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Bone marrow (BM) transplantation (BMT) represents a curative treatment for various hematological disorders. Prior to BMT, a large amount of the relevant anticancer drug needed to be administered to eliminate cancer cells. However, during this pre‑BMT cytotoxic conditioning regimen, hematopoietic stem cells in the BM and thymic epithelial cells were also destroyed. The T cell receptor (TCR) recognizes diverse pathogen, tumor and environmental antigens, and confers immunological memory and self‑tolerance. Delayed thymus reconstitution following pre‑BMT cytotoxic conditioning impedes de novo thymopoiesis and limits T cell‑mediated immunity. Several cytokines, such as RANK ligand, interleukin (IL)‑7, IL‑22 and stem cell factor, were recently reported to improve thymopoiesis and immune function following BMT. In the present study, it was found that the co‑transplantation of tonsil‑derived mesenchymal stromal cells (T‑MSCs) with BM‑derived cells (BMCs) accelerated the recovery of involuted thymuses in mice following partial pre‑BMT conditioning with busulfan‑cyclophosphamide treatment, possibly by inducing FMS‑like tyrosine kinase 3 ligand (FLT3L) and fibroblast growth factor 7 (FGF7) production in T‑MSCs. The co‑transplantation of T‑MSCs with BMCs also replenished the CD3+ cell population by inhibiting thymocyte apoptosis following pre‑BMT cytotoxic conditioning. Furthermore, T‑MSC co‑transplantation improved the recovery of the TCR repertoire and led to increased thymus‑generated T cell diversity.

Renal tumors account for approximately 7% of all childhood cancers. These include Wilms tumor (WT), clear cell sarcoma of the kidney (CCSK), malignant rhabdoid tumor (MRTK), renal cell carcinoma (RCC), congenital mesoblastic nephroma (CMN) and other rare tumors. We investigated the epidemiology of pediatric renal tumors in Korea.From January 2001 to December 2015, data of pediatric patients (0-18 years) newly-diagnosed with renal tumors at 26 hospitals were retrospectively analyzed.Among 439 patients (male, 240), the most common tumor was WT (n=342, 77.9%), followed by RCC (n=36, 8.2%), CCSK (n=24, 5.5%), MRTK (n=16, 3.6%), CMN (n=12, 2.7%), and others (n=9, 2.1%). Median age at diagnosis was 27.1 months (range 0-225.5) and median follow-up duration was 88.5 months (range 0-211.6). Overall, 32 patients died, of whom 17, 11, 1, and 3 died of relapse, progressive disease, second malignant neoplasm, and treatment-related mortality. Five-year overall survival and event free survival were 97.2% and 84.8% in WT, 90.6% and 82.1% in RCC, 81.1% and 63.6% in CCSK, 60.3% and 56.2% in MRTK, and 100% and 91.7% in CMN, respectively (p0.001).The pediatric renal tumor types in Korea are similar to those previously reported in other countries. WT accounted for a large proportion and survival was excellent. Non-Wilms renal tumors included a variety of tumors and showed inferior outcome, especially MRTK. Further efforts are necessary to optimize the treatment and analyze the genetic characteristics of pediatric renal tumors in Korea.

Reactive oxygen species (ROS) generated by neutrophils provide a frontline defence against invading pathogens. We investigated the supportive effect of tonsil-derived mesenchymal stem cells (TMSCs) on ROS generation from neutrophils using promyelocytic HL-60 cells. Methods: Differentiated HL-60 (dHL-60) cells were cocultured with TMSCs isolated from 25 independent donors, and ROS generation in dHL-60 cells was measured using luminescence. RNA sequencing and real-time PCR were performed to identify the candidate genes of TMSCs involved in augmenting the oxidative burst of dHL-60 cells. Transcriptome analysis of TMSCs derived from 25 independent donors revealed high levels of procollagen C-endopeptidase enhancer 2 (PCOLCE2) in TMSCs, which were highly effective in potentiating ROS generation in dHL-60 cells. In addition, PCOLCE2 knockdown in TMSCs abrogated TMSC-induced enhancement of ROS production in dHL-60 cells, indicating that TMSCs increased the oxidative burst in dHL-60 cells via PCOLCE2. Furthermore, the direct addition of recombinant PCOLCE2 protein increased ROS production in dHL-60 cells. These results suggest that PCOLCE2 secreted by TMSCs may be used as a therapeutic candidate to enhance host defences by increasing neutrophil oxidative bursts. PCOLCE2 levels in TMSCs could be used as a marker to select TMSCs exhibiting high efficacy for enhancing neutrophil oxidative bursts.

BACKGROUND: Mast cells are immune sentinels in the skin that respond to a wide range of pathological and environmental stimuli; they owe their function to the expression of Toll-like receptors (TLRs). We previously found that tonsil-derived mesenchymal stem cells (T-MSCs) were able to effectively attenuate TLR7-mediated skin inflammation in mice, which was accompanied by an increase in mast cell number. The present study investigated whether T-MSC extracellular vesicles, such as exosomes, are able to regulate mast cell activation in response to TLR7 stimulation. METHODS: The HMC-1 human mast cell line was treated with a TLR7 agonist in the presence or absence of T-MSC exosomes, and the levels of expressed inflammatory cytokines were assessed. Additionally, mice were repeatedly injected with a TLR7 agonist with or without interval treatments with T-MSC exosomes and assessed dermal distribution of mast cells and related immune cells. RESULTS: We showed that T-MSC exosomes containing microRNAs that target inflammatory cytokines significantly reduced the expression of inflammatory cytokines in TLR7 agonist-treated HMC-1 cells. In addition, T-MSC exosomes inhibited the increase in the number of both dermal mast cells and CD14-positive cells in TLR7 agonist-treated mice. CONCLUSION: Our data suggest that T-MSC exosomes have regulatory effects on mast cell activation under inflammatory conditions, including TLR7 stimulation.

Background Co-transplantation of bone marrow cells (BMCs) and mesenchymal stem cells (MSCs) is used as a strategy to improve the outcomes of bone marrow transplantation. Tonsil-derived MSCs (TMSCs) are a promising source of MSCs for co-transplantation. Previous studies have shown that TMSCs or conditioned media from TMSCs (TMSC-CM) enhance BMC engraftment. However, the factors in TMSCs that promote better engraftment have not yet been identified. Methods Mice were subjected to a myeloablative regimen of busulfan and cyclophosphamide, and the mRNA expression in the bone marrow was analyzed using an extracellular matrix (ECM) and adhesion molecule-targeted polymerase chain reaction (PCR) array. Nano-liquid chromatography with tandem mass spectrometry, real-time quantitative PCR, western blots, and enzyme-linked immunosorbent assays were used to compare the expression levels of metalloproteinase 3 (MMP3) in MSCs derived from various tissues, including the tonsils, bone marrow, adipose tissue, and umbilical cord. Recipient mice were conditioned with busulfan and cyclophosphamide, and BMCs, either as a sole population or with control or MMP3-knockdown TMSCs, were co-transplanted into these mice. The effects of TMSC-expressed MMP3 were investigated. Additionally, Enzchek collagenase and Transwell migration assays were used to confirm that the collagenase activity of TMSC-expressed MMP3 enhanced BMC migration. Results Mice subjected to the myeloablative regimen exhibited increased mRNA expression of collagen type IV alpha 1/2 (Col4a1 and Col4a2). Among the various extracellular matrix-modulating proteins secreted by TMSCs, MMP3 was expressed at higher levels in TMSCs than in other MSCs. Mice co-transplanted with BMCs and control TMSCs exhibited a higher survival rate, weight recovery, and bone marrow cellularity compared with mice co-transplanted with BMCs and MMP3-knockdown TMSCs. Control TMSC-CM possessed higher collagenase activity against collagen IV than MMP3-knockdown TMSC-CM. TMSC-CM also accelerated BMC migration by degrading collagen IV in vitro. Conclusions Collectively, these results indicate that TMSCs enhance BMC engraftment by the secretion of MMP3 for the modulation of the bone marrow extracellular matrix.

Located near the oropharynx, the tonsils are the primary mucosal immune organ. Tonsil tissue is a promising alternative source for the high-yield isolation of adult stem cells, and recent studies have reported the identification and isolation of tonsil-derived stem cells (T-SCs) from waste surgical tissue following tonsillectomies in relatively young donors (i.e., under 10 years old). As such, T-SCs offer several advantages, including superior proliferation and a shorter doubling time compared to bone marrow-derived mesenchymal stem cells (MSCs). T-SCs also exhibit multi-lineage differentiation, including mesodermal, endodermal (e.g., hepatocytes and parathyroid-like cells), and even ectodermal cells (e.g., Schwann cells). To this end, numbers of researchers have evaluated the practical use of T-SCs as an alternative source of autologous or allogenic MSCs. In this review, we summarize the details of T-SC isolation and identification and provide an overview of their application in cell therapy and regenerative medicine.

Since the discovery of stem cells and multipotency characteristics of mesenchymal stem cells (MSCs), there has been tremendous development in regenerative medicine. MSCs derived from bone marrow have been widely used in various research applications, yet there are limitations such as invasiveness of obtaining samples, low yield and proliferation rate, and questions regarding their practicality in clinical applications. Some have suggested that MSCs from other sources, specifically those derived from palatine tonsil tissues, that is, tonsil‐derived MSCs (TMSCs), could be considered as a new potential therapeutic tool in regenerative medicine due to their superior proliferation rate and differentiation capabilities with low immunogenicity and ease of obtaining. Several studies have determined that TMSCs have differentiation potential not only into the mesodermal lineage but also into the endodermal as well as ectodermal lineages, expanding their potential usage and placing them as an appealing option to consider for future studies in regenerative medicine. In this review, the differentiation capacities of TMSCs and their therapeutic competencies from past studies are addressed. stem cells 2019;37:1252–1260, Schematic illustration of lineage differentiation of tonsil‐derived mesenchymal stem cells.

BACKGROUND: The advantages of tonsil-derived mesenchymal stem cells (TMSCs) over other mesenchymal stem cells (MSCs) include higher proliferation rates, various differentiation potentials, efficient immune-modulating capacity, and ease of obtainment. Specifically, TMSCs have been shown to differentiate into the endodermal lineage. Estrogen deficiency is a major cause of postmenopausal osteoporosis and is associated with higher incidences of ischemic heart disease and cerebrovascular attacks during the postmenopausal period. Therefore, stem cell-derived, estrogen-secreting cells might be used for estrogen deficiency. METHODS: Here, we developed a novel method that utilizes retinoic acid, insulin-like growth factor-1, basic fibroblast growth factor, and dexamethasone to evaluate the differentiating potential of TMSCs into estrogen-secreting cells. The efficacy of the novel differentiating method for generation of estrogen-secreting cells was also evaluated with bone marrow- and adipose tissue-derived MSCs. RESULTS: Incubating TMSCs in differentiating media induced the gene expression of cytochrome P450 19A1 (CYP19A1), which plays a key role in estrogen biosynthesis, and increased 17β-estradiol secretion upon testosterone addition. Furthermore, CYP11A1, CYP17A1, and 3β-hydroxysteroid dehydrogenase type-1 gene expression levels were significantly increased in TMSCs. In bone marrow-derived and adipose tissue-derived MSCs, this differentiation method also induced the gene expression of CYP19A1, but not CYP17A1, suggesting TMSCs are a superior source for estrogen secretion. CONCLUSION: These results imply that TMSCs can differentiate into functional estrogen-secreting cells, thus providing a novel, alternative cell therapy for estrogen deficiency.

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&ndash, 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&ndash, 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.

The liver is an organ with remarkable regenerative capacity; however, once chronic fibrosis occurs, liver failure follows, with high mortality and morbidity rates. Continuous exposure to proinflammatory stimuli exaggerates the pathological process of liver failure; therefore, immune modulation is a potential strategy to treat liver fibrosis. Mesenchymal stem cells (MSCs) with tissue regenerative and immunomodulatory potential may support the development of therapeutics for liver fibrosis. Here, we induced hepatic injury in mice by injecting carbon tetrachloride (CCl4) and investigated the therapeutic potential of conditioned medium from tonsil-derived MSCs (T-MSC CM). In parallel, we used recombinant human IL-1Ra, which, as we have previously shown, is secreted exclusively from T-MSCs and resolves the fibrogenic activation of myoblasts. Hepatic inflammation and fibrosis were determined by histological analyses using H&E and Picro-Sirius Red staining. The results demonstrated that T-MSC CM treatment significantly reduced inflammation as well as fibrosis in the CCl4-injured mouse liver. IL-1Ra injection showed effects similar to T-MSC CM treatment, suggesting that T-MSC CM may exert anti-inflammatory and anti-fibrotic effects via the endogenous production of IL-1Ra. The expression of genes involved in fibrosis was evaluated, and the results showed significant induction of alpha-1 type I collagen, transforming growth factor beta, and tissue inhibitor of metalloproteases 1 upon CCl4 injection, whereas treatment with T-MSC CM or IL-1Ra downregulated their expression. Taken together, these data support the therapeutic potential of T-MSC CM and/or IL-1Ra for the alleviation of liver fibrosis, as well as in treating diseases involving organ fibrosis.

// Kyung-Ah Cho 1 , Minhwa Park 1 , Yu-Hee Kim 1 , Kyung-Ha Ryu 2 and So-Youn Woo 1 1 Department of Microbiology, College of Medicine, Ewha Womans University, Seoul, Republic of Korea 2 Department of Pediatrics, College of Medicine, Ewha Womans University, Seoul, Republic of Korea Correspondence to: So-Youn Woo, email: // Keywords : mesenchymal stem cells, osteoprotegerin, Th17 cells, osteoclasts, psoriasis, Immunology and Microbiology Section, Immune response, Immunity Received : February 20, 2017 Accepted : September 05, 2017 Published : September 28, 2017 Abstract Th17 cells play a critical role in several autoimmune diseases, including psoriasis and psoriatic arthritis (PsA). Psoriasis is a chronic inflammatory skin disease associated with systemic inflammation and comorbidities, such as PsA. PsA develops in nearly 70% of patients with psoriasis, and osteoclasts associated bone erosion is a hallmark of the disease. Thus far, the effect of Th17 cells on osteoclastogenesis via direct cell-to-cell interactions is less understood. In this study, we observed that Th17 cells directly promote osteoclast differentiation and maturation via expression of receptor activator of nuclear factor-κ β ligand (RANKL) in vitro . We investigated the impact of conditioned medium obtained from human palatine tonsil-derived mesenchymal stem cells (T-CM) on the interactions between osteoclasts and Th17 cells. T-CM effectively blunted the RANK-RANKL interaction between the osteoclast precursor cell line RAW 264.7 and Th17 cells via osteoprotegerin (OPG) activity. The frequency of tartrate-resistant acid phosphatase (TRAP)-positive cells in the bone marrow of an imiquimod (IMQ)-induced psoriasis mouse model was decreased following T-CM injection. Therefore, our data provide novel insight into the therapeutic potential of tonsil-derived mesenchymal stem cell-mediated therapy ( via OPG production) for the treatment of pathophysiologic processes induced by osteoclasts under chronic inflammatory conditions such as psoriasis.

Mesenchymal stem cells (MSCs) are often considered to be a good source for the development of regenerative medicine. Previously, we reported that tonsil‑derived MSC conditioned medium (T‑MSC CM) produces visceral fat reducing effects. As reduced visceral adiposity is closely associated with an increase in circulating adiponectin, the present study investigated the effects of T‑MSC CM on adiponectin production. T‑MSC CM was collected from previously isolated and characterized T‑MSCs and injected into senescence‑accelerated mouse prone 6 mice, which exhibit characteristics of aging and obesity. The results demonstrated a reduction in mouse weight and epididymal adipose tissue (eAT) mass following injection of T‑MSC CM. Significant increases in adiponectin expression in the eAT, and total and high molecular weight (HMW) adiponectin in the circulation were observed in the T‑MSC CM‑injected mice compared with control mice using reverse transcription‑quantitative polymerase chain reaction, western blot analysis and ELISA. In 3T3‑L1 adipocytes, T‑MSC CM treatment increased adiponectin secretion and multimerization, as detected using western blotting under non‑reducing and non‑heat‑denaturing conditions. Furthermore, glucose oxidase was used to induce oxidative stress in 3T3‑L1 adipocytes and it was observed that T‑MSC CM reduced reactive oxygen species production and the expression of certain oxidative stress markers. In addition, the results also demonstrated that the production of HMW adiponectin was increased, which indicates that T‑MSC CM may enhance adiponectin multimerization via amelioration of oxidative stress. Further studies are required to elucidate anti‑oxidant molecules secreted from T‑MSCs, and these results highlight the potential therapeutic relevance of T‑MSC CM for the treatment of obesity or obesity‑associated diseases.

It has been established that mesenchymal stem cells (MSCs) can have a suppressive effect on T cells, yet much remains unknown about the underlying mechanisms that support this effect. The T cell co-stimulatory pathway involving the programmed death-1 (PD-1) receptor and its ligand PD-L1 regulates T cell activation, tolerance, and subsequent immune-mediated tissue damage. In this study, human palatine tonsil-derived MSCs (T-MSCs) constitutively expressed PD-L1 and exhibited a suppressive activity that specifically targeted murine Th17 differentiation. Additionally, polyinosinic-polycytidylic acid (poly I:C), a Toll-like receptor 3 (TLR3) ligand, increased PD-L1 expression on T-MSCs. The elevated PD-L1 levels enhanced the suppressive functions of T-MSCs on Th17 differentiation. Therefore, pre-stimulation of T-MSCs with poly I:C may serve as an effective therapeutic priming step for modulating Th17-dominant immune responses.

Effector B cells are central contributors to the development of autoimmune disease by activating autoreactive T cells, producing pro-inflammatory cytokines and organizing ectopic lymphoid tissue. Conversely, IL-10-producing regulatory B (Breg) cells have pivotal roles in maintaining immunological tolerance and restraining excessive inflammation in autoinflammatory disease. Thus, regulating the equilibrium between antibody-producing effector B cells and Breg cells is critical for the treatment of autoimmune disease. In this study, we investigated the effect of human palatine tonsil-derived mesenchymal stem cells (T-MSCs) on estradiol (E2)-induced B-cell responses in vivo and in vitro. Transplantation of T-MSC into E2-treated mice alleviated B-cell-mediated immune responses and increased the population of IL-10-producing Breg cells. T-MSCs regulated the B-cell populations by producing Epstein–Barr virus (EBV)-induced 3 (EBI3), one of the two subunits of IL-35 that is the well-known inducer of Breg cells. We demonstrate a critical role of EBI3 (IL-35) in vitro by depleting EBI3 in T-MSCs and by adding exogenous IL-35 to the culture system. Taken together, our data suggest that IL-35-secreting MSCs may become an attractive therapeutic to treat B-cell-mediated autoimmune diseases via expanding Breg cells.

The application of mesenchymal stem cells (MSCs) for treating bone-related diseases shows promising outcomes in preclinical studies. However, cells that are isolated and defined as MSCs comprise a heterogeneous population of progenitors. This heterogeneity can produce variations in the performance of MSCs, especially in applications that require differentiation potential in vivo, such as the treatment of osteoporosis. Here, we aimed to identify genetic markers in tonsil-derived MSCs (T-MSCs) that can predict osteogenic potential. Using a single-cell cloning method, we isolated and established several lines of nondifferentiating (ND) or osteoblast-prone (OP) clones. Next, we performed transcriptome sequencing of three ND and three OP clones that maintained the characteristics of MSCs and determined the top six genes that were upregulated in OP clones. Upregulation of WNT16 and DCLK1 expression was confirmed by real-time quantitative PCR, but only WNT16 expression was correlated with the osteogenic differentiation of T-MSCs from 10 different donors. Collectively, our findings suggest that WNT16 is a putative genetic marker that predicts the osteogenic potential of T-MSCs. Thus, examination of WNT16 expression as a selection criterion prior to the clinical application of MSCs may enhance the therapeutic efficacy of stem cell therapy for bone-related complications, including osteoporosis.

Type 2 diabetes mellitus (T2DM) is a prevalent chronic metabolic disorder accompanied by high blood glucose, insulin resistance, and relative insulin deficiency. Endoplasmic reticulum (ER) stress induced by high glucose and free fatty acids has been suggested as one of the main causes of &beta, cell dysfunction and death in T2DM. Stem cell-derived insulin-secreting cells were recently suggested as a novel therapy for diabetes. In the present study, we demonstrate the therapeutic potential of tonsil-derived mesenchymal stem cells (TMSCs) to treat high-fat diet (HFD)-induced T2DM. To explore whether TMSC administration can alleviate T2DM, TMSCs were intraperitoneally injected in HFD-induced T2DM mice once every 2 weeks. TMSC injection markedly improved glucose tolerance and glucose-stimulated insulin secretion and prevented HFD-induced pancreatic &beta, cell hypertrophy and cell death. In addition, TMSC injection relieved the ER-stress response and preserved gene expression related to glucose sensing and insulin secretion. Moreover, administration of TMSC-derived conditioned medium induced similar therapeutic outcomes, suggesting paracrine effects. Finally, proteomic analysis revealed high secretion of insulin-like growth factor-binding protein 5 by TMSCs, and its expression was critical for the protective effects of TMSCs against HFD-induced glucose intolerance and ER-stress response in pancreatic islets. TMSC administration can alleviate HFD-induced-T2DM via preserving pancreatic islets and their function. These results provide novel evidence of TMSCs as an ER-stress modulator that may be a novel, alternative cell therapy for T2DM.

The author would like to correct the fifth, seventh and eighth co-authors’ name to Kyung-Ah Cho, Han Su Kim, Bo Young Park in the online published article.

Osteoporosis is a disease that affects 35% women and 20% men aged more than 65 years. Reduction in bone formation and increased bone resorption are known factors that drive osteoporosis, but recent studies suggest a positive correlation between bone marrow adipose tissue (MAT) and osteoporosis. Previously, we have observed that tonsil-derived mesenchymal stem cells (T-MSCs) reduce MAT in a mouse model of bone marrow depletion. That prompted us to investigate on the senile osteoporosis to characterize the bone-forming effect, as well as MAT-reducing effect of T-MSCs. In a mouse model of senescence-accelerated mouse prone 6 (SAMP6), we injected T-MSCs or T-MSC conditioned medium (CM) through tail vein and examined changes in bone microstructure using micro-CT scan and hematoxylin & eosin (H&E) staining. Biochemical markers of osteoporosis, deoxypyridinoline (DPD) and osteocalcin, were examined by ELISA. Results demonstrated attenuation in the progression of osteoporosis, in part, by sustaining osteocalcin production and by blocking MAT accumulation. Increase in matrix mineralization was determined using in vitro culture of murine preosteoblast cell line by treatment of T-MSC CM. Interestingly, T-MSC CM induced continuous weight loss and selectively reduced visceral adipose tissue mass. Finally, antiadipogenic effects of T-MSC CM were determined in vitro. In conclusion, regulation of bone together with MAT could be considered as a new therapeutic option for the treatment of senile osteoporosis and this report may provide a framework for future cell therapy using T-MSCs.

Mesenchymal stromal cells (MSCs) have therapeutic potential for repairing tissue damage and are involved in immune regulation. MSCs are predominantly isolated from bone marrow (BM), adipose tissue or placental tissue. Further to these well‑known sources, the isolation of MSCs from human tonsils was previously reported. The aim of the present study was to investigate a potential role for tonsil‑derived MSCs (T‑MSCs) in BM reconstitution and application towards supplementing hematopoiesis in a mouse model of BM transplantation (BMT). Eight‑week‑old BALB/c female mice received 80 mg/kg busulfan (Bu)/200 mg/kg cyclophosphamide (Cy) conditioning chemotherapy for BM ablation. Subsequently, human T‑MSCs were injected into the Bu/Cy‑treated mice with or without BM cells (BMCs) obtained from allogeneic C57BL/6 male mice. After 3 weeks, peripheral blood and BM was collected for analysis. The red blood cell count in the group that received BMCs had almost returned to normal, whereas mononuclear cell counts and BM cellularity were most improved in the T‑MSCs + BMCs group. These results indicate that the T‑MSCs enhanced myelopoiesis in the allogeneic BMT mouse model, as evidenced by the restoration of BM with hematopoietic cells, as well as increased myeloid colony formation in vitro. Therefore, T‑MSCs may provide a source of MSCs to facilitate myelopoiesis and megakaryocytosis following BMT.

The development of efficient and safe gene delivery carriers has been a major challenge in the clinical application of non-viral gene therapy. Herein, we report novel bioreducible poly(amido amine)s for the efficient delivery of genetic material such as plasmid DNA. A library of 34 different bioreducible polymer compounds was synthesized and screened to find lead materials for in vitro gene transfection. Our lead material (CBA-106) allows effortless polyplex formation with genetic materials by electrostatic interactions at the weight ratio of 1:5 (DNA/polymer). Polyplexes were further characterized by DLS and AFM analysis. Enhanced serum stability and bioreducibility under physiological conditions were confirmed, in addition to low cellular cytotoxicity. When compared with a commercially available gene delivery carrier (Lipofectamine 2000), CBA-1 06 shows comparable or even surpassing gene transfection efficiency. Furthermore, BMP-2 plasmids were efficiently delivered to tonsil-derived mesenchymal stem cells (TMSCs) for osteogenic commitment in vitro and in vivo. Taken together, our results clearly demonstrate the potential of novel bioreducible polymeric systems for gene delivery applications. We suggest that our system can provide a valuable platform for the broad application of gene regulation in cell therapy and regenerative medicine.

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/F‑12) supplemented with 1 ng/ml transforming growth factor-β, non-essential amino acids and insulin‑transferrin-selenium for 4 days followed by culture in myogenic induction medium [low-glucose DMEM containing 2% fetal bovine serum (FBS) and 10 ng/ml insulin‑like 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.

Graft-vs.-host disease (GVHD) is a severe and potentially life-threatening complication of hematopoietic stem cell transplantation. Approximately 50% of patients exhibiting GVHD will not benefit from conventional steroid treatment. Although several second‑line treatments are available for these patients, their prognoses remain poor due to the increased risk of infection, immunosuppression-mediated toxicity and incomplete GVHD remission, which occurs in the majority of cases. Mesenchymal stem cells (MSCs), a multipotent cell population, possess broad immunosuppressive activity and are a reportedly effective treatment of GVHD. However, the therapeutic effects of conditioned medium from MSCs on GVHD have not been demonstrated. In the present study, the efficacy of conditioned medium from human palatine tonsil‑derived MSCs (T‑MSC‑CM) was validated against GVHD in mice. The suppressive function of T‑MSC‑CM on immune cell chemotaxis was confirmed in vitro. A systemic infusion of T‑MSC‑CM in mice with GVHD resulted in prolonged survival, rapid recovery from weight loss and reduced pathological damage in numerous GVHD‑targeted organs. Furthermore, lymphocyte gene expression was significantly downregulated in GVHD mice administered T‑MSC‑CM. These results indicate that T‑MSC‑CM is a promising cellular agent to prevent or treat transplantation‑associated complications such as GVHD.

BACKGROUND: The liver is an organ with remarkable regenerative capacity; however, once chronic fibrosis occurs, liver failure follows, with high mortality and morbidity rates. Continuous exposure to proinflammatory stimuli exaggerates the pathological process of liver failure; therefore, immune modulation is a potential strategy to treat liver fibrosis. Mesenchymal stem cells (MSCs) with tissue regenerative and immunomodulatory potential may support the development of therapeutics for liver fibrosis. METHODS: Here, we induced hepatic injury in mice by injecting carbon tetrachloride (CCl(4)) and investigated the therapeutic potential of conditioned medium from tonsil-derived MSCs (T-MSC CM). In parallel, we used recombinant human IL-1Ra, which, as we have previously shown, is secreted exclusively from T-MSCs and resolves the fibrogenic activation of myoblasts. Hepatic inflammation and fibrosis were determined by histological analyses using H&E and Picro-Sirius Red staining. RESULTS: The results demonstrated that T-MSC CM treatment significantly reduced inflammation as well as fibrosis in the CCl(4)-injured mouse liver. IL-1Ra injection showed effects similar to T-MSC CM treatment, suggesting that T-MSC CM may exert anti-inflammatory and anti-fibrotic effects via the endogenous production of IL-1Ra. The expression of genes involved in fibrosis was evaluated, and the results showed significant induction of alpha-1 type I collagen, transforming growth factor beta, and tissue inhibitor of metalloproteases 1 upon CCl(4) injection, whereas treatment with T-MSC CM or IL-1Ra downregulated their expression. CONCLUSIONS: Taken together, these data support the therapeutic potential of T-MSC CM and/or IL-1Ra for the alleviation of liver fibrosis, as well as in treating diseases involving organ fibrosis.

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.

Human mesenchymal stem cells (MSCs) are adult multipotent cells that have plasticity and inhabit the stroma of diverse tissues. The potential utility of MSCs has been heavily investigated in the fields of regenerative medicine and cell therapy. However, MSCs represent diverse populations that may depend on the tissue of origin. Thus, the ability to identify specific MSC populations has remained difficult. Using RNA sequencing, we analyzed the whole transcriptomes of bone marrow-derived MSCs (BMs), adipose tissue-derived MSCs (AMs), and tonsil-derived MSCs (TMs). We categorized highly regulated genes from these MSC groups according to functional gene ontology (GO) classification. AMs and TMs showed higher expression of genes encoding proteins that function in protein binding, growth factor, or cytokine activity in extracellular compartments than BMs. Interestingly, TM were highly enriched for genes coding extracellular, protein-binding proteins compared with AMs. Functional Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis also showed differentially enriched signaling pathways between the three MSC groups. Further, we confirmed surface antigens expressed in common and in a tissue-specific manner on BMs, AMs, and TMs by flow cytometry analysis. This study provides comprehensive characteristics of MSCs derived from different tissues to better understand their cellular and molecular biology.

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.

Tonsil-derived (T-) mesenchymal stem cells (MSCs) display mutilineage differentiation potential and self-renewal capacity and have potential as a banking source. Diabetes mellitus is a prevalent disease in modern society, and the transplantation of pancreatic progenitor cells or various stem cell-derived insulin-secreting cells has been suggested as a novel therapy for diabetes. The potential of T-MSCs to trans-differentiate into pancreatic progenitor cells or insulin-secreting cells has not yet been investigated. We examined the potential of human T-MSCs to trans-differentiate into pancreatic islet cells using two different methods based on β-mercaptoethanol and insulin–transferin–selenium, respectively. First, we compared the efficacy of the two methods for inducing differentiation into insulin-producing cells. We demonstrated that the insulin–transferin–selenium method is more efficient for inducing differentiation into insulin-secreting cells regardless of the source of the MSCs. Second, we compared the differentiation potential of two different MSC types: T-MSCs and adipose-derived MSCs (A-MSCs). T-MSCs had a differentiation capacity similar to that of A-MSCs and were capable of secreting insulin in response to glucose concentration. Islet-like clusters differentiated from T-MSCs had lower synaptotagmin-3, -5, -7, and -8 levels, and consequently lower secreted insulin levels than cells differentiated from A-MSCs. These results imply that T-MSCs can differentiate into functional pancreatic islet-like cells and could provide a novel, alternative cell therapy for diabetes mellitus.

We previously isolated mesenchymal stromal cells from human tonsils (T-MSCs) and showed the potential of these cells to differentiate into the mesodermal lineage and acquire a follicular dendritic cell (FDC) phenotype under cytokine stimulation. Because these T-MSCs were originally isolated from inflamed tonsillar tissues, we were curious about their activation status in response to innate immune stimuli, such as Toll-like receptors (TLRs). Therefore, we analyzed the expression profile of TLRs in T-MSCs and stimulated the T-MSCs with TLR agonists. TLR3 stimuli induced C-C chemokine receptor type 6 expression in T-MSCs after 24h. Furthermore, results from cytokine arrays showed increases in epithelial neutrophil-activating peptide-78/C-X-C motif chemokine (CXCL) 5, granulocyte chemotactic protein-2/CXCL6, growth-related oncogene-α/CXCL1, interleukin-8/CXCL8, and interferon gamma-induced protein-10/CXCL10. CD54 expression was also increased after TLR3 stimulation. However, co-culturing T-MSCs with human B cells did not induce B-cell proliferation. This suggests that TLR3 stimulates the differentiation of T-MSCs into FDC-like cells and induces chemokine secretion, possibly by recruiting C-X-C chemokine receptor 2-expressing immune cells. In addition, T-MSCs also appeared to exert immunomodulatory effects by inhibiting B-cell proliferation, possibly by down-regulating CD18.

Mesenchymal stem cells (MSCs) are considered valuable sources for cell therapy because of their immune regulatory function. Here, we investigated the effects of tonsil-derived MSCs (T-MSCs) on the differentiation, maturation, and function of dendritic cells (DCs). We examined the effect of T-MSCs on differentiation and maturation of bone-marrow- (BM-) derived monocytes into DCs and we found suppressive effect of T-MSCs on DCs via direct contact as well as soluble mediators. Moreover, T cell proliferation, normally increased in the presence of DCs, was inhibited by T-MSCs. Differentiation of CD4+T cell subsets by the DC-T cell interaction also was inhibited by T-MSCs. The soluble mediators suppressed by T-MSCs were granulocyte-macrophage colony-stimulating factor (GM-CSF), RANTES, interleukin-6 (IL-6), and monocyte chemoattractant protein-1 (MCP-1). Taken together, T-MSCs exert immune modulatory function via suppression of the differentiation, maturation, and function of BM-derived DCs. Our data suggests that T-MSCs could be used as a novel source of stem cell therapy as immune modulators.

Our knowledge of the immunomodulatory role of mesenchymal stem cells (MSCs) in both the innate and adaptive immune systems has dramatically expanded, providing great promise for treating various autoimmune diseases. However, the contribution of MSCs to Th17-dominant immune disease, such as psoriasis and its underlying mechanism remains elusive. In this study, we demonstrated that human palatine tonsil-derived MSCs (T-MSCs) constitutively express both the membrane-bound and soluble forms of programmed death-ligand 1 (PD-L1), which enables T-MSCs to be distinguished from MSCs originating from other organs (i.e. bone marrow or adipose tissue). We also found that T-MSC-derived PD-L1 effectively represses Th17 differentiation via both cell-to-cell contact and a paracrine effect. Further, T-MSCs increase programmed death-1 (PD-1) expression on T-cells by secreting IFN-β, which may enhance engagement with PD-L1. Finally, transplantation of T-MSCs into imiquimod-induced psoriatic skin inflammation in mice significantly abrogated disease symptoms, mainly by blunting the Th17 response in a PD-L1-dependent manner. This study suggests that T-MSCs might be a promising cell source to treat autoimmune diseases such as psoriasis, via its unique immunoregulatory features. Copyright © 2017 John Wiley & Sons, Ltd.

Saturated free fatty acids (FFAs) act as lipid mediators and induce insulin resistance in skeletal muscle. Specifically, in obesity‐related diseases such as type 2 diabetes, FFAs directly reduce insulin sensitivity and glucose uptake in skeletal muscle. However, the knowledge of how FFAs mediate inflammation and subsequent tissue disorders, including fibrosis in skeletal muscle, is limited. FFAs are a natural ligand for toll‐like receptor 2 (TLR2) and TLR4, and induce chronic low‐grade inflammation that directly stimulates skeletal muscle tissue. However, persistent inflammatory stimulation in tissues could induce pro‐fibrogenic processes that ultimately lead to perturbation of the tissue architecture and dysfunction. Therefore, blocking the link between inflammatory primed skeletal muscle tissue and connective tissue might be an efficient therapeutic option for treating obesity‐induced muscle inactivity. In this study, we investigated the impact of conditioned medium obtained from human palatine tonsil‐derived mesenchymal stem cells (T‐MSCs) on the interaction between skeletal muscle cells stimulated with palmitic acid (PA) and fibroblasts. We found that PA‐treated skeletal muscle cells actively secreted interleukin‐1β (IL‐1β) and augmented the migration, proliferation and expression of fibronectin in L929 fibroblasts. Furthermore, T‐CM inhibited the skeletal muscle cell‐derived pro‐fibrogenic effect via the production of the interleukin‐1 receptor antagonist (IL‐1Ra), which is an inhibitor of IL‐1 signalling. Taken together, our data provide novel insights into the therapeutic potential of T‐MSC‐mediated therapy for the treatment of pathophysiological processes that occur in skeletal muscle tissues under chronic inflammatory conditions.

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) β(3) with anti-integrin α(v) β(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) β(3) and AMPK mediate CCN1-induced EC migration and tube formation independent of intracellular ATP levels alteration.

Although mesenchymal stem cells (MSC) isolated from bone marrow and adipose tissues are known to be subjected to in vitro culture-related alterations in their stem cell properties, such data have not been reported in human tonsil-derived MSC (T-MSC). Here, we investigated the culture-related changes of phenotypes, the senescence, and the differentiation potential of T-MSC. T-MSC were serially passaged by a standard protocol, and their characteristics were assessed, including MSC-specific surface antigen profiles, the senescence, and the differentiation potentials into adipocytes, chondrocytes and osteocytes. Up to at least passage 15, we found no alterations in either MSC-specific surface marker, CD14, CD34, CD45, CD73 and CD90, or the mRNA expression of embryonic stem cell gene markers, Nanog, Oct4-A and Sox-2. However, the expression of CD146, recently identified another MSC marker, dramatically decreased with increasing passages from ∼ 23% at passage 3 to ∼ 1% at passage 15. The average doubling time increased significantly from ∼ 38 h at passage 10 to ∼ 46 h at passage 15. From passage 10, the cell size increased slightly and SA-β-gal staining was evident. Both Alizarin Red S staining and osteocalcin expression showed that the osteogenic differentiation potential increased up to passage 10 and decreased thereafter. However, the adipogenic and chondrogenic differentiation potential decreased passage-dependently from the start, as evidenced by staining of Oil Red O and Alcian Blue, respectively. Consistent with a passage-dependent osteogenic differentiation, the expression of CCN1, an angiogenic protein known to be related to both senescence and osteogenesis, also increased up to passage 10. Furthermore, ectopic expression of small interfering RNA against CCN1 at passage 10 significantly reversed Alizarin Red S staining and osteocalcin expression. Altogether, our study demonstrates the characterization of long-term in vitro cultured T-MSC and that CCN1 may be involved in mediating a passage-dependent increase in osteogenic potential of T-MSC.

Acute liver failure, the fatal deterioration of liver function, is the most common indication for emergency liver transplantation, and drug-induced liver injury and viral hepatitis are frequent in young adults. Stem cell therapy has come into the limelight as a potential therapeutic approach for various diseases, including liver failure and cirrhosis. In this study, we investigated therapeutic effects of tonsil-derived mesenchymal stem cells (T-MSCs) in concanavalin A (ConA)- and acetaminophen-induced acute liver injury. ConA-induced hepatitis resembles viral and immune-mediated hepatic injury, and acetaminophen overdose is the most frequent cause of acute liver failure in the United States and Europe. Intravenous administration of T-MSCs significantly reduced ConA-induced hepatic toxicity, but not acetaminophen-induced liver injury, affirming the immunoregulatory capacity of T-MSCs. T-MSCs were successfully recruited to damaged liver and suppressed inflammatory cytokine secretion. T-MSCs expressed high levels of galectin-1 and -3, and galectin-1 knockdown which partially diminished interleukin-2 and tumor necrosis factor α secretion from cultured T-cells. Galectin-1 knockdown in T-MSCs also reversed the protective effect of T-MSCs on ConA-induced hepatitis. These results suggest that galectin-1 plays an important role in immunoregulation of T-MSCs, which contributes to their protective effect in immune-mediated hepatitis. Further, suppression of T-cell activation by frozen and thawed T-MSCs implies great potential of T-MSC banking for clinical utilization in immune-mediated disease.

Background We analyzed a nationwide registry of pediatric patients with hemophagocytic lymphohistiocytosis (HLH) in Korea to assess the clinical and genetic features and treatment outcomes in pediatric HLH. Methods The Korea Histiocytosis Working Party retrospectively analyzed data on 251 pediatric patients diagnosed with HLH between 1996 and 2011. Results In the study cohort, 25 cases were categorized with familial HLH, 64 with presumed secondary HLH, and 162 with unspecified HLH. Of 217 evaluable patients, 91 (42%) had concomitant Epstein–Barr virus infection. Of 238 evaluable patients, central nervous system (CNS) involvement, which was more frequent in the familial group, was evident in 81 cases (34%). Genetic tests revealed a predominant UNC13D mutation with a high incidence of two recurrent splicing mutations (c.118-308C>T and c.754-1G>C). The 5-yr overall survival rate was 68% (38% in the familial group and 81% in the presumed secondary group). The 5-yr overall survival rate among 32 patients who underwent allogeneic hematopoietic stem cell transplantation was 64%. In multivariate analysis, a younger age at diagnosis, severe transaminasemia, and a coagulation abnormality were independent prognostic factors for survival. Responses during initial treatments were also significant indicators of outcome. Conclusion Our study showed the unique predominance of a UNC13D mutation and vulnerability to Epstein–Barr virus infection in Korean children with HLH and emphasizes the prognostic significance of age, liver dysfunction, and treatment responses in this disease. A multicenter prospective trial that builds on the present results is warranted to identify subgroups of patients with a poor prognosis and identify optimal treatments.