Valproic Acid-Induced CCN1 Promotes Osteogenic Differentiation by Increasing CCN1 Protein Stability through HDAC1 Inhibition in Tonsil-Derived Mesenchymal Stem Cells.

Our previous study found that the level of CCN1 increases as osteogenic differentiation progresses in tonsil-derived mesenchymal stem cells (TMSCs). This study investigated how CCN1 is regulated through HDAC inhibition in TMSCs and their relationship with osteogenesis. Valproic acid (VPA) (1-5 mM), a well-known histone deacetylase (HDAC) inhibitor, strongly inhibited TMSC proliferation without altering MSC-specific surface markers, CD14, 34, 45, 73, 90 and 105. However, CD146 expression increased at 5 mM VPA. VPA increased osteogenic differentiation of TMSCs but decreased adipogenesis and chondrogenesis, as evidenced by the cell-specific staining of differentiation. The former was validated by the increased osteocalcin (OCN). The changes in CCN1 by VPA was biphasic; it increased until 48 h and decreased thereafter. Knockdown of CCN1 by using siRNA inhibited the osteogenic effect of VPA. VPA had no effect on CCN1 mRNA expression, but inhibition of protein synthesis by cycloheximide showed that VPA slowed down the CCN1 protein degradation. Moreover, overexpression of HDAC1 completely inhibited VPA-induced CCN1. Our results indicate that VPA inhibits the HDAC1, inducing CCN1 protein stability rather than gene expression, thereby promoting osteogenic differentiation of TMSCs. These findings present the noble implication of VPA as an inhibitor of HDAC1 to facilitate CCN1-induced osteogenic differentiation of MSCs.

Immunomodulatory Effect of Epidermal Growth Factor Secreted by Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells on Atopic Dermatitis.

Background and Objectives: Human mesenchymal stem cells (MSCs) are emerging as a treatment for atopic dermatitis (AD), a chronic inflammatory skin disorder that affects a large number of people across the world. Treatment of AD using human umbilical cord blood-derived MSCs (hUCB-MSCs) has recently been studied. However, the mechanism underlying their effect needs to be studied continuously. Thus, the objective of this study was to investigate the immunomodulatory effect of epidermal growth factor (EGF) secreted by hUCB-MSCs on AD. Methods and Results: To explore the mechanism involved in the therapeutic effect of MSCs for AD, a secretome array was performed using culture medium of hUCB-MSCs. Among the list of genes common for epithelium development and skin diseases, we focused on the function of EGF. To elucidate the effect of EGF secreted by hUCB-MSCs, EGF was downregulated in hUCB-MSCs using EGF-targeting small interfering RNA. These cells were then co-cultured with keratinocytes, Th2 cells, and mast cells. Depletion of EGF disrupted immunomodulatory effects of hUCB-MSCs on these AD-related inflammatory cells. In a Dermatophagoides farinae-induced AD mouse model, subcutaneous injection of hUCB-MSCs ameliorated gross scoring, histopathologic damage, and mast cell infiltration. It also significantly reduced levels of inflammatory cytokines including interleukin (IL)-4, tumor necrosis factor (TNF)-α, thymus and activation-regulated chemokine (TARC), and IL-22, as well as IgE levels. These therapeutic effects were significantly attenuated at all evaluation points in mice injected with EGF-depleted hUCB-MSCs. Conclusions: EGF secreted by hUCB-MSCs can improve AD by regulating inflammatory responses of keratinocytes, Th2 cells, and mast cells.

TGF-ß secreted by human umbilical cord blood-derived mesenchymal stem cells ameliorates atopic dermatitis by inhibiting secretion of TNF-α and IgE.

Human mesenchymal stem cells (MSCs) are promising therapeutics for autoimmune diseases due to their immunomodulatory effects. In particular, human umbilical cord blood-derived MSCs (hUCB-MSCs) have a prominent therapeutic effect on atopic dermatitis (AD). However, the underlying mechanism is unclear. This study investigated the role of transforming growth factor-beta (TGF-ß) in the therapeutic effect of hUCB-MSCs on AD. Small interfering RNA (siRNA)-mediated depletion of TGF-ß disrupted the therapeutic effect of hUCB-MSCs in a mouse model of AD by attenuating the beneficial changes in histopathology, mast cell infiltration, tumor necrosis factor-alpha (TNF-α) expression, and the serum IgE level. To confirm that hUCB-MSCs regulate secretion of TNF-α, we investigated whether they inhibit TNF-α secretion by activated LAD2 cells. Coculture with hUCB-MSCs significantly inhibited secretion of TNF-α by LAD2 cells. However, this effect was abolished by siRNA-mediated depletion of TGF-ß in hUCB-MSCs. TNF-α expression in activated LAD2 cells was regulated by the extracellular signal-related kinase signaling pathway and was suppressed by TGF-ß secreted from hUCB-MSCs. In addition, TGF-ß secreted by hUCB-MSCs inhibited maturation of B cells. Taken together, our findings suggest that TGF-ß plays a key role in the therapeutic effect of hUCB-MSCs on AD by regulating TNF-α in mast cells and maturation of B cells.

Optimization of Microenvironments Inducing Differentiation of Tonsil-Derived Mesenchymal Stem Cells into Endothelial Cell-Like Cells.

Background: Stem cell engineering is appealing consideration for regenerating damaged endothelial cells (ECs) because stem cells can differentiate into EC-like cells. In this study, we demonstrate that tonsil-derived mesenchymal stem cells (TMSCs) can differentiate into EC-like cells under optimal physiochemical microenvironments. Methods: TMSCs were preconditioned with Dulbecco's Modified Eagle Medium (DMEM) or EC growth medium (EGM) for 4 days and then replating them on Matrigel to observe the formation of a capillary-like network under light microscope. Microarray, quantitative real time polymerase chain reaction, Western blotting and immunofluorescence analyses were used to evaluate the expression of gene and protein of EC-related markers. Results: Preconditioning TMSCs in EGM for 4 days and then replating them on Matrigel induced the formation of a capillary-like network in 3 h, but TMSCs preconditioned with DMEM did not form such a network. Genome analyses confirmed that EGM preconditioning significantly affected the expression of genes related to angiogenesis, blood vessel morphogenesis and development, and vascular development. Western blot analyses revealed that EGM preconditioning with gelatin coating induced the expression of endothelial nitric oxide synthase (eNOS), a mature EC-specific marker, as well as phosphorylated Akt at serine 473, a signaling molecule related to eNOS activation. Gelatin-coating during EGM preconditioning further enhanced the stability of the capillary-like network, and also resulted in the network more closely resembled to those observed in human umbilical vein endothelial cells. Conclusion: This study suggests that under specific conditions, i.e., EGM preconditioning with gelatin coating for 4 days followed by Matrigel, TMSCs could be a source of generating endothelial cells for treating vascular dysfunction.

Preconditioning with interleukin-1 beta and interferon-gamma enhances the efficacy of human umbilical cord blood-derived mesenchymal stem cells-based therapy via enhancing prostaglandin E2 secretion and indoleamine 2,3-dioxygenase activity in dextran sulfate sodium-induced colitis.

Preconditioning with inflammatory cytokines has improved mesenchymal stem cells characteristics, including differentiation and immunomodulating functions. In this study, we developed a preconditioning combination strategy using interleukin-1beta (IL-1ß) and interferon-gamma (IFN-γ) to enhance the immuneregulatory ability of human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs). Our results showed that hUCB-MSCs preconditioned with IL-1ß and IFN-γ (primed hUCB-MSCs) created a statistically significant decrease in peripheral blood mononuclear cell proliferation, indicating that their immunosuppressive ability was increased. The secretion of PGE2, cyclooxygenase 2 mRNA expression, and indoleamine 2,3-dioxygenase (IDO) mRNA expression in primed hUCB-MSCs was significantly higher than those in the untreated hUCB-MSCs or the IL-1ß or IFN-γ only treated hUCB-MSCs. When inhibitors of IDO and PGE2 were treated, peripheral blood mononuclear cell proliferation, which is inhibited by primed hUCB-MSCs, was recovered. We found that Th1 T cell differentiation was also inhibited by PGE2 and IDO in the primed hUCB-MSCs, and Tregs differentiation was increased by PGE2 and IDO in the primed hUCB-MSCs. Furthermore, the primed hUCB-MSCs as well as supernatants increase CD4+ T cells migration. We demonstrated the therapeutic effects of primed hUCB-MSCs in dextran sulfate sodium-induced colitis model. In conclusion, we have demonstrated that primed hUCB-MSCs simultaneously enhance PGE2 and IDO and greatly improve the immunoregulatory capacity of MSCs, and we have developed an optimal condition for pretreatment of MSCs for the treatment of immune diseases. Our results raise the possibility that the combination of PGE2 and IDO could be therapeutic mediators for controlling immunosuppression of MSCs.

Therapeutic effect of long-interval repeated intravenous administration of human umbilical cord blood-derived mesenchymal stem cells in DBA/1 mice with collagen-induced arthritis.

Rheumatoid arthritis (RA) is a common inflammatory chronic disease. It has been reported that mesenchymal stem cells (MSCs) have the effect of immune suppression in collagen-induced arthritis (CIA) mice model. However, the in vivo therapeutic effect from the long-interval repeated intravenous administration of human umbilical cord blood-derived (hUCB)-MSCs had not been investigated in CIA mice model. This study was undertaken to investigate the effects of long-interval repeated intravenous administration of hUCB-MSCs at different doses in CIA mice model. Mice were intravenously injected with three different doses of hUCB-MSCs once every 2 weeks for three times. RA severity was assessed by clinical joint score and histologic analysis including hematoxylin and eosin staining, safranin-O staining, and toluidine blue staining. We used real-time polymerase chain reaction and flow cytometry to quantify differences in inflammatory cytokines and Tregs. Mice treated with hUCB-MSCs showed significant improvement in clinical joint score. Histologic analysis revealed that hUCB-MSCs definitely reduced joint inflammation, cartilage damage, and formation of pannus in multimedium and multihigh groups. These hUCB-MSCs also significantly decreased IL-1 beta protein levels in multimedium and multihigh groups and IL-6 protein levels in all hUCB-MSCs-treated groups. Furthermore, mRNA levels of IL-1 beta and IL-6 were decreased significantly in all hUCB-MSCs-treated groups, whereas the expression of anti-inflammatory cytokine IL-10 was increased in the multihigh group. Tregs known as suppressor T cells were also significantly increased in the multihigh group. Our findings suggest that long-interval repeated intravenous administration of hUCB-MSCs has therapeutic effects by improving symptoms of RA in CIA mice model in a dose-dependent manner.

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

BACKGROUND/AIMS: 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. METHODS: 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. RESULTS: 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 Ca²âº 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. CONCLUSION: 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 Ca²âº-dependent PP2B.

Association of serum mineral parameters with mortality in hemodialysis patients: Data from the Korean end-stage renal disease registry.

BACKGROUND: We investigated the associations between mineral metabolism parameters and mortality to identify optimal targets in Korean hemodialysis patients. METHODS: Among hemodialysis patients registered in the end-stage renal disease registry of the Korean Society of Nephrology between March 2012 and June 2017, those with serum calcium, phosphorus, and intact parathyroid hormone (iPTH) measured at enrollment were included. Association of serum levels of calcium, phosphorus, and iPTH with all-cause mortality was analyzed. RESULTS: Among 21,433 enrolled patients, 3,135 (14.6%) died during 24.8 ± 14.5 months of follow-up. After multivariable adjustment, patients in the first quintile of corrected calcium were associated with lower mortality (hazard ratio [HR], 0.84; 95% confidence interval [95% CI], 0.71-0.99; P = 0.003), while those in the fifth quintile were associated with higher mortality (HR, 1.39; 95% CI, 1.20-1.61; P < 0.001) compared with those in the third quintile. For phosphorus, only the lowest quintile was significantly associated with increased mortality (HR, 1.24; 95% CI, 1.08-1.43; P = 0.003). The lowest (HR, 1.18; 95% CI, 1.02-1.36; P = 0.026) and highest quintiles of iPTH (HR, 1.24; 95% CI, 1.05-1.46; P = 0.013) were associated with increased mortality. For target counts achieved according to the Kidney Disease Outcomes Quality Initiative guideline, patients who did not achieve any mineral parameter targets hadhigher mortality than those who achieved all three targets (HR, 1.37; 95% CI, 1.12-1.67; P = 0.003). CONCLUSION: In Korean hemodialysis patients, high serum calcium, low phosphorus, and high and low iPTH levels were associated with increased all-cause mortality.

HL156A, a novel pharmacological agent with potent adenosine-monophosphate-activated protein kinase (AMPK) activator activity ameliorates renal fibrosis in a rat unilateral ureteral obstruction model.

BACKGROUND: Renal fibrosis is characterized by excessive production and deposition of extracellular matrix (ECM), which leads to progressive renal failure. Adenosine-monophosphate-activated protein kinase (AMPK) is a highly conserved kinase that plays a key role in Smad-3 signaling. Here, we examined the effect of a novel AMPK activator, HL156A, on the inhibition of renal fibrosis in in vivo and in vitro models. METHODS: Unilateral ureteral obstruction (UUO) was induced in male Wistar rats. Rats with UUO were administered HL156A (20mg/kg/day), and then the kidneys were harvested 10 days after ligation for further analysis. RESULTS: In the rat UUO model, HL156A attenuated ECM protein deposition. After HL156A treatment, expressions of TGF-ß1, p-Smad3, α-SMA, fibronectin, and type IV collagen were suppressed, and E-cadherin expression was up-regulated. In the in vitro experiment, NRK52E cells were treated with HL156A before TGF-ß1 stimulation. The inhibitory effects of HL156A upon the signaling pathways and markers of the epithelial-to-mesenchymal transition (EMT) were analyzed. In TGF-ß1-treated NRK-52E cells, HL156A co-treatment inhibited the TGF-ß1-induced Smad3 signaling pathway and EMT markers. CONCLUSION: Taken together, the above findings suggest that HL156A, a novel AMPK activator, ameliorates renal fibrosis in vivo and in vitro.

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

BACKGROUND AND AIMS: Osteoporosis, 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. METHODS: We 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. RESULTS: Osteogenic 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. CONCLUSION: Our 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.

Esophageal Artery Pseudoaneurysm and Takayasu Arteritis in a Patient with Autosomal Dominant Polycystic Kidney Disease.

A 47-year-old female previously diagnosed with ADPKD visited the hospital due to sudden pain in her upper abdomen and back. Esophagogastroduodenoscopy, contrast-enhanced abdominal computed tomography (CT), and CT angiography identified an esophageal artery pseudoaneurysm and hematoma in the esophagus. Urgent angiography and embolization were performed. After the procedure, CT angiography and positron emission tomography were performed due to differences in blood pressure between the arms. The patient was also found to have Takayasu arteritis and subsequently received outpatient follow-up care. The possible mechanisms that cause vascular abnormalities in ADPKD patients include damaged vascular integrity due to abnormal polycystin expression caused by PKD mutations and connective tissue abnormalities. Further research is needed to confirm these mechanisms, and ADPKD patients should be assessed for vascular abnormalities.

Therapeutic potential of tonsil-derived mesenchymal stem cells in dextran sulfate sodium-induced experimental murine colitis.

The therapeutic potential of tonsil-derived mesenchymal stem cells (TMSC) prepared from human tonsillar tissue has been studied in animal models for several diseases such as hepatic injury, hypoparathyroidism, diabetes and muscle dystrophy. In this study, we examined the therapeutic effects of TMSC in a dextran sulfate sodium (DSS)-induced colitis model. TMSC were injected in DSS-induced colitis mice via intraperitoneal injection twice (TMSC[x2]) or four times (TMSC[x4]). Control mice were injected with either phosphate-buffered saline or human embryonic kidney 293 cells. Body weight, stool condition and disease activity index (DAI) were examined daily. Colon length, histologic grading, and mRNA expression of pro-inflammatory cytokines, interleukin 1ß (IL-1ß), IL-6, IL-17 and tumor necrosis factor α, and anti-inflammatory cytokines, IL-10, IL-11 and IL-13, were also measured. Our results showed a significant improvement in survival rates and body weight gain in colitis mice injected with TMSC[x2] or TMSC[x4]. Injection with TMSC also significantly decreased DAI scores throughout the experimental period; at the end of experiment, almost complete reversal of DAI scores to normal was found in colitis mice treated with TMSC[x4]. Colon length was also significantly recovered in colitis mice treated with TMSC[x4]. However, histopathological alterations induced by DSS treatment were not apparently improved by injection with TMSC. Finally, treatment with TMSC[x4] significantly reversed the mRNA levels of IL-1ß and IL-6, although expression of all pro-inflammatory cytokines tested was induced in colitis mice. Under our experimental conditions, however, no apparent alterations in the mRNA levels of all the anti-inflammatory cytokines tested were found. In conclusion, our findings demonstrate that multiple injections with TMSC produced a therapeutic effect in a mouse model of DSS-induced colitis.

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.

Tonsil-Derived Mesenchymal Stem Cells Differentiate into a Schwann Cell Phenotype and Promote Peripheral Nerve Regeneration.

Schwann cells (SCs), which produce neurotropic factors and adhesive molecules, have been reported previously to contribute to structural support and guidance during axonal regeneration; therefore, they are potentially a crucial target in the restoration of injured nervous tissues. Autologous SC transplantation has been performed and has shown promising clinical results for treating nerve injuries and donor site morbidity, and insufficient production of the cells have been considered as a major issue. Here, we performed differentiation of tonsil-derived mesenchymal stem cells (T-MSCs) into SC-like cells (T-MSC-SCs), to evaluate T-MSC-SCs as an alternative to SCs. Using SC markers such as CAD19, GFAP, MBP, NGFR, S100B, and KROX20 during quantitative real-time PCR we detected the upregulation of NGFR, S100B, and KROX20 and the downregulation of CAD19 and MBP at the fully differentiated stage. Furthermore, we found myelination of axons when differentiated SCs were cocultured with mouse dorsal root ganglion neurons. The application of T-MSC-SCs to a mouse model of sciatic nerve injury produced marked improvements in gait and promoted regeneration of damaged nerves. Thus, the transplantation of human T-MSCs might be suitable for assisting in peripheral nerve regeneration.

Myogenic differentiation potential of human tonsil-derived mesenchymal stem cells and their potential for use to promote skeletal muscle regeneration.

Stem cells are regarded as an important source of cells which may be used to promote the regeneration of skeletal muscle (SKM) which has been damaged due to defects in the organization of muscle tissue caused by congenital diseases, trauma or tumor removal. In particular, mesenchymal stem cells (MSCs), which require less invasive harvesting techniques, represent a valuable source of cells for stem cell therapy. In the present study, we demonstrated that human tonsil-derived MSCs (T-MSCs) may differentiate into myogenic cells in vitro and that the transplantation of myoblasts and myocytes generated from human T-MSCs mediates the recovery of muscle function in vivo. In order to induce myogenic differentiation, the T-MSC-derived spheres were cultured in Dulbecco's modified Eagle's medium/nutrient mixture F-12 (DMEM/F­12) supplemented with 1 ng/ml transforming growth factor-ß, non-essential amino acids and insulin­transferrin-selenium for 4 days followed by culture in myogenic induction medium [low-glucose DMEM containing 2% fetal bovine serum (FBS) and 10 ng/ml insulin­like growth factor 1 (IGF1)] for 14 days. The T-MSCs sequentially differentiated into myoblasts and skeletal myocytes, as evidenced by the increased expression of skeletal myogenesis-related markers [including α-actinin, troponin I type 1 (TNNI1) and myogenin] and the formation of myotubes in vitro. The in situ transplantation of T-MSCs into mice with a partial myectomy of the right gastrocnemius muscle enhanced muscle function, as demonstrated by gait assessment (footprint analysis), and restored the shape of SKM without forming teratomas. Thus, T-MSCs may differentiate into myogenic cells and effectively regenerate SKM following injury. These results demonstrate the therapeutic potential of T-MSCs to promote SKM regeneration following injury.

Expression of tenocyte lineage-related factors from tonsil-derived mesenchymal stem cells.

Human palatine tonsil-derived mesenchymal stem cells (TMSCs) are known to be a new source of progenitor cells. Using waste tissue after tonsillectomy as a cell provider can be the biggest benefit of TMSCs, compared with other stem cells. The purpose of this study was to investigate tenogenic differentiation of TMSCs and to access the differential effects of transforming growth factor beta 3 (TGF-ß3) on the tenogenesis of TMSCs. Human tonsil was obtained after tonsillectomy. Using a cytometric analysis, we were able to find that the TMSCs had typical mesenchymal stem cell markers: positive for CD73, CD90, and CD105, and negative for CD14, CD34, and CD45. Using TGF-ß3, the expressions of tenocyte-specific genes and proteins, such as collagen type 1 (COL1), tenomodulin (TNMD), and scleraxis (SCX), were measured by a quantitative polymerase chain reaction (PCR), immunofluorescence staining, immunohistochemistry and Western blot analyses. Quantitative PCR assay showed that TGF-ß3 significantly increased the expressions of tenocyte lineage marker genes, including COL1, TNMD, and SCX, at a 3-day treatment, compared with control. However, these increases were not found at long-term exposures (7 or 10 days), except that TNMD expression was maintained at 50 ng/mL at a 7-day exposure to TGF-ß3. Like genes, the protein expression levels of COL1, TNMD, and SCX were also induced in TGF-ß3-treated TMSCs in a 3-day treatment, which were maintained for 10 days, as evidenced by immunofluorescence staining, immunohistochemistry and Western blot analyses. This study demonstrated that TMSCs in tenogenic stimulation with TGF-ß3 have a high tenogenic differentiation potential.

Differentiated tonsil-derived mesenchymal stem cells embedded in Matrigel restore parathyroid cell functions in rats with parathyroidectomy.

Parathyroid cells release parathyroid hormone (PTH), which controls calcium homeostasis. Loss of parathyroid cells results in hypoparathyroidism and consequent low-turnover bone disease. Here, we investigated whether our recently-established human tonsil-derived mesenchymal stem cells (TMSC) restore in vivo parathyroid cell function in rats with parathyroidectomy (PTX). Compared with undifferentiated control TMSC, TMSC differentiated with activin A and soluble sonic hedgehog induced a significant release of PTH as early as day 7, with increased PTH release occurring in response to lower calcium levels and vice versa. Released PTH increased osteocalcin expression and alizarin red S staining in preosteoblastic cells, indicating its functional activity. PTX rats fed calcium-free diet only survived for ∼10 days. Subcutaneous injection with TMSC alone did not increase their survival rates, regardless of differentiation. However, survival rates increased for up to 28 days in response to TMSC embedded in Matrigel (TMSC-MA), showing 40% and 80% in control and differentiated TMSC-MA, respectively. When compared with continuous increases by control TMSC-MA, stable levels of secreted PTH and serum ionized calcium were found in PTX rats with differentiated TMSC-MA. This is the first report that differentiated TMSC resemble parathyroid cells and, if embedded in Matrigel, restore in vivo parathyroid function.

Massive Hemoptysis due to Endotracheal Hemangioma: A Case Report and Literature Review.

Tracheal hemangioma is a rare benign vascular tumor in adults. We reported a case of massive hemoptysis caused by a cavernous hemangioma in a 75-year-old man. This is the first report, to our knowledge, of a tracheal cavernous hemangioma that presented with massive hemoptysis. The lesion was removed with a CO2 laser under rigid laryngoscopy. Endovascular tumors, such as tracheobronchial hemangiomas, should be considered a diagnostic option in cases of massive hemoptysis without a significant underlying lung lesion.

Tonsil-derived mesenchymal stem cells ameliorate CCl4-induced liver fibrosis in mice via autophagy activation.

Liver transplantation is the treatment of choice for chronic liver failure, although it is complicated by donor shortage, surgery-related complications, and immunological rejection. Cell transplantation is an alternative, minimally invasive treatment option with potentially fewer complications. We used human palatine tonsil as a novel source of mesenchymal stem cells (T-MSCs) and examined their ability to differentiate into hepatocyte-like cells in vivo and in vitro. Carbon tetrachloride (CCl4) mouse model was used to investigate the ability of T-MSCs to home to the site of liver injury. T-MSCs were only detected in the damaged liver, suggesting that they are disease-responsive. Differentiation of T-MSCs into hepatocyte-like cells was confirmed in vitro as determined by expression of hepatocyte markers. Next, we showed resolution of liver fibrosis by T-MSCs via reduction of TGF-ß expression and collagen deposition in the liver. We hypothesized that autophagy activation was a possible mechanism for T-MSC-mediated liver recovery. In this report, we demonstrate for the first time that T-MSCs can differentiate into hepatocyte-like cells and ameliorate liver fibrosis via autophagy activation and down-regulation of TGF-ß. These findings suggest that T-MSCs could be used as a novel source for stem cell therapy targeting liver diseases.

CCN1 secreted by tonsil-derived mesenchymal stem cells promotes endothelial cell angiogenesis via integrin αv ß3 and AMPK.

CCN1 is highly expressed in cancer cells and has been identified in the secretome of bone marrow-derived mesenchymal stem cells (BM-MSC). Although secreted CCN1 is known to promote angiogenesis, its underlying mechanism remains unclear. Here, we examined whether our recently-established tonsil-derived MSC (T-MSC) secrete CCN1 and, if any, how CCN1 promotes the angiogenesis of human umbilical vein endothelial cells (HUVEC). Compared with untreated control T-MSC, a higher level of CCN1 was secreted by T-MSC treated with activin A and sonic hedgehog, drugs known to induce endodermal differentiation. Expectedly, conditioned medium collected from differentiated T-MSC (DCM) significantly increased HUVEC migration and tube formation compared with that from control T-MSC (CCM), and these stimulatory effects were reversed by neutralization with anti-CCN1 antibody. Treatment with recombinant human CCN1 (rh-CCN1) alone also mimicked the stimulatory effects of DCM. Furthermore, treatment with either DCM or rh-CCN1 increased the phosphorylation of AMP kinase (AMPK), and ectopic expression of siRNA of the AMPK gene inhibited all observed effects of both DCM and rh-CCN1. However, no alteration of intracellular ATP levels or phosphorylation of LKB1, a well-known upstream factor of AMPK activation, was observed under our conditions. Finally, the neutralization of integrin α(v) ß(3) with anti-integrin α(v) ß(3) antibody almost completely reversed the effects of CCN1 on AMPK phosphorylation, and EC migration and tube formation. Taken together, we demonstrated that T-MSC increase the secretion of CCN1 in response to endodermal differentiation and that integrin α(v) ß(3) and AMPK mediate CCN1-induced EC migration and tube formation independent of intracellular ATP levels alteration.