title: Ixmyelocel-T, an expanded multicellular therapy, contains a unique population of M2-like macrophages.
abstract: BACKGROUND
  M2 macrophages promote tissue repair and regeneration through various mechanisms including immunomodulation and scavenging of tissue debris. Delivering increased numbers of these cells to ischemic tissues may limit tissue injury and promote repair. Ixmyelocel-T is an expanded, autologous multicellular therapy cultured from bone-marrow mononuclear cells (BMMNCs). The purpose of this study was to characterize further a unique expanded population of M2-like macrophages, generated in ixmyelocel-T therapy.
  
  
  METHODS
  Approximately 50 ml of whole bone marrow was obtained from healthy donors and shipped overnight. BMMNCs were produced by using density-gradient separation and cultured for approximately 12 days to generate ixmyelocel-T. CD14+ cells were isolated from ixmyelocel-T with positive selection for analysis. Cell-surface phenotype was examined with flow cytometry and immunofluorescence, and expression of cytokines and chemokines was analyzed with enzyme-linked immunosorbent assay (ELISA). Quantitative real-time PCR was used to analyze expression of genes in BMMNCs, ixmyelocel-T, the CD14+ population from ixmyelocel-T, and M1 and M2 macrophages. Ixmyelocel-T was cultured with apoptotic BMMNCs, and then visualized under fluorescence microscopy to assess efferocytosis.
  
  
  RESULTS
  Macrophages in ixmyelocel-T therapy expressed surface markers of M2 macrophages, CD206, and CD163. These cells were also found to express several M2 markers, and few to no M1 markers. After stimulation with lipopolysaccharide (LPS), they showed minimal secretion of the proinflammatory cytokines interleukin-12 (IL-12) and tumor necrosis factor alpha (TNF-α) compared with M1 and M2 macrophages. Ixmyelocel-T macrophages efficiently ingested apoptotic BMMNCs.
  
  
  CONCLUSIONS
  Ixmyelocel-T therapy contains a unique population of M2-like macrophages that are characterized by expression of M2 markers, decreased secretion of proinflammatory cytokines after inflammatory stimuli, and efficient removal of apoptotic cells. This subpopulation of cells may have a potential role in tissue repair and regeneration.
authors.forename: Kelly J
authors.initials: KJ
authors.lastname: Ledford
authors.forename: Frank
authors.initials: F
authors.lastname: Zeigler
authors.forename: Ronnda L
authors.initials: RL
authors.lastname: Bartel
mesh_terms: D015703:Antigens, CD / Q000378:metabolism; D015214:Antigens, Differentiation, Myelomonocytic / Q000378:metabolism; D001854:Bone Marrow Cells / Q000166:cytology*; D002478:Cells, Cultured; D006801:Humans; D018664:Interleukin-12 / Q000378:metabolism; D037181:Lectins, C-Type / Q000378:metabolism; D018950:Lipopolysaccharide Receptors / Q000378:metabolism; D008070:Lipopolysaccharides / Q000633:toxicity; D008264:Macrophages / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D000090323:Mannose Receptor; D037241:Mannose-Binding Lectins / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D000076202:c-Mer Tyrosine Kinase; D000098902:CD163 Antigen
chemical_list: D015703:Antigens, CD; D015214:Antigens, Differentiation, Myelomonocytic; D000098902:CD163 Antigen; D037181:Lectins, C-Type; D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides; D000090323:Mannose Receptor; D037241:Mannose-Binding Lectins; D011518:Proto-Oncogene Proteins; D011956:Receptors, Cell Surface; D014409:Tumor Necrosis Factor-alpha; D018664:Interleukin-12; C097072:MERTK protein, human; D020794:Receptor Protein-Tyrosine Kinases; D000076202:c-Mer Tyrosine Kinase