Evaluation of putative cell surface markers that characterize human and murine adipose derived stem cells.

摘要

Mesenchymal stem cells (MSCs) have generated much interest because of the potential they possess in regenerative medicine. However, the biology of these cells is still poorly understood primarily because there are no specific markers available to identify them. Ease of harvest and abundance in tissues like bone marrow and adipose tissue makes the cells attractive for various applications in regenerative medicine. The application of these cells in regenerative medicine will require isolation and identification of the cells from a mixture of other cell types. Numerous surface antigens have been shown to be expressed on MSCs, however, these markers are not specific for MSCs alone. The present studies focused on cells harvested from murine and human adipose tissues and were directed toward assessing the effectiveness of a panel of surface antigens attributed to MSCs for stability in culture, identification of MSCs and application in isolating the cells from other cell types. Adipose derived stem cells (ADSCs) isolated from murine inguinal fat pads were sorted into cell subsets expressing CD73, CD90.2, and CD 105. The sorted and unsorted cells were assessed for in vitro and in vivo differentiation in order to determine the effectiveness of each marker to identify cells with high potential to differentiate toward osteogenic lineage. In vitro data showed that the CD73+ and CD105+ cell subsets displayed greater ability to differentiate toward the osteogenic lineage compared to the CD90.2+ subset. However, when the cell subsets were injected locally into mouse femur, no difference in osteogenic differentiation potential was observed. Therefore, this panel of surface antigens is not useful in identifying and isolating putative ADSCs from a variety of cell types. Human studies focused on assessing the effectiveness of early embryonic stem cell marker: Stage Specific Embryonic Antigen (SSEA-4). This antigen was shown to be expressed by MSCs isolated from bone marrow but its effectiveness in characterizing cells harvested from adipose tissue has not been established. Results showed that ADSCs sorted for SSEA-4 possessed a greater ability to differentiate toward the osteogenic, adipogenic, and neurogenic lineages in vitro. The evaluation of the sorted and unsorted cells also included the ability of cell subsets to heal critical sized defects in the calvarial. Data demonstrated that SSEA-4 Sorted and Unsorted ADSCs were able to regenerate a similar amount of bone in vivo. SSEA-4 Sorted and Unsorted ADSCs expressing BMP-2 demonstrated a similar rate of bone deposition; ADSCs expressing BMP-2 had the potential to regenerate a greater amount of bone than ADSCs which did not express the BMP-2 growth factor. Taken together, data indicate that SSEA-4 enriches for mesenchymal stem cells in vitro, however there is no significant difference between in vivo bone regeneration potential between Unsorted and SSEA-4 Sorted ADSCs. Because the cells are removed from their natural niche, the behavior of the cells in vitro compared to in vivo may be different due to culture conditions.;The rationale behind the present studies is to understand how the surface antigens can be utilized to isolate the cells with high osteogenic potential from a variety of other cells. It is well established that there are no specific markers available for the identification of the adult derived stem cells; analysis of the established surface antigens expressed by ADSCs with culturing offers means of assessing markers that can be applied to prospectively isolate a population of cells that exhibit high osteogenic potential as well as the potential to differentiate into other cell lineages. The data presented in this thesis have established that most of the surface markers attributed to mouse MSCs are unstable in culture; some markers are expressed at very high levels and are therefore inefficient in isolating cells that from other cell types. For human MSCs, the embryonic stem cell marker SSEA-4 was determined to significantly enrich for MSCs in vitro; however there was no significant difference for bone formation between Unsorted and SSEA-4 Sorted ADSCs in vivo.