title: Development of a surrogate potency assay to determine the angiogenic activity of Stempeucel®, a pooled, ex-vivo expanded, allogeneic human bone marrow mesenchymal stromal cell product.
abstract: Mesenchymal stromal cells (MSCs) have emerged as a more beneficial alternative to conventional therapy and may offer a potential cure for unmet medical needs. MSCs are known to possess strong immunomodulatory and anti-inflammatory properties. Moreover, they promote angiogenesis and tissue regeneration through the secretion of trophic factors. For these reasons, the past decade witnessed a sharp increase in the number of clinical trials conducted with stem cells for various vascular diseases requiring angiogenesis. In this study, we evaluated the in vitro angiogenic potency of Stempeucel®, which is an allogeneic pooled human bone marrow-derived mesenchymal stromal cell (phBMMSC) product. We previously established the safety of Stempeucel® in our pre-clinical studies, and clinical trials conducted for critical limb ischaemia and acute myocardial infarction.
  
  
  
  Because the proposed mechanism of action of phBMMSCs is mainly through the secretion of pro-angiogenic cytokines, we developed a surrogate potency assay by screening various batches of large-scale expanded phBMMSCs for the expression of angiogenic factors and cytokines through gene expression and growth factor analyses, followed by in vitro functional assays.
  
  
  
  The well characterized angiogenic vascular endothelial growth factor (VEGF) was selected and quantified in twenty six manufactured batches of phBMMSCs to establish consistency following the United States Food and Drug Administration recommendations. According to recommendations 21 CFR 211.165(e) and 211.194(a)(2), we also established and documented the specificity and reproducibility of the test methods employed through validation. Moreover, we also attempted to elucidate the mechanism of action of the cell population to ensure appropriate biological activity. The functional role of VEGF has been established through in vitro angiogenic assays and a dose-dependent correlation was observed with in vitro functional results.
  
  
  
  The data generated from this study suggest the selection of VEGF as a single surrogate marker to test the angiogenic potency of phBMMSCs. Our study reports the quantification of VEGF in twenty six batches of large-scale manufactured phBMMSCs, and a concentration-dependent correlation of secreted VEGF to endothelial cell functions of migration, proliferation and tube formation, in the conditioned medium obtained from nine phBMMSC batches. To our cognizance, this is the first study in which a single angiogenic factor (VEGF) has been qualified as a surrogate potency marker through all three in vitro functional assays to determine the angiogenic potency of the phBMMSC population.
authors.affiliation: Stempeutics Research Pvt. Ltd, Akshay Tech Park, #72 & 73, 2nd Floor, EPIP Zone, Phase 1, Bangalore, Whitefield, 560066, India.|Manipal University, Manipal, Karnataka, India.
authors.forename: Charan
authors.initials: C
authors.lastname: Thej
authors.affiliation: Stempeutics Research Pvt. Ltd, Akshay Tech Park, #72 & 73, 2nd Floor, EPIP Zone, Phase 1, Bangalore, Whitefield, 560066, India.
authors.forename: Balamurugan
authors.initials: B
authors.lastname: Ramadasse
authors.affiliation: Stempeutics Research Pvt. Ltd, Akshay Tech Park, #72 & 73, 2nd Floor, EPIP Zone, Phase 1, Bangalore, Whitefield, 560066, India.
authors.forename: Ankita
authors.initials: A
authors.lastname: Walvekar
authors.affiliation: Stempeutics Research Pvt. Ltd, Akshay Tech Park, #72 & 73, 2nd Floor, EPIP Zone, Phase 1, Bangalore, Whitefield, 560066, India. anish.majumdar@stempeutics.com.
authors.forename: Anish S
authors.initials: AS
authors.lastname: Majumdar
authors.affiliation: Stempeutics Research Pvt. Ltd, Akshay Tech Park, #72 & 73, 2nd Floor, EPIP Zone, Phase 1, Bangalore, Whitefield, 560066, India. sudha.b@stempeutics.com.
authors.forename: Sudha
authors.initials: S
authors.lastname: Balasubramanian
mesh_terms: D043925:Angiogenesis Inducing Agents / Q000737:chemistry / Q000276:immunology*; D001681:Biological Assay / Q000592:standards*; D001854:Bone Marrow Cells / Q000166:cytology / Q000276:immunology* / Q000378:metabolism; D002465:Cell Movement / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D054377:Chemokine CXCL12 / Q000235:genetics / Q000276:immunology; D017077:Culture Media, Conditioned / Q000737:chemistry; D015870:Gene Expression; D017228:Hepatocyte Growth Factor / Q000235:genetics / Q000276:immunology; D061307:Human Umbilical Vein Endothelial Cells / Q000166:cytology / Q000187:drug effects / Q000276:immunology; D006801:Humans; D015850:Interleukin-6 / Q000235:genetics / Q000276:immunology; D016209:Interleukin-8 / Q000235:genetics / Q000276:immunology; D045164:Mesenchymal Stem Cell Transplantation; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000276:immunology* / Q000378:metabolism; D061251:Primary Cell Culture; D012259:Ribonuclease, Pancreatic / Q000235:genetics / Q000276:immunology; D053773:Transforming Growth Factor beta1 / Q000235:genetics / Q000276:immunology; D014184:Transplantation, Homologous; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics / Q000276:immunology*
chemical_list: D043925:Angiogenesis Inducing Agents; C515255:CXCL12 protein, human; D054377:Chemokine CXCL12; D017077:Culture Media, Conditioned; C066855:HGF protein, human; C508600:IL6 protein, human; D015850:Interleukin-6; D016209:Interleukin-8; D053773:Transforming Growth Factor beta1; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D017228:Hepatocyte Growth Factor; C046366:angiogenin; D012259:Ribonuclease, Pancreatic
keywords: Angiogenesis; Endothelial tube formation; Pooled human bone marrow-derived mesenchymal stromal cell; Potency assay; Vascular endothelial growth factor