In vitro endothelial potential of human UC blood-derived mesenchymal stem cells  E.J. Gang, J.A. Jeong, S. Han, Q. Yan, C.-J. Jeon, H. Kim, PhD  Cytotherapy  Volume 8, Issue 3, Pages 215-227 (January 2006) DOI: 10.1080/14653240600735933 Copyright © 2006 International Society for Cellular Therapy Terms and Conditions

Figure 1 Morphologic, proliferative and immunophenotypic characterization of human UCB-derived adherent cells. (a) Morphology at a 40× magnification. (b) Growth curve measured by a trypan blue exclusion assay with three different cell cultures, and indicated by mean values of positive cells±SD. (c) Immunophenotype of an adherent cell population that was derived from human UCB. An open profile represents an isotype control for background fluorescence and a shaded one shows a positive signal. Cytotherapy 2006 8, 215-227DOI: (10.1080/14653240600735933) Copyright © 2006 International Society for Cellular Therapy Terms and Conditions

Figure 2 Immunophenotypic changes of UCB-derived MSC upon endothelial differentiation. The cells were incubated for up to 3 weeks with endothelial differentiation medium containing human VEGF, EGF and hydrocortisone. Expressions of endothelial lineage surface markers Flk-1, Flt-1, VE-Cadherin, vWF and VCAM-1 were analyzed by flow cytometry. Distribution of cells stained against corresponding Ab is shown as a shaded profile. An open profile represents an isotype control used to identify background fluorescence. Cytotherapy 2006 8, 215-227DOI: (10.1080/14653240600735933) Copyright © 2006 International Society for Cellular Therapy Terms and Conditions

Figure 3 RT-PCR and IF analyzes of endothelia-specific cell-surface markers. (a) Total RNA was extracted from the native and differentiated UCB-derived MSC. Gene expression of Flt-1, Flk-1, VE-cadherin, vWF, Tie-1 and Tie-2 was analyzed by RT-PCR. Messenger RNA of β-actin was used as an internal control. (b) UCB-derived MSC were cultured with endothelial differentiation medium for 2 weeks and stained with monoclonal mouse Ab raised against vWF and VE-cadherin, and then with FITC-conjugated anti-mouse IgG. After nuclei were stained with DAPI, the cells were observed by fluorescence microscopy (magnification 100×). Cytotherapy 2006 8, 215-227DOI: (10.1080/14653240600735933) Copyright © 2006 International Society for Cellular Therapy Terms and Conditions

Figure 4 Cytokine secretion profiling by Ab arrays. (a) Layouts of cytokine arrays with abbreviated names for cytokines. (b) Membrane hybridization signals of fresh endothelial medium. (c) Membrane hybridization signals from culture medium, with which UCB-derived MSC were incubated for 2 weeks. The full-name of cytokines are as follows: POS, positive controls; BNK, blank; NEG, negative controls; ANG, angiogenin; BNDF, brain-derived neurotrophic factor; BLC, B-lymphocyte chemoattractant; BMP, bone morphogenetic protein; CK, chemokine; CNTF, ciliary neuronotrophic factor; EGF, epidermal growth factor; FGF, fibroblast growth factor; FLT3L, fms-like tyrosine kinase-3 ligand; FKN, fractalkine; GCP, granulocyte chemotactic protein; GDNF, glial-derived neurotrophic factor; GM-CSF, granulocyte–macrophage colony-stimulating factor; IFN, interferon; IGFBP, insulin-like growth factor binding protein; IGF, insulin-like growth factor; IL, interleukin; IL-1RA, interleukin-1 receptor antagonist; LIGHT, homologous to lymophotoxin and identical with HVEM ligand; MCP, monocyte chemoattractant protein; M-CSF, macrophage colony-stimulating factor; MDC macrophage-derived chemokine; MIG, monokine induced by interferon-γ; MIP, macrophage inflammatory protein; NAP, neutrophil-activating peptide; NT, neurotrophin; PARC, pulmonary and activation-regulated chemokine; PDGF-BB, platelet-derived growth factor BB; RANTES, regulated upon activation, normal T-cell expressed and presumably secreted; SCF, stem cell factor; SDF, stromal cell-derived factor; TARC, thymus and activation-regulated chemokine; TGF, transforming growth factor; TNF, tumor necrosis factor; Acrp30, adipocyte complement-related protein of 30 kDa; AgRP, agouti-related protein homolog; AR, amphiregulin; Axl, AXL receptor tyrosine kinase; bFGF, basic fibroblast growth factor; BTC, betacellulin; CCL, CC-type chemokine ligand; CTACK, cuteaneous T-cell attracting chemokine; Dtk, developmental tyrosine kinase; EGF-R, epidermal growth factor-related protein; ENA, epithelial neutrophil-activating protein; Fas, Fas antigen; GCSF, granulocyte colony-stimulating factor; GITR-L, glucocorticoid-induced tumor necrosis factor receptor family-related gene ligand; GRO, growth-related oncogene; HCC, hemofiltrate CC chemokine; HGF, hepatocyte growth factor; ICAM, intercellular adhesion molecule; IGF-I SR, insulin-like growth factor I soluble receptor; IL-12p40, interleukin 12 p40 subunit; I-TAC, interferon-inducible T-cell alpha chemoattractant; Ltn, lymphotactin; MIF, mesoderm-inducing factor; MIP, macrophage inflammatory protein; MSP, macrophage-stimulating protein; OPG, osteoprotegerin; OSM, oncostatin M; PIGF, placental growth factor; spg130,; sTNFR, soluble TNF receptor; TECK, thymus-expressed chemokine; TIMP, tissue inhibitor of metalloproteinase; TPO, thrombopoietin; TRAIL, TNF-related apoptosis-inducing ligand; uPAR, urokinase plasminogen activator receptor; VEGF, vascular endothelial growth factor. Cytotherapy 2006 8, 215-227DOI: (10.1080/14653240600735933) Copyright © 2006 International Society for Cellular Therapy Terms and Conditions

Figure 5 LDL uptake and capillary network formation analyzes. (a) The native and differentiated UCB-derived MSC were incubated for 24h with DiI-labeled acetylated LDL. Cellular incorporation of LDL was measured by flow cytometry. A shaded profile indicates distribution of DiI-labeled cells while an open one represents a control. (b) After nuclei were stained with DAPI, the cells were visualized by fluorescence microscopy (magnification ×40). (c) Cells incubated for 2 weeks in endothelial differentiation medium were detached and replated into 96-well plates coated with ECMatrix gel 1h prior to cell seeding. Capillary structure formation was measured by in vitro angiogenesis assay kit. The vessel-like tube formed within 6h after plating and was photographed under a phase-contrast microscope with a magnification of ×40. Cytotherapy 2006 8, 215-227DOI: (10.1080/14653240600735933) Copyright © 2006 International Society for Cellular Therapy Terms and Conditions