Lentivirus transduction of human osteoclast precursor cells and differentiation into functional osteoclasts

Gene transfer into stem cells has been an ongoing priority as a treatment for genetic disease and cancer for more than two decades. Methods described herein, form the basis for providing the cell source to determine if osteoclast precursor cells (OcP) can be used as therapeutic gene delivery systems in vivo. Osteoclasts and tumor associated macrophages or OcP, support survival, tumor progression and osteolysis in bone cancers. Two sources of precursor cells are compared: CD14+ cells, the standard OcP, found abundantly in peripheral blood and CD34+ cells, hematopoietic stem cells that are rare, but which can be expanded into OcP. Our findings characterize cell yield at each step of the transduction process and thus provide essential data for planning future in vivo experiments. In addition we demonstrate that essential functions of OcP are preserved following lentiviral transduction. Specifically, neither the transduction method nor the lentiviral transduction influence the OcP's ability to form osteoclasts, express the marker gene, EGFP, or resorb bone. Finally, we conclude that CD34+ cells yield significantly more transduced cells and form functionally superior osteoclasts in vitro. This study represents a step towards considering human gene therapy for bone cancer by demonstrating successful transduction of human OcP for use as cellular delivery vehicles to sites of bone cancer.     

Osteoblasts/stromal cells stimulate osteoclast activation through expression of osteoclast differentiation factor/RANKL but not macrophage colony-stimulating factor: receptor activator of NF-kappa B ligand

This international, randomized, double-blind, placebo-controlled, parallel group, dose-ranging trial was designed to determine the efficacy of 2 years of therapy with a new matrix transdermal 17beta-estradiol (Menorest) in preventing bone loss in early postmenopausal women, and to identify an appropriate dose. Two hundred ninety-two ambulatory women with natural or surgical menopause for 1-6 years were randomized to receive patches delivering 17beta-estradiol 50, 75, or 100 microg/day twice weekly for 25 days per 28 day cycle (with dydrogesterone 10 mg twice daily from days 11 to 24) or placebo, for 24 months. The primary outcome measure was the percentage change from baseline in lumbar spine bone mineral density (BMD) at 2 years. Secondary endpoints were percentage changes from baseline in three sites of proximal femur BMD and total body BMD, and in biochemical bone turnover markers. At 2 years, the difference from placebo in percentage change from baseline of L1-4 spine BMD was 6.2%, 7.6%, and 7.8% in the 50, 75, and 100 microg/day groups, respectively. Lumbar spine bone increased in 65.5%, 76.8%, and 81.0% of patients in the respective active treatment groups, compared with 4.9% on placebo. BMD increased significantly relative to placebo in the femoral neck, trochanter, total hip, and total body. Serum osteocalcin, bone alkaline phosphatase and urinary type I collagen C-telopeptide decreased significantly and dose dependently in 17beta-estradiol patients vs. placebo. For example, at 2 years, the difference between placebo and the 50 microg/day group, expressed in percentage change from baseline, was 3.25% at the femoral neck, 3.92% at the trochanter, 3.52% for total hip, and 2.40% for the total body. Breast pain and skin reactions were more common in the actively treated groups, but tolerability was generally good. Therefore, after 2 years, 17beta-estradiol was well-tolerated and highly effective at doses of between 50 and 100 microg/day in preventing bone loss and reducing bone turnover in early postmenopausal women. The dose of 50 microg/day, the lowest dose tested, is a suitable dose. There was little clinical benefit of increasing the dosage from 75 to 100 microg/day.     

c-fos antisense DNA inhibits proliferation of osteoclast progenitors in osteoclast development but not macrophage differentiation in vitro

We previously reported that osteoclast formation in vitro, by coculture of mouse bone marrow and primary osteoblastic cells, occurs in two phases: proliferation of osteoclast progenitors followed by terminal differentiation into mature osteoclasts. Using this coculture system, we examined the effects of c-fos antisense and sense phosphorothioate oligonucleotides on osteoclast development and macrophage differentiation. Treatment with c-fos antisense for the first 4 days of coculture inhibited osteoclast formation in a dosedependent fashion. However, when c fos antisense was added during the second phase of coculture (4–6 days), osteoclast formation was unaffected. In contrast, c-fos antisense treatment had no effect on the appearance of F4/80 antigen-positive cells of the macrophage lineage in these cultures or on the induction by colony stimulating factor-1 of macrophage colony formation in cultures of mouse bone marrow cells in agar. Neither osteoclast differentiation nor macrophage appearance was inhibited by adding control c-fos sense in the cocultures. When c-fos antisense was added into an assay of bone resorption by mature osteoclasts, pit formation on dentine slices was unaffected. These results indicate that c-fos plays an important role in the proliferative phase of osteoclast progenitors in osteoclast development, but not in the terminal differentiation phase or in the bone resorbing activity of mature osteoclasts. c-fos antisense specifically inhibited osteoclast formation but had no effect on macrophage development.     

肝细胞生长因子诱导骨髓间充质干细胞向肝细胞分化的实验研究

目的探讨成年大鼠骨髓间充质干细胞(MSCs)在体外是否可定向诱导分化为肝细胞及其方法。方法采用梯度离心法,分离纯化SD大鼠的MSCs,流式细胞仪检测和碱性磷酸酶染色鉴定细胞类型。根据培养基中肝细胞生长因子(HGF)浓度不同,将MSCs分为4组进行诱导分化：A组0 ng／ml,B组10ng／ml,C组20ng／ml,D组40ng／ml。倒置显微镜连续观察细胞分化过程的形态学变化。于培养的1,3,7,14,21,28 d,分别以逆转录聚合酶链反应(RT-PCR)和细胞免疫组化检测各组细胞甲胎蛋白(AFP)、细胞角蛋白18(CK18)和白蛋白的基因和蛋白表达。结果分离纯化的SD大鼠MSCs的表面标志为CD29^ ,CD44^ ,CD34^-,CD45^-和CD90^ ,细胞的碱性磷酸酶染色阴性,细胞纯度达98％以上。C组与D组的MSCs,于培养第7天出现AFP基因表达,第14天表达增强,第28天表达减弱;第14天始出现白蛋白、CK18基因表达,而后持续。B组和A组在培养过程中,未出现AFP、CK18和白蛋白的表达。C组与D组MSCs的AFP细胞免疫组化染色培养第7天即出现阳性,第14天白蛋白和CK18免疫组化染色阳性。A组和B组的MSCs的AFP、白蛋白和CK18的免疫组化染色均阴性。结论成年大鼠MSCs在较高浓度的HGF的诱导下,可分化为肝细胞。     

Parathyroid hormone induces differentiation of mesenchymal stromal/stem cells by enhancing bone morphogenetic protein signaling

Parathyroid hormone (PTH) stimulates bone remodeling and induces differentiation of bone marrow mesenchymal stromal/stem cells (MSCs) by orchestrating activities of local factors such as bone morphogenetic proteins (BMPs). The activity and specificity of different BMP ligands are controlled by various extracellular antagonists that prevent binding of BMPs to their receptors. Low-density lipoprotein receptor-related protein 6 (LRP6) has been shown to interact with both the PTH and BMP extracellular signaling pathways by forming a complex with parathyroid hormone 1 receptor (PTH1R) and sharing common antagonists with BMPs. We hypothesized that PTH-enhanced differentiation of MSCs into the osteoblast lineage through enhancement of BMP signaling occurs by modifying the extracellular antagonist network via LRP6. In vitro studies using multiple cell lines, including Sca-1⁺CD45^–CD11b^–MSCs, showed that a single injection of PTH enhanced phosphorylation of Smad1 and could also antagonize the inhibitory effect of noggin. PTH treatment induced endocytosis of a PTH1R/LRP6 complex and resulted in enhancement of phosphorylation of Smad1 that was abrogated by deletion of PTH1R, β-arrestin, or chlorpromazine. Deletion of LRP6 alone led to enhancement of pSmad1 levels that could not be further increased with PTH treatment. Finally, knockdown of LRP6 increased the exposure of endogenous cell-surface BMP receptor type II (BMPRII) significantly in C2C12 cells, and PTH treatment significantly enhanced cell-surface binding of ¹²⁵I-BMP2 in a dose- and time-dependent manner, implying that LRP6 organizes an extracellular network of BMP antagonists that prevent access of BMPs to BMP receptors. In vivo studies in C57BL/6J mice and of transplanted green fluorescent protein (GFP)-labeled Sca-1⁺CD45^–CD11b^–MSCs into the bone marrow cavity of Rag2^−/− immunodeficient mice showed that PTH enhanced phosphorylation of Smad1 and increased commitment of MSCs to osteoblast lineage, respectively. These data demonstrate that PTH enhancement of MSC differentiation to the osteoblast lineage occurs through a PTH- and LRP6-dependent pathway by endocytosis of the PTH1R/LRp6 complex, allowing enhancement of BMP signaling. © 2012 American Society for Bone and Mineral Research.     

Regulation of osteoclast differentiation by fibroblast growth factor 2: stimulation of receptor activator of nuclear factor kappaB ligand/osteoclast differentiation factor expression in osteoblasts and inhibition of macrophage colony-stimulating factor function in osteoclast precursors.

This study investigated the mechanism of direct and indirect actions of fibroblast growth factor 2 (FGF-2) on osteoclast differentiation using two mouse cell culture systems. In the coculture system of osteoblasts and bone marrow cells, FGF-2 stimulated osteoclast formation. This effect was decreased markedly by osteoprotegerin (OPG) or NS-398, a selective cyclo-oxygenase 2 (COX-2) inhibitor. FGF-2 (> or = 10(-9) M) stimulated receptor activator of nuclear factor kappaB ligand/osteoclast differentiation factor (RANKL/ODF) messenger RNA (mRNA) expression from 2 h to 7 days in cultured osteoblasts. NS-398 did not affect the early induction but decreased the later one, indicating that the later effect is mediated by COX-2 induction in osteoblasts. To study the direct action of FGF-2 on osteoclast precursors, we used mouse macrophage-like cell line C7 cells that can differentiate into osteoclasts in the presence of soluble RANKL/ODF (sRANKL/ODF) and macrophage colony-stimulating factor (M-CSF). Although osteoblasts expressed all FGF receptors (FGFR-1 to -4), only FGFR-1 was detected in C7 cells at various differentiation stages. FGF-2 alone or in combination with sRANKL/ODF did not induce osteoclastogenesis from C7 cells; however, FGF-2 from lower concentrations (> or = 10(-11) M) significantly decreased osteoclast formation induced by M-CSF in the presence of sRANKL/ODF. FGF-2 did not alter mRNA levels of M-CSF receptor (Fms) or RANK in C7 cells. Immunoprecipitation/ immunoblotting analyses revealed that tyrosine phosphorylation of several cellular proteins including Fms in C7 cells induced by M-CSF was inhibited by FGF-2 in the presence of sRANKL/ODF. We conclude that FGF-2 regulates osteoclast differentiation through two different mechanisms: (1) an indirect stimulatory action via osteoblasts to induce RANKL/ODF partly through COX-2 induction and prostaglandin production and (2) a direct inhibitory action on osteoclast precursors by counteracting M-CSF signaling.     

不同浓度葡萄糖对大鼠骨髓破骨细胞分化的影响

目的观察不同浓度葡萄糖对大鼠骨髓破骨细胞（Osteoclasts OC）分化的影响，探讨糖尿病骨质疏松的发病机制。方法用M—CSF、RANKL诱导大鼠骨髓单个核细胞分化为OC，同时给予不同浓度的葡萄糖（0、5．5、15、25mmol／L）干预，通过观察抗酒石酸酸性磷酸酶（TRAP）染色阳性OC数、TRAP活性测定、OC膜表面RANK mRNA表达量来分析葡萄糖对破骨细胞分化的影响。结果①高浓度葡萄糖组（25mmol/L）培养7d时TRAP染色阳性OC数及TRAP活性测定均高于对照（0mmol／L）组、葡萄糖5．5mmol／L组（P〈0．01）；与对照组比较高浓度葡萄糖组培养3d时TRAP染色阳性OC数明显增多（P〈0．01）。②葡萄糖呈浓度依赖性上调OC膜表面RANK mRNA的表达，其中高浓度葡萄糖组培养的各时间点与其他3组比较差异有统计学意义（P〈0．01，P〈0．05）。结论①高浓度葡萄糖可促进破骨细胞的分化，其促进作用始于诱导分化的早期。②骨髓微环境中高浓度葡萄糖可引起OC分化增多，可能是糖尿病骨质疏松的发病机制之一。     

Shear stress induces endothelial differentiation from mouse embryo mesenchymal progenitor cells

Introduction. Hemodynamic factors play crucial roles in vascular development, remodeling, healing, and lesion formation. However, the underlying molecular mechanisms are largely unknown. The objective of this study was to test the hypothesis that shear stress could affect endothelial cell differentiation. Methods. A mouse emoryo mesenchymal progenitor cell line (C3H/10T1/2) was cultured under shear stress (15 dyn/cm²) for 0, 6, and 12 h. Expression of specific endothelial cell markers as well as a monocyte marker CD14 was determined by real-time PCR, flow cytometry, and fluorescence immunostaining. In addition, the change of cell morphology and the ability of acetylated LDL uptake were also investigated. Results. Under shear stress, CH3/10T1/2 cells showed a morphology change of lining up with the direction of flow. The mRNR levels of CD31, vWF, and VE-cadherin were increased by 757-, 108-, and 23-fold, respectively, in shear stress group as compared to static control group (P < 0.01). VEGFR-1 mRNA levels were also increased by 41% in shear stress group (P < 0.01). The flow cytometry study showed that shear stress increased CD31- and vWF-positive cells by 11- and 214-fold, respectively, as compared to controls (P < 0.01). However, CD14 mRNA was decreased by 74% in shear stress group as compared to controls (P < 0.01). In addition, the acetylated LDL uptake was increased by 10-fold in shear stress group as compared to controls (P < 0.01). All gene changes were also demonstrated by immunofluorescence staining. Conclusion. This is the first study to demonstrate that shear stress significantly induces expression of several endothelial cell markers at both mRNA and protein levels in a mouse emoryo mesenchymal progenitor cell line. In addition, shear stress changes cell morphology and increases the ability of acetylated LDL uptake. However, monocyte marker CD14 was significantly decreased after shear stress stimulation. This study suggests a novel mechanism of endothelial cell differentiation.     

Interleukin‐33 is expressed in differentiated osteoblasts and blocks osteoclast formation from bone marrow precursor cells

Since the hematopoetic system is located within the bone marrow, it is not surprising that recent evidence has demonstrated the existence of molecular interactions between bone and immune cells. While interleukin 1 (IL‐1) and IL‐18, two cytokines of the IL‐1 family, have been shown to regulate differentiation and activity of bone cells, the role of IL‐33, another IL‐1 family member, has not been addressed yet. Since we observed that the expression of IL‐33 increases during osteoblast differentiation, we analyzed its possible influence on bone formation and observed that IL‐33 did not affect matrix mineralization but enhanced the expression of Tnfsf11, the gene encoding RANKL. This finding led us to analyze the skeletal phenotype of Il1rl1‐deficient mice, which lack the IL‐33 receptor ST2. Unexpectedly, these mice displayed normal bone formation but increased bone resorption, thereby resulting in low trabecular bone mass. Since this finding suggested a negative influence of IL‐33 on osteoclastogenesis, we next analyzed osteoclast differentiation from bone marrow precursor cells and observed that IL‐33 completely abolished the generation of TRACP⁺ multinucleated osteoclasts, even in the presence of RANKL and macrophage colony‐stimulating factor (M‐CSF). Although our molecular studies revealed that IL‐33 treatment of bone marrow cells caused a shift toward other hematopoetic lineages, we further observed a direct negative influence of IL‐33 on the osteoclastogenic differentiation of RAW264.7 macrophages, where IL‐33 repressed the expression of Nfatc1, which encodes one of the key transciption factors of osteoclast differentiation. Taken together, these findings have uncovered a previously unknown function of IL‐33 as an inhibitor of bone resorption. © 2011 American Society for Bone and Mineral Research.     

Caffeine enhances osteoclast differentiation from bone marrow hematopoietic cells and reduces bone mineral density in growing rats

Caffeine‐containing beverage consumption has been associated with low bone mass and increased fracture risk in some, but not most, observational studies. The effects of caffeine on bone metabolism are still controversial. We investigated the effects of caffeine on the differentiation of bone progenitor cells and bone mineral density (BMD) by in vitro and in vivo experiments. Low‐concentration caffeine (0.005–0.1 mM) did not affect the bone marrow cell viability and alkaline phosphatase activity during osteoblast differentiation from bone marrow stromal cells, but it effectively enhanced the osteoclastogenesis from bone marrow hematopoietic cells and the bone resorption activity by pit formation assay. Moreover, caffeine effectively enhanced the receptor activator of NF‐κB ligand (RANKL), but reduced the osteoprotegerin protein expressions in osteoblast MC3T3‐E1 cells. Caffeine could also increase the cyclooxygenase‐2 (COX‐2) protein expression and prostaglandin (PG)E₂ production in cultured neonatal mouse calvariae. In animal study, BMD in lumbar vertebra, femur, or tibia was significantly lowered in growing rats supplemented with 0.2% caffeine in diets for 20 weeks compared with the control group. The calcium contents in tibia and femur of caffeine‐treated rats were also lower than that in the control group. The osteoclastogenesis of bone marrow cells isolated from caffeine‐treated rats was markedly enhanced as compared with the control group. Taken together, these results suggest that caffeine may reduce BMD in growing rats through the enhancement in osteoclastogenesis. Caffeine may possess the ability to enhance a COX‐2/PGE₂‐regulated RANKL‐mediated osteoclastogenesis. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29:954–960     

Cyclic strain induces vascular smooth muscle cell differentiation from murine embryonic mesenchymal progenitor cells

BACKGROUND: Hemodynamic forces play a crucial role in regulating vascular cell phenotype. However, the underlying molecular mechanisms are largely unknown. The objective of this study was to test our hypothesis that cyclic strain could affect smooth muscle cell (SMC) differentiation. METHODS: A murine embryonic mesenchymal progenitor cell line (C3H/10T1/2) was cultured with or without cyclic strain for 6 days. Changes in cell morphology were studied with fluorescence dye Calcein-AM staining. Expression of specific SMC markers, smooth muscle specific alpha-actin (alpha-SMA), and smooth muscle myosin heavy chain (SMMHC), was determined by real-time polymerase chain reaction (PCR) and Western blot. Transforming growth factor- beta (TGF-beta) was used as a positive control. RESULTS: With cyclic strain, CH3/10T1/2 cells demonstrated spindle-shaped morphology and parallel alignment. Cells exposed to cyclic strain illustrated significantly increased mRNA levels of alpha-SMA and SMMHC by 3- and 2-fold, respectively, compared with static cells (P<.05). In addition, cells cultured under cyclic strain with TGF-beta (2 ng/ml) supplementation demonstrated increased mRNA levels of alpha-SMA and SMMHC by 10- and 2-fold, respectively, compared with static cells (P<.05). Furthermore, protein levels of alpha-SMA and SMMHC were also significantly increased by more than 3-fold in cyclic strain-treated cells compared with static cultures (P<.05). TGF-beta synergistically enhanced the effect of cyclic strain on alpha-SMA mRNA expression in CH3/10T1/2 cells. CONCLUSIONS: This is the first study to demonstrate that cyclic strain significantly induces expression of two of the most important SMC markers in a murine embryonic mesenchymal progenitor cell line. Cyclic strain and TGF-beta have a synergistic effect on alpha-SMA mRNA expression.     

Expression of CD163 (hemoglobin scavenger receptor) in normal tissues, lymphomas, carcinomas, and sarcomas is largely restricted to the monocyte/macrophage lineage

CD163, a hemoglobin scavenger receptor, is expressed in monocytes and macrophages. We tested the expression of the CD163 protein in 1,105 human malignancies and normal tissues using tissue microarrays and conventional paraffin-embedded tissue sections. Besides staining nonneoplastic monocytes and histiocytes (tissue macrophages), membranous/cytoplasmic staining for CD163 was primarily limited to neoplasms with monocytic/histiocytic differentiation. CD163 reactivity was not observed in normal tissues, lymphomas, carcinomas, and in a majority of mesenchymal neoplasms, including follicular dendritic cell tumors (0 of 4), although it stained admixed histiocytes. Staining for CD163 was seen in Rosai-Dorfman disease (5 of 6), histiocytic sarcoma (3 of 4), littoral cell angioma (6 of 6), and Langerhans cell histiocytosis (3 of 5). A subset of atypical fibrous histiocytomas (9 of 16), benign fibrous histiocytomas (6 of 9), and atypical fibroxanthomas (1 of 3) also showed CD163 staining. Our studies also confirm earlier work showing that CD163 is expressed in acute myeloid leukemia with monocytic differentiation (AML, FAB subtype M5) (2 of 6), as well as a majority of giant cell tenosynovial tumors (7 of 8). Its limited range of expression and tissue specificity indicate that CD163 may have significant diagnostic utility in separating specific tumors with monocytic and histiocytic derivation from other entities in their differential diagnosis.     

The transition of cadherin expression in osteoblast differentiation from mesenchymal cells: consistent expression of cadherin-11 in osteoblast lineage.

Osteoblasts are derived originally from pluripotent mesenchymal stem cells on migration into the bone matrix. To elucidate the contribution of classical cadherins in this differentiation pathway, we developed a new protocol for their analysis and studied their specific expressions in various cell lines of the mesenchymal lineage, including osteoblasts. N-cadherin was expressed constitutively in all cell lines examined except an osteocyte-like cell line whereas cadherin-11 was expressed selectively in preosteoblast and preadipocyte cell lines. P-cadherin also was expressed in primary cultures of calvarial cells and mature osteoblasts at a relatively low level compared with N-cadherin and cadherin-11. M-cadherin was expressed only in a premyoblast cell line. We observed the transition of cadherin expression from M-cadherin to cadherin-11 in the premyoblast cell line when osteogenic differentiation was induced by treatment with bone morphogenetic protein 2 (BMP-2), while the expression of N-cadherin remained unchanged. In contrast, when a preadipocyte cell line, which shows a similar pattern of cadherin expression to osteoblasts, was induced to undergo adipogenic differentiation, the expression of N-cadherin and cadherin-11 was decreased. These observations characterize the cadherin expression profile of mesenchymal lineage cells, especially osteoblasts, which regularly express cadherin-11. Cadherin-11 may affect cell sorting, alignment, and separation through differentiation.