FACS analysis of undifferentiated MAPCs and MAPCs cultured with VEGF. MAPCs (after 40 population doublings; donor age, 28 years) were replated at 2 × 10⁴ cells/cm² in fibronectin-coated wells in serum-free defined medium without EGF or PDGF-BB, but with 10 ng/ml VEGF. Undifferentiated MAPCs at day 0, or VEGF-induced cells recovered after short trypsinization after 3, 9, or 14 days of culture, were stained with Ab’s against β2-microglobulin, HLA class I, MUC18, Flk1, Flt1, VCAM, CD62P, CD62E, vWF, CD31, CD34, CD36, AC133, VE-cadherin, or control IgG. Cells were analyzed by FACS. Plots show isotype control IgG staining profile (thin line) versus specific Ab staining profile (thick line). Each analysis shown is one representative example from a total of three donors. Values on x axes indicate intensity log. (a) Phenotype of undifferentiated MAPCs. MAPCs express low levels of β2-microglobulin, Flk1, Flt1, and AC133, but do not stain with any of the other anti-endothelial markers. (b) Phenotype of MAPCs cultured for 14 days with 10 ng/ml VEGF. MAPCs express high levels of most endothelial markers associated with endothelial cells, but lose expression of AC133. (c) Phenotype of MAPCs cultured for 3–9 days with 10 ng/ml VEGF. MAPCs lose expression of AC133 by day 3 of culture with VEGF, and acquire expression of Tek by day 3, and vWF, CD34, and MUC18 by day 9. β2-mic, β2-microglobulin; VE-cad, VE-cadherin.

Differentiation into endothelial cells requires absence of serum and high density of MAPCs. (a) MAPCs (after 65 population doublings; donor age, 22 years) were replated at 2 × 10⁴ cells/cm² in fibronectin-coated wells in serum-free medium with 10 ng/ml VEGF in the presence or absence of serum (2% FCS). After 9 days, cells were fixed with paraformaldehyde, permeabilized with Triton X-100, and stained with Ab’s against vWF and CD34. Representative example of three experiments, one from each of three different donors. (b) MAPCs (after 65 population doublings; donor age, 22 years) were replated at 2 × 10⁴ cells/cm² or ≤ 1 × 10⁴ cells/cm² in fibronectin-coated wells in serum-free defined medium with 10 ng/ml VEGF. After 9 days, cells were fixed with paraformaldehyde, permeabilized with Triton X-100, and stained with Ab’s against vWF and CD34. Representative example of three experiments from three donors. (c) MAPCs (after 45 population doublings; donor age, 28 years) were replated at 2 × 10⁴ cells/cm² in fibronectin-coated wells in serum-free defined medium with 10 ng/ml VEGF. After 9 days, endothelial cells were further expanded in 10% FCS with 10 ng/ml VEGF for more than 20 population doublings. Representative example of six experiments from six donors.

Further functional characterization of MAPC-derived endothelial cells. (a) Hypoxia upregulates Flk1 and Tek expression on MAPC-derived endothelial cells. MAPC-derived endothelial cells (after 30 population doublings; donor age, 33 years) were incubated at 37°C in 20% or 10% O₂ for 24 hours. Cells were recovered by trypsinization and stained with Ab’s against Flk 1, Tek, and IgG control, and were analyzed by flow cytometry. Plots show isotype control IgG staining profile (thin line) versus specific Ab staining profile (thick line). Representative example of more than three experiments from three donors. Number above plots shows mean fluorescence intensity (MFI) for the control IgG staining and the specific Ab staining. (b) Hypoxia upregulates VEGF production by MAPC-derived endothelial cells. MAPCs (after 30 population doublings; donor age, 45 years) and MAPC-derived endothelial cells were incubated at 37°C in 20% or 10% O₂ for 24 hours. Medium was collected, and VEGF levels were measured by ELISA. Results are shown as mean ± SEM of six experiments from three donors. (c) IL-1α induces expression of HLA-DR, a type of HLAclass II antigens and increases expression of adhesion receptors. MAPC-derived endothelial cells (after 40 population doublings, donor age = 31 years) were incubated with 75 ng/ml IL-1α in serum-free medium for 24 hours. Cells were stained with Ab’s against HLA-class I, HLA class II, β2-microglobulin, vWF, CD31, VCAM, CD62E, CD62P, or control Ab’s, and analyzed using FACS. Plots show isotype control IgG staining profile (thin line) versus specific Ab staining profile (thick line). Representative example of three experiments from three donors. Numbers above each plot show MFI for the control IgG staining and the specific Ab staining. Nl, normal; IL-1 induced populations.

Immunohistochemical evaluation of MAPC-derived endothelial cells. (a) MAPCs (after 65 population doublings; donor age, 22 years) were replated at 2 × 10⁴ cells/cm² in fibronectin-coated wells in serum-free defined medium with 10 ng/ml VEGF. After 14 days, cells were fixed with paraformaldehyde, permeabilized with Triton X-100, and stained with Ab’s against α_vβ₅ (scale bar = 50 μm), ZO-1, β-catenin, and γ-catenin. Cells were then evaluated by confocal fluorescence microscopy. Typical membrane staining is seen for the adhesion, α_vβ₅, and for the adhesion junction proteins ZO-1, β-catenin, and γ-catenin. Representative example from one of three total donors. Scale bar = 50 μm. (b) Morphology of MAPCs at day 0 (upper panel) and day 21 (lower panel) after VEGF treatment in bright-field microscopy. Scale bar = 25 μm.

Functional characterization of MAPC-derived endothelial cells. MAPCs (after 45 population doublings; donor age, 28 years) were replated at 2 × 10⁴ cells/cm² in fibronectin-coated wells in serum-free defined medium with 10 ng/ml VEGF for 14 days. (a) Histamine-mediated release of vWF from MAPC-derived endothelium. MAPC-derived endothelial cells were plated at 10⁴ cells/cm² in fibronectin-coated chamber slides. After 24 hours, cells were treated with 10 μM histamine in serum-free medium with VEGF for 25 minutes. Cells were then stained with Ab’s against vWF (FITC) and myosin (Cy3). vWF staining is found throughout the cytoplasm in untreated cells, but decreased and was detectable only in the perinuclear region following treatment with histamine. Staining with Ab’s against myosin shows cytoskeletal changes with increased numbers of myosin stress fibers, and widening of gap junctions. Representative example of three experiments from three donors. Scale bar = 60 μm. (b) MAPC-derived endothelium takes up a-LDL. MAPCs induced to differentiate with VEGF for 3, 7, and 9 days with VEGF were incubated with DiI-Ac-LDL. Cells were colabeled with Ab’s against Tek, Tie1, or vWF, and analyzed by confocal microscopy. After 3 days, we detected expression of Tek, but no uptake of a-LDL. After 7 days, cells expressed Tie1, but again did not take up a-LDL. However, acquisition of expression of vWF on day 9 was associated with uptake of a-LDL. Representative example of ten experiments from six donors. Scale bar = 100 μm. (c) Vascular tube formation by MAPC-derived endothelium. MAPC-derived endothelial cells were replated in Matrigel with VEGF. After 6 hours, typical vascular tubes could be seen. Representative example of six experiments from six donors. Scale bar = 200 μm.

Contribution of human MAPC-derived endothelial cells to neoangiogenesis in tumors and wound healing. (a–g) MAPC-derived endothelial cells (0.25 × 10⁶) (after 30–65 population doublings before differentiation) were injected intravenously into NOD-SCID mice after implantation of murine Lewis lung carcinoma spheroids (n = 5) from three donors, aged 19, 28, and 31 years). After 2 weeks, animals were sacrificed, and tumors were removed, sectioned, and stained with either anti-human β2-microglobulin–FITC or anti-mouse CD31–FITC and anti–vWF-Cy3. Shown are the 3D reconstructed figures of 350 images for either anti-human β2-microglobulin–FITC (c) or anti-mouse CD31–FITC (false colored as blue) (d), and merging of the two (e); anti–vWF-Cy3 (f); and merging of the three staining patterns (g). (a and b) Scale bar = 100 μm. (h) Wound healing. Ears of NOD-SCID mice used in the studies described in a were punched 3 and 5 days prior to intravenous injection of human MAPC-derived endothelial cells (a–c) or human foreskin fibroblasts (d–f). After 14 days, animals were sacrificed and ears were obtained and cryopreserved. Five-micrometer slides were stained with anti-human β2-microglobulin–FITC and anti–vWF-Cy3. Scale bar = 20 μm. C, cartilage; D, dermis. (i) Tumor angiogenesis is derived from endothelial cells generated in vivo from MAPCs. MAPCs (10⁶) (after 45 population doublings; donor age, 28 years) were injected intravenously into a NOD-SCID mouse (n = 1). After 12 weeks, the animal was sacrificed, at which time a thymic tumor was detected. Ten-micrometer slides were stained with anti-human β2-microglobulin–FITC and anti–vWF-Cy3. Shown is a highly vascularized area in the tumor stained with Ab’s against β2-microglobulin–FITC, vWF, and TOPRO-3 (not shown). Scale bar = 20 μm.