Isolation And Purification Of Adipose-derived Stem Cells With High Potential Of Adipogenic Differentiation

Background:Nowadays,autologous adipose tissue transplatation is the feasible way to repair soft tissue defects.But the result of transplatation is not satisfactory because of the later absorption.The emergence of tissue engineering offers unique therapeutic opportunities for the way to solve this problem.Tissue engineering combines cells,biomaterials and bioactive factors,and significant challenges remain with respect to cell sourcing and differentiation for application to engineered tissue repair.The multilineage potential of embryonic stem cells and adult stem cells from the bone marrow has been characterized extensively. Although embryonic stem cells potential is enormous,many ethical and political issues accompany their use.The clinical use of bone marrow-derived stem cells (BMSCs) also has presented problems including pain,morbidity and low cell number upon harvest.Since the new source of stem cells was isolated from adipose tissue, much of the work has focused on it.Compared with the other tissues,subcutaneous adipose depots are accessible,abundant and replenishable,thereby providing a potential adult stem cells reservoir for each individual.However,the components of cells,which were harvested from adipose tissue by collagenase digestion,were complicated and the multilineage potential of adipose derived stem cells(ASCs) was lower than BMSCs.For example,only 30%～40%of ASCs can be induced to adipocytes.So,it will be important to purify ASCs and enhance their efficiency ability of differentiation.Recent years,many groups working independently have shown that mature adipocytes can dedifferentiated to fibroblast-like cells though ceiling culture and the potential of adipogenic differentiation of the dedifferentiation adipocytes was higher than ASCs.Therefore,our study tried to screen the special surface markers relate to high potential of adipogenic by making a comparison between ASCs and dedifferentiation adipocytes on the expression of surface markers,then use the magnetic microbeads system to isolate the ASCs with high potential of adipogenic differentiation.Aim:To explore the surface markers associate with high potential of adipogenic differentiation and try to isolate ASCs with high potential of adipogenic by using magnetic microbeads system.Methods:1.Isolated ASCs from human adipose tissue,after proliferation and adipogenic induction of ASCs,the mature inductive adipocytes floating in the medium were cellected.Ceiling culture was used to culture adipocytes for 10d and then dedifferentiation adipocytes were obtained at the ceiling.Making a comparision between ASCs and dedifferentiation adipocytes in their reproductive activity, adipogenic differentiation potency and expression of surface markers.2.Used magnetic microbeads to isolate the cell population positive with the CD marker we had screened from stromal vascular fractions(SVF) and ASCs.The changes of the expression of CD markers of the separated cells were observed by flow cytometry analysis;red O oil stain and alizarin red stain were employed to exam the results of adipogenic induction and osteogenic induction respectively.Results:ASCs and dedifferentiation adipocytes were obtained successfully.The reproductive activity between the dedifferentiation and ASCs were similar.The potential of adipogenic differentiation of the former was stronger than the later and the expression of cell surface markers of both cells were approximately alike.But the CD54 positive expression of dedifferentiation adipocytes exceeded that of ASCs.CD54-PE positive cells(CD54~+) were isolated from SVF and ASCs using PE-coupled magnetic microbeads.Before separation,the CD54 positive expression of SVF was higher than ASCs.After separation,over 90%of both the two cell population was CD54 positive.The ASCs/CD54~+ cells remained high expression of CD54 while the SVF/CD54~+ cells were opposite after passage.The induction results showed that ASCs/CD54~+ population posses highest potential of adipogenic differentiation and intermediate potential of osteogenic differentiation whereas the SVF/CD54~+ cells showed the worst potential of differentiation on both of the two induction.Conclusion:1.This study compared the differences between dedifferentiation adipocytes and ASCs with respect to reproductive activity,potential of adipogenic differentiation and surface markers.The results suggested that dedifferentiation adipocytes,similar to stem cells,possess higher adipogenic differentiation ability than the later.The difference of both kinds of cells in expressing CD54 indicated that the higher expression of CD54 is related with its high potential of adipogenic differentiation.2.ASCs with high potential of adipogenic differentiation can be purified by CD54 magnetic microbeads.The separation of freshly isolated SVF by this method may lead to the negative result that the CD54 positive cells are still component complicated,even worsely lose most percentage of stem cells in them.Moreover,the expression of CD54 is not stable and fail to purify.In conclusion,CD54 cell perhaps serve as one of kinds surface molecules specifically expressed by stem cells with high potential of adipogenic differentiation and separation of CD54 positive cells from ASCs can be regarded as a way to purify the lineage of ASCs with high potential of ??ogenic differentiation.