Study On Differentiation Of CD34~+ Cells Into Endothelial Cells Under Different Conditions In Vitro

Objective To study the method of CD34+ cell isolated from human umbilical cord blood and induced to differentiate into endothelial cells in vitro. Then to explore whether the paracrine action of UC-MSCs can induce human umbilical cord blood derived CD34+ cells into endothelial cells in vitro. And to establish a effective method to isolate, culture and identify from HUVEC in vitro. Methods The cord blood and the umbilical cord from full-term section patients were collected in sterile condition immediately upon delivery. Mononuclear cells were isolated from fresh cord blood by 6% HES and density gradient centrifugation. CD34+ cells selected from mononuclear cells by the instruction of MACS. The umbilical cord was cut into pieces of 1mm3.UC were incubated with collagenase type IVand trypsin. The digested mixture was then passed through a filter to obtain cell suspension. Cells were plated in cell culture flask. Trypsin perfusion method was used to isolate HUVEC from fresh umbilical cord. HUVEC were observed under inverted phase contrast microscope. The surface markers were assessed by FACS. The experiment divided into CD34+ cells pure culture group, cytokine-induced group and two stem cells co-culture group with noncontact. A total of 5×105 CD34+ cells were plated on 6-well coated by fibronectin. A transwell insert was placed above the CD34+ cells layer and a total of 1×106 MSCs were plated in the upper well. The tow stem cells were cultured in M199 supplemented with 20%FBS, 1-glutamine 2mmol/L. The CD34+ cells' specific surface mark CD34 and endothelial specific surface marker were assessed by FACS analysis. Results In the experiment we dealt with the 20 fresh umbilical cord blood samples, with collected volume of 105.67±16.12ml. after MACS separation and purification, each cord blood specimen can be obtained CD34+ cells (10.98±5.88)×105. The average purity of enriched CD34+ cells as assessed by FACS was (95.02±3.82)%. Freshly isolated CD34+ cells were small and round which suspended in culture medium. In cytokine-induced group, attached spindle-like cells and a number of cell clusters appeared after 3 days culture of CD34+ cells and cord-like structure by 14 days. When the cells grow into large number, it displayed cobble-stone morphology. In two stem cells co-culture group Attached cord-like structure cells of CD34+ cells appeared after 7 days co-culture with non-contacted MSC, and when the CD34+ cells grew into large number, it formed colonies. MSCs were isolated from UC by collagenase and trypsin digestion sufficiently. FACS showed that the cells expressed high levers of CD105 97.4%, CD29 71.1%, CD49 80.0%, but did not express hematopoietic lineage marker CD34. The obtained HUVECs were small-round and clustering. The cells began adhere to the plate in tow hours, and attached cells were cord-like structure. The cells express endothelial specific markers, including CD 144, vWF and CD31. FACS analyses at 14 d of culture, the cells of pure culture group expressed CD 144, vWF, CD31, CD34, CD45, respectively (26.19±1.98)%, (11.46±2.04)%, (30.44±2.67)%, (51.96±5.80)%, (38.3±4.57)%. But the CD34+ cells of co-culture group expressed endothelial specific markers, including CD144(54.40±4.13)%, vWF(47.53±3.96)%, CD31(65.43±5.61)%, as compare with pure culture group(P<0.05), part expressed of CD34 and the leukocyte common antigen CD45 was negative expression. The results was consistent with human umbilical vein endothelial cells and the cytokine-induced group which CD144(69.16±1.99)%, vWF(57.49±3.67)%, CD31(78.38±7.20)%, as compare with pure culture group (P<0.05).Conclusions The two methods and the MACS are an effective ways to isolate CD34+cells from cord blood. The CD34+cells in cord blood which can differentiate into endothelial cells. The CD34+ cells can be used as a source of EPCs, and under the paracrine of UC-MSCs the cells also can be induced into endothelial cells. So in the process of promoting the differentiation of endothelial cell, we can substitute the paracrine factors of MSC for exogenous cytokines. And a large number of high purified endothelial cells could be acquired by digestion of trypsin. HUVEC can be proliferated and passaged successfully in vitro.