellular matrix (ECM) that is exceptionally supportive for neural cell growth. The neuropoietic activity of SB623 and parental MSCs has not been compared, and the SB623-derived neuropoietic mediators have not been identified.
  
  
  METHODS
  SB623 or parental MSCs were cocultured with rat embryonic brain cortex cells on cell-derived ECM in a previously characterized quantitative neuropoiesis assay. Changes in expression of rat neural differentiation markers were quantified by using rat-specific qRT-PCR. Human mediators were identified by using expression profiling, an enzymatic crosslinking activity, and functional interference studies by means of blocking antibodies, biologic inhibitors, and siRNA. Cocultures were immunolabeled for presynaptic vesicular transporters to assess neuronal specialization.
  
  
  RESULTS
  Among six MSC/SB623 pairs, SB623 induced expression of rat neural precursor, oligodendrocyte, and astrocyte markers on average 2.6 to 3 times stronger than did their parental MSCs. SB623 expressed significantly higher FGF2, FGF1, and BMP4, and lower FGFR1 and FGFR2 levels; and human FGF1, FGF2, BMPs, and HGF were implicated as neuropoietic mediators. Neural precursors grew faster on SB623- than on MSC-derived ECM. SB623 exhibited higher expression levels and crosslinking activity of tissue transglutaminase (TGM2). TGM2 silencing reduced neural precursor growth on SB623-ECM. SB623 also promoted the induction of GABA-ergic, but not glutamatergic, neurons more effectively than did MSCs.
  
  
  CONCLUSIONS
  These data demonstrate that SB623 cells tend to support neural cell growth more effectively than their parental MSCs and identify both soluble and insoluble mediators responsible, at least in part, for enhanced neuropoietic potency of SB623. The neuropoiesis assay is a useful tool for identifying beneficial factors produced by MSCs and their derivatives.
authors.forename: Irina
authors.initials: I
authors.lastname: Aizman
authors.forename: Brenna J
authors.initials: BJ
authors.lastname: Tirumalashetty
authors.forename: Michael
authors.initials: M
authors.lastname: McGrogan
authors.forename: Casey C
authors.initials: CC
authors.lastname: Case
mesh_terms: D000328:Adult; D000818:Animals; D001253:Astrocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D055415:Bone Morphogenetic Protein 4 / Q000235:genetics / Q000378:metabolism; D002540:Cerebral Cortex / Q000166:cytology; D017077:Culture Media, Conditioned / Q000494:pharmacology; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D005109:Extracellular Matrix / Q000378:metabolism*; D016220:Fibroblast Growth Factor 1 / Q000235:genetics / Q000378:metabolism; D016222:Fibroblast Growth Factor 2 / Q000235:genetics / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism; D017228:Hepatocyte Growth Factor / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008297:Male; D059630:Mesenchymal Stem Cells / Q000378:metabolism*; D058953:Neural Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D055495:Neurogenesis; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D051381:Rats; D051881:Receptor, Notch1 / Q000235:genetics* / Q000378:metabolism; D017468:Receptors, Fibroblast Growth Factor / Q000235:genetics / Q000378:metabolism; D011503:Transglutaminases / Q000235:genetics / Q000378:metabolism
chemical_list: D055415:Bone Morphogenetic Protein 4; D017077:Culture Media, Conditioned; C497761:NOTCH1 protein, human; D051881:Receptor, Notch1; D017468:Receptors, Fibroblast Growth Factor; C000717608:TGM2 protein, human; C000717609:Tgm2 protein, rat; D016222:Fibroblast Growth Factor 2; D016220:Fibroblast Growth Factor 1; D017228:Hepatocyte Growth Factor; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D011503:Transglutaminases; D019204:GTP-Binding Proteins