A S Virdi

Osteoprogenitor cells of mature human skeletal muscle tissue: an in vitro study

The presence of osteogenic progenitors in human skeletal muscle is suggested by the formation of ectopic bone in clinical and experimental conditions, but their direct identification has not yet been demonstrated. The aims of this study were to identify osteogenic progenitor cells in human skeletal muscle tissue and to expand and characterize them in culture. Specimens of gracilis and semitendinosus muscle were obtained from young adults and digested to separate the connective tissue and satellite cell fractions. The cells were cultured and characterized morphologically and immunohistochemically using antibodies known to be reactive with primitive osteoprogenitor cells, pericytes, intermediate filaments, and endothelial cells. Alkaline phosphatase activity and osteocalcin gene expression were also determined. In the early stages of culture, the connective tissue cells obtained were highly positive for primitive osteoprogenitor cell and for pericyte markers. Alkaline phosphatase activity was detectable at early stages of culture and rose as a function of time, whereas primitive osteoprogenitor cell markers declined and osteocalcin mRNA expression became detectable by reverse transcriptase-polymerase chain reaction (RT-PCR). It is shown that human skeletal muscle connective tissue contains osteogenic progenitor cells. Their identification as pericytes, perivascular cells with established osteogenic potential, suggests a cellular link between angiogenesis and bone formation in muscle tissue. These cells are easily cultured and expanded in vitro by standard techniques, providing an alternative source of osteogenic progenitor cells for possible cell-based therapeutic use in certain conditions.

Expression of estrogen receptor-alpha in cells of the osteoclastic lineage.

Abstract Estrogen deficiency at the menopause is associated with an increased rate of bone loss and subsequent risk of skeletal fracture. Whilst cells of the osteoblastic lineage are known to express estrogen receptors, the presence of estrogen receptors in osteoclasts remains controversial. We have examined expression of the classic estrogen receptor, estrogen receptor-alpha (ERα), during osteoclast differentiation. In situ mRNA hybridisation with a digoxygenin-labelled riboprobe to ERα mRNA, together with immunocytochemical analysis using a human ERα-specific monoclonal antibody demonstrated similar findings and confirmed the expression of ERα in chondroblasts and osteoblasts from human fetal bone and mineralising human bone marrow cultures. ERα expression was detected in human bone marrow cultures treated with 1,25(OH)2D3 and macrophage colony-stimulating factor and in macrophage cultures treated with 1,25(OH)2D3. However, in an in vitro model of human osteoclast formation, no ERα expression was observed in the osteoclasts that developed. The human preosteoclast TCG 51 cell line showed strong expression of ERα in contrast to the low levels observed in the more mature bone resorptive TCG 23 cell line. No expression was detectable in osteoclasts cultured from giant cell tumour of bone (GCTB) tissue or in osteoclasts in Pagetic, GCTB, or hyperparathyroid bone tissues. In conclusion, preosteoclasts express detectable levels of ERα, but osteoclast maturation and bone resorption is associated with loss of ERα expression. This indicates that ERα expression and regulation may play a role in osteoclast formation.

Phenotypic and molecular heterogeneity in fibrodysplasia ossificans progressiva.

Abstract. Fibrodysplasia (myositis) ossificans progressiva (FOP) is an extremely rare inherited disorder in which progressive ossification of major striated muscles, often following injury, is associated with abnormal skeletal patterning. Altered expression of bone morphogenetic proteins may be a contributory cause. To examine this hypothesis, we compared the patterns of expression of bone morphogenetic proteins (BMPs) mRNAs from lymphoblastoid cell lines from two small multigenerational families with autosomal dominant transmission of FOP. Although affected members of both families showed the characteristic phenotype of FOP, one family was more severely affected than the other. Expression of mRNAs for BMP-1, 2, 3, 5, and 6 mRNAs were not detected within the more severely affected family, but BMP-4 mRNA was expressed in affected but not unaffected members of this family. The results of linkage exclusion analysis using a highly polymorphic microsatellite marker near the BMP-4 gene were consistent with linkage of FOP and BMP-4 in this family. Within the less severely affected family, affected and unaffected members showed similar levels of mRNA expression of BMPs 1, 2, 4, and 5, and linkage of FOP to the BMP-4 gene was excluded. It is concluded that clinical, radiographic, and biochemical data in these two families with FOP establish clinical and molecular heterogeneity and also suggest the possibility of genetic heterogeneity.

Effects of interferon alpha on human osteoprogenitor cell growth and differentiation in vitro.

The specific effects of interferon alpha (IFNα), on the differentiation pathways of human osteogenic cells are not known. The aim of this study was to investigate possible effects of IFNα on osteogenic development by investigating cell differentiation, colony formation (colony forming unit‐fibroblastic, CFU‐F), cell proliferation, and gene expression, in particular bone morphogenetic protein (BMP) expression, of human bone marrow osteoprogenitor cells. Human bone marrow fibroblasts were cultured with or without the addition of IFNα (5–1,000 IU/ml) in the presence and absence of dexamethasone (10 nM) and ascorbate (100 μM), which are agents known to affect osteogenic differentiation. IFNα produced a significant dose‐dependent inhibition of cell proliferation and alkaline phosphatase specific activity at concentrations as low as 50 IU/ml. IFNα (50–1,000 IU/ml) inhibited the stimulation of alkaline phosphatase specific activity induced by ascorbate and dexamethasone. Examination of CFU‐F showed dose‐ and time‐dependent inhibitions of colony formation and reductions in both colony size and alkaline phosphatase‐positive CFU‐F colonies particularly at earlier times. Reactivity with an antibody specific for osteoprogenitors (HOP‐26), was reduced in IFNα‐treated cultures. Northern blot analysis showed a significant dose‐dependent up‐regulation of BMP‐2 mRNA, estrogen receptor alpha mRNA and osteocalcin mRNA expression in ascorbate/dexamethasone cultures. In contrast, IFNα significantly inhibited BMP‐2 mRNA expression in the absence of ascorbate and dexamethasone. In conclusion, IFNα inhibits human osteoprogenitor cell proliferation, CFU‐ F formation, HOP‐26 expression, and alkaline phosphatase specific activity and modulates BMP‐2 gene expression. These results suggest a role for IFNα in local bone turnover through the specific and direct modulation of osteoprogenitor proliferation and differentiation. J. Cell. Biochem. 74:372–385, 1999.

Retroviral marking of human bone marrow fibroblasts: in vitro expansion and localization in calvarial sites after subcutaneous transplantation in vivo

Amplification of multipotential stem cells, with or without ex vivo gene transfer, offers the potential for their use for beneficial repopulation of a host in which there is specific cellular deficiency or functional impairment. The aims of the current study were to immunoselect, genetically mark, and determine the fate of fibroblastic progenitor cells in vivo. A monoclonal antibody, HOP‐26, which has high reactivity with a cell surface antigen present on human osteoprogenitors in bone marrow fibroblast populations, was used to select these cells by immunopanning. Following culture in 10% FCS in αMEM containing ascorbate‐2‐phosphate and dexamethasone the amplified cells expressed the osteoblast phenotype as determined by expression of osteocalcin protein determined immunohistochemically, and Type I collagen and osteocalcin mRNA expressions determined by RT‐PCR analysis. The selected cells were genetically labeled using a murine leukemia virus (MuLV) encoding a reporter gene (lacZ) with a selective marker gene (neor) using a triple transient transfection protocol. Transfected cells were implanted in CB17 scid/scid mice by local subcutaneous injection over the calvariae. Localization of the genetically marked cells within the calvarial tissues was detected by β‐galactosidase histochemistry and immunocytochemistry. Genetically marked cells were observed within the periosteal layer in close association with the osteoblast layer, covering mineralized bone surfaces and within bone osteoid at 5 and 7 days after injection. This study demonstrates the successful selection, expansion, and retroviral‐marking of human osteoprogenitors and their migration and localization within calvariae of SCID mice following in vivo implantation. These basic studies indicate the migration of these cells to skeletal sites and support possibilities for future uses of human osteoprogenitors in therapy of bone deficiency diseases and the potential for development of gene therapy procedures in these conditions. J. Cell. Physiol. 186:201–209, 2001.

Immunohistochemical studies using BRL 12, a monoclonal antibody reacting specifically with osteogenic tissues.

A monoclonal antibody of immunoglobulin class G1 has been produced which reacts with a high molecular weight antigen apparently present exclusively in osteogenic tissues. Immunohistochemical studies have shown that the antigen is present throughout the mineralized matrix and in osteoid. None of the other tissues examined namely liver, intestine, kidney, spleen, thymus, heart, lung, skin, cartilage and skeletal muscle showed evidence of specific antibody binding. Immunohistochemical staining was also demonstrated in tissues developing from rabbit marrow cultured in vitro and in diffusion chambers in vivo. Temporal studies of antigen expression in the chambers indicated that the antigen occurs at sites of bone formation after the appearance of alkaline phosphatase but before the formation of a mineralized matrix. The results of these studies suggest that the monoclonal antibody recognises a product of differentiated osteoblasts. This antibody may therefore prove useful in studies of osteogenic differentiation.

Localisation of estrogen receptor mRNA in skeletal cells byin situ hybridisation

BOne loss in osteoporosis is due to an imbalance between the formation and destruction of bone. This can occur either at the level~ of the individual turnover units, the BMUs (imbalance), or at different sites (uncoupling). The size of the negative balance will depend both upon the bone loss at the individual site, and upon the number of sites formed and destroyed (turnover). These two processes are dependent upon systemic modulators, among others hormones, upon mechanical factors, and upon local eytokines. The cytokines which m ~ be relevant for bone formation are the IGFs, TGFI3, the BMP s, the FGFs, PDGF and prnstaglandins. Likely candidates for the resorption are the interleukins 1,4,6 and 11, TGF~t and 13, TNFct and 13, M-CSF, FGFs and prostaglandins. They are modulated among others by hormones such as PTH, PTHrP, calcitonin, 1,25-(OH)2 vitamin D, and oestrogens, and by mechanical strains. It appears that most of them act through the mediation of the osteoblasts whieh produce factors stimulating and inhibiting the recruitment and/or the activity of ostccelasts. Drugs can act either at the general level of turnover or at the level of the individual BMU. lnhihitors of bone resorption, such as oestrogens, bisphosphonates and caleitonin appear to act at both levels, although this can sometimes be due to the fact that both activities are linked. The inhibitory effect can be either directly on the osteoelast, as for ealcitonin, or mediated by the osteoblast through the production of inhibitor(s) of osteoclast differentiatio~ and/or activity, such as for oestrogens and bisphospbonates.~ Activators of formation can act either on the recruitment of new osteohlasts, on their survival, or on their activity.

Monoclonal Antibody BRL 12 Reacts with Bone Keratan Sulphate Proteoglycan

Our previous studies suggest that a monoclonal antibody, BRL 12, reacts with a specific product of differentiated cells of the osteoblastic lineage. In the present study, the bone constituent recognized by this antibody has been characterized by gel filtration, ion exchange chromatography, protein blotting and immunolocalization. Our findings show that the antibody reacts with an epitope associated with the core protein of rabbit keratan sulfate proteoglycan (KSPG), a molecule which shares considerable homology with the sialoprotein present in the bone tissue of other mammalian species.