rhBMP-2 Pre-Treated Human Periodontal Ligament Stem Cell Sheets Regenerate a Mineralized Layer Mimicking Dental Cementum.

The periodontal complex consisting of alveolar bone, cementum, and periodontal ligaments (PDL) supports human teeth through the systematic orchestration of mineralized tissues and fibrous tissues. Importantly, cementum, the outermost mineralized layer of dental roots, plays an essential role by bridging the inner ligaments from the dental root to the alveolar bone. When the periodontal complex is damaged, the regeneration of each component of the periodontal complex is necessary; however, it is still challenging to achieve complete functional regeneration. In this study, we tried to control the regeneration of cementum and PDL by using a human PDL stem cell (hPDLSC) sheet engineering technology with the pretreatment of recombinant human BMP-2 (rhBMP-2). Isolated hPDLSCs obtained from extracted human teeth were pretreated with rhBMP-2 for in vitro osteogenic differentiation and grafted on the micro/macro-porous biphasic calcium phosphate (MBCP) blocks, which represent dental roots. The MBCPs with hPDLSC sheets were implanted in the subcutaneous layer of immune-compromised mice, and rhBMP-2 pretreated hPDLSC sheets showed higher mineralization and collagen ligament deposition than the no-pretreatment group. Therefore, the rhBMP-2-hPDLSC sheet technique could be an effective strategy for the synchronized regeneration of two different tissues: mineralized tissue and fibrous tissues in periodontal complexes.

A Novel Immunomodulatory Mechanism Dependent on Acetylcholine Secreted by Human Bone Marrow-derived Mesenchymal Stem Cells.

BACKGROUND AND OBJECTIVES: Mesenchymal stem cells (MSCs) are used to treat autoimmune or inflammatory diseases. Our aim was to determine the immunomodulatory mechanisms elicited by MSCs during inflammation. METHODS AND RESULTS: We cocultured MSCs with peripheral blood mononuclear cells for a mixed lymphocyte reaction or stimulated them by phytohemagglutinin. Morphological changes of MSCs and secretion of acetylcholine (ACh) from MSCs were measured. The effects of an ACh antagonist and ACh agonist on lymphocyte proliferation and proinflammatory-cytokine production were determined. The inflammatory milieu created by immune-cell activation caused MSCs to adopt a neuronlike phenotype and induced them to release ACh. Additionally, nicotinic acetylcholine receptors (nAChRs) were upregulated in activated peripheral blood mononuclear cells. We observed that ACh bound to nAChR on activated immune cells and led to the inhibition of lymphocyte proliferation and of proinflammatory-cytokine production. MSC-mediated immunosuppression through ACh activity was reversed by an ACh antagonist called α-bungarotoxin, and lymphocyte proliferation was inhibited by an ACh agonist, ACh chloride. CONCLUSIONS: Our findings point to a novel immunomodulatory mechanism in which ACh secreted by MSCs under inflammatory conditions might modulate immune cells. This study may provide a novel method for the treatment of autoimmune diseases by means of MSCs.

Comparative study on metabolite level in tissue-specific human mesenchymal stem cells by an ultra-performance liquid chromatography quadrupole time of flight mass spectrometry.

Mesenchymal stem cells (MSCs) are a promising therapeutic option for cell-based therapy due to their immunomodulatory and regenerative properties. They can be isolated from various adult tissues, including bone marrow, fat, dental tissue, and glandular tissue. Although they share common characteristics, little is known about the biological differences between MSC populations derived from different tissues. In this study, we used MS to compare the endogenous metabolite level in the human MSCs originating from the bone marrow, adipose tissue, periodontal ligaments, and salivary glands. Using an optimized metabolomics technique, we verified that human MSCs exhibit differences in the endogenous metabolite level depending on their source material, while the multivariate analysis showed that 5 lysophosphatidylcholines and 3 lysophosphatidylethanolamines can serve as markers for the discrimination between MSC sources and may be related to differences in their differentiation capacity. These results may significantly contribute to further mechanistic studies on the MSCs and provide novel insights into the properties and optimal usage of MSCs from different tissues.

ICOSL expression in human bone marrow-derived mesenchymal stem cells promotes induction of regulatory T cells.

Mesenchymal stem cells (MSCs) can modulate lymphocyte proliferation and function. One of the immunomodulatory functions of MSCs involves CD4+CD25+FoxP3+ regulatory T cells (Tregs), which negatively regulate inflammatory responses. MSC-mediated Treg induction is supposed to be regulated by mechanisms requiring both soluble and cell contact-dependent factors. Although the involvement of soluble factors has been revealed, the contact-dependent mechanisms in MSC-mediated Treg induction remain unclear. We attempted to identify molecule(s) other than secreted factors that are responsible for MSC-mediated Treg induction and to uncover the underlying mechanisms. Under in vitro Treg-inducing conditions, ICOSL expression in MSCs coincided with Treg induction in co-cultures of MSCs with CD4+ T cells. When cultured in a transwell plate, MSCs failed to induce Tregs. Neutralization or knockdown of ICOSL significantly reduced Tregs and their IL-10 release. ICOSL overexpression in MSCs promoted induction of functional Tregs. ICOSL-ICOS signaling promoted Treg differentiation from CD4+ T cells through activation of the phosphoinositide 3-kinase-Akt pathway. MSCs primed with Interleukin-1ß significantly induced Tregs through ICOSL upregulation. We demonstrated that the Treg-inducing activity of MSCs is proportionate to their basal ICOSL expression. This study provides evidence that ICOSL expression in human MSCs plays an important role in contact-dependent regulation of MSC-mediated Treg induction.

Mesenchymal stromal cells inhibit CD25 expression via the mTOR pathway to potentiate T-cell suppression.

Mesenchymal stromal cells (MSCs) are known to suppress T-cell activation and proliferation. Several studies have reported that MSCs suppress CD25 expression in T cells. However, the molecular mechanism underlying MSC-mediated suppression of CD25 expression has not been fully examined. Here, we investigated the mTOR pathway, which is involved in CD25 expression in T cells. We showed that MSCs inhibited CD25 expression, which was restored in the presence of an inducible nitric oxide synthase (iNOS) inhibitor. Since CD25 mRNA expression was not inhibited, we focused on determining whether MSCs modulated components of the mTOR pathway in T cells. MSCs increased the phosphorylation of liver kinase B1 (LKB1) and AMP-activated protein kinase (AMPK) and decreased the phosphorylation of ribosomal protein S6 kinase 1 (S6K1) and eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1). In addition, the expression of 4E-BP1 increased dramatically in the presence of MSCs. An m7GTP pull-down assay showed increased binding of 4E-BP1 to the 5' cap-binding eukaryotic translation initiation factor 4E (eIF4E) complex in the presence of MSCs, which resulted in inhibition of mRNA translation. Treatment with 4EGI-1, a synthetic inhibitor of mRNA translation, also reduced CD25 expression in T cells. Polysome analysis confirmed decreased CD25 mRNA in the polysome-rich fraction in the presence of MSCs. Taken together, our results showed that nitric oxide, produced by MSCs, inhibits CD25 translation through regulation of the LKB1-AMPK-mTOR pathway to suppress T cells.

Single Cell Clones Purified from Human Parotid Glands Display Features of Multipotent Epitheliomesenchymal Stem Cells.

A better understanding of the biology of tissue-resident stem cell populations is essential to development of therapeutic strategies for regeneration of damaged tissue. Here, we describe the isolation of glandular stem cells (GSCs) from a small biopsy specimen from human parotid glands. Single colony-forming unit-derived clonal cells were isolated through a modified subfractionation culture method, and their stem cell properties were examined. The isolated clonal cells exhibited both epithelial and mesenchymal stem cell (MSC)-like features, including differentiation potential and marker expression. The cells transiently displayed salivary progenitor phenotypes during salivary epithelial differentiation, suggesting that they may be putative multipotent GSCs rather than progenitor cells. Both epithelial and mesenchymal-expressing putative GSCs, LGR5+CD90+ cells, were found in vivo, mostly in inter-secretory units of human salivary glands. Following in vivo transplantation into irradiated salivary glands of mice, these cells were found to be engrafted around the secretory complexes, where they contributed to restoration of radiation-induced salivary hypofunction. These results showed that multipotent epitheliomesenchymal GSCs are present in glandular mesenchyme, and that isolation of homogenous GSC clones from human salivary glands may promote the precise understanding of biological function of bona fide GSCs, enabling their therapeutic application for salivary gland regeneration.

Maintained Stemness of Human Periodontal Ligament Stem Cells Isolated After Prolonged Storage of Extracted Teeth.

BACKGROUND: Human periodontal ligament stem cells (hPDLSCs) are readily accessible and represent a promising source of mesenchymal stem cells (MSCs) for therapies. For expected clinical applications of stem cell therapies, prolonged maintenance of stemness of hPDLSCs after storage is crucial. Likewise, optimization of cell quality and standardization of manufacturing protocols require evaluation of hPDLSC characteristics after storage. The aim of this study is to evaluate maintenance of stemness of hPDLSCs isolated after prolonged storage of the periodontal ligament (PDL), by studying: 1) growth and proliferation; 2) immunophenotypes; and 3) differentiation capabilities of hPDLSCs. METHODS: After extraction of premolars (N = 10), hPDLSCs were isolated immediately (n = 5) or after 1 week of tooth storage in growth medium (n = 5). Both groups were evaluated for: 1) colony formation; 2) proliferation; and 3) immunophenotypes. Osteogenic, adipogenic, and chondrogenic differentiation capabilities of hPDLSCs were evaluated at different times and by real-time polymerase chain reaction (PCR). RESULTS: Storing PDLs attached to teeth did not affect stemness of hPDLSCs. Colony formation and proliferation properties of hPDLSCs from PDLs harvested immediately after extraction or after storage for 1 week were comparable, and there were no significant differences in immunophenotypic markers between the two groups. Osteogenic, adipogenic, and chondrogenic differentiation capabilities of hPDLSCs were unaltered after PDL storage. Real-time PCR analyses indicated that the expression of: 1) osteogenic (runt-related transcription factor 2, alkaline phosphatase, and osteocalcin); 2) adipogenic (peroxisome proliferator-activated receptor-γ and lipoprotein lipase); and 3) chondrogenic (aggrecan, collagen Type 2, and collagen Type 10) differentiation markers were similar for both groups. CONCLUSION: Within the limitations of the study, hPDLSCs stored for 1 week maintain the important characteristics of MSCs.

Evaluation of In Vivo Osteogenic Potential of Bone Morphogenetic Protein 2-Overexpressing Human Periodontal Ligament Stem Cells Combined with Biphasic Calcium Phosphate Block Scaffolds in a Critical-Size Bone Defect Model.

Human periodontal ligament stem cells (hPDLSCs) are considered potential cellular carriers for gene delivery in the field of tissue regeneration. This study tested the osseoregenerative potential of hPDLSCs transduced with replication-deficient recombinant adenovirus (rAd) containing the gene encoding bone morphogenetic protein-2 (BMP2; hPDLSCs/rAd-BMP2) in both in vivo and in vitro osteogenic environments. After the optimal condition for rAd-mediated transduction was determined, hPDLSCs were transduced to express BMP2. In vivo bone formation was evaluated in a critical-size rat calvarial bone defect model that more closely mimics the harsher in vivo milieu for bone regeneration than subcutaneous transplantation model. As support materials for bone regeneration, block-type biphasic calcium phosphate (BCP) scaffolds were combined with hPDLSCs and/or BMP2 and transplanted into critical-size bone defects in rats. Experimental groups were as follows: BCP scaffold control (group 1 [Gr1]), scaffold containing recombinant human BMP2 (rhBMP2; group 2 [Gr2]), scaffold loaded with normal hPDLSCs (group 3 [Gr3]), scaffold combined with both normal hPDLSCs and rhBMP2 (group 4 [Gr4]), and scaffold loaded with hPDLSCs transduced with rAd-BMP2 (hPDLSCs/rAd-BMP2; group 5 [Gr5]). Our data showed that new bone formation was highest in Gr2. Less mineralization was observed in Gr3, Gr4, and Gr5 in which hPDLSCs were transplanted. In vitro transwell assay demonstrated that hPDLSCs exert an inhibitory activity on BMP2-induced osteogenic differentiation. Our findings suggest that the in vivo bone regenerative potential of BMP2-overexpressing hPDLSCs could be compromised in a critical-size rat calvarial bone defect model. Thus, further investigations are required to elucidate the underlying mechanisms and to develop efficient techniques for improved tissue regeneration.

Galectin-9 is Involved in Immunosuppression Mediated by Human Bone Marrow-derived Clonal Mesenchymal Stem Cells.

Bone marrow-derived mesenchymal stem cells (MSCs) have immunomodulatory properties and can suppress exaggerated pro-inflammatory immune responses. Although the exact mechanisms remain unclear, a variety of soluble factors are known to contribute to MSC-mediated immunosuppression. However, functional redundancy in the immunosuppressive properties of MSCs indicates that other uncharacterized factors could be involved. Galectin-9, a member of the ß-galactoside binding galectin family, has emerged as an important regulator of innate and adaptive immunity. We examined whether galectin-9 contributes to MSC-mediated immunosuppression. Galectin-9 was strongly induced and secreted from human MSCs upon stimulation with pro-inflammatory cytokines. An in vitro immunosuppression assay using a knockdown approach revealed that galectin-9-deficient MSCs do not exert immunosuppressive activity. We also provided evidence that galectin-9 may contribute to MSC-mediated immunosuppression by binding to its receptor, TIM-3, expressed on activated lymphocytes, leading to apoptotic cell death of activated lymphocytes. Taken together, our findings demonstrate that galectin-9 is involved in MSC-mediated immunosuppression and represents a potential therapeutic factor for the treatment of inflammatory diseases.

Therapeutic effects of mouse bone marrow-derived clonal mesenchymal stem cells in a mouse model of inflammatory bowel disease.

Mouse bone marrow-derived clonal mesenchymal stem cells (mcMSCs), which were originated from a single cell by a subfractionation culturing method, are recognized as new paradigm for stem cell therapy featured with its homogenous cell population. Next to proven therapeutic effects against pancreatitis, in the current study we demonstrated that mcMSCs showed significant therapeutic effects in dextran sulfate sodium (DSS)-induced experimental colitis model supported with anti-inflammatory and restorative activities. mcMSCs significantly reduced the disease activity index (DAI) score, including weight loss, stool consistency, and intestinal bleeding and significantly increased survival rates. The pathological scores were also significantly improved with mcMSC. We have demonstrated that especial mucosal regeneration activity accompanied with significantly lowered level of apoptosis as beneficiary actions of mcMSCs in UC models. The levels of inflammatory cytokines including TNF-α, IFN-γ, IL-1ß, IL-6, and IL-17 were all significantly concurrent with significantly repressed NF-κB activation compared to the control group and significantly decreased infiltrations of responsible macrophage and neutrophil. Conclusively, our findings provide the rationale that mcMSCs are applicable as a potential source of cell-based therapy in inflammatory bowel diseases, especially contributing either to prevent relapse or to accelerate healing as solution to unmet medical needs in IBD therapy.

Manufacture of Clinical-Grade Human Clonal Mesenchymal Stem Cell Products from Single Colony Forming Unit-Derived Colonies Based on the Subfractionation Culturing Method.

Stem cell products derived from mesenchymal stem cells (MSCs) have been widely used in clinical trials, and a few products have been already commercialized. However, the therapeutic effects of clinical-grade MSCs are still controversial owing to mixed results from recent clinical trials. A potential solution to overcome this hurdle may be to use clonal stem cells as the starting cell material to increase the homogeneity of the final stem cell products. We have previously developed an alternative isolation and culture protocol for establishing a population of clonal MSCs (cMSCs) from single colony forming unit (CFU)-derived colonies. In this study, we established a good manufacturing practice (GMP)-compatible procedure for the clinical-grade production of human bone marrow-derived cMSCs based on the subfractionation culturing method. We optimized the culture procedures to expand and obtain a clonal population of final MSC products from single CFU-derived colonies in a GMP facility. The characterization results of the final cMSC products met our preset criteria. Animal toxicity tests were performed in a good laboratory practice facility, and showed no toxicity or tumor formation in vivo. These tests include single injection toxicity, multiple injection toxicity, biodistribution analysis, and tumorigenicity tests in vivo. No chromosomal abnormalities were detected by in situ karyotyping using oligo-fluorescence in situ hydridization (oligo-FISH), providing evidence of genetic stability of the clinical-grade cMSC products. The manufacture and quality control results indicated that our GMP methodology could produce sufficient clonal population of MSC products from a small amount of bone marrow aspirate to treat a number of patients.

Biomimetic chimeric peptide-tethered hydrogels for human mesenchymal stem cell delivery.

Here, we report a chimeric peptide-tethered fibrin hydrogel scaffold for delivery of human mesenchymal stem cells (hMSC). Osteopontin-derived peptide (OP) was used as an hMSC-tethering moiety. OP showed hMSC adhesion properties and enhanced hMSC proliferation. A natural fibrin-binding protein-derived peptide (FBP) was tested for its ability to tether hMSC to the fibrin gel matrix. FBP loading on fibrin gels was 8.2-fold higher than that of a scrambled peptide (scFBP). FBP-loaded fibrin gels were retained at injection sites longer than scFBP-loaded fibrin gels, showing a 15.9-fold higher photon intensity of fluorescent FBP-grafted fibrin gels than fluorescent scFBP-loaded fibrin gels 48 h after injection. On the basis of the fibrin gel-binding properties of FBP and the hMSC-binding and proliferation-supporting properties of OP, we constructed chimeric peptides containing FBP and OP linked with a spacer (FBPsOP). Four days after transplantation, the survival of hMSC in FBPsOP-grafted fibrin gels was 3.9-fold higher than hMSC in fibrin gels alone. Our results suggest the potential of FBPsOP-grafted fibrin gels as a bioactive delivery system for enhanced survival of stem cells.

Establishment and characterization of mesenchymal stem cell-like clonal stem cells from mouse salivary glands.

Successful therapy for radiation-induced salivary gland (SG) hypofunction is currently unavailable; however, tissue-specific stem cells are expected to be promising candidates for SG regeneration. Here, we present our method for the establishment of single cell-derived clonal stem cells from mouse SGs and describe their characteristics. Salivary gland-derived clonal stem cells (SGSCs) were isolated and expanded in vitro by a modified subfractionation culture method. The properties of SGSCs were examined with respect to their marker expression, gene expression, differentiation potential, and in vitro immunosuppressive activity relative to bone marrow-derived mesenchymal stem cells (BM-MSCs). SGSCs appeared to largely share the characteristics of BM-MSCs based on their marker expression, whereas they differentially expressed some genes, including AQP5, E-Cadherin, Laminin, ZO-1, and COL4. SGSCs showed the ability to differentiate into fat, bone, and cartilage cell types, as well as into α-amylase-producing and hepatocyte-like cells after appropriate induction. The in vitro immunosuppressive activity of SGSCs was found to be more potent than that of BM-MSCs. These results showed that SGSCs possess the properties of MSCs with some differential gene expression and they are salivary-specific stem cells with both epithelial and mesenchymal properties. The biological functions of SGSCs and their relevance to SG epithelial progenitor cells require further investigation.

Therapeutic effect of human clonal bone marrow-derived mesenchymal stem cells in severe acute pancreatitis.

Severe acute pancreatitis (SAP), a common necroinflammatory disease initiated by the premature activation of digestive enzymes within the pancreatic acinar cells, is associated with significant morbidity and mortality. In this study, we investigated whether human bone marrow-derived clonal mesenchymal stem cells (hcMSCs), isolated from human bone marrow aspirate according to our newly established isolation protocol, have potential therapeutic effects in SAP. SAP was induced by three intraperitoneal (i.p.) injections of cerulein (100 µg/kg) and sequential LPS (10 mg/kg) in Sprague-Dawley (SD) rats. hcMSCs (1 × 10(6)/head) were infused on 24 h after LPS injection via the tail vein. The rats were sacrificed 3 days after infusion of hcMSCs. We observed that infused hcMSCs reduced the levels of serum amylase and lipase. Infused hcMSCs ameliorated acinar cell necrosis, pancreatic edema, and inflammatory infiltration. Also, infused hcMSCs decreased the level of malondialdehyde, and increased the levels of glutathione peroxidase and superoxide dismutase. The number of TUNEL positive acinar cells was reduced after hcMSCs infusion. In addition, hcMSCs reduced the expression levels of pro-inflammation mediators and cytokines, and increased the expression of SOX9 in SAP. Taken together, hcMSCs could effectively relieve injury of pancreatitis as a promising therapeutics for SAP.

Pharmacokinetics and in vivo fate of intra-articularly transplanted human bone marrow-derived clonal mesenchymal stem cells.

In this study, we report the pharmacokinetics and in vivo fate of intra-articularly transplanted human mesenchymal stem cells (MSCs) in comparison with those of intravenously administered cells. Bone marrow-derived human clonal mesenchymal stem cells (hcMSCs) were transplanted to nude mice through intravenous or intra-articular routes. The numbers of hcMSCs in blood and tissue samples were measured by the quantitative real-time-polymerase chain reaction (qPCR) with human Alu (hAlu) as a detection marker. Following intra-articular transplantation, the blood levels of hcMSCs peaked 8 h postdose and gradually diminished, showing a 95-fold higher mean residence time than hcMSCs delivered through the intravenous route. Unlike intravenously administered hcMSCs, intra-articularly injected hcMSCs were mainly retained at injection joint sites where their levels 8 h postdose were 116-fold higher than those in muscle tissues. Regardless of injection routes, biodistribution patterns did not significantly differ between normal and osteoarthritis-induced mice. Quantitative analysis using hAlu-specific qPCR revealed that hcMSC levels in joint tissues were significantly higher than those in muscle tissues 120 days postdose. These dramatic differences in kinetic behavior and fate of intra-articularly transplanted hcMSCs compared with intravenously administered hcMSCs may provide insights useful for the development of human MSCs for arthritis therapeutics.

Non-invasive characterization of the adipogenic differentiation of human bone marrow-derived mesenchymal stromal cells by HS-SPME/GC-MS.

A non-invasive method to characterize human mesenchymal stromal cells during adipogenic differentiation was developed for the first time. Seven fatty acid methyl esters (FAMEs), including methyl laurate, methyl myristate, methyl palmitate, methyl linoleate, methyl oleate, methyl elaidate and methyl stearate, were used for characterizing adipogenic differentiation using headspace solid-phase microextraction (HS-SPME) which is a very simple and non-invasive method for the extraction of volatile compounds. Glassware was used for culturing mesenchymal stromal cells rather than the common plasticware to minimize contamination by volatile impurities. The optimal SPME fiber was selected by comparing diverse fibers containing two pure liquid polymers (PDMS and PA) and two porous solids (PDMS/DVB and CAR/PDMS). Using optimized procedures, we discovered that seven FAMEs were only detected in adipogenic differentiated mesenchymal stromal cells and not in the mesenchymal stromal cells before differentiation. These data could support the quality control of clinical mesenchymal stromal cell culture in the pharmaceutical industry in addition to the development of many clinical applications using mesenchymal stromal cells.

Isolation of adipose-derived stem cells by using a subfractionation culturing method.

OBJECTIVE: Adipose-derived stem cells (ASCs) isolated from subcutaneous adipose tissue have been tested in clinical trials. However, ASCs isolated by enzyme digestion and centrifugation are heterogeneous and exhibit wide variation in regenerative potential and clinical outcomes. Therefore, we developed a new method for isolating clonal ASCs (cASCs) that does not use enzyme digestion or centrifugation steps. RESEARCH DESIGN AND METHODS: In addition to cell surface markers and differentiation potential, we compared the mitogenic, paracrine and hair growth-promoting effects of ASCs isolated by the gradient centrifugation method (GCM) or by the new subfractionation culturing method (SCM). RESULTS: We selected three cASCs isolated by SCM that showed high rates of proliferation. The cell surface markers expressed by ASCs isolated by GCM or SCM were very similar, and SCM-isolated ASCs could potentially differentiate into different cell lineages. However, cASC lines exhibited better mitogenic and paracrine effects than ASCs isolated by GCM. The expression of Diras3, Myb, Cdca7, Mki67, Rrm2, Cdk1 and Ccna2, which may play a key role in cASC proliferation, was upregulated in cASCs. In addition, cASCs exhibited enhanced hair growth-promoting effects in dermal papilla cells and animal experiments. CONCLUSIONS: SCM generates a highly homogeneous population of ASCs via a simple and effective procedure that can be used in therapeutic settings.

MiR-124 inhibits myogenic differentiation of mesenchymal stem cells via targeting Dlx5.

MicroRNAs (miRNAs), including miR-1, miR-133, and miR-206, play a crucial role in muscle development by regulating muscle cell proliferation and differentiation. The aim of the present study was to define the effect of miR-124 on myogenic differentiation of mesenchymal stem cells (MSCs). The expression level of miR-124 in skeletal muscles was much lower than those in primary cultured bone marrow-derived MSCs and the bone, fat and brain tissues obtained from C57BL/6 mice. Myogenic stimuli significantly decreased the expression levels of miR-124 in mouse bone marrow-derived MSCs and C2C12 cells. Forced expression of miR-124 suppressed the expression of myogenic marker genes such as Myf5, Myod1, myogenin and myosin heavy chain and multinucleated myotube formation. Blockade of endogenous miR-124 with a hairpin inhibitor enhanced myogenic marker gene expression and myotube formation. During myogenic differentiation of MSCs and C2C12 cells, the levels of Dlx5, a known target of miR-124, were inversely regulated with those of miR-124. Furthermore, overexpression of Dlx5 increased myogenic differentiation, whereas knockdown of Dlx5 using siRNA inhibited myogenesis in C2C12 cells. These results suggest that miR-124 is a negative regulator of myogenic differentiation of MSCs and that upregulation of Dlx5 accompanied with downregulation of miR-124 by myogenic stimuli is necessary for the proper progression of myogenic differentiation.

Molecular Characterization of Neurally Differentiated Human Bone Marrow-derived Clonal Mesenchymal Stem Cells.

Bone marrow-derived mesenchymal stem cells (MSCs) are multipotent, with the ability to differentiate into different cell types. Additionally, the immunomodulatory activity of MSCs can downregulate inflammatory responses. The use of MSCs to repair injured tissues and treat inflammation, including in neuroimmune diseases, has been extensively explored. Although MSCs have emerged as a promising resource for the treatment of neuroimmune diseases, attempts to define the molecular properties of MSCs have been limited by the heterogeneity of MSC populations. We recently developed a new method, the subfractionation culturing method, to isolate homogeneous human clonal MSCs (hcMSCs). The hcMSCs were able to differentiate into fat, cartilage, bone, neuroglia, and liver cell types. In this study, to better understand the properties of neurally differentiated MSCs, gene expression in highly homogeneous hcMSCs was analyzed. Neural differentiation of hcMSCs was induced for 14 days. Thereafter, RNA and genomic DNA was isolated and subjected to microarray analysis and DNA methylation array analysis, respectively. We correlated the transcriptome of hcMSCs during neural differentiation with the DNA methylation status. Here, we describe and discuss the gene expression profile of neurally differentiated hcMSCs. These findings will expand our understanding of the molecular properties of MSCs and contribute to the development of cell therapy for neuroimmune diseases.

The molecular mechanism underlying the proliferating and preconditioning effect of vitamin C on adipose-derived stem cells.

Although adipose-derived stem cells (ASCs) show promise for cell therapy, there is a tremendous need for developing ASC activators. In the present study, we investigated whether or not vitamin C increases the survival, proliferation, and hair-regenerative potential of ASCs. In addition, we tried to find the molecular mechanisms underlying the vitamin C-mediated stimulation of ASCs. Sodium-dependent vitamin C transporter 2 (SVCT2) is expressed in ASCs, and mediates uptake of vitamin C into ASCs. Vitamin C increased the survival and proliferation of ASCs in a dose-dependent manner. Vitamin C increased ERK1/2 phosphorylation, and inhibition of the mitogen-activated protein kinase (MAPK) pathway attenuated the proliferation of ASCs. Microarray and quantitative polymerase chain reaction showed that vitamin C primarily upregulated expression of proliferation-related genes, including Fos, E2F2, Ier2, Mybl1, Cdc45, JunB, FosB, and Cdca5, whereas Fos knock-down using siRNA significantly decreased vitamin C-mediated ASC proliferation. In addition, vitamin C-treated ASCs accelerated the telogen-to-anagen transition in C3H/HeN mice, and conditioned medium from vitamin C-treated ASCs increased the hair length and the Ki67-positive matrix keratinocytes in hair organ culture. Vitamin C increased the mRNA expression of HGF, IGFBP6, VEGF, bFGF, and KGF, which may mediate hair growth promotion. In summary, vitamin C is transported via SVCT2, and increased ASC proliferation is mediated by the MAPK pathway. In addition, vitamin C preconditioning enhanced the hair growth promoting effect of ASCs. Because vitamin C is safe and effective, it could be used to increase the yield and regenerative potential of ASCs.