Role of human amnion-derived mesenchymal stem cells in promoting osteogenic differentiation by influencing p38 MAPK signaling in lipopolysaccharide -induced human bone marrow mesenchymal stem cells

Periodontitis is a chronic inflammatory disease induced by bacterial pathogens, which not only affect connective tissue attachments but also cause alveolar bone loss. In this study, we investigated the anti-inflammatory effects of Human amnion-derived mesenchymal stem cells (HAMSCs) on human bone marrow mesenchymal stem cells (HBMSCs) under lipopolysaccharide (LPS)-induced inflammatory conditions. Proliferation levels were measured by flow cytometry and immunofluorescence staining of 5-ethynyl-2′-deoxyuridine (EdU). Osteoblastic differentiation and mineralization were investigated using chromogenic alkaline phosphatase activity (ALP) activity substrate assays, Alizarin red S staining, and RT-PCR analysis of HBMSCs osteogenic marker expression. Oxidative stress induced by LPS was investigated by assaying reactive oxygen species (ROS) level and superoxide dismutase (SOD) activity. Here, we demonstrated that HAMSCs increased the proliferation, osteoblastic differentiation, and SOD activity of LPS-induced HBMSCs, and down-regulated the ROS level. Moreover, our results suggested that the activation of p38 MAPK signal transduction pathway is essential for reversing the LPS-induced bone-destructive processes. SB203580, a selective inhibitor of p38 MAPK signaling, significantly suppressed the anti-inflammatory effects in HAMSCs. In conclusion, HAMSCs show a strong potential in treating inflammation-induced bone loss by influencing p38 MAPK signaling.     

bFGF promotes adipocyte differentiation in human mesenchymal stem cells derived from embryonic stem cells

In this work we describe the establishment of mesenchymal stem cells (MSCs) derived from embryonic stem cells (ESCs) and the role of bFGF in adipocyte differentiation. The totipotency of ESCs and MSCs was assessed by immunofluorescence staining and RT-PCR of totipotency factors. MSCs were successfully used to induce osteoblasts, chondrocytes and adipocytes. MSCs that differentiated into adipocytes were stimulated with and without bFGF. The OD/DNA (optical density/content of total DNA) and expression levels of the specific adipocyte genes PPARγ2 (peroxisome proliferator activated receptor γ2) and C/EBPs were higher in bFGF cells. Embryonic bodies had a higher adipocyte level compared with cells cultured in plates. These findings indicate that bFGF promotes adipocyte differentiation. MSCs may be useful cells for seeding in tissue engineering and have enormous therapeutic potential for adipose tissue engineering.     

Involvement of PTP-RQ in differentiation during adipogenesis of human mesenchymal stem cells

Mesenchymal stem cells (MSCs) are self-renewable multipotent progenitor cells with the capacity to differentiate into several distinct mesenchymal lineages. While MSCs display significant potential in tissue engineering and therapeutic applications, the regulatory mechanisms underlying the differentiation of these cells are yet to be established. Phosphorylation is a post-translational modification that plays a significant role in diverse biological phenomena. In this study, to mine the protein tyrosine phosphatases (PTPs) involved in adipogenesis of human MSCs, differential expression of human PTPs was examined using RT-PCR analysis. Among the 107 human PTPs, PTP-RQ was dramatically downregulated during the early phase of adipogenesis. PTP-RQ is classified as a receptor-type III PTP with phosphatidylinositol phosphatase (PIPase) activity. Overexpression of PTP-RQ consistently led to reduced differentiation of MSCs into adipocytes via decreasing the phosphatidyl inositol phosphate level in cells, and consequently downregulating Akt/PKB phosphorylation. Our results collectively suggest that PTP-RQ is a useful target protein for regulating the differentiation of MSCs into adipocytes, and may be used to develop novel drugs for the treatment of obesity.     

Pulsed electromagnetic fields accelerate proliferation and osteogenic gene expression in human bone marrow mesenchymal stem cells during osteogenic differentiation

Osteogenesis is a complex series of events involving the differentiation of mesenchymal stem cells to generate new bone. In this study, we examined the effect of pulsed electromagnetic fields (PEMFs) on cell proliferation, alkaline phosphatase (ALP) activity, mineralization of the extracellular matrix, and gene expression in bone marrow mesenchymal stem cells (BMMSCs) during osteogenic differentiation. Exposure of BMMSCs to PEMFs increased cell proliferation by 29.6% compared to untreated cells at day 1 of differentiation. Semi‐quantitative RT‐PCR indicated that PEMFs significantly altered temporal expression of osteogenesis‐related genes, including a 2.7‐fold increase in expression of the key osteogenesis regulatory gene cbfa1, compared to untreated controls. In addition, exposure to PEMFs significantly increased ALP expression during the early stages of osteogenesis and substantially enhanced mineralization near the midpoint of osteogenesis. These results suggest that PEMFs enhance early cell proliferation in BMMSC‐mediated osteogenesis, and accelerate the osteogenesis. Bioelectromagnetics 31:209–219, 2010. © 2009 Wiley‐Liss, Inc.     

Synthetic nanostructures inducing differentiation of human mesenchymal stem cells into neuronal lineage

Human mesenchymal stem cells (hMSCs) have been shown to trans-differentiate into neuronal-like cells by culture in neuronal induction media, although the mechanism is not well understood. Topography can also influence cellular responses including enhanced differentiation of progenitor cells. As extracellular matrix (ECM) in vivo comprises topography in the nanoscale, we hypothesize that nanotopography could influence stem cell differentiation into specific non-default pathways, such as transdifferentiation of hMSCs. Differentiation and proliferation of hMSCs were studied on nanogratings of 350 nm width. Cytoskeleton and nuclei of hMSCs were aligned and elongated along the nanogratings. Gene profiling and immunostaining showed significant up-regulation of neuronal markers such as microtubule-associated protein 2 (MAP2) compared to unpatterned and micropatterned controls. The combination of nanotopography and biochemical cues such as retinoic acid further enhanced the up-regulation of neuronal marker expressions, but nanotopography showed a stronger effect compared to retinoic acid alone on unpatterned surface. This study demonstrated the significance of nanotopography in directing differentiation of adult stem cells.     

Effects of transforming growth factor-beta 1 and ascorbic acid on differentiation of human bone-marrow-derived mesenchymal stem cells into smooth muscle cell lineage

Bone-marrow-derived mesenchymal stem cells (MSCs) can differentiate into a variety of cell types including smooth muscle cells (SMCs). We have attempted to demonstrate that, following treatment with transforming growth factor-beta 1 (TGF-beta1) and ascorbic acid (AA), human bone-marrow-derived MSCs differentiate into the SMC lineage for use in tissue engineering. Quantitative polymerase chain reaction for SMC-specific gene (alpha smooth muscle actin, h1-calponin, and SM22alpha) expression was performed on MSCs, which were cultured with various concentrations of TGF-beta1 or AA. TGF-beta1 had a tendency to up-regulate the expression of SMC-specific genes in a dose-dependent manner. The expression of SM22alpha was significantly up-regulated by 30 muM AA. We also investigated the additive effect of TGF-beta1 and AA for differentiation into SMCs and compared this effect with that of other factors including platelet-derived growth factor BB (PDGF-BB). In addition to SMC-specific gene expression, SMC-specific proteins increased by two to four times when TGF-beta1 and AA were used together compared with their administration alone. PDGF did not increase the expression of SMC-specific markers. MSCs cultured with TGF-beta1 and AA did not differentiate into osteoblasts and adipocytes. These results suggest that a combination of TGF-beta1 and AA is useful for the differentiation of MSCs into SMCs for use in tissue engineering.     

1-磷酸鞘氨醇促进间充质干细胞向心肌分化

为研究1-磷酸鞘氨醇 (Sphingosine-1-phosphate,S1P) 对脐带间充质干细胞 (Umbilical cord mesenchymal stem cells,UC-MSCs) 和脂肪间充质干细胞 (Adipose derived mesenchymal stem cells,AD-MSCs) 向心肌分化的影响,探索其适宜的作用时间和浓度,将UC-MSCs和AD-MSCs接种到培养板,用添加不同浓度S1P的心肌细胞培养液诱导两种干细胞向心肌分化,诱导时间分为7 d、14 d和28 d。采用免疫荧光染色检测心肌特异性蛋白,α-肌动蛋白 (α-actin)、缝隙连接蛋白 (Connexin-43) 以及肌球蛋白重链 (MYH-6) 的表达,并通过共聚焦显微镜和荧光显微镜进行观察;采用MTT分析细胞的活性;膜片钳检测分化细胞的钙瞬变 (此为心肌细胞的功能性指标)。结果表明,S1P与心肌细胞培养液协同作用,能够促进UC-MSCs和AD-MSCs向心肌细胞的分化。并且,随着S1P浓度的增加,促分化作用增强,但细胞活性降低。S1P在心肌细胞培养液中的适宜作用时间为14 d,适宜作用浓度为0.5 μmol/L。而且联合心肌细胞培养液可以使UC-MSCs和AD-MSCs的心肌分化细胞产生钙瞬变,具有类似心肌细胞的功能性。S1P能够与心肌细胞培养液协同作用,促进UC-MSCs和AD-MSCs的心肌功能性分化。     

Basement membrane collagen type IV expression by human mesenchymal stem cells during adipogenic differentiation

During adipogenic differentiation human mesenchymal stem cells (hMSC) produce collagen type IV. In immunofluorescence staining differentiating hMSCs started to express collagen type IV when Oil Red O-positive fat droplets appeared intracellularly. Quantitative real time-polymerase chain reaction confirmed progressive increase of collagen type IV α1 and α2 mRNA levels over time, 18.6- and 12.2-fold by day 28, respectively, whereas the copy numbers of α3-α6 mRNAs remained rather stable and low. Type IV collagen was in confocal laser scanning microscopy seen around adipocytes, where also laminins and nidogen were found, suggesting pericellular deposition of all key components of the fully developed basement membrane. Immunofluorescence staining of matrix metalloproteinase-2 (MMP-2, 72 kD type IV collagenase, gelatinase A) and MMP-9 (92 kD type IV collagenase, gelatinase B) disclosed only faint staining of MSCs, but MMP-9 was strongly induced during adipogenesis, whereas MSC supernatants disclosed in zymography pro-MMP-2 and faint pro-MMP-9 bands, which increased over time, with partial conversion of pro-MMP-2 to its active 62 kD form. Differentiation was associated with increasing membrane type 1-MMP/MMP-14 and tissue inhibitor of metalloproteinase-2 (TIMP-2) staining, which may enable participation of type IV collagenases in basement membrane remodelling via ternary MT1-MMP/TIMP-2/MMP-2 or -9 complexes, focalizing the fully active enzyme to the cell surface. MMP-9, which increased more in immunofluorescence staining, was perhaps preferentially bound to cell surface and/or remodelling adipocyte basement membrane. These results suggest that upon MSC-adipocyte differentiation collagen type IV synthesis and remodelling become necessary when intracellular accumulation of fat necessitates a dynamically supporting and instructive, partly denatured adipogenic pericellular type IV collagen scaffold.     

Angiogenic CXC chemokine expression during differentiation of human mesenchymal stem cells towards the osteoblastic lineage

The potential role of ELR(+) CXC chemokines in early events in bone repair was studied using human mesenchymal stem cells (hMSCs). Inflammation, which occurs in the initial phase of tissue healing in general, is critical to bone repair. Release of cytokines from infiltrating immune cells and injured bone can lead to recruitment of MSCs to the region of repair. CXC chemokines bearing the Glu-Leu-Arg (ELR) motif are also released by inflammatory cells and serve as angiogenic factors stimulating chemotaxis and proliferation of endothelial cells. hMSCs, induced to differentiate with osteogenic medium (OGM) containing ascorbate, beta-glycerophosphate (beta-GP), and dexamethasone (DEX), showed an increase in mRNA and protein secretion of the ELR(+) CXC chemokines CXCL8 and CXCL1. CXCL8 mRNA half-life studies reveal an increase in mRNA stability upon OGM stimulation. Increased expression and secretion is a result of DEX in OGM and is dose-dependent. Inhibition of the glucocorticoid receptor with mifepristone only partially inhibits DEX-stimulated CXCL8 expression indicating both glucocorticoid receptor dependent and independent pathways. Treatment with signal transduction inhibitors demonstrate that this expression is due to activation of the ERK and p38 mitogen-activated protein kinase (MAPK) pathways and is mediated through the G(alphai)-coupled receptors. Angiogenesis assays demonstrate that OGM-stimulated conditioned media containing secreted CXCL8 and CXCL1 can induce angiogenesis of human microvascular endothelial cells in an in vitro Matrigel assay.     

人脐带间充质干细胞作为维持人胚胎干细胞生长饲养层细胞的研究

目的寻找可以维持人胚胎干细胞未分化生长的人源性细胞作为饲养层细胞,从而解决使用鼠源性细胞作为饲养层带来的安全问题。方法尝试以人脐带间充质干细胞作为饲养层细胞来培养人胚胎干细胞,检验其是否可以维持人胚胎干细胞的未分化生长状态。用胶原酶消化法分离人脐带间充质干细胞,光镜下观察细胞形态;流式细胞仪检测其表面标志;诱导人脐带间充质干细胞向成骨细胞和脂肪细胞进行分化。将人胚胎干细胞系H1接种于丝裂霉素C灭活后的人脐带间充质干细胞上,每隔5d进行一次传代。培养20代后,对人胚胎干细胞特性进行相关检测,包括细胞形态、碱性磷酸酶染色、相关多能性基因的表达、分化能力。结果从人脐带中分离出的间充质干细胞为梭形,呈平行排列生长或漩涡状生长;细胞高表达CD44、CD29、CD73、CD105、CD90、CD86、CD147、CD117,不表达CD14、CD38、CD133、CD34、CD45、HLA-DR;具有分化成脂肪细胞和成骨细胞的潜能。人胚胎干细胞在人脐带间充质干细胞饲养层上培养20代后,继续保持人胚胎干细胞的典型形态,碱性磷酸酶染色为阳性,免疫荧光染色显示OCT4、Nanog、SSEA4、TRA-1-81、TRA-1-60的表达为阳性,SSEA1表达为阴性,体外悬浮培养可以形成拟胚体。结论人脐带间充质干细胞可以作为人胚胎干细胞的饲养层细胞,支持其生长,并维持其未分化生长状态。     

Migration potential and gene expression profile of human mesenchymal stem cells induced by CCL25

Recruitment of mesenchymal stem cells (MSC) to tissue damages is a promising approach for in situ tissue regeneration. The physiological mechanisms and regulatory processes of MSC trafficking to injured tissue remain poorly understood. However, the pivotal role of chemokines in MSC recruitment has already been shown.The aim of this study was to determine the migratory potential and the gene expression profile of MSC stimulated with the CC chemokine CCL25 (TECK). Bone marrow derived human MSC were exposed to different doses of CCL25 in a standardized chemotaxis assay. Microarray gene expression profiling and pathway analysis were performed for CCL25 stimulated MSC.Maximum migration of MSC towards CCL25 was observed at 10³ nM. Microarray analysis revealed an induction of molecules directly involved in chemotaxis and homing of bone marrow cells (CXCL1-3, CXCL8, PDE4B), cytoskeletal and membrane reorganisation (CXCL8, PLD1, IGFBP1), cellular polarity (PLD1), and cell movement (CXCL1-3, CXCL6, CXCL8, PTGS2, PDE4B, TGM2). Respective chemokine secretion was confirmed by protein membrane-array analysis. The activation of CXCR2 ligands (CXCL1-3, CXCL5-6, CXCL8) and a LIF-receptor/gp130 ligand (LIF) indicated an involvement of the respective signaling pathways during initiation of chemotaxis and migration.These results suggest CCL25 as a new potential candidate for further in situ regeneration approaches.     

Trafficking and differentiation of mesenchymal stem cells

Mesenchymal stem cells (MSCs) are a heterogeneous population of stem/progenitor cells with pluripotent capacity to differentiate into mesodermal and non‐mesodermal cell lineages, including osteocytes, adipocytes, chondrocytes, myocytes, cardiomyocytes, fibroblasts, myofibroblasts, epithelial cells, and neurons. MSCs reside primarily in the bone marrow, but also exist in other sites such as adipose tissue, peripheral blood, cord blood, liver, and fetal tissues. When stimulated by specific signals, these cells can be released from their niche in the bone marrow into circulation and recruited to the target tissues where they undergo in situ differentiation and contribute to tissue regeneration and homeostasis. Several characteristics of MSCs, such as the potential to differentiate into multiple lineages and the ability to be expanded ex vivo while retaining their original lineage differentiation commitment, make these cells very interesting targets for potential therapeutic use in regenerative medicine and tissue engineering. The feasibility for transplantation of primary or engineered MSCs as cell‐based therapy has been demonstrated. In this review, we summarize the current knowledge on the signals that control trafficking and differentiation of MSCs. J. Cell. Biochem. 106: 984–991, 2009. © 2009 Wiley‐Liss, Inc.     

Cytoskeletal organization of human mesenchymal stem cells (MSC) changes during their osteogenic differentiation

Human MSCs have been studied to define the mechanisms involved in normal bone remodeling and the regulation of osteogenesis. During osteogenic differentiation, MSCs change from their characteristic fibroblast-like phenotype to near spherical shape. In this study, we analyzed the correlation between the organization of cytoskeleton of MSCs, changes in cell morphology, and the expression of specific markers (alkaline phosphatase activity and calcium deposition) of osteogenic differentiation. For osteoblastic differentiation, cells were cultured in a culture medium supplemented with 100 nM dexamethasone, 10 mM beta- glycerophosphate, and 50 microg/ml ascorbic acid. The organization of microfilaments and microtubules was examined by inmunofluorescence using Alexa fluor 594 phalloidin and anti alpha-tubulin monoclonal antibody. Cytochalasin D and nocodazole were used to alter reversibly the cytoskeleton dynamic. A remarkable change in cytoskeleton organization was observed in human MSCs during osteogenic differentiation. Actin cytoskeleton changed from a large number of thin, parallel microfilament bundles extending across the entire cytoplasm in undifferentiated MSCs to a few thick actin filament bundles located at the outermost periphery in differentiated cells. Under osteogenic culture conditions, a reversible reorganization of microfilaments induced by an initial treatment with cytochalasin D but not with nocodazole reduced the expression of differentiation markers, without affecting the final morphology of the cells. The results indicate that changes in the assembly and disassembly kinetics of microfilaments dynamic of actin network formation may be critical in supporting the osteogenic differentiation of human MSCs; also indicated that the organization of microtubules appears to have a regulatory role on the kinetic of this process.     

Age-related intrinsic changes in human bone-marrow-derived mesenchymal stem cells and their differentiation to osteoblasts

In vivo and in vitro studies indicate that a subpopulation of human marrow-derived stromal cells (MSCs, also known as mesenchymal stem cells) has potential to differentiate into multiple cell types, including osteoblasts. In this study, we tested the hypothesis that there are intrinsic effects of age in human MSCs (17-90 years). We tested the effect of age on senescence-associated beta-galactosidase, proliferation, apoptosis, p53 pathway genes, and osteoblast differentiation in confluent monolayers by alkaline phosphatase activity and osteoblast gene expression analysis. There were fourfold more human bone MSCs (hMSCs) positive for senescence-associated beta-galactosidase in samples from older than younger subjects (P < 0.001; n = 17). Doubling time of hMSCs was 1.7-fold longer in cells from the older than the younger subjects, and was positively correlated with age (P = 0.002; n = 19). Novel age-related changes were identified. With age, more cells were apoptotic (P = 0.016; n = 10). Further, there were age-related increases in expression of p53 and its pathway genes, p21 and BAX. Consistent with other experiments, there was a significant age-related decrease in generation of osteoblasts both in the STRO-1+ cells (P = 0.047; n = 8) and in adherent MSCs (P < 0.001; n = 10). In sum, there is an age-dependent decrease in proliferation and osteoblast differentiation, and an increase in senescence-associated beta-galactosidase-positive cells and apoptosis in hMSCs. Up-regulation of the p53 pathway with age may have a critical role in mediating the reduction in both proliferation and osteoblastogenesis of hMSCs. These findings support the view that there are intrinsic alterations in human MSCs with aging that may contribute to the process of skeletal aging in humans.