Expression of vascular cell adhesion molecule-1 indicates the differentiation potential of human bone marrow stromal cells

Bone marrow stromal cells (BMSCs) are a mixture of cells differing in differentiation potential including mesenchymal stem cells, and so far no CD antigens were found to be predictable for the differentiation property of each BMSC. Here we attempted to isolate differentiation-associated CD antigens using 100 immortalized human BMSC (ihBMSC) clones. Among 13 CD antigens analyzed, only CD106/Vascular cell adhesion molecule-1 (VCAM-1) showed a clear correlation with the differentiation potential of each clone; CD106-positive ihBMSC clones were less osteogenic and more adipogenic than CD106-negative clones. This association was confirmed in primary BMSCs sorted by CD106, showing that the CD106-positive fraction contained less osteogenic and more adipogenic cells than the CD106-positive fraction. The evaluation of CD106 fraction of BMSC strains in early passages predicted clearly the osteogenic and adipogenic potential after in vitro induction of differentiation, indicating the usefulness of CD106 as a differentiation-predicting marker of BMSC.     

Stromal stem cells from adipose tissue and bone marrow of age‐matched female donors display distinct immunophenotypic profiles

Adipose tissue is composed of lipid‐filled mature adipocytes and a heterogeneous stromal vascular fraction (SVF) population of cells. Similarly, the bone marrow (BM) is composed of multiple cell types including adipocytes, hematopoietic, osteoprogenitor, and stromal cells necessary to support hematopoiesis. Both adipose and BM contain a population of mesenchymal stromal/stem cells with the potential to differentiate into multiple lineages, including adipogenic, chondrogenic, and osteogenic cells, depending on the culture conditions. In this study we have shown that human adipose‐derived stem cells (ASCs) and bone marrow mesenchymal stem cells (BMSCs) populations display a common expression profile for many surface antigens, including CD29, CD49c, CD147, CD166, and HLA‐abc. Nevertheless, significant differences were noted in the expression of CD34 and its related protein, PODXL, CD36, CD 49f, CD106, and CD146. Furthermore, ASCs displayed more pronounced adipogenic differentiation capability relative to BMSC based on Oil Red staining (7‐fold vs. 2.85‐fold induction). In contrast, no difference between the stem cell types was detected for osteogenic differentiation based on Alizarin Red staining. Analysis by RT‐PCR demonstrated that both the ASC and BMSC differentiated adipocytes and osteoblast displayed a significant upregulation of lineage‐specific mRNAs relative to the undifferentiated cell populations; no significant differences in fold mRNA induction was noted between ASCs and BMSCs. In conclusion, these results demonstrate human ASCs and BMSCs display distinct immunophenotypes based on surface positivity and expression intensity as well as differences in adipogenic differentiation. The findings support the use of both human ASCs and BMSCs for clinical regenerative medicine. J. Cell. Physiol. 226: 843–851, 2011. © 2010 Wiley‐Liss, Inc.     

Neuronal differentiation of bone marrow-derived stromal stem cells involves suppression of discordant phenotypes through gene silencing

Tissue engineering involves the construction of transplantable tissues in which bone marrow aspirates may serve as an accessible source of autogenous multipotential mesenchymal stem cells. Increasing reports indicate that the lineage restriction of adult mesenchymal stem cells may be less established than previously believed, and stem cell-based therapeutics await the establishment of an efficient protocol capable of achieving a prescribed phenotype differentiation. We have investigated how adult mouse bone marrow-derived stromal cells (BMSCs) are guided to neurogenic and osteogenic phenotypes. Na?ve BMSCs were found surprisingly active in expression of a wide range of mRNAs and proteins, including those normally reported in terminally differentiated neuronal cells and osteoblasts. The na?ve BMSCs were found to exhibit voltage-dependent membrane currents similar to the neuronally guided BMSCs, although with smaller amplitudes. Once BMSCs were exposed to the osteogenic culture condition, the neuronal characteristics quickly disappeared. Our data suggest that the loss of discordant phenotypes during BMSC differentiation cannot be explained by the selection and elimination of unfit cells from the whole BMSC population. The percent ratio of live to dead BMSCs examined did not change during the first 8-10 days in either neurogenic or osteogenic differentiation media, and cell detachment was estimated at <1%. However, during this period, bone-associated extracellular matrix genes were selectively down-regulated in neuronally guided BMSCs. These data indicate that the suppression of discordant phenotypes of differentiating adult stem cells is achieved, at least in part, by silencing of superfluous gene clusters.     

LncRNA SNHG1 modulates adipogenic differentiation of BMSCs by promoting DNMT1 mediated Opg hypermethylation via interacting with PTBP1

Recent evidence indicates that the abnormal differentiation of bone marrow‐derived mesenchymal stem cells (BMSCs) plays a pivotal role in the pathogenesis of osteoporosis. LncRNA SNHG1 has been found to be associated with the differentiation ability of BMSCs. In this study, we aimed to elucidate the role of lncRNA SNHG1 and its associated pathway on the differentiation of BMSCs in osteoporosis. Mice that underwent bilateral ovariectomy (OVX) were used as models of osteoporosis. Induced osteogenic or adipogenic differentiation was performed in mouse BMSCs. Compared to sham animals, lncRNA SNHG1 expression was upregulated in OVX mice. Also, the in vitro expression of SNHG1 was increased in adipogenic BMSCs but decreased in osteogenic BMSCs. Moreover, overexpression of SNHG1 enhanced the adipogenic capacity of BMSCs but inhibited their osteogenic capacity as determined by oil red O, alizarin red, and alkaline phosphatase staining, while silencing of SNHG1 led to the opposite results. LncRNA SNHG1 interacting with the RNA‐binding polypyrimidine tract‐binding protein 1 (PTBP1) promoted osteoprotegerin (Opg) methylation and suppressed Opg expression via mediating DNA methyltransferase (DNMT) 1. Furthermore, Opg was showed to regulate BMSC differentiation. Knockdown of SNHG1 decreased the expressions of adipogenic related genes but increased that of osteogenic related genes. However, the knockdown of Opg partially reversed those effects. In summary, lncRNA SNHG1 upregulated the expression of DNMT1 via interacting with PTBP1, resulting in Opg hypermethylation and decreased Opg expression, which in turn enhanced BMSC adipogenic differentiation and contributed to osteoporosis.     

大鼠骨髓间充质干细胞的原代培养及鉴定

摘要 目的：研究大鼠BMSCs（骨髓间充质干细胞）原代培养与纯度鉴定的方法。方法：无菌环境中,从SD大鼠股骨与胫骨端采集骨髓,先行酶消化,利用全骨髓细胞悬液贴壁法对提取BMSCs实施传代培养,选取生长良好的第3代细胞进行鉴定;对BMSCs实施成脂与成骨诱导分化,同时经由油红O（ORO）与茜素红（ARS）染色法对诱导分化效果加以鉴定;借助流式细胞术（FCM）对CD34、CD44与CD90这3类BMSCs表面标志物的表达展开分析。结果：BMSCs是长梭状贴壁细胞,生长状态为纤维细胞样漩涡状;在第3代BMSCs传代期间,其第1-3 d发展至生长潜伏期,呈较慢速的生长;第3-5 d发展至对数生长期,呈高速生长;待至第7 d长速增殖最大,速度停止上升进入平缓期;BMSCs成骨、成脂诱导结束后,对其诱导分化鉴定发现：细胞出现明显形态学变化,通过ORO对脂肪染色,细胞显示橘红色;待成骨诱导培养结束,通过ARS对钙盐染色,显示红色,且出现矿化结节沉积,说明BMSCs具有良好的成骨、成脂分化能力;FCM测定发现：CD34表达呈阴性（1.09 %）,CD90（96.8 %）与CD44（92.4 %）皆呈阳性,与BMSCs表型相符。结论：经由全骨髓黏附培养技术有效分离BMSCs,且完成培养。     

The effect of different implant biomaterials on the behavior of canine bone marrow stromal cells during their differentiation into osteoblasts

We investigated the effects of different implant biomaterials on cultured canine bone marrow stromal cells (BMSC) undergoing differentiation into osteoblasts (dBMSC). BMSC were isolated from canine humerus by marrow aspiration, cultured and differentiated on calcium phosphate scaffold (CPS), hydroxyapatite, hydroxyapatite in gel form and titanium mesh. We used the MTT method to determine the effects of osteogenic media on proliferation. The characteristics of dBMSC were assessed using alizarin red (AR), immunocytochemistry and osteoblastic markers including alkaline phosphatase/von Kossa (ALP/VK), osteocalcin (OC) and osteonectin (ON), and ELISA. The morphology of dBMSC on the biomaterials was investigated using inverted phase contrast microscopy and scanning electron microscopy. We detected expression of ALP/VK, AR, OC and ON by day 7 of culture; expression increased from day 14 until day 21. CPS supported the best adhesion, cell spreading, proliferation and differentiation of BMSCs. The effects of the biomaterials depended on their surface properties. Expression of osteoblastic markers showed that canine dBMSCs became functional osteoblasts. Tissue engineered stem cells can be useful clinically for autologous implants for treating bone wounds.     

Loss of Lgr4 inhibits differentiation,migration and apoptosis,and promotes proliferation in bone mesenchymal stem cells

The key signaling networks regulating bone marrow mesenchymal stem cells (BMSCs) are poorly defined. Lgr4, which belongs to the leucine-rich repeat-containing G protein-coupled receptor (LGR) family, is widely expressed in multiple tissues from early embryogenesis to adulthood. We investigated whether Lgr4 functions in BMSCs and in osteogenesis, adipogenesis, and skeletal myoblasts, using mice with a β-geo gene trap inserted into the Lgr4 gene. Abundant Lgr4 expression was detected in skeletal, adipose and muscular tissue of Lgr4^+/– mice at E16.5 by β-gal staining, and Lgr4-deficiency promoted BMSC proliferation (16 ± 4 in wild-type [WT] and 28 ± 2 in Lgr4^−/−) using colony forming units-fibroblast assay, while suppressing BMSC migration (from 103 ± 18 in WT to 57 ± 10 in Lgr4^−/−) by transwell migration assay and apoptosis ratio (from 0.0720 ± 0.0123 to 0.0189 ± 0.0051) by annexin V staining assay. Deletion of Lgr4 decreased bone mass (BV/TV from 19.16 ± 2.14 in WT mice to 10.36 ± 1.96 in KO) and fat mass through inhibiting BMSC differentiation to osteoblasts or adipocytes. Furthermore, LGR4-regulated osteogenic, adipogenic, and myogenic gene expression. Importantly, our data showed that loss of Lgr4-inhibited fracture healing by suppressing osteoblast differentiation. Moreover, deletion of Lgr4 in BMSCs-delayed fracture healing following stem cell therapy by BMSC transplantation. Together, our results demonstrated that LGR4 is essential for mesoderm-derived tissue development and BMSC differentiation, demonstrating that LGR4 could be a promising drug target for related diseases and a critical protein for stem cell therapy.     

Serum‐free medium with osteogenic supplements induces adipogenesis in rat bone marrow stromal cells

Adult BMSCs (bone marrow stromal cells) contain MSCs (mesenchymal stem cells). MSCs can differentiate into osteoblasts, chondrocytes, adipocytes and myoblasts and are thus considered useful in tissue engineering for therapeutic and clinical purposes. FCS (fetal calf serum) is usually included in the differentiation medium, but the clinical application of FCS may pose a problem for some patients. To improve the efficiency and safety of BMSC cultivation, the effect of SF (serum‐free) conditions on the osteogenic differentiation of rat BMSCs was examined. In the presence of 10% FCS and osteogenic supplements, the cells formed mineralized von Kossa‐positive deposits. Under SF conditions with osteogenic supplementation, however, the cells possessed cytosolic lipid vacuoles that could be stained with Oil Red O. The mRNA expression of PPARγ (peroxisome proliferator‐activated receptor γ), an adipogenic marker, increased under the SF condition with osteogenic supplements. These data indicate that rat BMSCs differentiate into adipocytes under SF conditions even with osteogenic supplementation and that FCS is needed to induce proper osteogenic differentiation in rat BMSCs.     

Morphologic and transcriptomic comparison of adipose- and bone-marrow-derived porcine stem cells cultured in alginate hydrogels

Advances in bioengineering, material chemistry, and developmental biology have led to the design of three-dimensional (3D) culture systems that better resemble the surrounding structure and chemistry of the in situ niches of cells in tissues. This study was designed to characterize and compare porcine adipose-derived stem cells (ADSC) and bone-marrow-derived stem cells (BMSC) induced to differentiate toward osteogenic and adipogenic lineages in vitro by using a 3D alginate hydrogel. The morphology and gene expression of the two cell populations during differentiation were analyzed. Both ADSC and BMSC showed morphological evidence of osteogenic and adipogenic differentiation. Expression patterns of genes characteristic of the onset of osteogenic differentiation (ALP, COL1A1, SPARC, SPP1) were low at the beginning of culture and generally increased during the period of differentiation up to 28 days in culture. Expression of genes associated with adipogenic differentiation (ACSL1, ADFP, ADIPOQ, CD36, DBI, DGAT2, PPARG, SCD) was consistently increased in ADSC cultured in alginate hydrogel relative to the start of differentiation. However, adipogenic gene expression of BMSC cultured in alginate hydrogel was more limited when compared with that of ADSC. Evaluation of cell numbers (via the MTT staining assay) suggested a greater viability of BMSC under osteogenic conditions in alginate hydrogels than under adipogenic conditions, whereas ADSC had greater viability under adipogenic conditions than under osteogenic conditions. This study thus provides an important initial evaluation of ADSC and BMSC seeded and differentiated toward the osteogenic and adipogenic cell lineages in a 3D alginate hydrogel in vitro.     

Sodium-dependent vitamin C transporter SVCT2: Expression and function in bone marrow stromal cells and in osteogenesis

Ascorbic acid (Vitamin C) has a critical role in bone formation and osteoblast differentiation, but very little is known about the molecular mechanisms of ascorbic acid entry into bone marrow stromal cells (BMSCs). To address this gap in knowledge, we investigated the identity of the transport system that is responsible for the uptake of ascorbic acid into bone marrow stromal cells (BMSCs). First, we examined the expression of the two known isoforms of the sodium-coupled ascorbic acid transporter, namely SVCT1 and SVCT2, in BMSCs (Lin ? ve Sca1 + ve) and bone at the mRNA level. Only SVCT2 mRNA was detected in BMSCs and bone. Uptake of ascorbic acid in BMSCs was Na⁺-dependent and saturable. In order to define the role of SVCT2 in BMSC differentiation into osteoblasts, BMSCs were stimulated with osteogenic media for different time intervals, and the activity of SVCT2 was monitored by ascorbic acid uptake. SVCT2 expression was up-regulated during the osteogenic differentiation of BMSCs; the expression was maximal at the earliest phase of differentiation. Subsequently, osteogenesis was inhibited in BMSCs upon knock-down of SVCT2 by lentivirus shRNA. We also found that the expression of the SVCT2 could be negatively or positively modulated by the presence of oxidant (Sin-1) or antioxidant (Ascorbic acid) compounds, respectively, in BMSCs. Furthermore, we found that this transporter is also regulated with age in mouse bone. These data show that SVCT2 plays a vital role in the osteogenic differentiation of BMSCs and that its expression is altered under conditions associated with redox reaction. Our findings could be relevant to bone tissue engineering and bone related diseases such as osteoporosis in which oxidative stress and aging plays important role.     

Alkbh1‐mediated DNA N6‐methyladenine modification regulates bone marrow mesenchymal stem cell fate during skeletal aging

ObjectivesDNA N6‐methyladenine (N6‐mA) demethylase Alkbh1 participates in regulating osteogenic differentiation of mesenchymal stem cell (MSCs) and vascular calcification. However, the role of Alkbh1 in bone metabolism remains unclear.Materials and MethodsBone marrow mesenchymal stem cells (BMSCs)‐specific Alkbh1 knockout mice were used to investigate the role of Alkbh1 in bone metabolism. Western blot, qRT‐PCR, and immunofluorescent staining were used to evaluate the expression of Alkbh1 or optineurin (optn). Micro‐CT, histomorphometric analysis, and calcein double‐labeling assay were used to evaluate bone phenotypes. Cell staining and qRT‐PCR were used to evaluate the osteogenic or adipogenic differentiation of BMSCs. Dot blotting was used to detect the level of N6‐mA in genomic DNA. Chromatin immunoprecipitation (Chip) assays were used to identify critical targets of Alkbh1. Alkbh1 adeno‐associated virus was used to overexpress Alkbh1 in aged mice.ResultsAlkbh1 expression in BMSCs declined during aging. Knockout of Alkbh1 promoted adipogenic differentiation of BMSCs while inhibited osteogenic differentiation. BMSC‐specific Alkbh1 knockout mice exhibited reduced bone mass and increased marrow adiposity. Mechanistically, we identified optn as the downstream target through which Alkbh1‐mediated DNA m6A modification regulated BMSCs fate. Overexpression of Alkbh1 attenuated bone loss and marrow fat accumulation in aged mice.ConclusionsOur findings demonstrated that Alkbh1 regulated BMSCs fate and bone‐fat balance during skeletal aging and provided a potential target for the treatment of osteoporosis.     

Utility of tricalcium phosphate and osteogenic matrix cell sheet constructs for bone defect reconstruction

AIM: To determine the effects of transplanting osteogenic matrix cell sheets and beta-tricalcium phosphate (TCP) constructs on bone formation in bone defects.METHODS: Osteogenic matrix cell sheets were prepared from bone marrow stromal cells (BMSCs), and a porous TCP ceramic was used as a scaffold. Three experimental groups were prepared, comprised of TCP scaffolds (1) seeded with BMSCs; (2) wrapped with osteogenic matrix cell sheets; or (3) both. Constructs were implanted into a femoral defect model in rats and bone growth was evaluated by radiography, histology, biochemistry, and mechanical testing after 8 wk.RESULTS: In bone defects, constructs implanted with cell sheets showed callus formation with segmental or continuous bone formation at 8 wk, in contrast to TCP seeded with BMSCs, which resulted in bone non-union. Wrapping TCP constructs with osteogenic matrix cell sheets increased their osteogenic potential and resulting bone formation, compared with conventional bone tissue engineering TCP scaffolds seeded with BMSCs. The compressive stiffness (mean ± SD) values were 225.0 ± 95.7, 30.0 ± 11.5, and 26.3 ± 10.6 MPa for BMSC/TCP/Sheet constructs with continuous bone formation, BMSC/TCP/Sheet constructs with segmental bone formation, and BMSC/TCP constructs, respectively. The compressive stiffness of BMSC/TCP/Sheet constructs with continuous bone formation was significantly higher than those with segmental bone formation and BMSC/TCP constructs.CONCLUSION: This technique is an improvement over current methods, such as TCP substitution, and is useful for hard tissue reconstruction and inducing earlier bone union in defects.     

GPX7 Facilitates BMSCs Osteoblastogenesis via ER Stress and mTOR Pathway

Emerging evidence indicates extensive oxidative stress is a consequence of obesity which impairs bone formation. Glutathione peroxidase 7 (GPX7) is a conserved endoplasmic reticulum (ER) retention protein, lacking of which causes accumulation of reactive oxygen species (ROS) and promotes adipogenesis. Since the imbalance between osteogenic and adipogenic differentiation of bone marrow mesenchymal stem cell (BMSC) leads to severe bone diseases such as osteoporosis, it is critical to investigate the potential protective role of Gpx7 in osteogenesis. Here, we provide evidence that deficiency of Gpx7 reduces osteogenesis, but increases adipogenesis in both human BMSCs (hBMSCs) and mouse mesenchymal stem cell line. Interestingly, further studies indicate this defect can be alleviated by the ER stress antagonist, but not the ROS inhibitor, unveiling an unexpected finding that, unlike adipogenesis, lacking of Gpx7 inhibits osteogenesis mediating by induced ER stress instead of enhanced ROS. Furthermore, the mTOR signalling pathway is found down-regulation during osteogenic differentiation in Gpx7-deficient condition, which can be rescued by relief of ER stress. Taken together, for the first time we identify a novel function of Gpx7 in BMSCs’ osteogenic differentiation and indicate that Gpx7 may protect against osteoporotic deficits in humans through ER stress and mTOR pathway interplay.     

The effect of implantation on scaffoldless three-dimensional engineered bone constructs

Our laboratory has previously developed scaffoldless engineered bone constructs (EBC). Bone marrow stromal cells (BMSC) were harvested from rat femur and cultured in medium that induced osteogenic differentiation. After reaching confluence, the monolayer of cells contracted around two constraint points forming a cylinder. EBCs were placed in small diameter (0.5905 × 0.0625 in.) or large diameter (0.5905 × 0.125 in.) silicone tubing and implanted intramuscularly in the hind limb of a rat. Bone mineral content (BMC) of the EBC was analyzed before implantation and at 1 and 2 mo following implantation and compared to that of native femur bone at different stages of development. Negligible BMC was observed in E-20 femur or EBCs prior to implantation. One-month implantation in both small and large tubing increased BMC in the EBC. BMC of EBC from large tubing was greater than in 14 d rat neonatal femurs, but was 2% and 3% of BMC content in adult bone after 1 and 2 mo of implantation, respectively. Alizarine Red and osteopontin staining of the EBCs before and after implantation confirmed increased bone mineralization in the implanted EBCs. Implanted EBCs also had extensive vascularization. Our data suggest that BMSC can be successfully used for the generation of scaffoldless EBC, and this model can be potentially used for the generation of autologous bone transplants in humans.     

Comparison of gingiva‐derived and bone marrow mesenchymal stem cells for osteogenesis

Presently, bone marrow is considered as a prime source of mesenchymal stem cells; however, there are some drawbacks and limitations. Compared with other mesenchymal stem cell (MSC) sources, gingiva‐derived mesenchymal stem cells (GMSCs) are abundant and easy to obtain through minimally invasive cell isolation techniques. In this study, MSCs derived from gingiva and bone marrow were isolated and cultured from mice. GMSCs were characterized by osteogenic, adipogenic and chondrogenic differentiation, and flow cytometry. Compared with bone marrow MSCs (BMSCs), the proliferation capacity was judged by CCK‐8 proliferation assay. Osteogenic differentiation was assessed by ALP staining, ALP assay and Alizarin red staining. RT‐qPCR was performed for ALP, OCN, OSX and Runx2. The results indicated that GMSCs showed higher proliferative capacity than BMSCs. GMSCs turned more positive for ALP and formed a more number of mineralized nodules than BMSCs after osteogenic induction. RT‐qPCR revealed that the expression of ALP, OCN, OSX and Runx2 was significantly increased in the GMSCs compared with that in BMSCs. Moreover, it was found that the number of CD90‐positive cells in GMSCs elevated more than that of BMSCs during osteogenic induction. Taking these results together, it was indicated that GMSCs might be a promising source in the future bone tissue engineering.     

The effect of combined regulation of the expression of peroxisome proliferator-activated receptor-γ and calcitonin gene-related peptide on alcohol-induced adipogenic differentiation of bone marrow mesenchymal stem cells

Studies have shown that alcohol can upregulate the expression of peroxisome proliferator-activated receptor-γ (PPARγ) gene in bone marrow mesenchymal stem cells (BMSCs). High expression of PPARγ can promote adipogenic differentiation of BMSCs, and reduce their osteogenic differentiation. Abnormal proliferation of adipocytes and fatty accumulation in osteocytes can result in high intraosseous pressure and disturbance of blood circulation in the femoral head, which induces osteonecrosis of the femoral head (ONFH). Downregulation of PPARγ is efficient in inhibiting adipogenesis and maintaining osteogenesis of BMSCs, which might potentially reduce the incidence of ONFH. Calcitonin gene-related peptide (CGRP) is a neuropeptide gene which has been closely associated with bone regeneration. In this study, we aimed to observe the effect of combined regulation of the expression of PPARγ and CGRP genes on alcohol-induced adipogenic differentiation of BMSCs. Our results demonstrated that simultaneous downregulation of PPARγ and upregulation of CGRP was efficient in suppressing adipogenic differentiation of BMSCs and promoting their osteogenic differentiation. These findings might enlighten a novel approach for the prevention of ONFH.