title: Expansion and angiogenic potential of mesenchymal stem cells from patients with critical limb ischemia.
abstract: BACKGROUND
  Critical limb ischemia (CLI) is a life- and limb-threatening condition affecting 1% to 10% of the population with peripheral arterial disease. Traditional revascularization options are not possible for up to 50% of CLI patients, in which case, the use of cellular therapies, such as bone marrow-derived mesenchymal stem cells (MSCs), hold great promise as an alternative revascularization therapy. However, no randomized, controlled phase 3 trials to date have demonstrated an improvement in limb salvage with cellular therapies. This may be due to poor cell quality (ie, inability to generate a sufficient number of angiogenic MSCs) or to the inadequate retention and viability of MSCs after delivery, or both. Because concerns remain about the expansion and angiogenic potential of autologous MSCs in the CLI population, the objective of this study was to examine the effect of our novel culture media supplement, pooled human platelet lysate (PL), in lieu of the standard fetal bovine serum (FBS), to improve the expansion potential of MSCs from CLI patients. We also characterized the in vitro angiogenic activity of MSCs from the tibia of amputated CLI limbs compared with MSCs from healthy donors.
  
  
  METHODS
  MSCs were obtained from the tibia of four CLI patients (ISC) and four ISC patients with diabetes mellitus (ISC+DM) undergoing major amputation. Healthy MSCs were aspirated from the iliac crest of four young and healthy donors. MSCs were isolated and expanded in culture with PL or FBS. MSCs from passage 3 to 6 were used for phenotypic marker expression and for adipogenic and osteogenic differentiation and were tested for their in vitro angiogenic activity on human microdermal endothelial cells. In parallel MSCs were cultured to passage 11 for population-doubling calculations.
  
  
  RESULTS
  MSCs from ISC and ISC+DM patients and from healthy patients exhibited appropriate expression of cell surface markers and differentiation capacity. Population doublings were significantly greater for PL-stimulated compared with FBS-stimulated MSCs in all groups. Biologically active amounts of angiogens were identified in the secretome of all MSCs without consistent trends among groups. PL expansion did not adversely affect the angiogenic activity of MSCs compared with FBS. The ISC and ISC+DM MSCs demonstrated angiogenic effects on endothelial cells similar to those of healthy and ISC MSCs.
  
  
  CONCLUSIONS
  PL promotes the rapid expansion of MSCs from CLI and healthy persons. Importantly, MSCs expanded from CLI patients demonstrate the desired angiogenic activity compared with their healthy counterparts. We conclude that autologous MSCs from CLI patients can be sufficiently expanded with PL and be expected to deliver requisite angiogenic effects in vivo. We expect the improved expansion of ISC and ISC+DM with PL to be helpful in improving the successful delivery of autologous MSCs to patients with CLI.
authors.affiliation: Department of Surgery, Emory University School of Medicine, Atlanta, Ga; Surgical and Research Services, Atlanta Veterans Affairs Medical Center, Atlanta, Ga; Parker H. Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, Ga. Electronic address: lbrewst@emory.edu.
authors.forename: Luke
authors.initials: L
authors.lastname: Brewster
authors.affiliation: Department of Surgery, Emory University School of Medicine, Atlanta, Ga.
authors.forename: Scott
authors.initials: S
authors.lastname: Robinson
authors.affiliation: Department of Surgery, Emory University School of Medicine, Atlanta, Ga.
authors.forename: Ruoya
authors.initials: R
authors.lastname: Wang
authors.affiliation: Parker H. Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, Ga.
authors.forename: Sarah
authors.initials: S
authors.lastname: Griffiths
authors.affiliation: Department of Surgery, Emory University School of Medicine, Atlanta, Ga.
authors.forename: Haiyan
authors.initials: H
authors.lastname: Li
authors.affiliation: Department of Biology, Morehouse College, Atlanta, Ga.
authors.forename: Alexandra
authors.initials: A
authors.lastname: Peister
authors.affiliation: Parker H. Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, Ga; Department of Hematology and Medical Oncology, Emory University School of Medicine, Atlanta, Ga.
authors.forename: Ian
authors.initials: I
authors.lastname: Copland
authors.affiliation: Parker H. Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, Ga; Wallace H. Coulter Department of Biomedical Engineering at Georgia Institute of Technology, Atlanta, Ga.
authors.forename: Todd
authors.initials: T
authors.lastname: McDevitt
mesh_terms: D050156:Adipogenesis; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000671:Amputation, Surgical; D042501:Angiogenic Proteins / Q000378:metabolism; D015415:Biomarkers / Q000378:metabolism; D016022:Case-Control Studies; D002450:Cell Communication; D019070:Cell Lineage; D049109:Cell Proliferation; D002469:Cell Separation / Q000379:methods; D002478:Cells, Cultured; D018920:Coculture Techniques; D016638:Critical Illness; D003925:Diabetic Angiopathies / Q000175:diagnosis / Q000473:pathology* / Q000503:physiopathology / Q000601:surgery; D042783:Endothelial Cells / Q000378:metabolism; D005260:Female; D006801:Humans; D007511:Ischemia / Q000175:diagnosis / Q000473:pathology* / Q000503:physiopathology / Q000601:surgery; D035002:Lower Extremity / Q000098:blood supply*; D008297:Male; D059630:Mesenchymal Stem Cells / Q000378:metabolism / Q000473:pathology*; D008875:Middle Aged; D018919:Neovascularization, Physiologic; D010012:Osteogenesis; D010641:Phenotype; D013977:Tibia / Q000378:metabolism / Q000473:pathology* / Q000601:surgery; D013997:Time Factors; D055815:Young Adult
chemical_list: D042501:Angiogenic Proteins; D015415:Biomarkers