Characteristics of adipose-derived stem cells isolated from buccal fat pads using CD 271 cell sorting technique

Abstract

Objective To compare the characteristics of ASCs isolated from intra-oral buccal fat pads using CD 271 Magnetic-Activated Cell Sorting (MACS) and plastic adherence (PA). Materials and Methods The buccal fat tissue was harvested from ten patients whom undergone orthognathic surgeries. ASCs were isolated from the tissue using PA (Group A) and MACS; CD 271+ (Group B) and CD271- (Group C), (n=5/group). Colony forming unit fibroblasts (CFU-f) of the cells in each group were evaluated within 20 days of culture. The immune-phenotyping markers following the International Society for Cellular Therapy (ISCT) protocols including CD90, CD73 and CD105 as the positive markers and CD14, CD20, CD34, CD45 as the negative markers were analyzed by flow cytometry analysis. Gingival fibroblast was served as a negative control group. For multi- differentiation analysis, the cells of each group were cultured in the inductive medium. The adipogenic differentiation was assessed by Oil Red O staining. The chondrogenic differentiation was assessed by alcian blue staing. The osteogenic differentiation were assessed by ELISA to measure the level of alkaline phosphatase activity (ALP) and Osteocalcin (OCN). Results CFU-f formed in the Group B cells, but were not detected in the other groups. The cells of groups A-C could express the immunophenotyping markers of mesenchymal stem cells including CD 73, 90 and 105. No statistical difference was detected among the groups. It was noted that CD 73 was detected at the highest levels followed by CD 105 and CD 90 respectively. The cells of the control group expressed those markers remarkably less than the experiment groups (significant differences were found in CD 73 and CD 105, p < 0.05). In addition, the cells of all groups expressed hematopoietic stem cell markers including CD 14, 20, 34 and 45 at very low levels. The cells of groups A-C demonstrated adipogenic, chondrogenic and osteogenic differentiation when cultured in the inductive conditions. There was no significant difference of those properties among the groups. Conclusion CD 271 is considered as a proper marker for sorting ASCs from buccal fat tissue. However, it cannot be use as the sole marker. Although the ASCs expressed CD 90 at the lowest levels, they still had osteogenic differentiation capacity. Therefore, they can be used as a stem cell source to repair bone defects.