Comparative analysis of gene expression identifies distinct molecular signatures of bone marrow- and periosteal-skeletal stem/progenitor cells

Periosteum and bone marrow (BM) both contain skeletal stem/progenitor cells (SSCs) that participate in fracture repair. However, the functional difference and selective regulatory mechanisms of SSCs in different locations are unknown due to the lack of specific markers. Here, we report a comprehensive gene expression analysis of bone marrow SSCs (BM-SSCs), periosteal SSCs (P-SSCs), and more differentiated osteoprogenitors by using reporter mice expressing Interferon-inducible Mx1 and NestinGFP, previously known SSC markers. We first defined that the BM-SSCs can be enriched by the combination of Mx1 and NestinGFP expression, while endogenous P-SSCs can be isolated by positive selection of Mx1, CD105 and CD140a (known SSC markers) combined with the negative selection of CD45, CD31, and osteocalcinGFP (a mature osteoblast marker). Comparative gene expression analysis with FACS-sorted BM-SSCs, P-SSCs, Osterix+ preosteoblasts, CD51+ stroma cells and CD45+ hematopoietic cells as controls revealed that BM-SSCs and P-SSCs have high similarity with few potential differences without statistical significance. We also found that CD51+ cells are highly heterogeneous and have little overlap with SSCs. This was further supported by the microarray cluster analysis, where the two SSC populations clustered together but are separate from the CD51+ cells. However, when comparing SSC population to controls, we found several genes that are uniquely upregulated in endogenous SSCs. Amongst these genes, we found KDR (aka Flk1 or VEGFR2) to be most interesting and discovered that it is highly and selectively expressed in P-SSCs. This finding suggests that endogenous P-SSCs are functionally very similar to BM-SSCs with undetectable significant differences in gene expression but there are distinct molecular signatures in P-SSCs, which can be useful to specify P-SSC subset in vivo.