TacGhee Yi

Immunomodulatory properties of mesenchymal stem cells and their therapeutic applications

Mesenchymal stem cells (MSCs) are adult stem cells that can be isolated from most adult tissues, including bone marrow, adipose, liver, amniotic fluid, lung, skeletal muscle and kidney. The term MSC is currently being used to represent both mesenchymal stem cells and multipotent mesenchymal stromal cells. Numerous reports on systemic administration of MSCs leading to functional improvements based on the paradigm of engraftment and differentiation have been published. However, it is not only difficult to demonstrate extensive engraftment of cells, but also no convincing clinical results have been generated from phase 3 trials as of yet and prolonged responses to therapy have been noted after identification of MSCs had discontinued. It is now clear that there is another mechanism by which MSCs exert their reparative benefits. Recently, MSCs have been shown to possess immunomodulatory properties. These include suppression of T cell proliferation, influencing dendritic cell maturation and function, suppression of B cell proliferation and terminal differentiation, and immune modulation of other immune cells such as NK cells and macrophages. In terms of the clinical applications of MSCs, they are being tested in four main areas: tissue regeneration for cartilage, bone, muscle, tendon and neuronal cells; as cell vehicles for gene therapy; enhancement of hematopoietic stem cell engraftment; and treatment of immune diseases such as graft-versus-host disease, rheumatoid arthritis, experimental autoimmune encephalomyelitis, sepsis, acute pancreatitis and multiple sclerosis. In this review, the mechanisms of immunomodulatory effects of MSCs and examples of animal and clinical uses of their immunomodulatory effects are described.

Intraglandular transplantation of bone marrow-derived clonal mesenchymal stem cells for amelioration of post-irradiation salivary gland damage

OBJECTIVES External irradiation in head and neck cancers may induce irreversible hyposalivation and consequent xerostomia, stemming from radiation damage to salivary glands (SGs). As cell-based therapy has been reported to be able to repair or restore damaged SG tissues, we attempted to determine whether bone marrow-derived clonal mesenchymal stem cells (BM-cMSCs) can ameliorate irradiation-induced salivary gland damage via a murine model. METHODS External irradiation at a dose of 15Gy was delivered to the neck fields of C57BL/6 mice. We directly administered either homologous mouse BM-cMSCs labeled with PKH26 (treatment group) or PBS (control group) into SGs 24h after irradiation. Salivary flow rate (SFR) and lag time of salivation were measured at 12weeks after transplantation. At 4 and 12weeks post-transplantation, we performed morphological, histological, and immunofluorescent examinations. Transdifferentiation of administered BM-cMSCs into salivary epithelial cells was observed by confocal microscopy. RESULTS SFR was significantly increased in BM-cMSCs-transplanted mice compared with PBS-injected mice at 12weeks after transplantation. Administration of BM-cMSCs preserved the microscopic morphologies of SGs, with more functional acini in BM-cMSC-transplanted SGs than in PBS-injected SGs. Immunofluorescent staining revealed less apoptotic cells and increased microvessel density in BM-cMSC-transplanted SGs compared with PBS-injected SGs. PKH-26 labeled BM-cMSCs were detected in transplanted SGs at 4weeks after transplantation and in vivo transdifferentiation of BM-cMSCs into acinar cells was also observed. CONCLUSION This study suggests that BM-cMSCs can ameliorate salivary damage following irradiation and can be used as a source of cell-based therapy for restoration of irradiation-induced salivary hypofunction.

Trichostatin A-mediated upregulation of p21 WAF1 contributes to osteoclast apoptosis

Histone deacetylase inhibitors (HDIs), a new class of anti-cancer agents, have been reported to suppress formation of osteoclast precursors and their fusion into multinucleated cells. However, little is known about the effect of HDIs on mature osteoclasts, which may have significance for their therapeutic use. Here, we demonstrate a novel action of HDIs on osteoclast apoptosis. Primary multinucleated mature osteoclasts were prepared from mouse bone marrow cells. Treatment of osteoclasts with the HDI trichostatin A (TSA) caused apoptosis, as confirmed by annexin V staining and caspase activation. TSA caused the upregulation of p21WAF1 in osteoclasts. To understand the role of p21WAF1 upregulation in TSA-treated osteoclasts, shRNA against p21WAF1-containing lentivirus was introduced into osteoclasts. The suppression of p21WAF1 decreased TSA-directed osteoclast apoptosis. Collectively, our results provide evidence that TSA causes osteoclast apoptosis, which involves, in part, TSA-induced upregulation of p21WAF1, and strongly supports HDIs as potential therapeutic agents for excessive bone resorption.

Msx2 mediates the inhibitory action of TNF-α on osteoblast differentiation

TNF-α, a proinflammatory cytokine, inhibits osteoblast differentiation under diverse inflammatory conditions; however, the underlying mechanisms in terms of the TNF-α signaling pathway remain unclear. In this study, we examined the role of Msx2 in TNF-α-mediated inhibition of alkaline phosphatase (ALP) expression and the signaling pathways involved. TNF-α down-regulated ALP expression induced by bone morphogenetic protein 2 (BMP2) in C2C12 and Runx2-/- calvarial cells. Over-expression of Msx2 suppressed BMP2-induced ALP expression. Furthermore, TNF-α induced Msx2 expression, and the knockdown of Msx2 by small interfering RNAs rescued ALP expression, which was inhibited by TNF-α. TNF-α activated the NF-κB and the JNK pathways. Inhibition of NF-κB or JNK activation reduced the inhibitory effect of TNF-α on ALP expression, whereas TNF-α-induced Msx2 expression was only suppressed by the inhibition of the NF-κB pathway. Taken together, these results indicate that Msx2 mediates the inhibitory action of TNF-α on BMP2-regulated osteoblast differentiation and that the TNF-α-activated NF-κB pathway is responsible for Msx2 induction.

Characterization of mouse clonal mesenchymal stem cell lines established by subfractionation culturing method

AIM To characterize single-cell-derived mouse clonal mesenchymal stem cells (mcMSCs) established with bone marrow samples from three different mouse strains. METHODS We established mcMSC lines using subfractionation culturing method from bone marrow samples obtained from long bones. These lines were characterized by measuring cell growth, cell surface epitopes, differentiation potential, lineage-specific gene expression and T-cell suppression capability. Nonclonal MSCs isolated by the conventional gradient centrifugation method were used as controls. RESULTS All mcMSC lines showed typical nonclonal MSC-like spindle shape morphology. Lines differed in optimal growth density requirement. Cell surface epitope profiles of these mcMSC lines were similar to those of nonclonal MSCs. However, some lines exhibited different expression levels in a few epitopes, such as CD44 and CD105. Differentiation assays showed that 90% of the mcMSC lines were capable of differentiating into adipogenic and/or chondrogenic lineages, but only 20% showed osteogenic lineage differentiation. T-cell suppression analysis showed that 75% of the lines exhibited T-cell suppression capability. CONCLUSION mcMSC lines have similar cell morphology and cell growth rate but exhibit variations in their cell surface epitopes, differentiation potential, lineage-specific gene expression and T-cell suppression capability.

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.

Tumor necrosis factor-α enhances the transcription of smad ubiquitination regulatory factor 1 in an activating protein-1- and runx2-dependent manner†

Smad ubiquitination regulatory factor 1 (Smurf1) is an E3 ubiquitin ligase that negatively regulates osteoblast differentiation. Although tumor necrosis factor‐α (TNF‐α) has been shown to increase Smurf1 expression, the details of the regulatory mechanisms remain unclear. Here, we investigated the molecular mechanism by which TNF‐α stimulates Smurf1 expression in C2C12 and primary cultured mouse calvarial cells. TNF‐α treatment rapidly induced the activation of NF‐κB and MAPKs. Smurf1 induction by TNF‐α was blocked by the inhibition of JNK or ERK, while the inhibition of NF‐κB and p38 MAPK had no effect on Smurf1 induction. TNF‐α treatment or c‐Jun overexpression enhanced the activity of a luciferase reporter that contained a 2.7 kb mouse Smurf1 promoter sequence. Site‐directed mutagenesis of the Smurf1 reporter and chromatin immunoprecipitation analysis demonstrated that the activating protein‐1 (AP‐1) binding motif at −922 bp on the mouse Smurf1 promoter mediated TNF‐α/JNK/AP‐1‐stimulated Smurf1 transcription. Interestingly, Smurf1 expression was not observed in Runx2‐null mouse calvarial cells. When Runx2 was ectopically expressed in these cells, the basal and TNF‐α‐induced expression of Smurf1 was restored. Overexpression of Runx2 transactivated the Smurf1 promoter in a dose‐dependent manner. Reporter and chromatin immunoprecipitation assays demonstrated that the Runx2‐binding motif at −202 bp functioned in Runx2‐mediated Smurf1 expression. ERK activation by TNF‐α treatment or constitutively active MEK1 overexpression increased Smurf1 expression in a Runx2‐dependent manner. These results suggest that the JNK/AP‐1 and ERK/Runx2 signaling pathways mediate TNF‐α‐dependent Smurf1 transcription. J. Cell. Physiol.

Intracavernous Delivery of Clonal Mesenchymal Stem Cells Restores Erectile Function in a Mouse Model of Cavernous Nerve Injury

INTRODUCTION Recently, much attention has focused on stem cell therapy; bone marrow-derived stem cells (BMSCs) are one of the most studied mesenchymal stem cells used in the field of erectile dysfunction (ED). However, a major limitation for the clinical application of stem cell therapy is the heterogeneous nature of the isolated cells, which may cause different treatment outcomes. AIM We investigated the effectiveness of mouse clonal BMSCs obtained from a single colony by using subfractionation culturing method (SCM) for erectile function in a mouse model of cavernous nerve injury (CNI). METHODS Twelve-week-old C57BL/6J mice were divided into four groups: sham operation group, bilateral CNI group receiving a single intracavernous (IC) injection of phosphate-buffered saline (20 μL) or clonal BMSCs (3 × 10(5) cells/20 μL), and receiving a single intraperitoneal (IP) injection of clonal BMSCs (3 × 10(5) cells/20 μL). MAIN OUTCOME MEASURES The clonal BMSC line was analyzed for cell-surface epitopes by using fluorescence-activated cell sorting and for differentiation potential. Two weeks after CNI and treatment, erectile function was measured by electrically stimulating the cavernous nerve. The penis was harvested for histologic examinations and Western blot analysis. RESULTS Clonal BMSCs expressed cell surface markers for mesenchymal stem cells and were capable of differentiating into several lineages, including adipogenic, osteogenic, and chondrogenic cells. Both IC and IP injections of clonal BMSCs significantly restored cavernous endothelial and smooth muscle content, and penile nNOS and neurofilament content in CNI mice. IC injection of clonal BMSCs induced significant recovery of erectile function, which reached 90-100% of the sham control values, whereas IP injection of clonal BMSCs partially restored erectile function. CONCLUSION We established a homogeneous population of mouse clonal BMSCs using SCM; clonal BMSCs successfully restored erectile function in CNI mice. The homogeneous nature of clonal mesenchymal stem cells may allow their clinical applications.

Mesenchymal Stem Cell Lines Isolated by Different Isolation Methods Show Variations in the Regulation of Graft-versus-host Disease

Since the discovery of the immunomodulation property of mesenchymal stem cells (MSCs) about a decade ago, it has been extensively investigated whether MSCs can be used for the treatment of immune-related diseases, such as graft-versus-host disease (GvHD). However, how to evaluate the efficacy of human MSCs for the clinical trial is still unclear. We used an MHC-mismatched model of GvHD (B6 into BALB/c). Surprisingly, the administration of the human MSCs (hMSCs) could reduce the GvHD-related mortality of the mouse recipients and xenogeneically inhibit mouse T-cell proliferation and IFN-γ production in vitro. We recently established a new protocol for the isolation of a homogeneous population of MSCs called subfractionation culturing methods (SCM), and established a library of clonal MSC lines. Therefore, we also investigated whether MSCs isolated by the conventional gradient centrifugation method (GCM) and SCM show different efficacy in vivo. Intriguingly, clonal hMSCs (hcMSCs) isolated by SCM showed better efficacy than hMSCs isolated by GCM. Based on these results, the MHC-mismatched model of GvHD may be useful for evaluating the efficacy of human MSCs before the clinical trial. The results of this study suggest that different MSC lines may show different efficacy in vivo and in vitro.

Bone marrow-derived clonal mesenchymal stem cells as a source of cell therapy for promoting vocal fold wound healing.

Objectives: We investigated whether mouse bone marrow-derived clonal mesenchymal stem cells (BM-cMSCs) could promote vocal fold (VF) wound healing by using a xenograft animal model. Methods: Homogeneous BM-cMSCs isolated by a subfractionation culturing method from the bone marrow aspirates of green fluorescent protein transgenic mice were injected into the VFs of rabbits immediately after direct mechanical injury. Macroscopic, biomechanical (rheometric), histologic, immunohistochemical, and transcriptional evaluations were performed on the scarred VFs 1 to 3 months after injury. Engraftment of the implanted BM-cMSCs was determined by detection of green fluorescent protein cells in the recipient VF by confocal microscopy. Results: The BM-cMSC-treated VFs showed improved morphological properties and viscoelasticity as compared to control VFs injected with phosphate-buffered saline solution. Histologic and immunohistochemical evaluations showed less excessive collagen deposition and increased density of glycosaminoglycans in the BM-cMSC-treated VFs as compared to the control VFs at 3 months after injury (p = 0.003 and p = 0.037, respectively). BM-cMSC transplantation led to a significant attenuation of fibronectin (p = 0.036) and transforming growth factor β1 (p = 0.042) messenger RNA expression at 1 month after injury. Green fluorescent protein-expressing BM-cMSCs engrafted in recipient VFs were found at 1 month after implantation. Conclusions: BM-cMSCs appeared to survive in the injured xenogeneic VFs after transplantation for up to 1 month and favorably enhanced the wound healing of VFs after injury. We conclude that BM-cMSCs are a possible source of cell therapy for vocal fold regeneration.