Human Tonsil-derived Mesenchymal Stem Cells Research Articles

Therapeutic Effect of Schwann Cell-Like Cells Differentiated from Human Tonsil-Derived Mesenchymal Stem Cells on Diabetic Neuropathy in db/db Mice.

Diabetic neuropathy (DN) is the most common complication of diabetes, and approximately 50% of patients with this disease suffer from peripheral neuropathy. Nerve fiber loss in DN occurs due to myelin defects and is characterized by symptoms of impaired nerve function. Schwann cells (SCs) are the main support cells of the peripheral nervous system and play important roles in several pathways contributing to the pathogenesis and development of DN. We previously reported that human tonsil-derived mesenchymal stem cells differentiated into SCs (TMSC-SCs), named neuronal regeneration-promoting cells (NRPCs), which cells promoted nerve regeneration in animal models with peripheral nerve injury or hereditary peripheral neuropathy. In this study, NRPCs were injected into the thigh muscles of BKS-db/db mice, a commonly used type 2 diabetes model, and monitored for 26weeks. Von Frey test, sensory nerve conduction study, and staining of sural nerve, hind foot pad, dorsal root ganglia (DRG) were performed after NRPCs treatment. Von Frey test results showed that the NRPC treatment group (NRPC group) showed faster responses to less force than the vehicle group. Additionally, remyelination of sural nerve fibers also increased in the NRPC group. After NRPCs treatment, an improvement in response to external stimuli and pain sensation was expected through increased expression of PGP9.5 in the sole and TRPV1 in the DRG. The NRPCs treatment may alleviate DN through the remyelination and the recovery of sensory neurons, could provide a better life for patients suffering from complications of this disease.

Oral ulcer treatment using human tonsil-derived mesenchymal stem cells encapsulated in trimethyl chitosan hydrogel: an animal model study

BackgroundOral ulcers are a common side effect of chemotherapy and affect patients’ quality of life. While stem cell transplantation is a potential treatment for oral ulcers, its efficacy is limited as the stem cells tend to remain in the affected area for a short time. This study aims to develop a treatment for oral ulcers by using trimethyl chitosan (TMC) hydrogel with human tonsil-derived stem cells (hTMSCs) to increase the therapeutic effect of stem cells and investigate their effectiveness.MethodsAnimals were divided into four experimental groups: Control, TMC hydrogel, hTMSCs, and hTMSCs loaded in TMC hydrogel (Hydrogel + hTMSCs) (each n = 8). Oral ulcers were chemically induced by anesthetizing the rats followed by injection of dilute acetic acid in the right buccal mucosa. After confirming the presence of oral ulcers in the animals, a single subcutaneous injection of 100 µL of each treatment was applied to the ulcer area. Histological analyses were performed to measure inflammatory cells, oral mucosal thickness, and fibrosis levels. The expression level of inflammatory cytokines was also measured using RT-PCR to gauge therapeutic the effect.ResultsThe ulcer size was significantly reduced in the TMC hydrogel + hTMSCs group compared to the control group. The stem cells in the tissue were only observed until Day 3 in the hTMSCs treated group, while the injected stem cells in the TMC Hydrogel + hTMSCs group were still present until day 7. Cytokine analysis related to the inflammatory response in the tissue confirmed that the TMC Hydrogel + hTMSCs treated group demonstrated superior wound healing compared to other experimental groups.ConclusionThis study has shown that the adhesion and viability of current stem cell therapies can be resolved by utilizing a hydrogel prepared with TMC and combining it with hTMSCs. The combined treatment can promote rapid healing of oral cavity wounds by enhancing anti-inflammatory effects and expediting wound healing. Therefore, hTMSC loaded in TMC hydrogel was the most effective wound-healing approach among all four treatment groups prolonging stem cell survival. However, further research is necessary to minimize the initial inflammatory response of biomaterials and assess the safety and long-term effects for potential clinical applications.

Far-infrared radiation emitted from eco-friendly carbon fiber composite sheet induced osteogenesis of human tonsil-derived mesenchymal stem cells

Far-infrared (FIR) therapy has been extensively studied because of its numerous benefits in improving human health. A planar heating element device was designed to assess the in vitro efficacy of FIR therapy by using a flexible carbon fiber composite sheet (CFS) with versatile designs and shapes. The physical properties were optimized for in vitro assays, and low hygroscopicity and stable thermal performance were obtained through thermoplastic polyurethane (TPU) lamination. The CFS evenly released FIR at temperatures ranging from 25 to 80 °C. There was no significant effect on cell viability under most conditions. However, the FIR emitted from the CFS at 70 and 80 °C for 2 h increased apoptotic cell death. The FIR irradiation between 60 and 80 °C for 1 h or less effectively induced osteogenesis of the tonsil-derived mesenchymal stem cells (TMSCs) without inducing cytotoxicity. The most significant osteogenic induction occurred at 60 and 70 °C, as evidenced by increased calcium mineralization and osteogenic markers. This study demonstrated that CFS efficiently generates the FIR, which induces the osteogenicity of TMSCs at a non-cytotoxic level, suggesting the potential applicability of the CFS device for treating bone diseases such as osteoporosis.

Preclinical Efficacy of Peripheral Nerve Regeneration by Schwann Cell-like Cells Differentiated from Human Tonsil-Derived Mesenchymal Stem Cells in C22 Mice.

Charcot-Marie-Tooth disease (CMT) is a hereditary disease with heterogeneous phenotypes and genetic causes. CMT type 1A (CMT1A) is a type of disease affecting the peripheral nerves and is caused by the duplication of the peripheral myelin protein 22 (PMP22) gene. Human tonsil-derived mesenchymal stem cells (TMSCs) are useful for stem cell therapy in various diseases and can be differentiated into Schwann cell-like cells (TMSC-SCs). We investigated the potential of TMSC-SCs called neuronal regeneration-promoting cells (NRPCs) for peripheral nerve and muscle regeneration in C22 mice, a model for CMT1A. We transplanted NRPCs manufactured in a good manufacturing practice facility into the bilateral thigh muscles of C22 mice and performed behavior and nerve conduction tests and histological and ultrastructural analyses. Significantly, the motor function was much improved, the ratio of myelinated axons was increased, and the G-ratio was reduced by the transplantation of NRPCs. The sciatic nerve and gastrocnemius muscle regeneration of C22 mice following the transplantation of NRPCs downregulated PMP22 overexpression, which was observed in a dose-dependent manner. These results suggest that NRPCs are feasible for clinical research for the treatment of CMT1A patients. Research applying NRPCs to other peripheral nerve diseases is also needed.

Tensin Regulates Fundamental Biological Processes by Interacting with Integrins of Tonsil-Derived Mesenchymal Stem Cells.

Human tonsil-derived mesenchymal stem cells (TMSCs) have a superior proliferation rate and differentiation potential compared to adipose-tissue-derived MSCs (AMSCs) or bone-marrow-derived MSCs (BMSCs). TMSCs exhibit a significantly higher expression of the tensin3 gene (TNS3) than AMSCs or BMSCs. TNS is involved in cell adhesion and migration by binding to integrin beta-1 (ITG β1) in focal adhesion. Here, we investigated the roles of four TNS isoforms, including TNS3 and their relationship with integrin in various biological processes of TMSCs. Suppressing TNS1 and TNS3 significantly decreased the cell count. The knockdown of TNS1 and TNS3 increased the gene and protein expression levels of p16, p19, and p21. TNS1 and TNS3 also have a significant effect on cell migration. Transfecting with siRNA TNS3 significantly reduced Oct4, Nanog, and Sox-2 levels. Conversely, when TNS4 was silenced, Oct4 and Sox-2 levels significantly increase. TNS1 and TNS3 promote osteogenic and adipogenic differentiation, whereas TNS4 inhibits adipogenic differentiation of TMSCs. TNS3 is involved in the control of focal adhesions by regulating integrin. Thus, TNS enables TMSCs to possess a higher proliferative capacity and differentiation potential than other MSCs. Notably, TNS3 plays a vital role in TMSC biology by regulating ITGβ1 activity.

Conditioned medium from human tonsil-derived mesenchymal stem cells inhibits glucocorticoid-induced adipocyte differentiation.

Obesity, which has become a major global health problem, involves a constitutive increase in adipocyte differentiation signaling. Previous studies show that mesenchymal stem cells (MSCs) induce weight loss and glycemic control. However, the mechanisms by which MSCs regulate adipocyte differentiation are not yet known. In this study, we investigated the effects of conditioned medium obtained from human tonsil-derived MSCs (T-MSC CM) on adipocyte differentiation. We found that T-MSC CM attenuated adipocyte differentiation from early stages via inhibiting glucocorticoid signaling. T-MSC CM also increased the phosphorylation of p38 mitogen-activated protein kinase and glucocorticoid receptors and decreased the subsequent nucleus translocation of glucocorticoid receptors. Chronic treatment of mice with synthetic glucocorticoids induced visceral and bone marrow adipose tissue expansion, but these effects were not observed in mice injected with T-MSC CM. Furthermore, T-MSC CM injection protected against reductions in blood platelet counts induced by chronic glucocorticoid treatment, and enhanced megakaryocyte differentiation was also observed. Collectively, these results demonstrate that T-MSC CM exerts inhibitory effects on adipocyte differentiation by regulating glucocorticoid signal transduction. These findings suggest that the therapeutic application of T-MSC CM could reduce obesity by preventing adipose tissue expansion.

Promotion of Platelet Production by Co-Transplantation of Mesenchymal Stem Cells in Bone Marrow Transplantation.

Therapeutic strategies that can promote platelet production are in demand to enhance clinical outcomes of bone marrow transplantation (BMT). Our research group has studied human tonsil-derived mesenchymal stem cells (T-MSCs) and their effectiveness in promoting bone marrow (BM) engraftment. Here, we analyzed the effects of T-MSCs on platelet production and hemostasis. Donor BM cells (BMCs) were isolated from C57BL/6 mice and transplanted with or without T-MSCs to BALB/c recipient mice. Mice were sacrificed and blood cells were counted using an Auto Hematology Analyzer. Femur sections were stained with CD41 antibody to analyze megakaryocytes in the BM. Growth factor secretion from MSCs was analyzed using the Quantibody Array. Effects of T-MSC conditioned medium (CM) on megakaryopoiesis were investigated using the MegaCult assay. In a mouse model of BMT, T-MSC CM was injected with or without anti-placental growth factor (α-PlGF) blocking antibody, and blood cell numbers and coagulation were analyzed. T-MSC co-transplantation increased percent survival of BMT mice. Platelet numbers were significantly lower in the BMC-only group, whereas T-MSC co-transplantation restored circulating platelets to levels similar to those of the control group. Significantly reduced numbers of CD41 + megakaryocytes in Bu-Cy and BMC groups were increased by T-MSC co-transplantation. PlGF secretion from T-MSCs were detected and enhanced megakaryopoiesis, platelet production, and coagulation by T-MCS CM were disrupted in the presence of the α-PlGF blocking antibody. We demonstrated the effectiveness of T-MSC co-transplantation in promoting platelet production and coagulation after BMT. These findings highlight the potential therapeutic relevance of T-MSCs for preventing thrombocytopenia after BMT.

Anti-Aging Effects of Nanovesicles Derived from Human Tonsil-Derived Mesenchymal Stem Cells

Growing evidence has demonstrated that biomimetic nanovesicles produced from specific cells show bioactive properties such as anti-tumor or anti-inflammatory activities. However, the properties of these nanovesicles are very diverse, depending on their cell sources. In this study, human tonsil-derived mesenchymal stem cells (TMSCs) were used in the production of functional biomimetic nanovesicles with anti-senescence. TMSCs were isolated from human tonsil tissue obtained by tonsillectomy. TMSC-derived nanovesicles (TMSC-NVs) were produced by serial extrusion using a mini-extruder. Western blotting and particle analysis were performed for characterization of TMSC-NVs. They were applied to both replicative and ultraviolet B-induced senescent human dermal fibroblasts in vitro. Following six days of treatment, analysis of the proliferation and senescence level of fibroblasts was performed using cell counting and senescence-associated β-galactosidase assay, respectively. Treatment with TMSC-NVs enhanced the cell proliferation and reduced the activity of senescence-associated β-galactosidase in both replicative and ultraviolet B-induced senescent cells. Treatment with TMSC-NVs resulted in increased expression of extracellular matrix and anti-oxidant genes. Treatment with TMSC-NVs resulted in reduced expression of vinculin in focal adhesion. These results show that TMSC-NVs have an effect on recovering from cellular senescence by oxidative stress and can be applied as useful materials for the development of skin rejuvenation.

Conditioned Medium from Human Tonsil-Derived Mesenchymal Stem Cells Enhances Bone Marrow Engraftment via Endothelial Cell Restoration by Pleiotrophin.

Cotransplantation of mesenchymal stem cells (MSCs) with hematopoietic stem cells (HSCs) has been widely reported to promote HSC engraftment and enhance marrow stromal regeneration. The present study aimed to define whether MSC conditioned medium could recapitulate the effects of MSC cotransplantation. Mouse bone marrow (BM) was partially ablated by the administration of a busulfan and cyclophosphamide (Bu–Cy)-conditioning regimen in BALB/c recipient mice. BM cells (BMCs) isolated from C57BL/6 mice were transplanted via tail vein with or without tonsil-derived MSC conditioned medium (T-MSC CM). Histological analysis of femurs showed increased BM cellularity when T-MSC CM or recombinant human pleiotrophin (rhPTN), a cytokine readily secreted from T-MSCs with a function in hematopoiesis, was injected with BMCs. Microstructural impairment in mesenteric and BM arteriole endothelial cells (ECs) were observed after treatment with Bu–Cy-conditioning regimen; however, T-MSC CM or rhPTN treatment restored the defects. These effects by T-MSC CM were disrupted in the presence of an anti-PTN antibody, indicating that PTN is a key mediator of EC restoration and enhanced BM engraftment. In conclusion, T-MSC CM administration enhances BM engraftment, in part by restoring vasculature via PTN production. These findings highlight the potential therapeutic relevance of T-MSC CM for increasing HSC transplantation efficacy.

Human Tonsil-Derived Mesenchymal Stem Cells-Loaded Hydroxyapatite-Chitosan Patch for Mastoid Obliteration.

Canal wall down (CWD) mastoidectomy is a surgical procedure that is essential to eradicate chronic ear diseases but results into bone defects due to the resulting mastoid cavity. This study investigated the ability of human tonsil-derived mesenchymal stem cells (hTMSCs) combined with a hydroxyapatite (HAp)-chitosan patch to promote osteogenesis in the treatment of postoperative temporal bone defects using an animal model. At 12 weeks postsurgery, the obliteration ratio and bone formation in the tympanic bulla of rats that were grafted with hTMSCs and a hydroxyapatite (HAp)-chitosan patch or not were compared using histopathological observation and computed tomography (CT) imaging. The group of rats that was administered the Patch group and the group of rats that was administered hTMSCs + Patch group both showed significantly remarkable obliteration ratios and bone formation compared with the Sham and hTMSCs groups. Moreover, the hTMSCs + Patch group showed bone formation in both the periphery and the central region of the tympanic bulla cavity, indicating prominently enhanced osteogenesis compared to the Patch group. Altogether, the results showed that combining hTMSCs with a HAp-chitosan patch accelerated osteogenesis for reconstruction of postoperative temporal bone defects. These findings demonstrate that the hTMSCs + Patch scaffolds are promising for treatment of the bone defects in the tissue engineering.

Differentiation of Motor Neuron-Like Cells from Tonsil-Derived Mesenchymal Stem Cells and Their Possible Application to Neuromuscular Junction Formation.

Human tonsil-derived mesenchymal stem cells (T-MSCs) are newly identified MSCs and present typical features of MSCs, including having the differentiation capacity into the three germ layers and excellent proliferation capacity. They are easily sourced and are useful for stem cell therapy in various disease states. We previously reported that T-MSCs could be differentiated into skeletal myocytes and Schwann-like cells; therefore, they are a promising candidate for cell therapies for neuromuscular disease. Motor neurons (MNs), which regulate spontaneous behavior, are affected by a wide range of MN diseases (MNDs) for which there are no effective remedies. We investigated the differentiation potential of MN-like cells derived from T-MSCs (T-MSC-MNCs) for application to therapy of MNDs. After the process of MN differentiation, the expression of MN-related markers, including Islet 1, HB9/HLXB9 (HB9), and choline acetyltransferase (ChAT), was increased when compared with undifferentiated T-MSCs. The secretion of acetylcholine to the conditioned medium was significantly increased after MN differentiation. We cocultured T-MSC-MNCs and human skeletal muscle cells, and confirmed the presence of the acetylcholine receptor clusters, which demonstrated the formation of neuromuscular junctions. The potential functional improvements afforded by these T-MSC-MNCs could be useful in the treatment of MNDs caused by genetic mutation, viral infection, or environmental problems.

Far-Infrared Irradiation Inhibits Adipogenic Differentiation and Stimulates Osteogenic Differentiation of Human Tonsil-Derived Mesenchymal Stem Cells: Role of Protein Phosphatase 2B.

Far-infrared (FIR) irradiation has been reported to exhibit various biological effects including improvement of cardiovascular function. However, its effect on the differentiation of stem cells has not been studied. Using tonsil-derived mesenchymal stem cells (TMSC), we examined whether and how FIR irradiation affects adipogenic or osteogenic differentiation. TMSC were exposed to FIR irradiation (3-25 μm wavelength) for various times (0, 30, or 60 min), and then adipogenic or osteogenic differentiation was induced for 14 days with its respective commercially available differentiation medium. At the end of the differentiation, the cells were stained using Oil red O or Alizarin red S solution, and the expression of differentiation-specific proteins was analyzed by western blotting. FIR irradiation did not alter cell viability or the expression of MSC-specific surface antigens (CD14, CD34, CD45, CD73, CD90, and CD105) in TMSC. However, FIR irradiation significantly inhibited adipogenic differentiation of TMSC, as evidenced by decreased Oil red O staining as well as protein expression of peroxisome proliferator-activated receptor γ and fatty acid binding protein 4. In contrast, FIR irradiation induced osteogenic differentiation, as evidenced by increased Alizarin red S staining as well as protein expression of osteocalcin and alkaline phosphatase. Treatment with heat alone did not inhibit the adipogenic differentiation of TMSC, suggesting that the inhibitory effect on adipogenic differentiation was not due to heat induced by FIR irradiation. However, heat alone did stimulate osteogenic differentiation, but to a lesser extent than FIR irradiation. Furthermore, FIR irradiation increased intracellular Ca²⁺ levels and the activity of protein phosphatase 2B (PP2B) in TMSC. Treatment with cyclosporin A, a specific PP2B inhibitor, reversed the inhibitory effect of FIR irradiation on adipogenic differentiation of TMSC, but had no effect on osteogenic differentiation. Our data demonstrate that FIR irradiation inhibits adipogenic differentiation but enhances osteogenic differentiation of TMSC; the inhibitory effect on adipogenic differentiation is non-thermal and mediated at least in part by activation of Ca²⁺-dependent PP2B.

Development of a three-dimensionally printed scaffold grafted with bone forming peptide-1 for enhanced bone regeneration with in vitro and in vivo evaluations

Defects in bone are some of the most difficult injuries to treat. Biomimetic scaffolds represent a promising approach for successful bone tissue regeneration. In this study, a three-dimensional (3D) scaffold with osteo-inductive functionality was designed and assayed both in-vitro and in-vivo. Bone formation peptide-1 (BFP1), an osteo-promoting specific peptide, was covalently bound to a 3D printed polycaprolactone (PCL) scaffold using polydopamine (DOPA). The amount of BFP1 immobilized on the surface was found to increase depending on the BFP1 concentration of the loading solution. To observe the biological effects of the 3D scaffolds, human tonsil-derived mesenchymal stem cells (hTMSCs) were isolated. The cells were cultured on the scaffolds and observed to rapidly differentiate into osteoblast-like cells with osteo-promoting capabilities. The scaffolds were implanted in a rabbit calvarial defect model for 8 weeks and successfully stimulated both vessel and bone regeneration. Osteo-promoting 3D scaffolds may provide a safer and more efficient approach for bone repair and remodelling in regenerative medicine.

Differentiation of Human Tonsil-Derived Mesenchymal Stem Cells into Schwann-Like Cells Improves Neuromuscular Function in a Mouse Model of Charcot-Marie-Tooth Disease Type 1A.

Charcot-Marie-Tooth disease type 1A (CMT1A) is the most common inherited motor and sensory neuropathy, and is caused by duplication of PMP22, alterations of which are a characteristic feature of demyelination. The clinical phenotype of CMT1A is determined by the degree of axonal loss, and patients suffer from progressive muscle weakness and impaired sensation. Therefore, we investigated the potential of Schwann-like cells differentiated from human tonsil-derived stem cells (T-MSCs) for use in neuromuscular regeneration in trembler-J (Tr-J) mice, a model of CMT1A. After differentiation, we confirmed the increased expression of Schwann cell (SC) markers, including glial fibrillary acidic protein (GFAP), nerve growth factor receptor (NGFR), S100 calcium-binding protein B (S100B), glial cell-derived neurotrophic factor (GDNF), and brain-derived neurotrophic factor (BDNF), which suggests the differentiation of T-MSCs into SCs (T-MSC-SCs). To test their functional efficiency, the T-MSC-SCs were transplanted into the caudal thigh muscle of Tr-J mice. Recipients’ improved locomotive activity on a rotarod test, and their sciatic function index, which suggests that transplanted T-MSC-SCs ameliorated demyelination and atrophy of nerve and muscle in Tr-J mice. Histological and molecular analyses showed the possibility of in situ remyelination by T-MSC-SCs transplantation. These findings demonstrate that the transplantation of heterologous T-MSC-SCs induced neuromuscular regeneration in mice and suggest they could be useful for the therapeutic treatment of patients with CMT1A disease.

Double intratibial injection of human tonsil-derived mesenchymal stromal cells recovers postmenopausal osteoporotic bone mass

Background and aimsOsteoporosis, which is a disease characterized by weakening of the bone, affects a large portion of the senior population. The current therapeutic options for osteoporosis have side effects, and there is no effective treatment for severe osteoporosis. Thus, we urgently need new treatment strategies, such as topical therapies and/or safe and effective stem cell therapies. MethodsWe investigated the therapeutic potential of directly injecting human tonsil-derived mesenchymal stem cells (TMSC) into the right proximal tibias of ovariectomized postmenopausal osteoporosis model mice. Injections were given once (1×) or twice (2×) during the 3-month experimental period. At the end of the experiment, micro-computed tomographic images revealed some improvement in the proximal tibias and more significant improvement in the femoral heads of treated mice. ResultsOsteogenic effect was qualitatively and quantitatively more pronounced in TMSC/2×-treated mice. Furthermore, TMSC/2× mice exhibited significant recovery of the serum osteocalcin level, which is pathologically elevated in osteoporosis, and increased serum alkaline phosphatase, which indicates bone formation. TMSC therapy was generally well tolerated and caused no apparent toxicity in the experimental mice. Moreover, TMSC therapy reduced visceral fat. ConclusionOur results demonstrate that double injection of TMSC directly into the proximal tibia triggers recovery of osteoporosis, and thus could be a potential therapeutic approach for severe bone loss.

Tonsil-derived mesenchymal stem cell-embedded in situ crosslinkable gelatin hydrogel therapy recovers postmenopausal osteoporosis through bone regeneration.

We investigated therapeutic potential of human tonsil-derived mesenchymal stem cells (TMSC) subcutaneously delivered to ovariectomized (OVX) mice for developing more safe and effective therapy for osteoporosis. TMSC were isolated from tonsil tissues of children undergoing tonsillectomy, and TMSC-embedded in situ crosslinkable gelatin-hydroxyphenyl propionic acid hydrogel (TMSC-GHH) or TMSC alone were delivered subcutaneously to the dorsa of OVX mice. After 3 months, three-dimensionally reconstructed micro-computed tomographic images revealed better recovery of the femoral heads in OVX mice treated with TMSC-GHH. Serum osteocalcin and alkaline phosphatase were also recovered, indicating bone formation only in TMSC-GHH-treated mice, and absence in hypercalcemia or other severe macroscopic deformities showed biocompatibility of TMSC-GHH. Additionally, visceral fat reduction effects by TMSC-GHH further supported their therapeutic potential. TMSC provided therapeutic benefits toward osteoporosis only when embedded in GHH, and showed potential as a supplement or alternative to current therapies.

Enhanced Osteogenic Commitment of Human Mesenchymal Stem Cells on Polyethylene Glycol-Based Cryogel with Graphene Oxide Substrate.

Graphene oxide (GO) is considered a comparatively recent biomaterial with enormous potential because of its nontoxicity, high dispersity, and enhanced interaction with biomolecules. These characteristics of GO can promote the interactions between the substrates and cell surfaces. In this study, we incorporated GO in a cryogel-based scaffold system to observe their influence on the osteogenic responses of human tonsil-derived mesenchymal stem cells (hTMSCs). Compared to polyethylene glycol (PEG)-based cryogel scaffold, GO-embedded PEG-based (PEGDA-GO) cryogels not only showed improved cell attachment and focal adhesion kinase (FAK) signaling activation but also enhanced cell viability. Taken together, we demonstrated that PEGDA-GO cryogels can stimulate osteogenic differentiation under an osteoinductive condition and enhance osteogenic phenotypes compared to the control group. In summary, we demonstrate that GO embedded in cryogels system is an effective biofunctionalizing scaffold to control osteogenic commitment of stem cells.

Chondroitin Sulfate-Based Biomineralizing Surface Hydrogels for Bone Tissue Engineering.

Chondroitin sulfate (CS) is the major component of glycosaminoglycan in connective tissue. In this study, we fabricated methacrylated PEGDA/CS-based hydrogels with varying CS concentration (0, 1, 5, and 10%) and investigated them as biomineralizing three-dimensional scaffolds for charged ion binding and depositions. Due to its negative charge from the sulfate group, CS exhibited an osteogenically favorable microenvironment by binding charged ions such as calcium and phosphate. Particularly, ion binding and distribution within negatively charged hydrogel was dependent on CS concentration. Furthermore, CS dependent biomineralizing microenvironment induced osteogenic differentiation of human tonsil-derived mesenchymal stem cells in vitro. Finally, when we transplanted PEGDA/CS-based hydrogel into a critical sized cranial defect model for 8 weeks, 10% CS hydrogel induced effective bone formation with highest bone mineral density. This PEGDA/CS-based biomineralizing hydrogel platform can be utilized for in situ bone formation in addition to being an investigational tool for in vivo bone mineralization and resorption mechanisms.

Autophagy induction in the skeletal myogenic differentiation of human tonsil-derived mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are capable of self-renewal and differentiation and are thus a valuable source for the replacement of diseased or damaged organs. Previously, we reported that the tonsils can be an excellent reservoir of MSCs for the regeneration of skeletal muscle (SKM) damage. However, the mechanisms involved in the differentiation from tonsil-derived MSCs (T-MSCs) to myocytes via myoblasts remain unclear. To clarify these mechanisms, we analyzed gene expression profiles of T-MSCs during differentiation into myocytes compared with human skeletal muscle cells (hSKMCs). Total RNA was extracted from T-MSCs, T-MSC-derived myoblasts and myocytes, and hSKMCs and was subjected to analysis using a microarray. Microarray analysis of the three phases of myogenic differentiation identified candidate genes associated with myogenic differentiation. The expression pattern of undifferentiated T-MSCs was distinguishable from the myogenic differentiated T-MSCs and hSKMCs. In particular, we selected FNBP1L, which among the upregulated genes is essential for antibacterial autophagy, since autophagy is related to SKM metabolism and myogenesis. T-MSCs differentiated toward myoblasts and skeletal myocytes sequentially, as evidenced by increased expression of autophagy-related markers (including Beclin-1, LC3B and Atg5) and decreased expression of Bcl-2. Furthermore, we reconfirmed that autophagy has an effect on the mechanism of skeletal myogenic differentiation derived from T-MSCs by treatment with 5-azacytidine and bafilomycin A1. These data suggest that the transcriptome of the T-MSC-derived myocytes is similar to that of hSKMCs, and that autophagy has an important role in the mechanism of myogenic differentiation of T-MSCs.

Role of Fibroblast Growth Factor-5 on the Proliferation of Human Tonsil-Derived Mesenchymal Stem Cells.

Human mesenchymal stem cells (MSCs) are a promising tool for therapeutic applications in cell-based therapy and regenerative medicine, and MSCs from the human palatine tonsils have recently been used as a new tissue source. However, the understanding of the proliferation and differentiation capacity of tonsil-derived MSCs (T-MSCs) is limited. In this study, we compared the proliferative potential of T-MSCs with those of bone marrow MSCs (BM-MSCs) and adipose tissue-derived MSCs (A-MSCs). Additionally, we investigated the underlying mechanism of T-MSC function. We showed that T-MSCs proliferated faster than A-MSCs and BM-MSCs in methylthiazolyl diphenyl-tetrazolium (MTT) assays, cell count assays, and cell cycle distribution analyses. DNA microarray and real-time PCR analyses revealed that the expression of fibroblast growth factor-5 (FGF5) was significantly elevated in T-MSCs compared with those in A-MSCs and BM-MSCs. Cell growth curves showed a difference in cell growth between untreated cells and siFGF5-treated T-MSCs. The administration of recombinant human FGF5 (rhFGF5) to the cells transfected with siFGF5 led to a significant increase in the proliferation rates. The administration of rhFGF5 to T-MSCs led to an increase in the levels of phosphorylated ERK1/2. However, treatment with siFGF5 resulted in an overall decrease in the level of phosphorylated ERK1/2. The osteogenic differentiation of T-MSCs was reduced following siFGF5 transfection, and it recovered to near-normal levels when rhFGF5 was added. These findings indicate that T-MSCs show significantly higher proliferative potential compared with those of BM-MSCs and A-MSCs. FGF5 facilitates cell proliferation through ERK1/2 activation, and it influences the osteogenic differentiation of T-MSCs.