Characterization of early mouse mesenchymal stem/progenitor cells for generating cartilage and study their role in endochondral ossification

Mesenchymal stem/ stromal cells (MSCs) isolated from mice have allowed to address some of the major issues that could not be investigated by using human MSCs. For this thesis, a thorough characterization of a previously described mouse MSC population, namely PαS MSCs (identified by expression of both PDGFRα and Sca1 antigens) was done. Using a developmental approach, the MSC-like cells were also identified in embryonic stages. We identified 4 subpopulations of PαS cells, based on the expression of the CD90 and CD73 markers. The flow-cytometry based analysis revealed different time points of the emergence of the different subpopulations of these cells during ontogeny. Using a Sox9-GFP reporter line, we were able to correlate these changes with the predominant chondrogenic and osteogenic processes during progression of mouse development and endochondral ossification.
A developmental paradigm was used to characterize the “stem” like properties of PαS subpopulations in vitro from postnatal day P2, since a significant increase of this population was observed from E18.5 until P2 after birth. The conditions of the in vitro trilineage differentiation and chondrogenic differentiation were refined to achieve better results by mimicking the molecular pathways operating during limb development. Two subsets of PαS cells were discriminated by the expression of CD90 marker (PαS+CD90+ and PαS+CD90- subsets). These subsets were assessed for their in vivo fate by engrafting cartilage templates generated from these cells subcutaneously in nude mice. Strikingly, the cartilage models generated from the PαS+CD90+ subset remodeled completely to undergo endochondral ossification and the resulting bone ossicles were invaded by host-derived bone marrow cells. On the other hand, the cartilage models generated from the PαS+CD90- subset appeared to remodel only partially. No endochondral ossification was observed, but the expression of Sox9 persisted, which pointed to maintenance of chondrogenic potential. As in vivo functions of MSCs may depend on their interaction with HSCs in the stem cell niche, cartilage templates generated from PαS subsets isolated at embryonic day E18.5 were also engrafted. This developmental stage coincides with migration of the first HSCs from fetal liver to the bone marrow. The remodeling of cartilage into bone was reduced and only small bone ossicles formed when cartilage templates from PαS+CD90+ cells were implanted.
Thus, this study focuses on extensive characterization of PαS cells to identify new subpopulations and refining the conditions of multilineage differentiation in these subpopulations with a developmental approach. Emergence of these subpopulations during mouse development to contribute towards cartilage and bone lineage was determined by studying in detail their in vivo fates during endochondral ossification.