Culture And Identification Of Human Endothelial Progenitor Cells From Cord Blood In Vitro

In 1997, endothelial progenitor cells (EPCs) were first isolated by Asahara and his colleagues from the peripheral blood . The EPC is a kind of precursor cell that can differentiate into vascular endothelial cell directly and is closely related with angiogenesis after birth, which is an important physiological and pathological significance. A growing amount of evidence demonstrates that EPCs have the capacity to migrate to the peripheral circulation and to differentiate into mature endothelial cells, and these cells have been also termed angioblasts. EPCs are involved in adult neovasculogenesis and have important physiological and pathophysiological significance. Diabetic retinopathy(DR) is one of the most common diabetic vascular complications and is indeed associated with alterations of the number and the function of EPCs. These cells are recruited to the foci of injury, where they can differentiate into endothelial cells that are capable of engrafting with the native tissue. This phenomenon suggests that EPCs mobilization to the sites of injury may participate in local tissue repair. The abundance of circulating EPCs, although low in basal conditions, increases several fold after mobilization with cytokines and growth factors. This study is designed to isolate EPCs from umbilical cord blood, and observe the surface markers of EPCs in the process of its proliferation and differentiation in vitro, which may provide the feasible research methods and the new source of cell transplantation for the future treatment of Diabetic retinopathy experimentally. This observation provides a basis for clinical application of EPC transplantation. MethodsMNCs were isolated by Percoll density gradient centrifugation from cord blood, and then suspended in Medium 199 supplemented with Fetal Calf Serum (FCS ) or Autologous Serum (AS) for culturing, differentiating, proliferating and identification. The expressions of specific antigens on cell surface were analysed by immunohistochemistry or immunofluorescent staining and the positive rate was detected by flow cytometry analysis.Results1 During culture, Attached cells appeared after 24-48 hours culture and became spindle-shaped gradually. Attached cells formed several cord-like sructure after 10 days culture and displayed endothelium-like and cobble-stone morphology with outgrowth, which is the character of endothelial cells.2 On the 7th day, the density of the adherent cells in the holes with FN is significantly more than the holes with free FN.3 On the 14th day, the attached cells cultured with FCS-M199 were positive for the cell markers CD31 and vWF(92.7±2.2%,73.3±4.2% respectivly). The adherent cells cultured with AS-M199 were positive for the cell markers CD31 and vWF(90.8±2.8%,72.7±4.1% respectivly).4 On the 7th day(,83.0±4.3)% of the attached cells cultured with FCS-M199 were positive for the double marker DiI-acLDL/ FITC-UEA-Ⅰ. The red fluorescent expressed by EPCs labeled with DiI-acLDL can be sustained more than 6 Weeks in vitro. (80.7±5.9)% of the adherent cells cultured with AS-M199 were positive for the double marker DiI-acLDL/ FITC-UEA-Ⅰ.5 On the 7th day,flow cytometric analysis showed positive staining of attached cell for CD34,CD133 and VEGFR2 (17.8%±3.7%,22.1%±4.4%,81.5%±5.0% respectively),while CD133 disappeared in the 14th day. Conclusions:1. We confirmed a feasible method to induce endothelial progenitor cells: isolating mononuclear cells by gradient centrifugation , then inducing with VEGF,bFGF and BPE.2. Fibronectin (FN) as a component of the extracellular matrix played an indispensable role in the early identification and EPCs adhesion.3. EPCs can be induced successfully with M199 which contained autologous serum(AS).4. DiI-acLDL can be used as an ideal tracer for the transplantation of EPCs in vivo.