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Blood, Vol. 109, Issue 11, 4761-4768, June 1, 2007
In vivo vasculogenic potential of human blood-derived endothelial progenitor cells
Blood Melero-Martin et al.
109: 4761
Supplemental materials for: Melero-Martin et al, Vol 109, Issue 11, 4761-4768
Files in this Data Supplement:
- Document 1. Methods for phenotypic characterization of EPCs (PDF, 76.7 KBKB)
- Figure S1. Isolation of cord blood EPCs (PDF, 95.1 KB) -
Cord blood–derived EPCs (cbEPCs) were obtained from the mononuclear cell (MNC) fractions of 25 mL umbilical cord blood samples. MNCs were seeded on 1% gelatin-coated tissue culture plates using EBM-2 supplemented with SingleQuots (-hydrocortisone), 20% FBS, 1× glutamine-penicillin-streptomycin (GPS), and 15% autologous plasma. Unbound cells were removed at 48 hours and the bound fraction was maintained in culture using EBM-2 supplemented with 20% FBS, SingleQuots (-hydrocortisone), and 1× GPS (EBM-2/20%). Colonies of endothelial-like cells were allowed to grow until confluent, trypsinized, and purified using CD31-coated magnetic beads. CD31-selected cbEPCs were serially passaged and cultured on fibronectin (FN)–coated (1 µg/cm2) plates at 5 × 103 cell/cm2 in EBM-2/20%.
- Figure S2. Phenotypic characterization of cbEPCs at different passages (PDF, 462 KB) -
CD31-selected cbEPCs were evaluated at passages 4, 6, 9, 12, and 15. HDMECs and HSVSMCs served as positive and negative controls, respectively. (A) Cytometric analysis of cultured cbEPCs for endothelial markers CD34, VEGF-R2, CD31, VWF, CD105, and neuropilin-1. Solid gray histograms represent cells stained with fluorescent antibodies. Isotype-matched controls are overlaid in a black line on each histogram. (B) Phase microscopy pictures of cultured cbEPCs grown in confluent monolayer and showing characteristic cobblestone morphology. (C) RT-PCR analysis of cbEPCs for endothelial markers CD34, CD105, CD31, VE-cadherin, VWF, and eNOS. (D) Up-regulation of E-selectin, ICAM-1, and VCAM-1 in cultured cbEPCs in response to TNF- . Solid gray histograms represent cells stained with fluorescent antibodies, while black lines correspond to the isotype-matched control fluorescent antibodies.
- Figure S3. Further phenotypic characterization of cbEPCs (PDF, 99.2 KB) -
CD31-selected cbEPCs were evaluated at passage 6. HDMECs served as positive control, while HSVSMCs and BMMSCs served as negative controls. (A) Cytometric analysis of cultured cbEPCs for VEGF receptors neuropilin-1 (NRP-1) and Flt-1 (VEGF-R1). Solid gray histograms represent cells stained with fluorescent antibodies. Isotype-matched controls are overlaid in a black line on each histogram. (B) Western blotting of cultured cbEPCs showing positive reaction for CD31 and negative reaction for smooth muscle/mesenchymal cell receptor PDGF-Rβ. (C) Indirect immunofluorescence of cultured cbEPCs grown in confluent monolayer showing positive staining for CD31 and negative staining for smooth muscle/mesenchymal cell markers -SMA and calponin.
- Figure S4. Species specificity of anti–human CD31 and anti–α-SMA antibodies (PDF, 131 KB) -
(A) The specificity of the anti–human CD31 antibody was confirmed by the negative reaction obtained when mouse lung tissue sections were stained. The -SMA antibody was not human specific, as shown by the positive staining of mouse lung tissue sections. (B) Immunohistochemical staining of the Matrigel implants (prepared as described in Figure 2 of the article) using human-specific CD31 antibody and -SMA antibody reveals the presence of human lumenal structures throughout the xenografts. Higher magnifications of a mouse vessel adjacent to the implant shows negative reaction for CD31, confirming the specificity of the human CD31 antibody. All images are representative of implants harvested from four different animals.
- Figure S5. Isolation of adult peripheral blood EPCs (PDF, 125 KB) -
(A) Adult peripheral blood–derived EPCs (adult EPCs) were obtained from the mononuclear cell (MNC) fractions of 50 mL peripheral blood samples from healthy volunteer donors. MNCs were seeded on 1% gelatin-coated tissue culture plates using EBM-2 supplemented with SingleQuots (-hydrocortisone), 20% FBS, 1× GPS, and 15% autologous plasma. Unbound cells were removed at 4 days and the bound fraction was maintained in culture using EBM-2 supplemented with 20% FBS, SingleQuots (-hydrocortisone), and 1× GPS (EBM-2/20%). Colonies of endothelial-like cells were harvested using cloning rings, expanded, and purified using CD31-coated magnetic beads. CD31-selected adult EPCs were serially passaged and cultured on fibronectin (FN)–coated (1 µg/cm2) plates in EBM-2/20%. (B) Cytometric analysis of cultured adult EPCs for endothelial markers CD31, VEGF-R2, CD146, and CD34, and hematopoietic/monocytic markers CD14 and CD45 at passages 6 (P6) and 9 (P9). Solid gray histograms represent cells stained with fluorescent antibodies. Isotype-matched controls are overlaid in a black line on each histogram.
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