Primitive adult hematopoietic stem cells can function as osteoblast precursors

Abstract

Osteoblasts are continually recruited from stem cell pools to maintain bone. Although their immediate precursor is a plastic-adherent mesenchymal stem cell able to generate tissues other than bone, increasing evidence suggests the existence of a more primitive cell that can differentiate to both hematopoietic and mesenchymal cells. We show here that the "side population" (SP) of marrow stem cells, defined by their ability to rapidly expel a DNA-binding dye and to regenerate the hematopoietic compartment, can differentiate to osteoblasts through a mesenchymal intermediate. When transplanted into lethally irradiated mice, single gene-marked murine SP cells reconstituted depleted osteoprogenitor pools, such that a large proportion of the osteogenic cells in the epiphysis of long bone carried the donor SP cell marker. These findings suggest that the developmental capacity of SP cells is not restricted to the hematopoietic lineages but extends to osteogenic differentiation. This property not only elucidates a previously unrecognized step in osteoblast development, but also has intriguing implications for the use of SP cells in clinical orthopedics and stem cell-based disorders of bone.

Fig. 1.

Bone-like nodules are derived from donor SP cells in cultures treated with osteogenic medium. Nodules were stained for both β-galactosidase (a and b, blue) and mineral deposits (a, red-brown) and costained for alkaline phosphatase, a marker of differentiated osteoblasts (c and d, green fluorescence). A bright-field image of a typical nodule is shown in e. This nodule was later stained for alkaline phosphatase (f) and the CD45 surface antigen (g, red). h is a composite of f and g. (Magnification, ×10.)

Fig. 2.

A single transplanted SP can differentiate to osteogenic MSCs. Cultures of bone marrow from mice undergoing single SP-cell transplantation were established and subsequently treated with osteoinductive medium (α-MEM containing 100 nM dexamethasone and 50 μg/ml ascorbic acid). After 2 weeks, cultures were stained for mouse osteocalcin, a recognized marker of osteoblasts. Individual bone-like nodules (≈10–20) in the cultures were photographed and stained for β-galactosidase (A, blue-green). B includes an overlay of A showing the results of osteocalcin staining (green). Black arrows denote individual cells staining for both β-galactosidase and osteocalcin, and red arrows indicate peripheral cells staining for β-galactosidase only. The major fraction of cells in the apex of any nodule staining positively for β-galactosidase reacted with both reagents.

Fig. 3.

Detection of donor SP cell-derived osteoblasts lining the trabeculae of hind-limb bone. Serial hind-limb sections from transplanted mice were stained with an antibody against β-galactosidase, detected with horseradish peroxidase (brown) and hematoxylin (blue). Positively staining osteogenic cells were readily apparent in trabecular bone (arrows) whether mice were transplanted with marked SP cells (a1–a3) or whole bone marrow (b1–b3). a3 duplicates a2 using polarized light, and b3 shows the results with an anti-osteocalcin antibody. c2 (five sections away from the section depicted in c1) and c3 show representative bone sections from nontransplanted C57BL/6 CD45.1 mice stained with anti-β-galactosidase or an anti-human mitochondrial antigen, respectively. Although osteogenic cells were evident in trabecular bone, they lacked staining altogether. c1 is an adjacent section stained with hematoxylin and eosin. d1 and d2 show representative bone sections of mice transplanted with either SP cells or whole bone marrow, respectively, and stained with anti-β-galactosidase antibody. Red arrows in d1a and d2 indicate the position of osteocytes staining positively for β-galactosidase. (Magnification, ×40, except d1a, ×100.)

Fig. 4.

Osteocalcin analysis of bone from C57BL/6 CD45.1 mice transplanted with marked SP cells for the presence of marked osteoblasts. Mice were irradiated and transplanted with SP cells derived from C57BL/6 CD45.2 Rosa26 mice. Paraffin-embedded sections of normal long bone were then costained with an anti-β-galactosidase antibody and an antibody against mouse osteocalcin, a definitive marker of osteoblasts. A shows a representative field from a section stained with the anti-β-galactosidase antibody and Vector Nova Red (red-brown). B shows the same field after removal of the mounting medium and staining with the antibody against mouse osteocalcin and Vector SG (black). Bone sections from nontransplanted mice were also double stained and shown to be negative for β-galactosidase, and other murine tissues failed to react with the anti-osteocalcin antibody (not shown). (Magnification, ×40.)