Characteristics Of Human Adipose-derived Stem Cells And The Ability Of Differentiate Into Endothelial Cells In Vitro

PartⅠThe biological study of human adipose-derived stem cells (hADSCs) Objective To isolate culcure human adipose-derived stem cells (hADSCs) from adipose tissue and investigate their biological characteristics.Methods ADSC cells were obtained by collagenase digestion and culcured.Cell grows and variation of morphology was observed by inverted phase contrast microscope . The cellular proliferation activity was assessed by MTT method.Flow cytometry were used to analyse cell cycle and the expressions of immunophenotype (CD31,CD34,CD44,CD29).Results1. In the adipose tissue,there was a population of cells with high potential to proliferate through generations. The primarily cultured cells adhered to the wall about six hours and exhibited a fibroblast-like morphology after culturing for 12 hours.2. The cell proliferation activity was higher in ADSC cells,which was not degrade obviously with successive passages.3. ADSC cells also showed characteristic stem cell cycle.The proportion of ADSCs in proliferative phase was large.4. ADSC cells of morphous and phenotype were very similar as bone marrow–derived stromal cells (BMSC).The immunophenotype phenotype of the adipose-derived cells based on flow cytometry changed progressively with adherence and passage.Stromal cell–associated markers(CD44,CD29) increased significantly with successive passages. Endothelial cell–associated marker(CD31)did not change significantly with serial passage(p>0.05).The stem cell–associated marker (CD34)was at peak levels in the early-passage ADSCs and remained present,although at reduced levels,through out the culture period(p<0.05).PartⅡHuman adipose-derived stem cells differentiate into endothelial cells in vitro Objective To study on induction and differentiation condition of endothelial cells from ADSCs when cultured with VEGF in vitro ,which can offer the cytological basis for clinical application and tissue engineering .Methods ADSC cells were obtained by collagenase digestion.ADSCs were cultured in two kinds of cell culture condition :low concentration induction group which added VEGF 20ng/ml and b-FGF 10ng/ml;high concentration induction group which added VEGF50ng/ml and b-FGF10ng/ml .The antigens expression of endothelial-specific markers(CD34,CD31)were detected by flow cytometry. The ability of ADSC to engulf DiI-ac-LDL and bind FITC-Letin was observed under a fluorescence microscope.Ⅷfactor was detected by immunofluorescence.The expression of CD133 and CD144 was detected by RT-PCR .Results1. Endothelial cell–associated markers(CD31,CD34) increased significantly when cultured with VEGF. The positive rates of CD31 and CD34 were higher induced for eight days than induced for four days (p<0.05). There was no significant difference between expression of CD31 and CD34 of two concentration induction groups after induced eight days(p>0.05).2. Differentiated ADSCs displayed red fluorescence when phagocytosising DiI-ac-LDL and green fluorescence when binding FITC-Lectin.3. Induced ADCS cells expressedⅧfactor and displayed green fluorescence under fluorescent microscopy.4. The expression of CD144 and CD133 was higher than blank control group (p<0.05). The expression of CD133 of differentiated ADSCs inducing for four days was higher than inducing for eight days. With the increasing of inducing time,the expression of CD144 of differentiated ADSCs was increased.Conclusions1. ADSCs can be obtained from adipose tissue by collagenase digestion.,exhibite a fibroblast-like morphology and has strong proliferation capacity,which was similar to bone marrow–derived stromal cells (BMSC). ADSCs express stromal cell–associated markers which are assayed by flow cytometer.2. ADSCs can be differentiated into endothelial-like cells in the presence of VEGF,b-FGF, and showed expression of endothelial cell markers.ADSCs may be a potential source of endothelial cells and offer the ideal seed cells for tissue engineering.