Abstract
Primary osteogenic cells have been known to reside within the CD45-CD31-Ter119-Sca-1- cell fraction, particularly in the CD51+ subpopulation. However, detailed determination of the frequency of osteogenic cells within this Sca-1- cell population remains yet to be determined. In addition, it is not clear that other cell surface markers can be used to further sub-fractionate this Sca-1-CD51+ osteogenic cell population and to define their developmental stages. In this report, both Sca-1-CD24med and Sca-1- CD24-/lo cells have been shown to be two small subsets of the Sca-1-CD51+ cell fraction. These two cell fractions show subtle difference in the expression level of osteogenic marker genes such as Osx and Opn, and in vitro proliferate rate. All these observations suggest that they may be at different developmental stages of osteogenesis. The Sca-1-CD24med cell fraction is enriched for the more mature osteolineage cells than the Sca-1-CD24-/lo counterpart. In contrast, most of the Sca-1-CD24hi and Sca-1+CD24-/lo cells do not contain CFU-ALP nor express osteogenic gene markers. The high proliferation ability and osteo-adipogenic differentiation potentials confirm that the Sca-1+CD24-/lo cells are the multipotential mesenchymal stromal cells. The determination of individual stromal cell subpopulations will lead to a better understanding in the hierarchical organization of these osteolineage cells.
Highlights
Bone is a highly organized tissue, comprised of a calcified connective tissue matrix and specific bone cells, including bone progenitors, osteoblasts, and osteocytes
During flow analysis and cell sorting, the cellular subpopulations within this CD31/CD45/Ter119(triple negative) stromal cell fraction were focused. This stromal cell fraction was coined as endosteal bone marrow (EBM) cell fraction throughout the study
In this report, based on the results of (i) flow cytometric analysis of stromal cell subpopulations with Stem cell antigen-1 (Sca-1) and CD24, (ii) in vitro expansion capacity and differentiation assays (iii) primary adherent Colony forming unit (CFU) assay, and (iv) osteogenic marker gene profiling in primary cells, we have identified there are one single marrow stromal cells (MSCs) population (Sca-1+CD24-/ lo) and two subsets of committed osteogenic cells (Sca-1-CD24med and Sca-1-CD24-/lo) in the mouse endosteal bone (Figure 5)
Summary
Bone is a highly organized tissue, comprised of a calcified connective tissue matrix and specific bone cells, including bone progenitors, osteoblasts, and osteocytes. Osteoblasts are derived from multipotent marrow stromal cells (MSCs) through a series of proliferation and differentiation steps before expressing recognizable specific osteoblast marker genes. MSCs have been identified as Lin-CD45-CD31Sca-1+CD51+ [4,5], CD45-Ter119-Sca-1+ PDGFR-α+ [6], and CD45CD31-Ter119-Sca-1+ ALCAM- [7]. These cells have high proliferation capacity (self-renewal); multiple cell lineage differentiation potential (multipotential), and the lack of gene expression for osteogenic differentiation, such as Runt related transcription factor 2 (Runx2), Osteocalcin (Ocn), Osteopontin (Opn)
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