Umbilical cord blood-selected CD133(+) cells exhibit vasculogenic functionality in vitro and in vivo.

BACKGROUND AIMS: Current clinical trials utilize non-selected bone marrow (BM) mononuclear cells (MNC) to augment vasculo genesis within ischemic vascular beds. Recent reports have identified a diminished number and function of hemat-opoietic stem cells (HSC) from aged and diseased patients. Umbilical cord blood (UCB) provides a potential robust allo-geneic source of HSC for therapeutic vasculogenesis. METHODS: MNC and magnetically isolated CD133(+) cells were assessed for viability (trypan blue) and surface phenotype (flow cytometry). To test in vivo functionality of the cells, NOD/SCID mice underwent ligation of the right femoral artery followed immediately by cell injection. Blood flow recovery, necrosis, BM engraftment of human cells and histologic capillary density were determined. Cells were tested for potential mechanisms mediating the in vivo effects, including migration, cytokine secretion and angiogenic augmentation (Matrigel assays). RESULTS: Surface expression analysis showed CD31 (PECAM) expression was greatly increased in UCB CD133(+) cells compared with BM MNC. At 28 days, perfusion ratios were highest in animals receiving UCB CD133(+) cells, while animals receiving BM CD133(+) cells and BM MNC demonstrated perfusion ratios statistically higher than in animals treated with cytokine media alone. Animals receiving CD133(+) cells showed a statistically higher capillary density, reduced severe digit necrosis and increased engraftment in the BM than animals treated with unselected BM MNC. In vitro studies showed equivalent migration to stromal-derived factor-1 (SDF-1), increased production of tumor necrosis factor alpha (TNF-alpha) and increased branch points with the co-incubation of CD133(+) cells with human umbilical vein endothelial cells (HUVEC) in the Matrigel angiogenesis assay. CONCLUSIONS: Taken together, UCB CD133(+) cells exhibit robust vasculogenic functionality compared with BM MNC in response to ischemia.

Influence of human leucocyte antigen disparity and graft lymphocytes on allogeneic engraftment and survival after umbilical cord blood transplant in adults.

The dose of graft-nucleated cells and CD34(+) haematopoietic progenitor cells are predictors of allogeneic engraftment and survival in umbilical cord blood (UCB) recipients. In this single institution prospective phase II trial, flow cytometric analyses of CD34(+) progenitor and lymphocyte populations in unmodified single unit human leucocyte antigen (HLA)-disparate UCB grafts infused into 31 consecutive adults (median age 41 years, range 20-64) receiving myeloablative conditioning were compared with clinical outcomes. Median infused UCB graft-nucleated cells and CD34(+) dose was 2.2 x 10(7)/kg and 1.2 x 10(5)/kg respectively. Day to absolute neutrophil count >/=0.5 x 10(9)/l with full donor chimerism averaged 27 d (range 12-41). Univariate analyses demonstrated that UCB graft-infused cell doses of CD34(+) (P = 0.015), CD3(+) (P = 0.024) and CD34(+)HLADR(+)CD38(+) progenitors (P = 0.043) correlated with neutrophil engraftment. This same analysis did not demonstrate a correlation between CD34(+) (P = 0.11), CD3(+) (P = 0.28) or CD34(+)HLADR(+)CD38(+) (P = 0.108) cell dose and event-free survival (EFS). High-resolution matching for HLA-class II (DRB1) resulted in improved EFS (P = 0.02) and decreased risk for acute graft-versus-host disease (GVHD) (P = 0.004). Early mortality (prior to post-transplant day +28) occurred in three patients, while 26 patients achieved myeloid engraftment. These results suggest that UCB graft matching at DRB1 is an important risk factor for acute GVHD and survival, while higher UCB graft cell doses of CD34(+), committed CD34(+) progenitors and CD3(+) T cells favourably influence UCB allogeneic engraftment.

The safety of autologous intracoronary stem cell injections in a porcine model of chronic myocardial ischemia.

BACKGROUND: Intracoronary mononuclear cell therapy may produce angiogenesis in chronic myocardial ischemia. Potential complications include periprocedural infarction secondary to: reduced coronary blood flow; hyperviscosity from the cell preparation; or microvascular dysfunction. To date, no studies to evaluate these potential complications have been reported. The objective of this report was to study the safety and feasibility of intracoronary injections of autologous bone marrow mononuclear cells in a porcine chronic myocardial ischemia model. METHODS: Domestic pigs (n = 5) underwent ameroid cuff placement of the left circumflex artery. Bone marrow-derived mononuclear cells [15 x 10(6) cells] labeled with CM dioctadecyl tetramethylindocarbocyanine were given by intracoronary injection. Animals were sacrificed, and hearts and vital organs were inspected grossly and by histopathology, and bone marrow underwent immunofluorescence microscopy. RESULTS: Troponin I levels, gross inspection and histopathology did not reveal evidence of myocardial infarction. Labeled cells were observed in perivascular structures in myocardium at the injection site in all animals and in the spleen from one animal. Bone marrow aspirates indicated labeled cells. CONCLUSIONS: Intracoronary injection of autologous mononuclear cells in a porcine chronic myocardial ischemia model appears safe. Intracoronary injection resulted in cell localization in the perivascular areas of myocardium supplied by the injected vessel. Cell localization was observed only in the spleen in just one animal. Labeled cells were identified in bone marrow aspirates from three animals following injection, suggesting a role for bone marrow engraftment and repopulation as a possible mechanism for progenitor cell localization in myocardium.

Direct comparison of umbilical cord blood versus bone marrow-derived endothelial precursor cells in mediating neovascularization in response to vascular ischemia.

Endothelial precursor cells (EPCs) cultured from adult bone marrow (BM) have been shown to mediate neovasculogenesis in murine models of vascular injury. We sought to directly compare umbilical cord blood (UCB)- and BM-derived EPC surface phenotypes and in vivo functional capacity. UCB and BM EPCs derived from mononuclear cells (MNC) were phenotyped by surface staining for expression of stromal (Stro-1, CXCR4, CD105, and CD73), endothelial (CD31, CD146, and vascular endothelial [VE]-cadherin), stem cell (CD34 and CD133), and monocyte (CD14) surface markers and analyzed by flow cytometry. The nonobese diabetic/severe combined immunodeficiency murine model of hind-limb ischemia was used to analyze the potential of MNCs and culture-derived EPCs from UCB and BM to mediate neovasculogenesis. Histologic evaluation of the in vivo studies included capillary density as a measure of neovascularization. Surface CXCR4 expression was notably higher on UCB-derived EPCs (64.29%+/-7.41%) compared with BM (19.69%+/-5.49%; P=.021). Although the 2 sources of EPCs were comparable in expression of endothelial and monocyte markers, BM-derived EPCs contained higher proportions of cells expressing stromal cell markers (CD105 and CD73). Injection of UCB- or BM-derived EPCs resulted in significantly improved perfusion as measured by laser Doppler imaging at days 7 and 14 after femoral artery ligation in nonobese diabetic/severe combined immunodeficiency mice compared with controls (P<.05). Injection of uncultured MNCs from BM or UCB showed no significant difference from control mice (P=.119; P=.177). Tissue samples harvested from the lower calf muscle at day 28 demonstrated increased capillary densities in mice receiving BM- or UCB-derived EPCs. In conclusion, we found that UCB and BM-derived EPCs differ in CXCR4 expression and stromal surface markers but mediate equivalent neovasculogenesis in vivo as measured by Doppler flow and histologic analyses.

Effect of ibuprofen on neutrophil migration in vivo in cystic fibrosis and healthy subjects.

Long-term treatment with ibuprofen twice daily, at doses that achieve peak plasma concentration (Cmax) >50 microg/ml, slows progression of lung disease in patients with cystic fibrosis (CF). Previous data suggest that Cmax >50 microg/ml is associated with a reduction in neutrophil (PMN) migration into the lung and that lower concentrations are associated with an increase in PMN migration. To estimate the threshold concentration at which ibuprofen is associated with a decrease in PMN migration in vivo, we measured the PMN content of oral mucosal washes in 35 healthy (age 19-40 years) and 16 CF (age 18-32 years) subjects who took ibuprofen twice daily for 10 days in doses that achieved Cmax 8 to 90 microg/ml. Cmax >50 microg/ml was associated with a 31 +/- 7% (mean +/- S.E.M.) reduction in PMNs in CF (n = 11, p < 0.001) and 25 +/- 6% reduction in PMNs in healthy subjects (n = 16, p < 0.001). Increasing concentrations above 50 microg/ml was not associated with a greater decrease in PMNs. The reduction in PMN migration was consistently present 12 h after a dose, but not after 24 h. Cmax <50 microg/ml was associated with an increase in PMNs of approximately 40%. These results suggest that Cmax >50 microg/ml and twice daily dosing of ibuprofen are required to decrease PMN migration, and reinforce the current recommendation that pharmacokinetics should be performed in CF patients prescribed ibuprofen.