Identification of stromal cell precursors in human bone marrow by a novel monoclonal antibody, STRO-1

Murine IgM monoclonal antibody STRO-1 identifies a cell surface antigen expressed by stromal elements in human bone marrow (BM). STRO-1 binds to approximately 10% of BM mononuclear cells, greater than 95% of which are nucleated erythroid precursors, but does not react with committed progenitor cells (colony-forming unit granulocyte-macrophage [CFU-GM], erythroid bursts [BFU-E], and mixed colonies [CFU-Mix]). Fibroblast colony-forming cells (CFU-F) are present exclusively in the STRO-1+ population. Dual-color cell sorting using STRO-1 in combination with antibody to glycophorin A yields a population approximately 100-fold enriched in CFU-F in the STRO-1+/glycophorin A+ population. When plated under long-term BM culture (LTBMC) conditions, STRO-1+ cells generate adherent cell layers containing multiple stromal cell types, including adipocytes, smooth muscle cells, and fibroblastic elements. STRO-1+ cells isolated from LTBMC at later times retain the capacity to generate adherent layers with a cellular composition identical to that of the parent cultures. The STRO-1-selected adherent layers are able to support the generation of clonogenic cells and mature hematopoietic cells from a population of CD34+ cells highly enriched in so-called long-term culture-initiating cells. We conclude that antibody STRO-1 binds to BM stromal elements with the capacity to transfer the hematopoietic microenvironment in vitro.     

CD34 expression by stromal precursors in normal human adult bone marrow.

Normal bone marrow cells were isolated by fluorescence-activated cell sorting (FACS) on the basis of CD34 antigen expression and then assayed in vitro for colonies of fibroblastic cells (fibroblast colony-forming units [CFU-F]). Greater than 95% of detectable CFU-F were recovered in the CD34+ population, while their numbers were markedly depleted in the CD34- population. Additional experiments showed that the majority of CFU-F exhibited high forward and perpendicular light scatter and low-density CD34 antigen. Growth of sorted cells in medium optimized for long-term marrow culture (LTMC) produced a complex mixture of adherent stromal elements including fibroblasts, adipocytes, smooth muscle cells, and macrophages. Monoclonal antibody STRO-1, which identifies bone marrow stromal cells, reacted with approximately 5% of CD34+ cells, which included all CFU-F and stromal precursors in LTMC. Experiments using soybean agglutinin (SBA) further showed that these stromal elements were restricted to a population of bone marrow cells with the phenotype CD34+/SBA+. These properties of stromal precursors are quite distinct from those of primitive hematopoietic progenitors, showing that although the precursors of the hematopoietic and stromal systems share expression of CD34, they are otherwise phenotypically distinct cell types.     

High concentrations of dexamethasone suppress the proliferation but not the differentiation or further maturation of human osteoblast precursors in vitro: relevance to glucocorticoid-induced osteoporosis

OBJECTIVE: The use of glucocorticoids (GCs) in the treatment of RA is a frequent cause of bone loss. In vitro, however, this same class of steroids has been shown to promote the recruitment and/or maturation of primitive osteogenic precursors present in the colony forming unit-fibroblastic (CFU-F) fraction of human bone and marrow. In an effort to reconcile these conflicting observations, we investigated the effects of the synthetic GC dexamethasone (Dx) on parameters of growth and osteogenic differentiation in cultures of bone marrow stromal cells derived from a large cohort of adult human donors (n=30). METHODS: Marrow suspensions were cultured in the absence and presence of Dx at concentrations between 10 pm and 1 microm. After 28 days we determined the number and diameter of colonies formed, the total number of cells, the surface expression of receptors for selected growth factors and extracellular matrix proteins and, based on the expression of the developmental markers alkaline phosphatase (AP) and the antigen recognized by the STRO-1 monoclonal antibody, the proportion of cells undergoing osteogenic differentiation and their extent of maturation. RESULTS: At a physiologically equivalent concentration, Dx had no effect on the adhesion of CFU-F or on their subsequent proliferation, but did promote their osteogenic differentiation and further maturation. These effects were independent of changes in the expression of the receptors for fibroblast growth factors, insulin-like growth factor 1, nerve growth factor, platelet-derived growth factors and parathyroid hormone/parathyroid hormone-related protein, but were associated with changes in the number of cells expressing the alpha(2) and alpha(4), but not beta(1), integrin subunits. At supraphysiological concentrations, the effects of Dx on the osteogenic recruitment and maturation of CFU-F and their progeny were maintained but at the expense of a decrease in cell number. CONCLUSIONS: A decrease in the proliferation of osteogenic precursors, but not in their differentiation or maturation, is likely to be a key factor in the genesis of GC-induced bone loss.     

The growth factor requirements of STRO-1-positive human bone marrow stromal precursors under serum-deprived conditions in vitro

Factors that regulate the growth and development of primitive bone marrow stromal cell precursors are not well defined. We have examined 25 purified recombinant growth factors for their ability to initiate and support clonogenic growth of fibroblast colony-forming cells (CFU- F) from adult human bone marrow. Assays were performed using bone marrow mononuclear cells (BMMNC) enriched in CFU-F by magnetic- activated cell sorting (MACS) using the monoclonal antibody (MoAb) STRO- 1. A serum-deprived assay was developed to avoid components of fetal calf serum (FCS) that may mask or otherwise modify the response of CFU- F to exogenously added factors. L-ascorbate and the glucocorticoid dexamethasone were found to be essential for CFU-F colony development under serum-deprived conditions. Importantly, clonogenic growth of CFU- F in this culture system was absolutely dependent on an exogenous source of growth factor. Platelet-derived growth factor-BB (PDGF) and epidermal growth factor (EGF) demonstrated the greatest ability to support colony growth. Colony formation was dose-dependent, with half- maximal colony numbers at approximately 0.2 ng/mL for either factor and plateau numbers at concentrations in excess of 1.0 ng/mL. Simultaneous addition of PDGF and EGF had no effect on the number of colonies initiated but resulted in dose-dependent increases in mean colony diameter that were significant (P < or = .05) when compared with the effect of either factor alone or with the size of colonies elicited in control cultures by 20% FCS. Fluorescence-activated cell sorting (FACS) of BMMNC using MoAbs to the alpha chain of the PDGF receptor and to the EGF receptor in combination with the Moab STRO-1 demonstrated constitutive expression of both receptors by greater than 90% on CFU-F. Receptors for insulin-like growth factor-1 (IGF-1) and nerve growth factor (NGF) were also detected on STRO-1+ CFU-F, but in vitro both IGF- 1 and NGF did not support colony growth. This report demonstrates the development of a simple, reproducible, and stringent culture system for the growth and assay of stromal precursors under serum-deprived conditions and represents an important prerequisite for future studies of the role of growth factors in the regulation of stromal cell proliferation, differentiation, and development.     

Characterization and purification of osteogenic cells from murine bone marrow by two-color cell sorting using anti-Sca-1 monoclonal antibody and wheat germ agglutinin

Osteogenic cells were sorted from bone marrow of 5-fluorouracil (5-FU)- treated mice based on light scatter characteristics, Sca-1 expression, and their binding to wheat germ agglutinin (WGA). Four sort gates were established using forward (FSC) and perpendicular (SSC) light scatter and were denominated as FSChigh SSClow, FSClow SSChigh, FSClow SSClow, and FSChigh SSChigh cell. Cells from the FSChigh SSChigh gate, but not from the other gates, synthesized alkaline phosphatase, collagen, and osteocalcin and formed a mineralized matrix in culture. The number of osteoprogenitor cells was significantly enriched after depleting the 5- FU bone marrow from cells of the lymphoid and myeloid lineage, eg, T cells, B cells, natural killer cells, granulocytes, macrophages, and erythrocytes. Approximately 95% of the FSChigh SSChigh cell population of this "lineage-negative" (Lin-) marrow expressed the Sca-1 antigen (Sca-1+) and bound WGA. Three additional sort windows were established based on WGA binding intensity and were denominated as Sca-1+ WGAdull, Sca-1+ WGAmedium, and Sca-1+ WGAbright. Cells from the Sca-1+ WGAbright gate, but not from the other gates, synthesized bone proteins and formed a mineralized matrix. However, they lost this capacity upon subcultivation. Further immunophenotypic characterization showed that FSChigh SSChigh Lin- Sca-1+ WGAbright cells expressed stromal (KM16) and endothelial (Sab-1 and Sab-2) markers, but not hematopoietic surface markers such as c-kit and Thy1.2. Sorted FSChigh SSChigh Lin- Sca-1+ WGAbright cells form three-dimensional nodules that stain with the von Kossa technique and contain osteoblast and osteocyte-like cells.     

Dexamethasone recruitment of self-renewing osteoprogenitor cells in chick bone marrow stromal cell cultures.

Bone marrow stromal cells are a mixed population that contribute to the formation of the hematopoietic microenvironment. The osteogenic lineage includes populations of cells that, in culture, form discrete nodules of mineralized tissue when grown in the presence of ascorbic acid and sodium beta-glycerophosphate. We have used nodule formation to assay for the self-renewal capacity of osteoprogenitor cells in chick bone marrow cultures. To examine the regulatory influence of dexamethasone (Dx), first subcultures were grown continuously or split 1:1 at repeated subculture. Cells in continuous culture exhibited less than two population doublings, while cellular proliferation and alkaline phosphatase area were inhibited by 10(-8) mol/L Dx. Cells in split (redistributed) cultures exhibited up to 14 population doublings and cellular proliferation was also inhibited by Dx. In contrast with continuous cultures, redistributed cultures treated with Dx had increased alkaline phosphatase area and 15-fold larger amounts of mineralized tissue formation than controls. Osteogenesis was sustained for up to four subcultures and the ratio of mineralized tissue area to alkaline phosphotase positive cell area was at most 0.55. These data indicate that the osteogenic lineage of bone marrow stromal cells contains self-renewing progenitors that are recruited by Dx in culture and that at a maximum, only 55% of the alkaline phosphatase-positive cell population contributes to osteogenesis.     

Myeloma plasma cells alter the bone marrow microenvironment by stimulating the proliferation of mesenchymal stromal cells

Multiple myeloma is an incurable hematologic cancer characterized by the clonal proliferation of malignant plasma cells within the bone marrow. Numerous studies suggest that the myeloma plasma cells occupy and alter the stromal tissue of the bone marrow as a means of enhancing their survival and growth. However, the nature and magnitude of the changes to the stromal cell tissue remain to be determined. In this study, we used mesenchymal stromal cell and osteoblast-related cell surface marker expression (STRO-1 and alkaline phosphatase, respectively) and flow cytometry to enumerate mesenchymal stromal cell and osteoblast numbers in bone marrow recovered from myeloma patients at the time of diagnosis. Using this approach, we identified an increase in the number of STRO-1 positive colony forming mesenchymal stromal cells and a concomitant decrease in alkaline phophatase osteoblasts. Notably, this increase in mesenchymal stromal cell numbers correlated closely with plasma cell burden at the time of diagnosis. In addition, in comparison with the osteoblast population, the STRO-1+ mesenchymal stromal cell population was found to express higher levels of plasma cell- and osteoclast-activating factors, including RANKL and IL-6, providing a mechanism by which an increase in mesenchymal stromal cells may promote and aid the progression of myeloma. Importantly, these findings were faithfully replicated in the C57BL/KaLwRij murine model of myeloma, suggesting that this model may present a unique and clinically relevant system in which to identify and therapeutically modulate the bone microenvironment and, in turn, alter the progression of myeloma disease.     

Characterization of a 5-fluorouracil-enriched osteoprogenitor population of the murine bone marrow

In the presence of beta-glycerophosphate and vitamin C, cultures of normal mouse bone marrow cells form three-dimensional structures that stain positive with the Von Kossa technique and express alkaline phosphatase (ALP), collagen type I, and osteocalcin. Little is known about the characteristics and frequency of the cells that contribute to this phenomenon. Most likely, mature osteoblastic cells do not contribute to the nodule formation because no osteocalcin expressing cells are detected in the flushed marrow by in situ hybridization. Limiting dilution analysis shows that, in normal bone marrow, 1 of 2.2 x 10(5) cells has the potency to form a bone nodule and to express ALP, collagen, and osteocalcin in a temporal fashion. Upon in vivo treatment with 5-fluorouracil (5-FU), this frequency increases 12-fold, eg, 1 in 1.75 x 10(4) cells shows osteogenic activity. In comparison, fibroblast colony forming cells occur at a frequency of 1 of 2.5 x 10(4) or 1 of 5 x 10(3) plated cells in normal or 5-FU-treated marrow, respectively. Using density centrifugation, the majority of the osteoprogenitor cells in 5-FU marrow are found in the low-density (1.066 to 1.067 g/mL) fractions. In addition, these cells bind to nylon wool but not to plastic and aggregate in the presence of wheat germ agglutinin and soybean agglutinin. Scanning and transmission electron microscopy shows that the bone nodules in 5-FU marrow cultures are composed of fibroblastoid cells embedded in a mineralized collagen matrix. In conclusion, our results show that a quiescent cell population in the murine bone marrow with fibroblastoid characteristics contributes to the formation of bone-like nodules in vitro.     

Effects of hydrochlorothiazide and furosemide diuretics on human bone marrow stromal osteoprogenitor cells

Thiazide diuretics have been shown to decrease bone loss and improve bone mineral density, while long-term furosemide therapy has been suggested to decrease bone mineral content. However, the direct effects of these diuretics on osteoblastic cells are not well established. Some investigators have reported direct effects of thiazides on osteoblastic cells but the results remain controversial, and there are few data about the direct effect of furosemide on osteoblastic cells. We investigated the effects of hydrochlorothiazide (HCTZ) and furosemide on proliferation, alkaline phosphatase activity, osteocalcin, and interleukin-6/interleukin-11 (IL-6/IL-11) secretion in cultured normal human bone marrow stromal osteoprogenitor cells (hBMSCs). Treatment with HCTZ or furosemide for 24 hours in the concentration range of 10(-6) to 10(-4) mol/L did not affect 3H-thymidine incorporation in hBMSCs. Cellular alkaline phosphatase activity and osteocalcin production were not changed significantly by treatment with HCTZ or furosemide (up to 10(-4) mol/L) during culture. There was also no significant difference in IL-6 and IL-11 production in hBMSCs. These results suggested that HCTZ or furosemide had no significant direct effect on proliferation, alkaline phosphatase activity, osteocalcin, and IL-6/IL-11 production in hBMSCs, and the effects of these diuretics on bone mass may be related to the indirect action on calcium balance.     

Long-term culture in dexamethasone unmasks an abnormal phenotype in osteoblasts isolated from osteoporotic subjects

We have shown that osteoblastic cells derived from trabecular bone explants of osteoporotic subjects (OP cells) exhibited an altered alkaline phosphatase (ALP) response to 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] compared to control (CON) cells. Our hypothesis that OP cells have other intrinsic abnormalities was investigated using our cell models representing two different stages of differentiation. OP and CON cells were cultured in the absence (-DEX) or presence (+DEX) of 10 nM dexamethasone (DEX) in 10% fetal calf serum (FCS) prior to exposure to serum-free medium containing 1 nM of PTH and/or 17-beta estradiol (E2). Both OP and CON cells responded to DEX with a two-fold increase in basal ALP activity. While E2 or PTH+E2 had no effect on OP cells, both treatments inhibited ALP activity in CON cells (p<0.05). OP and CON cells grown in DEX also expressed PTH-stimulated adenylate cyclase (AC) activities higher than those of (-DEX) cells. OP+DEX cells, however, exhibited activities which were 8-fold higher than those of CON+DEX cells (p<0.001). In OP+DEX cells, E2 stimulated basal AC activity (p<0.05) but did not affect PTH-stimulated activity. In contrast, in CON+DEX cells, E2 had no effect on basal activity but inhibited PTH-stimulated AC activity (p<0.001). Osteocalcin production was 4-fold lower in OP+DEX cells compared to OP-DEX and CON cells (p<0.05) while osteocalcin mRNA levels were significantly lower in OP+DEX and CON+/-DEX cells compared to OP-DEX cells (p<0.05). E2 did not affect osteocalcin protein or mRNA levels in either OP or CON cells. No differences in mRNA levels were found for estrogen receptor-alpha (ER-a) in OP+/-DEX cells whereas these levels were significantly higher in CON+DEX compared to CON-DEX cells (p<0.05). These results indicate that DEX amplified the differences between OP and CON cells and confirm the presence of intrinsic osteoblastic abnormalities in patients with osteoporosis that persist in culture.     

Characterization of fibroblastic stromal cells from murine bone marrow

Several properties of fibroblastic colony-forming units (CFU-F) from murine bone marrow and their in vitro progeny were evaluated. CFU-F had a high buoyant density relative to total bone marrow cells; they were noncycling in situ and adhered to nylon wool. The fibroblastic cells stained positively for fibronectin, lipid, alkaline phosphatase, and nonspecific esterase, while phagocytosis assays were negative, and ultrastructural analysis failed to reveal desmosomes. These properties contrasted bone-marrow-derived fibroblastic cells to both endothelial cells and macrophages. Fibroblastic cells derived from several hemopoietic organs and skin were screened for antigenic determinants present on hemopoietic cells using monoclonal antibodies. Mac-1 and B220 were absent from all fibroblastic cells studied, whereas the Forsmann and Pgp-1 antigens were always present. Thy-1 was not detected on bone-marrow-derived fibroblasts, but was present on fibroblastic cells derived from other sources. T200 was found on all hemopoietic organ-derived fibroblastic cells, but not on those derived from blood and skin. Thus, analysis of antigenic determinants allowed distinction between fibroblastic cells from different organs.     

Osteogenic progenitor cells in rat bone marrow stromal populations exhibit self-renewal in culture

Marrow stromal cells are a heterogeneous population, comprising a variety of lineages including osteogenic cells. In the presence of ascorbic acid, sodium beta-glycerophosphate, and dexamethasone, rat bone marrow stromal cells form discrete nodules of mineralized, bonelike tissue. We used nodule formation by rat bone marrow stromal cells to assay for the self-renewal capacity of osteogenic progenitor cell populations. Cultures were subcultured every 5 days up to six times. Osteogenesis was assayed from second to sixth subcultures by counting the number and measuring the areas of mineralized nodules formed in cultures grown with 10(-8) mol/L dexamethasone. Nodule number and area decreased progressively between second and sixth subcultures. Alkaline phosphatase activity associated with individual cells and measured videodensitometrically decreased exponentially between the second and sixth subculture. The number of cells with alkaline phosphatase activity also decreased with progressive subculturing. The proportions of 3H-thymidine-labeled cells after continuous labeling from the beginning of the culture period showed 90% labeling for cells with alkaline phosphatase activity and fibroblastlike cells. Cultures labeled for only the first 3 days exhibited higher labeling of alkaline phosphatase-positive cells than fibroblastlike cells (P less than .05). Cultures that were flash-labeled at the end of the culture period demonstrated low labeling indices for cells with alkaline phosphatase activity and up to 10-fold higher labeling indices for fibroblastlike cells. Separate cultures treated with a cytocidal dose of high specific activity 3H-thymidine did not form nodules. These results indicate that osteogenic progenitor cells or another cell type required for nodules to develop must divide early in culture if nodule formation is to occur, and that osteoprogenitor cells express a limited capacity for self-renewal.     

Differentiation of normal human bone cells by transforming growth factor-beta and 1,25(OH)2 vitamin D3.

To investigate the role of transforming growth factor-beta 1 (TGF beta) in bone metabolism, the effects of this agent on the differentiation characteristics of human bone cells were studied in vitro. Human bone cells were isolated from femoral head samples by collagenase digestion. Differentiation characteristics included alkaline phosphatase activity, osteocalcin production, and mRNA levels for alkaline phosphatase, type I alpha 2-procollagen, and osteocalcin. The effect of TGF beta on alkaline phosphatase was not constant, but varied with the incubation conditions. At high cell density and in the presence of serum, TGF beta decreased alkaline phosphatase activity. However, at low cell density and under serum-free conditions, TGF beta stimulated alkaline phosphatase activity. The addition of 1,25(OH)2 vitamin D3 also stimulated alkaline phosphatase. The combination of the two agents gave a greater increase in activity than the sum of the activities when the two agents were given alone. The percentage of cells that stain positively for alkaline phosphatase changed in parallel with the change in specific activity. The percentage of positive cells increased from 17% to 64%, while the specific activity increased from 22 to 169 mU/mg protein. To investigate the mechanism of this stimulation, mRNA levels were measured at 24 hours. Individually, TGF beta and 1,25(OH)2D3 increased message levels for alkaline phosphatase and type I procollagen, but the greatest effect was produced by the combination of the two factors. 1,25(OH)2D3 increased osteocalcin mRNA levels, but TGF beta markedly inhibited this stimulation. TGF beta also inhibited production of osteocalcin by the human bone cells. TGF beta appears to modulate differentiation of human bone cells in combination with 1,25(OH)2D3 and other factors.     

Vitamin K stimulates osteoblastogenesis and inhibits osteoclastogenesis in human bone marrow cell culture

Accumulating evidence indicates that menaquinone-4 (MK-4), a vitamin K(2) with four isoprene units, inhibits osteoclastogenesis in murine bone marrow culture, but the reason for this inhibition is not yet clear, especially in human bone marrow culture. To clarify the inhibitory mechanism, we investigated the differentiation of colony-forming-unit fibroblasts (CFU-Fs) and osteoclasts in human bone marrow culture, to learn whether the enhancement of the differentiation of CFU-Fs from progenitor cells might relate to inhibition of osteoclast formation. Human bone marrow cells were grown in alpha-minimal essential medium with horse serum in the presence of MK-4 until adherent cells formed colonies (CFU-Fs). Colonies that stained positive for alkaline phosphatase activity (CFU-F/ALP(+)) were considered to have osteogenic potential. MK-4 stimulated the number of CFU-F/ALP(+) colonies in the presence or absence of dexamethasone. The stimulation was also seen in vitamin K(1) treatment. These cells had the ability to mineralize in the presence of alpha-glycerophosphate. In contrast, both MK-4 and vitamin K(1) inhibited 1,25 dihydroxyvitamin D(3)-induced osteoclast formation and increased stromal cell formation in human bone marrow culture. These stromal cells expressed ALP and Cbfa1. Moreover, both types of vitamin K treatment decreased the expression of receptor activator of nuclear factor kappaB ligand/osteoclast differentiation factor (RANKL/ODF) and enhanced the expression of osteoprotegerin/osteoclast inhibitory factor (OPG/OCIF) in the stromal cells. The effective concentrations were 1.0 microM and 10 microM for the expression of RANKL/ODF and OPG/OCIF respectively. Vitamin K might stimulate osteoblastogenesis in bone marrow cells, regulating osteoclastogenesis through the expression of RANKL/ODF more than through that of OPG/OCIF.     

Transplantation of bone marrow fibroblastoid stromal cells in mice via the intravenous route

Kinetics of fibroblastic colony-forming cells (CFU-F) were studied in mouse bone marrow after lethal total body irradiation and intravenous bone marrow transplantation. After an initial decrease, CFU-F numbers recovered, and plateaued 5 weeks post-treatment at 10% of normal values. Using chromosome-marked donor bone marrow cells we found that 1 day after transplantation 72% of donor CFU-F had reached the recipient's bone marrow, indicating a highly specific lodgment of CFU-F. Three months after transplantation donor CFU-F were still detectable and comprised about half of the femoral CFU-F population. It is concluded that CFU-F, a component of the haemopoietic microenvironment, are transplantable via the intravenous route.     

Human mulipotential mesenchymal/stromal stem cells are derived from a discrete subpopulation of STRO-1bright/CD34 /CD45(-)/glycophorin-A-bone marrow cells

Magnetic and flow cytometry-based methods were used to characterize clonogenic stromal cells in human bone marrow. STRO-1(bright) stromal cells were found to lack expression of CD34, CD45 and glycophorin-A markers associated with hematopoietic progenitor cells. These studies support the view that these are two distinct stem cell compartments in adult bone marrow.     

Presence of alkaline-phosphatase-positive reticulum cells in fetal liver and bone marrow of mice and their absence in yolk sac

One of the conspicuous stromal cells in hematopoietic parenchyma of the bone marrow in adult mice is the alkaline-phosphatase-positive reticulum cell (A1-RC). To determine whether it is present in the fetal yolk sac, liver, and bone marrow of mice, these tissues were examined at the gestational ages when they are sites of hematopoiesis. Fetuses of nine, 16 and 18 days of gestation were fixed and embedded in plastic, and 2-micron sections were stained for alkaline phosphatase and examined by light microscopy. No cells with the characteristics of the A1-RC were seen in the blood islands of the yolk sac, the site of erythropoiesis. Because of the high resolution of this method, however, alkaline phosphatase activity was observed on the border of the adjacent endodermal cells for the first time. By 16 days, the liver contained A1-RC interspersed with hematopoietic cells, as well as alkaline phosphatase activity in bile canaliculi. We found that the A1-RC is the predominant stromal cell of bone marrow at 18 days of gestation. We conclude that, as maturation proceeds, the hematopoietic stromal environment becomes more similar to that of the adult.     

Interleukin-10 inhibits the osteogenic activity of mouse bone marrow

Murine bone marrow cells synthesize bone proteins, including alkaline phosphatase (ALP), collagen type I, and osteocalcin, and form a mineralized extracellular matrix when cultured in the presence of beta- glycerophosphate and vitamin C. Interleukin-10 (IL-10) suppressed the synthesis of these bone proteins and mineralization without affecting cell proliferation. In addition, mRNA levels for the latter proteins were reduced in IL-10-treated cultures. This inhibitory effect was most outspoken when IL-10 was added before ALP activity peaked, eg, day 15 of culture. No significant effect was observed when IL-10 was added at later time points. This finding suggests that IL-10 acts at osteogenic differentiation stages that precede ALP expression but is ineffective on cells that progressed beyond this maturation stage. Likewise, IL-10 appeared to be unable to block both ALP activity and collagen synthesis in the preosteosteoblastic cell lines MN7 and MC3T3 that constitutively synthesize these proteins. Whereas IL-10 did not alter the number of fibroblast colony-forming cells of the marrow, it significantly reduced their osteogenic differentiation potential. In contrast to control cultures, IL-10-treated stroma was unable to either synthesize osteocalcin or to mineralize when subcultured over a 25-day period in the absence of IL-10. The inhibitory activity of IL-10 coincided with significant changes in stroma morphology. Whereas control cultures contained mainly flat adherent polygonal cells, significant numbers of rounded semiadherent to nonadherent cells were observed in the presence of IL-10. Scanning and transmission electron microscopy showed that, in contrast to control cultures, IL-10-treated stromas completely lacked a mineralized extracellular matrix. Collectively, these data suggest that IL-10 may have important regulatory effects on bone biology because of its capacity to downregulate early steps of osteogenic differentiation.