טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentSabbah Nadin
SubjectIdentifying Genotypic and Phenotypic Features and
Characterization of Human Peripheral Blood Derived
Endothelial Progenitor Cells (EPCs)
DepartmentDepartment of Medicine
Supervisor Dr. Hadar Zigdon


Abstract

Endothelial progenitor cells (EPCs) are rare, circulating subpopulation of mononuclear cells (MNCs) derived from the bone-marrow. Circulating EPCs have the ability to induce and modulate vasculogenesis and angiogenesis. Since their amount and function are regulated by systemic and environmental factors, the therapeutic potential of each individual patient's EPCs is unknown. Therefore, it is essential to predict the potency of individual patients' EPCs to function in-vivo.  

The aim of this study was to identify and characterize the genetic and functional in-vitro variations between human EPCs isolated from unrelated healthy donors. These differences are likely to be correlated with in-vivo function.

Our study cohort included 5 males and 5 females between the ages of 24-43 years old. Human EPCs were isolated from peripheral blood and cultured. In order to identify EPCs, cells were dyed using specific bio-markers; flow cytometry analysis revealed that more than 99%  of the cells were CD31, 60-98% were CD34, KDR and CXCR4. Cells showed low (0-10%) percentages of CD14 and CD45. Cell growth rate was evaluated by XTT proliferation assay, showing that EPCs of all donors were at their log phase after 48 hours of seeding. Proliferation rate was different between the highest and lowest performing donors (p<0.0001). EPCs condition medium enhanced the migration of mesenchymal stem cells (compared to endothelial growth medium without EPCs). Again, significant difference was found between donors with highest vs. lowest chemotactic activity (p<0.01). Real-Time PCR was performed to evaluate expression levels of: SDF-1, VEGF-A, CCL2, PDGFB, KDR and CXCR4. Significant difference was noted between donors with highest vs. lowest expression levels of CXCR4 (p<0.001). Interestingly, a positive correlation was found between the expression levels of SDF-1 and its receptor, CXCR4 (R=0.948, p<0.0001). As well as, positive correlation was noted between SDF-1, CXCR4 and EPCs proliferation capacity (R=0.736, *p<0.05 and R=0.8, p<0.01, respectively). Neither age nor gender of the donors influenced EPCs performance (e.g. proliferation rate, chemo-attractive capacity and gene expression). In order to investigate EPCs’ angiogenic capacity, cells were loaded on β-tri-calcium phosphate (β-TCP) scaffold and transplanted into subcutaneous pouches in a nude mouse model. Blood vessels in the scaffold were counted using immunostaining with anti-mouse CD31. Mean blood vessel density was higher in the EPC- transplants (134.1±16.8) vs. control (79.4±14.0), p<0.0001. The results showed significant difference between donors with highest vs. lowest blood vessel density (p<0.0001). Nevertheless, donors with the lowest blood vessel density were significantly higher compared to the control (p<0.05). Finally, using a multiple regression model, we were able to build a mathematical equation that assists in predicting blood vessel density by two independent variables: i) amount of MSCs that migrated toward EPCs condition medium. ii) expression level of KDR gene.

In summary, human EPCs derived from peripheral blood of healthy donors share common phenotypic properties, but differ in their in-vitro and in-vivo functions. Expression of KDR and the chemo-attractive ability of EPCs can be used to predict the in-vivo angiogenic capacity. These results pave the way for standardized clinical use of human EPCs