Kazuhito Satomura

Single‐Colony Derived Strains of Human Marrow Stromal Fibroblasts Form Bone After Transplantation In Vivo

Populations of marrow stromal fibroblasts (MSFs) can differentiate into functional osteoblasts and form bone in vivo. It is not known, however, what proportion of MSF precursor cells, colony forming units‐fibroblast (CFU‐Fs), have osteogenic potential. In the present study, analysis of bone formation in vivo by single‐colony derived strains of human marrow stromal fibroblasts (HMSFs) has been performed for the first time. Each strain originated from an individual CFU‐F and underwent four passages in vitro prior to subcutaneous implantation into immunodeficient mice within vehicles containing hydroxyapatite‐tricalcium phosphate ceramic. Multicolony derived HMSF strains were also transplanted to serve as positive controls. After 8 weeks, abundant bone formation was found in the transplants of all multicolony derived HMSF strains, whereas 20 out of 34 (58.8%) single‐colony derived strains from four donors formed bone. Immunostaining with antibody directed against human osteonectin and in situ hybridization for human‐specific alu sequences demonstrated that cells forming new bone were of human origin and were vital for at least 45 weeks post‐transplantation. Both the incidence of bone‐forming colonies and the extent of bone formation by single‐colony derived HMSF strains were increased by cultivation with dexamethasone and ascorbic acid phosphate. Other factors, including type of transplantation vehicle, morphology, size, and structure of the original HMSF colonies showed no obvious correlation with the incidence or extent of bone formation. Hematopoietic tissue within the newly formed bone was developed in the transplants exhibiting exuberant bone formation. These results provide evidence that individual human CFU‐Fs have osteogenic potential and yet differ from each other with respect to their osteogenic capacity.

Targeted disruption of the biglycan gene leads to an osteoporosis-like phenotype in mice

The resilience and strength of bone is due to the orderly mineralization of a specialized extracellular matrix (ECM) composed of type I collagen (90%) and a host of non-collagenous proteins that are, in general, also found in other tissues. Biglycan (encoded by the gene Bgn) is an ECM proteoglycan that is enriched in bone and other non-skeletal connective tissues. In vitro studies indicate that Bgn may function in connective tissue metabolism by binding to collagen fibrils and TGF-ß (Refs 5,6), and may promote neuronal survival. To study the role of Bgn in vivo, we generated Bgn-deficient mice. Although apparently normal at birth, these mice display a phenotype characterized by a reduced growth rate and decreased bone mass due to the absence of Bgn. To our knowledge, this is the first report in which deficiency of a non-collagenous ECM protein leads to a skeletal phenotype that is marked by low bone mass that becomes more obvious with age. These mice may serve as an animal model to study the role of ECM proteins in osteoporosis.

Bone formation in vivo: comparison of osteogenesis by transplanted mouse and human marrow stromal fibroblasts.

BACKGROUND Marrow stromal fibroblasts (MSFs) are known to contain bone precursor cells. However, the osteogenic potential of human MSFs has been poorly characterized. The aim of this study was to compare the osteogenic capacity of mouse and human MSFs after implantation in vivo. METHODS After in vitro expansion, MSFs were loaded into a number of different vehicles and transplanted subcutaneously into immunodeficient mice. RESULTS Mouse MSFs transplanted within gelatin, polyvinyl sponges, and collagen matrices all formed a capsule of cortical-like bone surrounding a cavity with active hematopoiesis. In transplants of MSFs from transgenic mice harboring type I procollagen-chloramphenicol acetyltransferase constructs, chloramphenicol acetyltransferase activity was maintained for up to 14 weeks, indicating prolonged bone formation by transplanted MSFs. New bone formation by human MSFs was more dependent on both the in vitro expansion conditions and transplantation vehicles. Within gelatin, woven bone was observed sporadically and only after culture in the presence of dexamethasone and L-ascorbic acid phosphate magnesium salt n-hydrate. Consistent bone formation by human MSFs was achieved only within vehicles containing hydroxyapatite/tricalcium phosphate ceramics (HA/TCP) in the form of blocks, powder, and HA/TCP powder-type I bovine fibrillar collagen strips, and bone was maintained for at least 19 weeks. Cells of the new bone were positive for human osteonectin showing their donor origin. HA/TCP powder, the HA/TCP powder-type I bovine fibrillar collagen strips, and HA/TCP powder held together with fibrin were easier to load and supported more extensive osteogenesis than HA/TCP blocks and thus may be more applicable for therapeutic use. CONCLUSIONS In this article, we describe the differences in the requirements for mouse and human MSFs to form bone, and report the development of a methodology for the consistent in vivo generation of extensive bone from human MSFs.

Repair of craniotomy defects using bone marrow stromal cells.

BACKGROUND Techniques used to repair craniofacial skeletal defects parallel the accepted surgical therapies for bone loss elsewhere in the skeleton and include the use of autogenous bone and alloplastic materials. Transplantation of a bone marrow stromal cell population that contains osteogenic progenitor cells may be an additional modality for the generation of new bone. METHODS Full thickness osseous defects (5 mm) were prepared in the cranium of immunocompromised mice and were treated with gelatin sponges containing murine alloplastic bone marrow stromal cells derived from transgenic mice carrying a type I collagen-chloramphenicol acetyltransferase reporter gene to follow the fate of the transplanted cells. Control surgical sites were treated with spleen stromal cells or gelatin sponges alone, or were left untreated. The surgical defects were analyzed histologically for percent closure of the defect at 2, 3, 4, 6, and 12 weeks. RESULTS Cultured bone marrow stromal cells transplanted within gelatin sponges resulted in osteogenesis that repaired greater than 99.0+/-2.20% of the original surgical defect within 2 weeks. In contrast, cranial defects treated with splenic fibroblasts, vehicle alone, or sham-operated controls resulted in minimal repair that was limited to the surgical margins. Bone marrow stromal cells carrying the collagen transgene were immunodetected only in the newly formed bone and thus confirmed the donor origin of the transplanted cells. CONCLUSIONS These studies demonstrate that mitotically expanded bone marrow cells can serve as an abundant source of osteoprogenitor cells that are capable of repairing craniofacial skeletal defects in mice without the addition of growth or morphogenetic factors.

Osteogenic imprinting upstream of marrow stromal cell differentiation

Five spontaneously transformed cell lines were established from a population of murine bone marrow stromal cells (BMSCs) and the expression profiles of phenotype‐characteristic genes, patterns of in vitro differentiation, and osteogenic capacity after in vivo transplantation were determined for each. All the clones expressed stable levels of cbfa1, the osteogenic “master” gene, whereas the levels of individual phenotypic mRNAs were variable within each, suggestive of both maturational and phenotypic plasticity in vitro. Varying levels of collagen type I and alkaline phosphatase (AP) were expressed in all the clonal lines. The clonal lines with proven in vivo osteogenic potential (3 out of 5) had a high proliferation rate and expressed bone sialoprotein (BSP), whereas the two nonosteogenic clones proliferated more slowly and never expressed BSP. Bone nodules were only observed in 2 out of 3 of the osteogenic lines, and only 1 out of three formed cartilage‐like matrix in vitro. There was no evidence of chondrogenesis in the nonosteogenic lines. By contrast, LPL was expressed in two osteogenic and in two nonosteogenic lines. These results demonstrate the presence of multipotential and restricted progenitors in the murine stromal system. cbfa1, collagen type I, and AP expression were common to all, and therefore presumably early, basic traits of stromal cell lines that otherwise significantly differ with respect to growth and differentiation potential. This finding suggests that an osteogenic imprinting lies upstream of diversification, modulation, and restriction of stromal cell differentiation potential. J. Cell. Biochem. 78:391–403, 2000. Published 2000 Wiley‐Liss, Inc.

Receptor tyrosine kinase expression in human bone marrow stromal cells

Bone marrow stromal cells (BMSCs) are a heterogeneous population of cells derived from colony‐forming units‐fibroblastic (CFU‐Fs). These cells reside in the bone marrow cavity and are capable of differentiating into several cell phenotypes including osteoblasts, chondroblasts, hematopoiesis‐supporting stromal cells, and adipocytes. However, the factors that regulate the proliferation and differentiation of the BMSC population are for the most part unknown. Since many members of the receptor tyrosine kinase (RTK) family have been shown to participate in growth control of various mesenchymal cell populations, in this study we examined the expression and function of RTKs in the BMSC population. Degenerate oligonucleotides corresponding to two conserved catalytic domains of the RTK family and RT‐PCR were used initially to determine which RTKs are expressed in the human BMSC (hBMSC) system. After subcloning the amplification product generated from mRNA of a multicolony‐derived hBMSC strain, PDGF receptor (β), EGF receptor, FGF receptor 1, and Axl were identified by DNA sequencing of 26 bacterial colonies. Furthermore, PDGF and EGF were found to enhance BMSC growth in a dose‐dependent manner and to induce tyrosine phosphorylation of intracellular molecules, including the PDGF and EGF receptors themselves, demonstrating the functionality of these receptors. On the other hand, bFGF was found to have little effect on proliferation or tyrosine phosphorylation. Since single colony‐derived hBMSC strains are known to vary from one colony to another in colony habit (growth rate and colony structure) and the ability to form bone in vivo, the expression levels of these RTKs were determined in 18 hBMSC clonal strains by semiquantitative RT‐PCR and were found to vary from one clonal strain to another. While not absolutely predictive of the osteogenic capacity of individual clonal strains, on average, relatively high levels of PDGF‐receptor were found in bone‐forming strains, while on average, nonbone‐forming strains had relatively high levels of EGF‐receptor. Taken together, these results indicate that RTKs play a role in the control of hBMSC proliferation, and that the differential pattern of RTK expression may be useful in correlating the biochemical properties of individual clonal strains with their ability to produce bone in vivo. J. Cell. Physiol. 177:426–438, 1998.

Expression pattern of macrophage migration inhibitory factor during embryogenesis.

Although macrophage migration inhibitory factor (MIF) was originally identified as a lymphokine that inhibits the migration of macrophages, its ubiquitous expression suggests it may have a role beyond the immune system. Here we report a detailed characterization of MIF expression during mouse embryogenesis. The MIF expression pattern was found to parallel tissues specification and organogenesis.

Immortalization and Characterization of Bone Marrow Stromal Fibroblasts from a Patient with a Loss of Function Mutation in the Estrogen Receptor‐α Gene

A male patient with abnormal postpubertal bone elongation was shown earlier to have a mutation in both alleles of the estrogen receptor, resulting in a nonfunctional gene. Marrow stromal fibroblasts (MSFs) derived from this patient were called HERKOs (human estrogen receptor knock outs), and in order to obtain continuous HERKO cell lines, they were immortalized using a recombinant adenovirus‐origin‐minus SV40 virus. MSFs are unique cells because they support hematopoesis and contain a mixed population of precursor cells for bone, cartilage, and fat. Three established cell lines (HERKO2, HERKO4, and HERKO7) were characterized and compared with the heterogeneous population of nonimmortalized HERKOs for their osteogenic potential. We performed Northern analysis of matrix genes implicated in bone development and metabolism and an in vivo bone formation assay by transplanting the cells subcutaneously into immunodeficient mice. All three HERKO lines expressed high amounts of collagen 1A1, osteopontin, osteonectin, fibronectin, decorin, biglycan, and alkaline phosphatase. Except for osteopontin, expression of these genes was slightly lower compared with nonimmortalized HERKOs. In the in vivo bone formation assay, the heterogeneous population of nonimmortalized HERKOs formed bone with high efficiency, while the HERKO lines induced a high‐density, bone‐like matrix. Finally, all HERKO cell types secreted high levels of insulin‐like growth factor I and interleukin‐6 into the culture medium relative to cells of normal human subjects. In summary, these lines of HERKO cells retain several of the phenotypic traits of MSFs after immortalization, including matrix and cytokine production, and provide a valuable source of a unique human material for future studies involving estrogen action in bone and bone marrow metabolism.