טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentKawar Jaraisy Rawan
SubjectRole of Human Endothelial Progenitor Cells (EPC) in
Angiogenesis and Osteogensis
DepartmentDepartment of Medicine
Supervisor Dr. Hadar Zigdon


Abstract

Endothelial progenitor cells (EPCs) are subpopulation of circulating cells that can be good candidates for regenerative medicine. Previous studies in animal models showed that local transplantation of EPCs into bone defects enhance bone formation.
Since bone regeneration relays on adequate blood supply, we hypothesis that the role of EPCs in osteogenesis may be associated with improved angiogenesis.  Additionally, EPCs may contribute to osteogenesis by differentiating into osteogenic cells and or recruiting host osteogenic cells into the regenerated site. The aim of this study was to understand the role of EPCs in ectopic bone formation. Specifically to investigate the direct and indirect (paracrine) involvement of EPCs in angiogenesis and osteogenesis.

Human EPCs (hEPCs) were isolated cultured and characterized. In order to investigate the role of EPCs in ectopic bone formation, cells were seeded onto synthetic β-tri-calcium phosphate (β-TCP) scaffold and transplanted into subcutaneous pouches in nude mice. Before transplantation, cells were labeled with DII (membrane dye). Mice were allocated to control (β-TCP without cells, n=12) and test (β-TCP with hEPCs, n=18) groups. Mice were sacrificed after ten days, three and eight weeks. In vivo imaging system-(IVIS) and confocal microscopy were used to track the implanted cells in the implantation site.  Following sacrifice, transplants were prepared for histological analysis to evaluate ectopic mineralization and angiogenesis. Blood vessels density was quantified using anti-mouse-CD31. The direct involvement of transplanted hEPCs in blood vessels formation was analyzed using anti-human-CD31 and anti-Human Nuclear Antigen (HNA). The recruitment of host EPCs and MSCs was evaluated by anti-mouse CD31 and the anti-mouse CD73 respectively. Furthermore, a double staining immunohistochemistry was performed in order to evaluate the proximity between the recruited and transplanted cells. In order to shade light on the angiogenic and osteogenic mechanism of hEPCs, Real- Time PCR was held to evaluate the expression of specific angiogenic and osteogenic genes in cultured hEPCs.

FACS analysis of primary cultured hEPCs revealed that more than 95% were CD31. Cells were positive for CD34 and KDR, and negative for CD45, CD14. Blood vessel density was six fold higher in the test group ten days after transplantation (p = 0.047) and ectopic mineralization was found after 3 and 8 weeks. Labelled hEPCs were observed in the implantation site adjacent to newly formed vessels (according to confocal). Additionally, immunohistochemichal staining with HNA and human CD31 showed integration of the transplanted hEPCs into the lumen of newly formed vessels. hEPCs transplantation accelerated recruitment of resident CD73  and CD31 cells, and proximity was found between hEPCs  (HNA) and recruited (CD73) cells. Finally, Real-Time PCR of EPCs revealed high expression levels of angiogenic and osteogenic related genes such as: CXCL12, CXCR4, PDGFB, PDGFBR, VEGFA, and BMP4.

Local transplantation of hEPCs enhanced angiogenesis and ectopic mineralization. hEPCs contributed to angiogenesis  and osteogenesis by integration to the newly formed blood vessels walls and by recruitment of resident CD73 and CD31. A possible explanation for both mechanisms can be attributed to high expression of angiogenic and osteogenic genes in cultured hEPCs.