Role of epidermal growth factor receptor in osteoblastic differentiation of rat bone marrow stromal cells

In an attempt to understand the role of epidermal growth factor (EGF) and its receptor (EGF-R) in osteoblastic cell differentiation, the changes in [¹²⁵I]-EGF binding capacity, synthesis of EGF-R protein, and expression of EGF-R mRNA were investigated during osteoblastic differentiation of cultured bone marrow stromal cells which were collected from the femora of young adult rats. In addition, the ability of EGF to suppress osteoblastic differentiation was also studied. Dexamethasone at a concentration of 0.1 mM increased the expression of osteoblastic markers by bone marrow stromal cells cultured in alpha-modified minimum essential medium (-MEM) con taining 1% fetal bovine serum (FBS), 50 mg/ml ascorbic acid, and 10 mM -glycerophosphate, as revealed by elevated alkaline phosphatase activity, an increase in osteopontin mRNA expression, and bone nodule formation. This osteoblastic differentiation was accompanied by a decreased expression of EGF-R mRNA, decreased synthesis of EGF-R protein, and a decreased number of EGF-binding sites without any change in affinity. When these cells were incubated with dexamethasone and EGF in combination throughout the culture, they exhibited significantly lower levels of all osteoblastic markers than did dexamethasonetreated cells, indicating suppression of osteoblastic differentiation by EGF. In contrast, EGF treatment of the cells induced expression of EGF-R mRNA. Thus, a decrease in EGF binding associated with osteoblastic differentiation could lead to decreased responsiveness of bone marrow cells to EGF, whereas the EGF-induced increase in expression of EGF-R could facilitate the inhibition of cell differentiation by EGF. These findings suggested that upregulation of EGF-R on bone marrow stromal cells antagonizes their differentiation, and thus possibly functions as a negative regulator of osteoblastic differentiation.     

Hemopoietic functions of marrow-derived osteogenic cells

Summary Osteoblasts, members of the marrow stromal cellular network, may play an active role in the hemopoietic microenvironment as well as in bone remodeling. In this study, we examined the extent to which marrow-derived osteogenic cells (MBA-15) possess various stromal functions. This marrow stromal-derived cell line was shown by us to exhibit osteoblastic characteristics in culture and to form bone in vivo. These cells are shown here to constitutively produce and secrete cytokines identified as M-CSF, GM-CSF, and IL-6. MBA-15 cells modulate growth of normal and malignant myeloid and lymphoid cells as well as leukemia cell lines in vitro. Cell-cell interactions were studied in co-cultures with adherent MBA-15 cells and the target hemopoietic cells. Growth inhibition effects, observed under various experimental conditions, can be attributed to the presence of different soluble and membrane-bound inhibitory activities produced by MBA-15 cells. Thus, MBA-15 cells spontaneously produce both stimulators and inhibitors that can affect myeloid and lymphoid cell growth. Marrow osteogenic cells may therefore participate in the stromal regulation of hemopoiesis.     

Bone-like nodules formed by human bone marrow stromal cells: comparative study and characterization

Autologous bone marrow stromal cells have been proposed as an adjuvant in the treatment of bone nonunion. This cell therapy would require the establishment of culture conditions that permit the rapid expansion of these cells ex vivo while retaining their potential for further differentiation. Our aim was to achieve a full differentiation process using human bone marrow aspirates. We first analyzed the effects of mineralization medium (with ascorbic acid and phosphate) and dexamethasone (dex) during the primary culture of human bone marrow stromal (HBMS) cells on the proliferation/differentiation behavior of first-passage cells. The most appropriate schedule was then selected to further characterize this differentiation model. We showed that primary culture of HBMS cells in proliferation medium (DMEM supplemented with 10% fetal calf serum), with a 48-h treatment by mineralization medium and dex resulted in a better osteoblastic differentiation of first-passage cells than primary culture carried out in mineralization medium with or without dex. We showed that culture of HBMS cells under these conditions (primary culture in proliferation medium, followed by subculture in mineralization medium) led to the formation of specifically mineralized bone-like nodules similar to the ones observed with rat bone marrow stromal cells. Our nodules exhibited three distinct cell types, reproducing in vitro a tissue-like structure. This treatment demonstrated an optimal proliferation and expression of osteoblastic markers such as alkaline phosphatase, osteocalcin, and type I collagen. The primary culture allowed the multiplication of the number of adherent progenitor cells at the initial time of plating by a mean factor of 44,000, which was found to be negatively correlated with age. Thus, this differentiation model could provide a new tool to elaborate an autologous cell therapy designed to enhance osteogenesis.     

Role of the cholesterol biosynthetic pathway in osteoblastic differentiation of marrow stromal cells.

Cholesterol is an important molecule that plays a key role in regulating cellular differentiation and function. Although the possible role of lipids has been implicated in regulating osteoblastic cells, the role of cholesterol in that process is not well defined. In this study we have examined the role of the cellular cholesterol biosynthetic pathway on osteoblastic differentiation of marrow stromal cells (MSCs). Treatment of pluripotent mouse MSCs M2-10B4 with inhibitors of the cholesterol biosynthetic pathway mevastatin or mevinolin inhibited the maturation of these cells into functional osteoblastic cells. This was determined by the inhibition of the activity and expression of alkaline phosphatase (ALP), a key enzyme involved in differentiation and mineralization of osteoblastic cell cultures, as well as inhibition of mineralization. Mevastatin treatment did not affect expression of the osteoblast-specific gene osteocalcin (OCN). Furthermore, promoter-reporter studies in MSCs showed that mevastatin inhibited activity of the ALP gene promoter, suggesting regulation by derivatives of the cholesterol biosynthetic pathway. The effects of mevastatin and mevinolin were reversed by mevalonate but not by geranylgeraniol or farnesol, intermediates in the cholesterol biosynthetic pathway. Altogether, these results suggest that products of the cholesterol biosynthetic pathway are important for proper development of MSCs into functional osteoblastic cells capable of forming a mineralized matrix. Identification of those molecules may provide new therapeutic approaches to prevent the decline in osteoblastic activity in osteoporosis and aging.     

酒精对骨髓基质细胞增殖及分化的影响

目的 观察酒精对骨髓基质细胞增殖及分化的作用．探讨骨质疏松的病理学机理。方法 以0.09mol/L酒精加入骨髓基质细胞培养物中，测定增殖的骨髓基质细胞数及培养液中骨钙素含量。通过苏丹Ⅲ脂肪细胞染色计数观察酒精作用时间对脂肪细胞分化的影响。结果 实验组骨髓基质细胞数及培养液中骨钙素含量明显低于对照组，脂肪细胞的数量随酒精作用时间延长而增多。结论 酒精抑制骨髓基质细胞增殖及向成骨方向分化，促进其向脂肪细胞分化，这可能与酒精中毒引起继发性骨质疏松时骨量减少、髓内脂肪组织增多有关。     

Characterization of vitamin D receptor immunoreactivity in human bone cells

The present study examined the expression of the vitamin D receptor (VDR) in adult human bone by immunohistochemical analysis. Antiserum from goats immunized with an N-terminal rat VDR peptide was purified by affinity chromatography. The purified antiserum recognized both endogenous rat and recombinant human VDR in Western blots. The purified antiserum was also able to specifically supershift the recombinant human VDR when analyzed in mobility shift assays. Immunohistochemical analysis of MG-63 cells, a human osteoblastic cell line known to express the VDR, revealed prominent staining over the nuclei of these cells. Immunostaining was greatly attenuated in the presence of an excess of the immunizing peptide. Analysis of bone biopsy samples from 16 normal human subjects immunostained for VDR protein showed strong, immunopositive staining over bone cells, particularly osteoblasts, in keeping with prior studies. In addition, there was significant immunoreactivity observed in nuclei of osteoclasts, lining cells and scattered bone marrow stromal cells of the adult human bone. Results showed that 298 osteoblasts out of 808 (36.9%) examined were immunopositive. It was also observed that 29 osteoclasts out of 125 (23%) contained VDR immunoreactivity. The ability to detect VDR in osteoclasts and stromal cell populations suggests that in addition to regulating osteoblast function, these other cell types are also direct targets of the hormone's action. These results demonstrate the utility of this purified antiserum in detecting the VDR in a variety of molecular techniques and should prove useful in examining receptor expression in various pathological conditions.     

Histomorphometric Analysis of Heterotopic Bone Formed by Stromal-Osteogenic Subpopulations

MBA-15.4 and MBA-15.6 cell lines are marrow stromal clonal subpopulations and represent various stages of differentiation of the osteoblastic family. These cells vary in terms of morphology, proliferation rate, synthesis of matrix proteins as collagen and noncollagenous proteins, and by their responses to hormones and growth factors. Their differential properties directly reflect the clonal cells' ability to form bone in vivo. When the cells were transplanted at an ectopic site, under the kidney capsule, MBA-15.4 line formed small foci of bone whereas MBA-15.6 cell line formed massive woven bone during the same period of time. In this study, we focused on the histomorphometric analysis of ectopic ossicles formed by the clonal cell lines. Assessments of bone mass changes involved measurements of cellular components, osteoid, and formation of primary bone. The bony tissue formed was condensed, no hemopoiesis was noted, and the ossicle was not remodeled. The histology studies were used for quantitative analysis of the ossicle formation and describe the dynamics of ossicles formed by the individual cell types. Received: 9 August 1995 / Accepted: 12 April 1996     

Estrogen regulation of growth and alkaline phosphatase expression by cultured human bone marrow stromal cells.

Estrogen has been reported to regulate the growth and differentiation of cultured murine osteoprogenitor cells in bone marrow stroma. This study tested the ability of 17beta-estradiol (E2) to regulate growth and expression of alkaline phosphatase (ALP), an osteoblastic differentiation marker, in strains of normal human bone marrow stromal cells derived from different donors. In eight strains examined, E2 at 1 and 10 nM produced at most modest effectxs on growth and ALP activity. Growth inhibition, seen in 4 of the 8 strains, was more common than stimulation (2 of the 8 strains); the greatest observed E2 effect was an inhibition of ca. 50%. E2 altered ALP activity less dramatically than cell growth. Differences from control in total ALP per culture were seen in only two strains: one was a reduction, one an increase. Colony forming assays were used to determine if E2 changed the proportion of ALP-expressing cells in marrow stromal cell cultures. In contrast to growth experiments, ALP expression under colony forming conditions (200 cells per 35 mm-diameter well) was dependent on the type of serum supplementation used. Under permissive conditions using medium supplemented with 10% charcoal-treated fetal bovine serum, 10 nM E2 increased the number of ALP-positive colonies (cfu-ap) but not the total number of colonies formed (cfu-f). When cells cultured in the presence or absence of 10 nM E2 were replated at colony forming densities, significantly higher proportions of cfu-ap were found in 2 of 6 strains examined, while pretreatment with E2 affected the number of cfu-f in only 1 of the 6 strains. Similar results were obtained when colony formation was carried out in the presence of dexamethasone and ascorbate, although these agents themselves increased the formation of both cfu-f and cfu-ap. These results show that the direct effects of E2 on human marrow stromal cells are small and vary depending on the cell strain and on the experimental conditions; however, the E2 actions observed in this study were consistent with reports that E2 exerts direct actions on osteoblasts and osteoblast progenitor cells that favor rather than suppress their phenotypic expression.     

Establishment of an osteogenic cell line derived from adult mouse bone marrow stroma by use of a recombinant retrovirus

Summary In order to characterize fibroblastic colonyforming units (CFU-F) from murine bone marrow in relation to osteogenesis, adherent cells of 7-day-old BALB/c mouse bone marrow cultures were infected with a recombinant retrovirus (N2/fosB) containing the bacterial neomycin resistance gene. One of the G418-resistant clones, MN7, was selected for further analysis on the basis of its high expression of the bone-specific alkaline phosphatase. The cells have now been in culture for more than 1 year and maintain a stable phenotype. The osteogenic nature of the immortalized clone MN7 was demonstrated as follows: (1) Mineralization was detected by ⁸⁵Sr uptake and with the Von Kossa staining method only after in vitro cultivation on a collagen type I matrix. (2) Osteoblastic phenotype markers, including the synthesis of type I collagen, osteonectin, and the bonespecific isoenzyme of alkaline phosphatase were expressed in vitro. (3) MN7 cells responded to bone effectors such as parathyroid hormone and 1,25-dihydroxyvitamin D₃. (4) Intraperitoneal injection of MN7 cells into 1-day-old BALB/c mice produced typical osteosarcomas in all animals. We conclude that MN7, derived entirely in vitro from a stromal CFU-F colony, represents a stable murine osteosarcoma cell line expressing the osteoblastic phenotype and provides the first direct evidence needed to establish adult mouse marrow-derived, nonhematopoietic stromal cells as osteoprogenitors.     

Atherogenic diet and minimally oxidized low density lipoprotein inhibit osteogenic and promote adipogenic differentiation of marrow stromal cells.

In osteoporosis, the bone marrow stroma osteogenic cell population declines and adipocyte numbers increase. We recently showed that oxidized lipids inhibit differentiation of preosteoblasts. In this report, we assess the effect of minimally oxidized low density lipoprotein (MM-LDL) on osteoblastic differentiation of murine marrow stromal cells, M2-10B4. MM-LDL, but not native LDL, inhibited stromal cell osteoblastic differentiation as demonstrated by inhibition of alkaline phosphatase activity, collagen I processing, and mineralization, through a mitogen-activated protein kinase-dependent pathway. In addition, marrow stromal cells from C57BL/6 mice fed a high fat, atherogenic diet failed to undergo osteogenic differentiation in vitro. The ability of MM-LDL to regulate adipogenesis was also assessed. Treatment of M2-10B4 as well as 3T3-L1 preadipocytes with MM-LDL, but not native LDL, promoted adipogenic differentiation in the presence of peroxisome proliferator-activated receptor (PPAR) gamma agonist thiazolidinediones, BRL49653 and ciglitizone. Based on promoter-reporter construct experiments, MM-LDL may be acting in part through activating PPARalpha. These observations suggest that LDL oxidation products promote osteoporotic loss of bone by directing progenitor marrow stromal cells to undergo adipogenic instead of osteogenic differentiation. These data lend support to the "lipid hypothesis of osteoporosis."     

Interleukin-1beta and tumor necrosis factor-alpha, but not interleukin-6, stimulate osteoprotegerin ligand gene expression in human osteoblastic cells.

Recent studies have identified osteoprotegerin ligand (OPG-L) as the essential factor required for osteoclastogenesis, and that the effects are prevented by its soluble receptor, osteoprotegerin (OPG). However, there are limited data at present on the regulation of OPG-L expression in human osteoblastic cells by other cytokines. Because interleukin (IL)-1beta, tumor necrosis factor (TNF)-alpha, and IL-6 all increase osteoclastogenesis, we assessed whether OPG-L mRNA steady-state levels were regulated by these cytokines in human osteoblastic cells. By northern analysis, IL-1beta (5 nmol/L) and TNF-alpha (9 nmol/L) increased OPG-L mRNA steady-state levels by up to two- to three-fold in normal marrow stromal cells (MS), an immortalized marrow stromal cell line (hMS), and the osteosarcoma cell line, MG-63, whereas IL-6 (2 nmol/L, with or without its soluble receptor) had no effect on OPG-L mRNA levels in any of these cells. IL-1beta and TNF-alpha increased OPG-L mRNA steady-state levels in the normal MS cells and the hMS cell line in a time- and dose-dependent fashion by up to 4.1-fold and up to 2.6-fold, respectively. Our data are thus consistent with the hypothesis that the proinflammatory and bone-resorbing cytokines, IL-1beta and TNF-alpha, but not IL-6, may stimulate osteoclastogenesis by inducing the expression of OPG-L.     

Marrow stromal cells for cellular cardiomyoplasty: feasibility and potential clinical advantages

OBJECTIVES: Marrow stromal cells are mesenchymal stem cells able to differentiate into cardiomyocytes in vitro. We tested the hypothesis that marrow stromal cells, when implanted into myocardium, can undergo milieu-dependent differentiation and express cardiomyogenic phenotypes in vivo. METHODS: Isogenic adult rats were used as donors and recipients to simulate autologous transplantation. Marrow stromal cells isolated from donor leg bones were culture-expanded, labeled with 4;,6-diamidino-2-phenylindole, and then injected into the myocardium of the recipients. The hearts were harvested from 4 days to 12 weeks after implantation, and the implant sites were examined to identify the phenotypes of the labeled marrow stromal cells. RESULTS: Viable cells labeled with 4;, 6-diamidino-2-phenylindole can be identified in host myocardium at all time points after implantation. Implanted marrow stromal cells show the growth potential in a myocardial environment. After 4 weeks, donor cells derived from marrow stromal cells demonstrate myogenic differentiation with the expression of sarcomeric myosin heavy chain and organized contractile proteins. Positive staining for connexin 43 indicates the formation of gap junctions, which suggests that cells derived from marrow stromal cells, as well as native cardiomyocytes, are connected by intercalated disks. CONCLUSIONS: Different cell sources have been used as donor cells for cellular cardiomyoplasty. Our findings indicate that marrow stromal cells can also be used as donor cells. In an appropriate microenvironment they will exhibit cardiomyogenic phenotypes and may replace native cardiomyocytes lost by necrosis or apoptosis. Because marrow stromal cells can be obtained repeatedly by bone marrow aspiration and expanded vastly in vitro before being implanted or used as autologous implants, and because their use does not call for immunosuppression, the clinical use of marrow stromal cells for cellular cardiomyoplasty appears to be most advantageous.     

Role of stromal cells in osteoclast differentiation in bone marrow

Bone marrow stromal cells have been considered to play an important role in osteoclast differentiation. However, the interaction of these cells in vivo has not been clearly demonstrated. To clarify this, we examined the distribution of alkaline phosphatase (ALPase) and tartrate-resistant acid phosphatase (TRAPase) activities as markers of osteoblastic and osteoclastic cells, respectively. Rat tibiae were fixed and embedded in Technovit 8100 or paraffin. ALPase and TRAPase activities were detected simultaneously on a plastic section by the azo-dye method. ALPase activity was detected on the plasma membranes of osteoblasts and some bone marrow fibroblastic stromal cells. These ALPase-positive cells were connected to each other by cytoplasmic processes, forming a cellular network in bone marrow. The ALPase activity of fibroblastic stromal cells tended to be stronger in those cells close to the bone surface than in the cells in the center of bone marrow. Reticular fibers in bone marrow were found to form a network. The ALPase-positive fibroblastic stromal cells may be reticular cells, because the localization of those cells was in accord with the localization of reticular fibers. The TRAPase-positive mononuclear cells and osteoclasts were mostly observed to be associated with the intensely ALPase-positive fibroblastic stromal cells. Immunoreactivity of osteoclast differentiation factor (ODF) was found in the fibroblastic stromal cells. These findings suggest that the network of ALPase-positive fibroblastic stromal cells in bone marrow serves as a guide for the migration of osteoclast precursor cells toward the bone surface, and may control the differentiation and activity of osteoclasts.     

Dentin matrix protein 1 expression during osteoblastic differentiation, generation of an osteocyte GFP-transgene

Our previous studies have demonstrated that promoter-green fluorescent protein (GFP) transgenes can be used to identify and isolate populations of cells at the preosteoblastic stage (pOBCol3.6GFP) and at the mature osteoblastic stage (pOBCol2.3GFP) in living primary bone cell cultures. This strategy forms the basis for appreciating the cellular heterogeneity of lineage and relating gene function to cell differentiation. A weakness of this approach was the lack of a selective marker for late osteoblasts and mature osteocytes in the mineralized matrix. In this study, we have examined the expression of DMP-1 mRNA in murine marrow stromal and calvarial osteoblast cultures, and in bone, and calvaria in vivo. Furthermore, we have generated transgenic mice utilizing a mouse DMP1 cis-regulatory system to drive GFP as a marker for living osteocytes. Transgene expression was directed to mineralized tissues and showed a high correlation with the expression of the endogenous gene. Osteocyte-restricted expression of GFP was observed in histological sections of femur and calvaria and in primary cell cultures. Generation of this transgenic model will facilitate studies of gene expression and biological functions in these terminally differentiated bone cells.     

Stromal cell activity in bone marrow from the tibia and iliac crest of patients with rheumatoid arthritis

Bone marrow aspirates were obtained from the iliac crest and tibial epiphysis in 23 patients with rheumatoid arthritis (RA) who were undergoing total knee arthroplasty. The number of fibroblast colony-forming units (CFU-F), which contain osteogenic precursor cells, and alkaline phosphatase (ALP) activity, as a marker of the osteoblastic phenotype, were compared between the iliac and tibial marrow for each patient. The prevalence of CFU-F in tibial marrow was similar to that in iliac marrow (96% vs 100%, respectively). However, the average number of CFU-F per 4 × 10⁵ bone marrow mononuclear cells was significantly lower in tibial marrow than in iliac marrow (8.2 vs 28.1, respectively; P < 0.01). Although ALP activity was detected in all iliac and tibial marrow specimens, it was significantly lower in tibial marrow compared with iliac marrow (3.7 vs 11.9 nmol/min/mg protein, respectively; P < 0.01). In addition, there was a significant correlation between the patient's age and the number of CFU-F in iliac marrow (r = −0.547; P < 0.01), although there was no correlation in tibial marrow. These results demonstrate that the osteogenic activity of bone marrow varies at different sites in patients with RA. The data may also contribute to further investigation into the differential effects of various disease processes on systemic as well as local stromal cell activity in bone marrow. Received: April 18, 2000 / Accepted: July 11, 2000