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.     

Mesenchymal stem cells from the bone marrow stroma: basic biology and potential for cell therapy

Mesenchymal stem cells (MSCs) can be isolated from the bone marrow stroma where they constitute an adult somatic stem cell population distinct from hemapoietic stem cells. MSCs are multipotent cells in that they have the capacity to generate progeny that can differentiate into multiple cell lineages. MSCs can be explanted in vitro from bone marrow aspirates and expanded in culture where they can be induced to terminally differentiate into osteoblasts, chrondrocytes, adipocytes, tenocytes and heamapoteic supporting tissue. This ability to differentiate has also been demonstrated in vivo following transplantation into rodents. Recent work has shown that MSCs may have a broader capacity for differentiation than was previously envisioned. In some circumstances, this increased potential for differentiation may make MSCs viable alternatives to embryonic stem cells. Accordingly, the multipotential capacity of MSCs, their accessible origin, and high ex vivo expansive potential, makes these cells attractive as tools for tissue engineering and cell-based therapy. This review will explore the basic biology of MSCs derived from adult tissues and consider their isolation, culture, and differentiation. In addition, we will examine some of the potential clinical applications of these cells and consider the future perspectives of their use.     

Identification and characterization of mouse bone marrow stromal cell lines immortalized by temperature-sensitive SV40 T antigen: supportive activity for osteoclast differentiation

Osteoblasts are derived from mesenchymal/stromal cells in bone marrow, and gain the ability to support osteoclastogenesis during differentiation though the expression of receptor activator of NF-kappaB ligand (RANKL) and macrophage-colony stimulating factor (M-CSF). However, the properties (differentiation stage and expression of osteoblast marker genes) of stromal or osteoblastic cells that have the capacity to support osteoclast differentiation are unclear. Therefore, we sought to establish and characterize bone marrow-derived stromal cell lines (TSB) from temperature-sensitive SV40 T-antigen transgenic mice to define them at the clonal level. Of the 24 randomly selected cell lines, only 2 cell lines, TSB13 and TSB20, could support osteoclast differentiation in the presence of 1alpha,25(OH)(2)D(3). In both cell lines, RANKL mRNA was induced and osteoprotegerin (OPG) mRNA was decreased in response to treatment with 1alpha,25(OH)(2)D(3) for 2 days. Other RNA expression analyses of osteoblast-specific marker genes demonstrated the following characteristics of TSB13 and TSB20: (1) alkaline phosphatase (ALP) and type I collagen genes are expressed; (2) osteocalcin and osteopontin genes are expressed at low levels, and their expression levels are upregulated after induction of differentiation by a temperature shift from 33 degrees C to 37 degrees C, or 1alpha,25(OH)(2)D(3) treatment. Consequently, the long-term culture of TSB13 and TSB20 cell lines strongly stimulated osteocalcin expression and effectively induced calcified nodule formation in the presence of phosphate. The results suggest that the supportive cells for osteoclastogenesis are restricted to a specialized population of bone marrow stromal cells, and the high ratio of RANKL vs. OPG expression found in this population after 1alpha,25(OH)(2)D(3) treatment might be a general property of osteoclast-supporting cells.     

Differential gene expression in cultured osteoblasts and bone marrow stromal cells from patients with Paget's disease of bone.

Paget's disease is a focal condition of bone. To study changes in cells within pagetic lesions, we cultured osteoblasts and stromal cells from 22 patients and compared gene expression in these cells to cells from healthy bone. We identified several differentially regulated genes, and we suggest that these changes could lead to the formation of the lesions. INTRODUCTION: Paget's disease is a focal condition of bone of unknown cause. Although it is regarded as primarily an osteoclast disorder, the tight coupling of the activity of osteoclasts and osteoblasts suggests that the osteoblast could play a key role in its pathogenesis. The aim of the study was to identify possible changes in pagetic osteoblasts and stromal cells that might contribute to the development of pagetic lesions. MATERIALS AND METHODS: Candidate genes were identified based on known bone cell regulators, supplemented with microarray analysis. Gene expression was determined by real-time PCR in primary cultures of osteoblasts and bone marrow stromal cells from pagetic patients and control subjects. Concentrations of secreted proteins were determined by ELISA. RESULTS: Dickkopf1 mRNA and protein levels were increased in both pagetic osteoblast and stromal cell cultures, and interleukin (IL)-1 and IL-6 were overexpressed in pagetic osteoblasts. These changes parallel recent findings in myeloma bone disease, which shares some clinical similarities with Paget's disease. Alkaline phosphatase was overexpressed, and bone sialoprotein and osteocalcin were underexpressed in pagetic osteoblasts, consistent with their circulating levels in pagetic patients. It is hypothesized that overexpression of Dickkopf1, IL-1, and IL-6 would result in stimulation of osteoclast proliferation and inhibition of osteoblast growth, leading to the development of the characteristic lytic bone lesions. By stimulating osteoblast differentiation, Dickkopf1 and IL-6 may also promote mineralization, leading to the conversion of lytic lesions to sclerotic. CONCLUSIONS: These findings suggest that dysregulated gene expression in pagetic osteoblasts could cause the changes in bone cell number and function characteristic of Paget's disease.     

Enhancement of fractionated bone marrow transplantation on hematopoietic cell homing in a mouse allogeneic model

BACKGROUND: The conventional method of bone mar row transplantation (BMT) requires a large quantity of bone marrow cells (BMC) transplanted by a single injection. However, the homing efficiency of BMC is low, because the emptying of available niches might be a continuous process after stem-cell death after irradiation. In this article, the death character of CD34 stem cells was directly detected, and a novel method of fractionated (Fr) BMT that aimed to better use each niche that was continuously emptied was developed. METHODS: Apoptosis and necrosis were detected using trivariate fluorescence-activated cell sorting after labeling cells with phycoerythrin-CD34-fluorescein isothiocyanate-Annix V-7-aminoactinomycin D. Fr-BMT was conducted in a model of allogeneic BMT, where the total dosage of BMC was divided into four doses, and each dose was administrated for 4 consecutive days at 24-hr intervals after lethal irradiation. RESULTS: CD34 cells underwent a continuous death process after lethal irradiation and presented three death peaks within a week. Fr-BMT decreased the BMC transplanted but increased the accumulatively homed BMC and enhanced the survival rates of recipients. CONCLUSIONS: Fr-BMT is able to enhance hematopoietic cell homing and engraftment, and this method might prove to be a solution to the previous clinical problem that a single unit of umbilical cord blood is not sufficient for engraftment in adults.     

Successful generation of donor specific hematopoietic stem cell lines from co-cultured bone marrow with human embryonic stem cell line: a new methodology

We report the generation of 30 healthy human embryonic stem cell (h-ESC) lines from 33 voluntary oocyte donors using a donor somatic cell nuclear transfer (SCNT) technique on 190 oocytes. Our aim was to coculture them with their own bone marrow (BM) to generate hematopoietic progenitor cells for therapeutic purposes. Pluripotency and undifferentiated stage were confirmed using molecular cell surface markers. Normal karyotype of these cell lines was confirmed. Here we demonstrate that SCNT-h-ESCs differentiate to hematopoietic precursors when cocultured with unmodified, nonirradiated donor BM. We did not use any xenogeneic material for this hematopoietic differentiation. Hematopoietic precursors derived from them expressed cell surface antigens CD45/34. When further cultured with hematopoietic growth factors these hematopoietic precursors formed characteristic myeloid, erythroid, and megakaryocyte lineages. Phenotypic CD34+ cells derived from NT-h-ESCs were functionally similar to their counterparts in primary hematopoietic tissues like BM, umbilical cord, and blood. More terminally differentiated hematopoietic cells derived from h-ESCs under these culture conditions also expressed normal surface antigens like glycophorin A on erythroid cells, CD15 on myeloid cells, and CD41 on megakaryocytes. We report generation of hematopoietic progenitor cells from h-ESC lines by a SCNT technique, with differentiation into further lineages with structural and functional similarities to their adult counterparts in vivo. This novel alternative source of CD34+ stem cells from h-ESC lines generated without any xenogeneic material might be used to create transplantation tolerance, to implement regenerative medicine, and to treat autoimmune disorders.     

Study of gelatinized marrow stroma osteoblasts and true bone ceramic active bone

Trueboneceramic(TBC)isacomparativelygoodframematerialinbonetissueengineeringbecauseofitsadvantagesofnaturalbonetrabeculestructure, easydegradation, nonimmunogenicity, easymanufacture, abundantresources, andlowcost, etc1.Owingtoitsfragileness, nonductility, andasmoothsurfaceunfitforseedcelladhesion, itisstilldifficultforwidespreadclinicaluse. Bytakingadvantageofthepeculiaritiesofsodiumalginatethatwilltransformfromliquidstateintogelatinationstateandproducelateralconjunctionwhencombiningwithbiva…     

A quadripotential mesenchymal progenitor cell isolated from the marrow of an adult mouse.

Adult marrow contains mesenchymal progenitor cells (MPCs) that have multiple differentiation potentials. A conditionally immortalized MPC clone, BMC9, has been identified that exhibits four mesenchymal cell phenotypes: chondrocyte, adipocyte, stromal (support osteoclast formation), and osteoblast. The BMC9 clone, control brain fibroblasts and another marrow-derived clone, BMC10, were isolated from a transgenic mouse (H-2Kb-tsA58) containing a gene for conditional immortality. To test for chondrogenic potential, cells were cultured in defined medium containing 10 ng/ml transforming growth factor beta and 10-7 M dexamethasone in 15-ml polypropylene tubes ("aggregate cultures"). Adipogenic potential was quantitated by flow cytometry of Nile Red-stained cells cultured for 1 and 2 weeks in medium containing isobutyl methylxanthine, indomethacin, insulin, and dexamethasone. Support of osteoclast formation was measured by quantitating multinucleated tartrate-resistant acid phosphatase-positive cells in spleen cell cocultures of test clones (immortomouse clones and positive control ST2 cells) cultured in the presence of 10-7 M vitamin D3 and 150 mM ascorbate-2-phosphate. In vivo osteogenic potential was assayed by histologic examination of bone formation in subcutaneous implants, into athymic mouse hosts, of a composite of cells combined with porous calcium phosphate ceramics. The bone marrow-derived clone BMC9 has the potential to express each of the four mesenchymal characteristics tested, while brain fibroblasts, tested under identical conditions, did not exhibit any of these four mesenchymal characteristics. BMC10 cells exhibited osteogenic and chondrogenic phenotypes, but showed only minimal expression of adipocytic or osteoclast-supportive phenotypes. Clone BMC9 is, minimally, a quadripotential MPC isolated from the marrow of an adult mouse that can differentiate into cartilage and adipose, support osteoclast formation, and form bone. The BMC9 clone is an example of an adult-derived multipotential progenitor cell that is situated early in the mesenchymal lineage.     

Differential expression of Galalpha1,3Gal epitopes on fetal and adult porcine hematopoietic cells

Galα1-3Gal (Gal) is the major epitope on pig tissues bound by human natural antibodies. Xenogeneic hematopoietic cell transplantation is being investigated to induce immunological tolerance to xenografts. We have investigated the level of Gal expression on pig hematopoietic cells. Cells were collected from pig fetal liver and bone marrow (BM), and also from adult BM and peripheral blood, before and after treatment with pig-specific hematopoietic growth factors. Fluorescent activated cell sorting (FACS) analysis was performed with the M86 monoclonal antibody (specific for Gal), lineage markers, and biotinylated stem cell factor (SCF) to detect c-kit expression. In fetal pig BM and liver, there was no significant difference in Gal expression between monocytes/macrophages (myeloid cells) and lymphocytes. In adult hematopoietic cells from all sources, Gal-positive subpopulations in T cells showed weak expression of Gal, whereas B cells demonstrated higher expression, and myeloid cells showed highest expression. Adult BM and mobilized peripheral blood progenitor cells contained small populations with very low or negligible expression of Gal. A very small population of c-kit-positive cells, indicating progenitor cells, were Gal-negative. The small Gal-negative population that exists in progenitor cells might explain why some pig colony forming units (CFU) can be resistant to human serum.     

Differentiation of transplanted bone marrow cells in the adult mouse brain.

BACKGROUND: Bone marrow transplantation is reportedly effective in preventing the progression of neurological deterioration in lysosomal storage disorders, although the mechanism underlying the therapeutic effects remains to be elucidated. Recent research on stem cell biology suggests that bone marrow cells contain nonhematopoietic stem cells, including brain precursor cells. To evaluate the contribution of bone marrow cells as carriers for cell and gene therapy of neurological disorders, we studied the fate of transplanted bone marrow cells in the adult mouse brain. METHODS: Bone marrow cells were genetically marked with a retroviral vector containing the green fluorescence protein gene and then transplanted into irradiated mice by either systemic infusion or direct injection. To identify cell types, brain sections were stained with specific antibodies against neuronal cell markers-neuron specific enolase for neurons, glial fibrillary acidic protein (GFAP) for astrocytes, carbonic anhydrase II (CAII) for oligodendrocytes, and ionized calcium binding adaptor molecule 1 (Iba1) for microglia-and then examined under a confocal microscope. RESULTS: Twenty-four weeks after systemic infusion, transplanted cells expressed Iba1 but none of the other brain cell markers. Conversely, 12 weeks after direct injection, transplanted cells were stained with antibodies against GFAP, CAII, and Iba1. CONCLUSIONS: Bone marrow contains cells capable of differentiating into oligodendrocytes, astrocytes, and microglia when exposed to the brain microenvironment. Autologous bone marrow cells may be useful as carriers for ex vivo gene therapy for lysosomal disorders with neurological symptoms.     

Parathyroid hormone regulates the distribution and osteoclastogenic potential of hematopoietic progenitors in the bone marrow

Parathyroid hormone (PTH) increases both the number of osteoclast in bone and the number of early hematopoietic stem cells (HSCs) in bone marrow. We previously characterized the phenotype of multiple populations of bone marrow cells with in vitro osteoclastogenic potential in mice. Here we examined whether intermittent administration of PTH influences these osteoclast progenitor (OCP) populations. C57BL/6 mice were treated with daily injections of bPTH(1–34) (80 µg/kg/day) for 7 or 14 days. We found that PTH caused a significant increase in the percentage of TN/CD115⁺CD117^high and TN/CD115⁺CD117^int cells (p < .05) in bone marrow on day 7. In contrast, PTH decreased the absolute number of TN/CD115⁺CD117^low cells by 39% on day 7 (p < .05). On day 14, there was no effect of PTH on osteoclast progenitor distribution in vivo. However, PTH treatment for 7 and 14 days did increase receptor activator of NF‐κB ligand (RANKL)– and macrophage colony‐stimulating factor (M‐CSF)–stimulated in vitro osteoclastogenesis and bone resorption in TN/CD115⁺ cells. In the periphery, 14 days of treatment increased the percentage and absolute numbers of HSCs (Lin^?CD117⁺Sca‐1⁺) in the spleen (p < .05). These data correlated with an increase in the percent and absolute numbers of HSCs in bone marrow on day 14 (p < .05). Interestingly, the effects on hematopoietic progenitors do not depend on osteoclast resorption activity. These results suggest that in vivo PTH treatment increased in vitro osteoclastogenesis and resorption without altering the number of osteoclast precursors. This implies that in vivo PTH induces sustained changes, possibly through an epigenetic mechanism, in the in vitro responsiveness of the cells to M‐CSF and RANKL. © 2011 American Society for Bone and Mineral Research.     

Trabecular bone turnover, bone marrow cell development, and gene expression of bone matrix proteins after low calcium feeding in rats

Low-calcium-fed animals have been accepted as one of the experimental models showing a reduction in bone mass. However, the effects of short-term low-calcium feeding on bone turnover, the development of osteoprogenitor cells, and gene expression of bone matrix proteins have not been reported. In this study, we examined the effect of a low-calcium diet on rat tibia and analyzed the changes in the bone by histomorphometry, bone marrow cell culture, and in situ and Northern hybridization of the bone matrix proteins. Rats were fed either a low-calcium diet (0.05% Ca) or a normal calcium diet (0.5% Ca) using the pair feeding technique. They were killed at day 0, 12 h, and days 1, 2, and 3. In the low-calcium group, the serum parathyroid hormone (PTH) level was temporarily increased in 12 h after feeding the low-calcium diet. Bone mineral density in the trabecular bone was significantly decreased from 1 day after the low-calcium feeding, but cortical bone did not show any changes during the experimental period. The bone volume per tissue volume in the proximal tibia also decreased from day 1 in the low-calcium group. The number of osteoclasts and osteoblasts on the trabecular bone surface was increased in the low-calcium group compared with the normal-calcium group. An ex vivo study showed that the number of progenitors of osteoclasts and osteoblasts in bone marrow was also increased in the low-calcium group of rats. The localization of type I collagen mRNA was observed in osteoblasts in the low-calcium group. The Northern hybridization study showed that the gene expression of type I collagen, osteopontin, and osteocalcin was increased at day 3 in the low-calcium group. These results indicated that the trabecular bone surface quickly responded to the low-calcium feeding and that bone remodeling activity was activated probably by PTH. The changes in bone marrow cell populations and the gene expression of bone matrix proteins are closely associated with increased bone turnover induced by the low-calcium diet, resulting in rapid bone loss of the trabecular bone.     

髓内脂肪细胞对骨髓基质细胞成骨能力的影响

目的探讨在骨髓腔中,来源于骨髓基质细胞的脂肪细胞对骨髓基质细胞成骨能力的影响。方法将骨髓基质细胞以1×106个/ml浓度接种于24孔培养板中,共分为5组,分别加入0、103、104、105、106/ml浓度的脂肪细胞悬液,建立脂肪细胞和骨髓基质细胞共培养体系。共培养12d,每4d取细胞样本,检测细胞内碱性磷酸酶活性,利用原位杂交方法检测I型胶原mRNA表达,3H脯氨酸掺入实验检测骨髓基质细胞胶原合成能力。结果经过12d培养,不同时段的共培养体系中,随着脂肪细胞浓度上升,骨髓基质细胞内碱性磷酸酶相对活性下降,各实验组明显低于对照组(P<0.05或P<0.01);I型胶原mRNA表达减弱,对照组染色最深,实验组着色较淡;各实验组3H脯氨酸掺入实验min-1值均低于同期对照组(P<0.05或P<0.01)。结论髓内脂肪细胞干扰了骨髓基质细胞的成骨能力,可能与原发性骨质疏松症的发病有关。     

Effects of lead on osteoclast-like cell formation in mouse bone marrow cell cultures

To examine an effect of lead (Pb) on the process of osteoclast-like cell formation from its progenitors, we used a mouse bone marrow culture system in which osteoclast-like multinucleated cells (MNCs) were formed in response to bone-resorbing agents. In a 9-day culture period, Pb dose-dependently stimulated MNC formation over the concentration range 2–10 M, whereas at 40 M Pb, MNC formation declined. In an 11-day culture period, MNC formation reached a maximum at 5 M Pb and decreased with increasing concentration of Pb at 10–40 M. Pb-stimulated MNC formation was inhibited by both indomethacin and SC19220, an antagonist of prostaglandin E₂ (PGE₂) receptor. Pb stimulated the production of PGE₂ in marrow cell cultures, suggesting that Pb-stimulated MNC formation is dependent on the production of PGE₂. 3-Isobutyl-1-methylxanthine potentiated Pb-stimulated MNC formation and 2,5-dideoxyadenosine, an inhibitor of adenylate cyclase, inhibited it. A calcium ionophore A23187 increased Pb-induced MNC formation and verapamil, a calcium channel blocker, depressed it. It is possible that a PGE₂-induced increase in the levels of cyclic adenosine 3,5-monophosphate (cAMP) and calcium ions in marrow cells is involved in Pb-induced MNC formation. Pb and parathyroid hormone showed a synergistic stimulation on MNC formation. From these results, Pb is thought to induce osteoclast-like cell formation by a mechanism involving PGE₂ which increases the intracellular levels of cAMP and calcium ions.     

胶质细胞源性神经营养因子(GDNF)在大鼠骨髓基质干细胞中的表达

目的构建表达外源性胶质细胞源性神经营养因子(GDNF)的转基因大鼠骨髓基质干细胞(BMSCs),拟应用于脊髓损伤的基因治疗研究。方法提取新生大鼠肾组织mRNA,用逆转录PCR法扩增GDNF全长cDNA,构建其真核表达载体PEG-GFP-GDNF,酶切及测序鉴定;然后转染原代培养的大鼠BMSCs,荧光免疫细胞化学及细胞形态学检测GDNF的表达。结果扩增到全长687bp序列准确的GDNF编码片段,并在GDNF转基因原代大鼠BMSCs中检测到重组GDNF的表达。结论体外可构建成功表达GDNF的转基因BMSCs,有望应用于脊髓损伤等中枢神经系统疾病的治疗研究。     

Prostasin基因在前列腺细胞株中表达情况研究

目的 探讨Prostasin在前列腺癌中的功能。方法 运用RT-PCR方法检测人前列腺增生细胞株BPH-1，无转移能力前列腺癌细胞株LNCaP，及有转移能力的前列腺癌细胞株PC-3、DU-145中Prostasin基闪表达情况。结果 Prostasin基斟在BPH-l和LNCaP中正常表达，在PC-3、DU-145中低表达。BPH-l中Prostasin的表达与DU-145和PC-3比较差异有显著性，同样LNCaP中Prostasin的表达与DU-145和PC-3比较差异亦有显著性（P〈0．01）。BPH-1与LNCaP之间表达差异无显著性，DU-145和PC-3之间表达差异亦无显著性，（P〉0.05）。结论 ProStasin可能是前列腺癌的转移抑制剂。