|
|
Blood, Vol. 111, Issue 3, 1302-1305, February 1, 2008
Differential in vivo potential of endothelial progenitor cells from human umbilical cord blood and adult peripheral blood to form functional long-lasting vessels
Blood Au et al.
111: 1302
Supplemental materials for: Au et al
Files in this Data Supplement:
- Document 1. Supplemental materials and methods (PDF, 80.7 KB)
- Figure S1 (JPG, 121 KB)
-
Peripheral blood derived endothelial cells (a) and cord blood derived endothelial cells (b) expressed endothelial markers including CD31, VE-cadherin, von Willebrand Factor (vWF) and Tie2, as determined by immunohistochemistry (CD31, VE-cadherin, vWF, and Tie2, yellow; DAPI, blue). PB-EPCs (c) and CB-EPCs (d) were positive for Dil-AcLDL uptake. Scale bars, 50 µm.
- Figure S2. After ex vivo expansion of CB-EPCs, over 99% of the EPCs that are used for in vivo implantation are CD31+CD45− endothelial cells (JPG, 46 KB)
-
These cells are positive for the endothelial-selective markers VEGFR2 and Tie2 and negative for the monocyte-specific marker CD14 and progenitor/stem cell marker CD133.
- Figure S3. PB-EPCs or CB-EPCs were mixed with 10T1/2 cells in a fibronectin/collagen gel, and implanted into cranial windows in SCID mice (JPG, 29.3 KB)
-
Images were taken at periodic time points with multi-photon laser scanning microscope for in vivo dynamics of vascularization by the implanted endothelial cells. The vessel density of unperfused blood vessels was quantified. There was a gradual reduction in the density of unperfused vessels in both groups of animals implanted with PB-EPCs only and PB-EPCs with 10T1/2 cells (A). There was no significant difference between the two groups. In the group implanted with CB-EPC only, there was a rapid decrease in the density of unperfused vessels (B). In the group co-implanted with CB-EPCs and 10T1/2 cells, there was a gradual decrease in the density of unperfused vessels and some of the unperfused vessels persisted even at late time points.
- Figure S4 (JPG, 56.4 KB)
-
Peripheral blood derived EPCs were incorporated either alone (a) or with 10T1/2 cells (b) and cord blood derived EPCs were similarly incorporated either alone (c) or with 10T1/2 cells (d) in a Type I collagen gel/fibronectin matrix. The EPC-derived cell formed vessel-like structure after 4 days in culture. The presence of 10T1/2 cells promoted a much higher number of the vessel-like structure. In contrast to the in vitro data, the presence of 10T1/2 cells had no effect on engineered vessel density in vivo. The lack of a salutary effect of 10T1/2 cells in vivo could potentially be due to poor viability of implanted PB-EPCs rather than a defect in 10T1/2 cells recruitment. Scale bars, 100 µm.
- Figure S5 (JPG, 54.6 KB)
-
Whole mount staining of the implanted collagen gel revealed that the CB-EPCs (EGFP+) at day 87 after implantation maintained the expression of VE-Cadherin in vivo (EGFP, green; VE-Cadherin, red).
- Figure S6. Whole mount staining of collagen gel with CB-EPCs (EGFP+) and 10T1/2 cells (DsRed+) at day 33 after implantation (JPG, 108 KB)
-
10T1/2 cells proliferated and became part of the stroma in vivo. Some of the 10T1/2 cells functioned as perivascular cells around blood vessels. Only the perivascular portion of 10T1/2 cells expressed smooth muscle markers, SM22α (a) and Desmin (b) in vivo (EGFP, green; DsRed, red, SM22α and Desmin, blue).
- Figure S7. Peripheral blood (A) and cord blood (B) derived endothelial cells were stained for Ki-67, a marker for proliferating cells (Ki-67, yellow; DAPI, blue) (JPG, 41.8 KB)
-
Quantification of Ki-67 positive cells showed that a higher percentage of CB-EPCs was undergoing proliferation. Scale bars, 50 µm, A, B; *P<0.01.
- Figure S8. PB (A) and CB (B) derived endothelial cells were exposed to serum free media for 48 hours and the cells were then stained for TUNEL, a marker for apoptotic cells (TUNEL, red; DAPI, blue) (JPG, 25.9 KB)
-
Scale bars, 100 µm, A, B.
- Figure S9. Red blood cell velocities of CB-EPC derived vessels and host brain vessels were measured by line-scan method and they were plotted against vessel diameters (JPG, 36.3 KB)
-
- Video 1. Z-stack images showing that EGFP+ cells served as conduit for blood flow (AVI, 3.30 MB)
-
CB-EPCs labeled with EGFP were co-implanted with 10T1/2 cells onto a cranial window in SCID mouse and images were taken 77 days after implantation. (Green, CB derived endothelial cell expressing enhanced green fluorescent protein (EGFP); red, functional blood vessels contrast-enhanced by rhodamine-dextran.)
| |
