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.     

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.     

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.     

Effects of miR-31 on the osteogenesis of human mesenchymal stem cells

Exploring the molecular mechanisms that regulate the osteogenesis of human mesenchymal stem cells (hMSCs) will bring us more efficient methods for improving the treatment of bone-related diseases. In this study, we analyzed the effects of miR-31 on the osteogenesis of hMSCs. The overexpression of miR-31 repressed the osteogenesis of hMSCs, whereas the downregulation enhanced this process. SATB2 was testified to be a direct target of miR-31, and its effects on the osteogenesis were also described. Most importantly, the knockdown of SATB2 attenuated miR-31’s osteogenic effects. Taken together, our findings suggest that miR-31 regulates the osteogenesis of hMSCs by targeting SATB2.     

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.     

Morphological examination during in vitro cartilage formation by human mesenchymal stem cells

The formation of the skeleton through endochondral ossification is one of the most complex processes in development. One approach to resolving this complexity is to examine simplified systems. In vitro cartilage formation by mesenchymal stem cells (MSCs) is observed when the cells are cultured as a micromass. Several studies have confirmed the molecular events, showing the usefulness of these cells as a differentiation model. We have elucidated the process of cartilage formation in MSCs from the morphological point of view by light and transmission electron microscopy and immunohistochemical examination. The morphology of the MSCs changed from spherical to spindle-shaped, and the cells aggregated and formed junctional complexes during Day 1. At Day 7, three layers were observed. The superficial zone consisted of several layers of elongated cells with junctional complexes. The middle zone was composed of apoptotic bodies, and the deep zone was occupied by chondrocyte-like cells excreting extracellular matrices. At Day 14, the middle zone had disappeared, and the chondrocyte-like cells in the deep zone were detected within cartilage lacuna. They were covered by cartilage matrices containing collagen types I, II, and X and chondroitin sulfate. By Day 21, the outer layer consisting of spindle-shaped cells had disappeared in places. As the pellet grew, the outer layer seemed to be unable to stretch to maintain a constant covering around the pellet. Our findings have thus revealed that MSCs change their morphology depending upon their microenvironment during differentiation. In vitro cartilage formation by MSCs makes it possible to clarify the detailed morphological events that occur during chondrogenesis. S. Ichinose and I. Sekiya contributed equally to this study     

Multipotent mesenchymal stem cells from adult human eye conjunctiva stromal cells

Abstract Identification of mesenchymal stem cells (MSCs) derived from alternative sources has provided an exciting prospect for intensive investigation. This work focused on characterizing a new source of MSCs from stromal cells from human eye conjunctiva. In this study, after conjunctiva biopsies and culture of stromal segment of this tissue, fibroblast-like (SH2⁺, SH3⁺, CD29⁺, CD44⁺, CD166⁺, CD13⁺) human stromal cells, which can be differentiated toward the osteogenic, adipogenic, chondrogenic, and neurogenic lineages, were obtained. These cells expressed Oct-4, Nanog, Rex-1 genes, and some lineage-specific markers like cardiac actin and Keratin. Taken together, the results indicate that conjunctiva stromal-derived cells are a new source of multipotent MSCs and despite originating from an adult source, they express undifferentiated stem cell markers.     

Comparison of metabolite profiles in U87 glioma cells and mesenchymal stem cells

Gas chromatography-mass spectrometry (GC-MS) profiles were generated from U87 glioma cells and human mesenchymal stem cells (hMSC). 37 metabolites representing glycolysis intermediates, TCA cycle metabolites, amino acids and lipids were selected for a detailed analysis. The concentrations of these metabolites were compared and Pearson correlation coefficients were used to calculate the relationship between pairs of metabolites. Metabolite profiles and correlation patterns differ significantly between the two cell lines. These profiles can be considered as a signature of the underlying biochemical system and provide snap-shots of the metabolism in mesenchymal stem cells and tumor cells.     

Alpha-smooth muscle actin expression and structure integrity in chondrogenesis of human mesenchymal stem cells

The expression of alpha-smooth muscle actin (SMA) by human mesenchymal stem cells (hMSCs) during chondrogenesis was investigated by the use of pellet culture. Undifferentiated hMSCs expressed low but detectable amounts of SMA and the addition of transforming growth factor β1 (TGF-β1) to the culture medium increased SMA expression in a dose-dependent manner. Differentiation in pellet culture was rapidly induced in the presence of TGF-β1 and was accompanied by the development of annular layers at the surface of the pellet. These peripheral layers lacked expression of glycosaminoglycan and type II collagen during early differentiation. Progress in differentiation increased the synthesis of glycosaminoglycan and type II collagen and the expression of SMA in these layers. Double-staining for type II collagen and SMA by immunofluorescence demonstrated the differentiation of hMSCs into cells positive for these two proteins. The addition of cytochalasin D, a potent inhibitor of the polymerization of actin microfilaments, caused damage to the structural integrity and surface smoothness of the chondrogenic pellets. The SMA-positive cells in the peripheral layers of the chondrogenic pellets mimic those within the superficial layer of articular cartilage and are speculated to play a major role in cartilage development and maintenance.This work was supported by grants R92-001-1 and R92-001-2 from the Veterans General Hospital, Taipei, and grant NSC-92-2314-B-075-022 from the National Science Council, Taiwan.     

Kojyl cinnamate ester derivatives promote adiponectin production during adipogenesis in human adipose tissue-derived mesenchymal stem cells

The subcutaneous fat tissue mass gradually decreases with age, and its regulation is a strategy to develop anti-aging compounds to ameliorate the photo-aging of human skin. The adipogenesis of human adipose tissue-mesenchymal stem cells (hAT-MSCs) can be used as a model to discover novel anti-aging compounds. Cinnamomum cassia methanol extracts were identified as adipogenesis-promoting agents by natural product library screening. Cinnamates, the major chemical components of Cinnamomum cassia extracts, promoted adipogenesis in hAT-MSCs. We synthesized kojyl cinnamate ester derivatives to improve the pharmacological activity of cinnamates. Structure–activity studies of kojyl cinnamate derivatives showed that both the α,β-unsaturated carbonyl ester group and the kojic acid moiety play core roles in promoting adiponectin production during adipogenesis in hAT-MSCs. We conclude that kojyl cinnamate ester derivatives provide novel pharmacophores that can regulate adipogenesis in hAT-MSCs.     

Neuronal differentiation of human mesenchymal stem cells: changes in the expression of the Alzheimer's disease-related gene seladin-1

Seladin-1 (SELective Alzheimer's Disease INdicator-1) is an anti-apoptotic gene, which is down-regulated in brain regions affected by Alzheimer's disease (AD). In addition, seladin-1 catalyzes the conversion of desmosterol into cholesterol. Disruption of cholesterol homeostasis in neurons may increase cell susceptibility to toxic agents. Because the hippocampus and the subventricular zone, which are affected in AD, are the unique regions containing stem cells with neurogenic potential in the adult brain, it might be hypothesized that this multipotent cell compartment is the predominant source of seladin-1 in normal brain. In the present study, we isolated and characterized human mesenchymal stem cells (hMSC) as a model of cells with the ability to differentiate into neurons. hMSC were then differentiated toward a neuronal phenotype (hMSC-n). These cells were thoroughly characterized and proved to be neurons, as assessed by molecular and electrophysiological evaluation. Seladin-1 expression was determined and found to be significantly reduced in hMSC-n compared to undifferentiated cells. Accordingly, the total content of cholesterol was decreased after differentiation. These original results demonstrate for the first time that seladin-1 is abundantly expressed by stem cells and appear to suggest that reduced expression in AD might be due to an altered pool of multipotent cells.     

WNT/beta-catenin pathway up-regulates Stat3 and converges on LIF to prevent differentiation of mouse embryonic stem cells

Embryonic stem (ES) cells rely on growth factors provided by feeder cells or exogenously to maintain their pluripotency. In order to identify such factors, we have established sub-lines of STO feeder cells which exhibit variable ability in supporting ES cell self-renewal. Functional screening identifies WNT5A and WNT6 as STO cell-produced factors that potently inhibit ES cell differentiation in a serum-dependent manner. Furthermore, direct activation of beta-catenin without disturbing the upstream components of the WNT/beta-catenin pathway fully recapitulates the effect of WNTs on ES cells. Importantly, the WNT/beta-catenin pathway up-regulates the mRNA for Stat3, a known regulator of ES cell self-renewal in the mouse. Finally, LIF is able to mimic the serum effect to act synergistically with WNT proteins to inhibit ES cell differentiation. Therefore, our study reveals part of the molecular mechanisms by which the WNT/beta-catenin pathway acts to prevent ES cell differentiation through convergence on the LIF/JAK-STAT pathway at the level of STAT3.