Dual Targeting of EZH2 Degradation and EGFR/HER2 Inhibition for Enhanced Efficacy against Burkitt's Lymphoma.

EZH2, a histone methyltransferase, contributes significantly to cancer cell survival and proliferation. Although various EZH2 inhibitors have demonstrated promise in treating lymphoma, they have not fully managed to curb lymphoma cell proliferation despite effective reduction of the H3K27me3 mark. We used MS1943, an EZH2 selective degrader, which successfully diminishes EZH2 levels in lymphoma cells. Additionally, lapatinib, a dual inhibitor of the epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2) tyrosine kinases, targets a receptor protein that regulates cell growth and division. The overexpression of this protein is often observed in lymphoma cells. Our study aims to combine these two therapeutic targets to stimulate apoptosis pathways and potentially suppress Burkitt's lymphoma cell survival and proliferation in a complementary and synergistic manner. We observed that a combination of MS1943 and lapatinib induced apoptosis in Daudi cells and caused cell cycle arrest at the S and G2/M phases in both Ramos and Daudi cells. This strategy, using a combination of MS1943 and lapatinib, presents a promising therapeutic approach for treating lymphoma and potentially Burkitt's lymphoma.

Overcoming the therapeutic limitations of EZH2 inhibitors in Burkitt's lymphoma: a comprehensive study on the combined effects of MS1943 and Ibrutinib.

Enhancer of zeste homolog 2 (EZH2) and Bruton's tyrosine kinase (BTK) are both key factors involved in the development and progression of hematological malignancies. Clinical studies have demonstrated the potential of various EZH2 inhibitors, which target the methyltransferase activity of EZH2, for the treatment of lymphomas. However, despite their ability to effectively reduce the H3K27me3 levels, these inhibitors have shown limited efficacy in blocking the proliferation of lymphoma cells. To overcome this challenge, we employed a hydrophobic tagging approach utilizing MS1943, a selective EZH2 degrader. In this study, we investigated the inhibitory effects of two drugs, the FDA-approved EZH2 inhibitor Tazemetostat, currently undergoing clinical trials, and the novel drug MS1943, on Burkitt's lymphoma. Furthermore, we assessed the potential synergistic effect of combining these drugs with the BTK inhibitor Ibrutinib. In this study, we evaluated the effects of combination therapy with MS1943 and Ibrutinib on the proliferation of three Burkitt's lymphoma cell lines, namely RPMI1788, Ramos, and Daudi cells. Our results demonstrated that the combination of MS1943 and Ibrutinib significantly suppressed cell proliferation to a greater extent compared to the combination of Tazemetostat and Ibrutinib. Additionally, we investigated the underlying mechanisms of action and found that the combination therapy of MS1943 and Ibrutinib led to the upregulation of miR29B-mediated p53-upregulated modulator of apoptosis PUMA, BAX, cleaved PARP, and cleaved caspase-3 in Burkitt's lymphoma cells. These findings highlight the potential of this innovative therapeutic strategy as an alternative to traditional EZH2 inhibitors, offering promising prospects for improving treatment outcomes in Burkitt's lymphoma.

Biomimetic hydrogel blanket for conserving and recovering intrinsic cell properties.

BACKGROUND: Cells in the human body experience different growth environments and conditions, such as compressive pressure and oxygen concentrations, depending on the type and location of the tissue. Thus, a culture device that emulates the environment inside the body is required to study cells outside the body. METHODS: A blanket-type cell culture device (Direct Contact Pressing: DCP) was fabricated with an alginate-based hydrogel. Changes in cell morphology due to DCP pressure were observed using a phase contrast microscope. The changes in the oxygen permeability and pressure according to the hydrogel concentration of DCP were analyzed. To compare the effects of DCP with normal or artificial hypoxic cultures, cells were divided based on the culture technique: normal culture, DCP culture device, and artificial hypoxic environment. Changes in phenotype, genes, and glycosaminoglycan amounts according to each environment were evaluated. Based on this, the mechanism of each culture environment on the intrinsic properties of conserving chondrocytes was suggested. RESULTS: Chondrocytes live under pressure from the surrounding collagen tissue and experience a hypoxic environment because collagen inhibits oxygen permeability. By culturing the chondrocytes in a DCP environment, the capability of DCP to produce a low-oxygen and physical pressure environment was verified. When human primary chondrocytes, which require pressure and a low-oxygen environment during culture to maintain their innate properties, were cultured using the hydrogel blanket, the original shapes and properties of the chondrocytes were maintained. The intrinsic properties could be recovered even in aged cells that had lost their original cell properties. CONCLUSIONS: A DCP culture method using a biomimetic hydrogel blanket provides cells with an adjustable physical pressure and a low-oxygen environment. Through this technique, we could maintain the original cellular phenotypes and intrinsic properties of human primary chondrocytes. The results of this study can be applied to other cells that require special pressure and oxygen concentration control to maintain their intrinsic properties. Additionally, this technique has the potential to be applied to the re-differentiation of cells that have lost their original properties.

BAFF blockade attenuates acute graft-versus-host disease directly via the dual regulation of T- and B-cell homeostasis.

Introduction: B-cell-activating factor (BAFF) is associated with donor-specific antibodies and chronic graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT). However, the effects of BAFF on T-cell physiological function have not been fully elucidated in acute GVHD. Methods: We examined the effects of belimumab, a monoclonal antibody targeting BAFF, for the treatment of acute GVHD. We examined the effects of T cells and B cells separately when inducing GVHD in mouse model. Results: Therapeutic functional manipulation of endogenous BAFF can improve acute GVHD during the early post-transplant period. In this study, BAFF was shown to increase the proportions of CD4+IL-17+, CD4+IL-6+ Th17, and CD4+IFN-γ+ Th1 cells and to reduce the proportion of regulatory T (Treg) cells. Furthermore, the belimumab therapy group showed increased B220+IgD+IgM+ mature B cells but decreased B220+IgD-IgM- memory B cells, B220+Fas+GL-7+ germinal center formation, and B220+IgD-CD138+ plasma cells. These results indicate that BAFF can alleviate acute GVHD by simultaneously regulating T and B cells. Interestingly, the BAFF level was higher in patients with acute GVHD after HSCT compared with patients receiving chemotherapy. Conclusion: This study suggests that BAFF blockade might modulate CD4 +T-cell-induced acute GVHD early after allo-HSCT and the possibility of simultaneously controlling chronic GVHD, which may appear later after allo-HSCT.

Human Palatine Tonsils Are Linked to Alzheimer's Disease through Function of Reservoir of Amyloid Beta Protein Associated with Bacterial Infection.

Amyloid-ß (Aß)-peptide production or deposition in the neuropathology of Alzheimer's disease (AD) was shown to be caused by chronic inflammation that may be induced by infection, but the role of pathogenic-bacteria-related AD-associated Aß is not yet clearly understood. In this study, we validated the hypothesis that there is a correlation between the Aß-protein load and bacterial infection and that there are effects of bacteria, Staphylococcus aureus (S. aureus), on the Aß load in the inflammatory environment of human tonsils. Here, we detected Aß-peptide deposits in human tonsil tissue as well as tissue similar to tonsilloliths found in the olfactory cleft. Interestingly, we demonstrated for the first time the presence of Staphylococcus aureus (S. aureus) clustered around or embedded in the Aß deposits. Notably, we showed that treatment with S. aureus upregulated the Aß-protein load in cultures of human tonsil organoids and brain organoids, showing the new role of S. aureus in Aß-protein aggregation. These findings suggest that a reservoir of Aß and pathogenic bacteria may be a possible therapeutic target in human tonsils, supporting the treatment of antibiotics to prevent the deposition of Aß peptides via the removal of pathogens in the intervention of AD pathogenesis.

Protective Effect of Human-Neural-Crest-Derived Nasal Turbinate Stem Cells against Amyloid-ß Neurotoxicity through Inhibition of Osteopontin in a Human Cerebral Organoid Model of Alzheimer's Disease.

The aim of this study was to validate the use of human brain organoids (hBOs) to investigate the therapeutic potential and mechanism of human-neural-crest-derived nasal turbinate stem cells (hNTSCs) in models of Alzheimer's disease (AD). We generated hBOs from human induced pluripotent stem cells, investigated their characteristics according to neuronal markers and electrophysiological features, and then evaluated the protective effect of hNTSCs against amyloid-ß peptide (Aß1-42) neurotoxic activity in vitro in hBOs and in vivo in a mouse model of AD. Treatment of hBOs with Aß1-42 induced neuronal cell death concomitant with decreased expression of neuronal markers, which was suppressed by hNTSCs cocultured under Aß1-42 exposure. Cytokine array showed a significantly decreased level of osteopontin (OPN) in hBOs with hNTSC coculture compared with hBOs only in the presence of Aß1-42. Silencing OPN via siRNA suppressed Aß-induced neuronal cell death in cell culture. Notably, compared with PBS, hNTSC transplantation significantly enhanced performance on the Morris water maze, with reduced levels of OPN after transplantation in a mouse model of AD. These findings reveal that hBO models are useful to evaluate the therapeutic effect and mechanism of stem cells for application in treating AD.

Therapeutic Potential of Human Nasal Inferior Turbinate-Derived Stem Cells: Microarray Analysis of Multilineage Differentiation.

INTRODUCTION: Human nasal inferior turbinate-derived stem cells (hNTSCs) are attractive sources of adult stem cells for medical application because they can be easily obtained and cultivated with a highly proliferative capacity. The ability of hNTSCs to differentiate into chondrocytes, osteocytes, and neural cells makes them potential replacement therapeutic candidates in intractable disease. Nevertheless, detailed expression pattern of genes associated with trilineage differentiation (osteogenesis, chondrogenesis, and neurogenesis) in hNTSCs has not been revealed yet. METHODS: In this study, we aimed to evaluate gene expression patterns of various transcription factors and marker genes associated with a particular lineage (osteogenesis, chondrogenesis, and neurogenesis) of differentiation of hNTSCs by DNA microarrays. RESULTS: In microarrays, 36 transcripts such as E2F transcription factor 1, activating transcription factor 5, and AKR1B10 were upregulated and 36 transcripts such as CA9, PPFIA4, HAS2, and COL4A4 were downregulated in osteogenically differentiated hNTSCs. In chondrogenically differentiated hNTSCs, 3 transcripts (NUDT14, CPA4, and heparin-binding epidermal growth factor-like growth factor) were upregulated and 82 transcripts such as PTGS1, CLEC2D, and TET1 were downregulated. In neurally differentiated hNTSCs, 61 transcripts such as insulin-like growth factor-binding protein-1, nerve growth factor receptor, FGF1, OLFML1, and EPGN were upregulated and 98 transcripts such as ACAN, RUNX2, and C21orf96 were downregulated. In gene ontology (GO) analysis, cell signal-related GO terms were highly expressed. By contrast, catalysis GO terms and GO terms related to oxidoreductase were overrepresented in chondrogenically differentiated hNTSCs and osteogenically differentiated hNTSCs, respectively. CONCLUSION: Considering overall results, hNTSCs-specific genetic information may promote further studies on intracellular mechanisms defining key features of these cells.

Potential application of human neural crest-derived nasal turbinate stem cells for the treatment of neuropathology and impaired cognition in models of Alzheimer's disease.

BACKGROUND: Stem cell transplantation is a fascinating therapeutic approach for the treatment of many neurodegenerative disorders; however, clinical trials using stem cells have not been as effective as expected based on preclinical studies. The aim of this study is to validate the hypothesis that human neural crest-derived nasal turbinate stem cells (hNTSCs) are a clinically promising therapeutic source of adult stem cells for the treatment of Alzheimer's disease (AD). METHODS: hNTSCs were evaluated in comparison with human bone marrow-derived mesenchymal stem cells (hBM-MSCs) according to the effect of transplantation on AD pathology, including PET/CT neuroimaging, immune status indicated by microglial numbers and autophagic capacity, neuronal survival, and cognition, in a 5 × FAD transgenic mouse model of AD. RESULTS: We demonstrated that hNTSCs showed a high proliferative capacity and great neurogenic properties in vitro. Compared with hBM-MSC transplantation, hNTSC transplantation markedly reduced Aß42 levels and plaque formation in the brains of the 5 × FAD transgenic AD mice on neuroimaging, concomitant with increased survival of hippocampal and cortex neurons. Moreover, hNTSCs strongly modulated immune status by reducing the number of microglia and the expression of the inflammatory cytokine IL-6 and upregulating autophagic capacity at 7 weeks after transplantation in AD models. Notably, compared with transplantation of hBM-MSCs, transplantation of hNTSCs significantly enhanced performance on the Morris water maze, with an increased level of TIMP2, which is necessary for spatial memory in young mice and neurons; this difference could be explained by the high engraftment of hNTSCs after transplantation. CONCLUSION: The reliable evidence provided by these findings reveals a promising therapeutic effect of hNTSCs and indicates a step forward the clinical application of hNTSCs in patients with AD.

DNMT3A haploinsufficiency causes dichotomous DNA methylation defects at enhancers in mature human immune cells.

DNMT3A encodes an enzyme that carries out de novo DNA methylation, which is essential for the acquisition of cellular identity and specialized functions during cellular differentiation. DNMT3A is the most frequently mutated gene in age-related clonal hematopoiesis. As such, mature immune cells harboring DNMT3A mutations can be readily detected in elderly persons. Most DNMT3A mutations associated with clonal hematopoiesis are heterozygous and predicted to cause loss of function, indicating that haploinsufficiency is the predominant pathogenic mechanism. Yet, the impact of DNMT3A haploinsufficiency on the function of mature immune cells is poorly understood. Here, we demonstrate that DNMT3A haploinsufficiency impairs the gain of DNA methylation at decommissioned enhancers, while simultaneously and unexpectedly impairing DNA demethylation of newly activated enhancers in mature human myeloid cells. The DNA methylation defects alter the activity of affected enhancers, leading to abnormal gene expression and impaired immune response. These findings provide insights into the mechanism of immune dysfunction associated with clonal hematopoiesis and acquired DNMT3A mutations.

Human mesenchymal stem cells derived from umbilical cord and bone marrow exert immunomodulatory effects in different mechanisms.

BACKGROUND: Mesenchymal stem cells (MSCs) are an attractive tool to treat graft-versus-host disease because of their unique immunoregulatory properties. Although human bone marrow-derived MSCs (BM-MSCs) were the most widely used MSCs in cell therapy until recently, MSCs derived from human umbilical cords (UC-MSCs) have gained popularity as cell therapy material for their ethical and noninvasive collection. AIM: To investigate the difference in mechanisms of the immunosuppressive effects of UC-MSCs and BM-MSCs. METHODS: To analyze soluble factors expressed by MSCs, such as indolamine 2,3-dioxygenase, cyclooxygenase-2, prostaglandin E2 and interleukin (IL)-6, inflammatory environments in vitro were reconstituted with combinations of interferon-gamma (IFN-γ), tumor necrosis factor alpha and IL-1ß or with IFN-γ alone. Activated T cells were cocultured with MSCs treated with indomethacin and/or anti-IL-10. To assess the ability of MSCs to inhibit T helper 17 cells and induce regulatory T cells, induced T helper 17 cells were cocultured with MSCs treated with indomethacin or anti-IL-10. Xenogeneic graft-versus-host disease was induced in NOG mice (NOD/Shi-scid/IL-2Rγnull) and UC-MSCs or BM-MSCs were treated as cell therapies. RESULTS: Our data demonstrated that BM-MSCs and UC-MSCs shared similar phenotypic characteristics and immunomodulation abilities. BM-MSCs expressed more indolamine 2,3-dioxygenase after cytokine stimulation with different combinations of IFN-γ, tumor necrosis factor alpha-α and IL-1ß or IFN-γ alone. UC-MSCs expressed more prostaglandin E2, IL-6, programmed death-ligand 1 and 2 in the in vitro inflammatory environment. Cyclooxygenase-2 and IL-10 were key factors in the immunomodulatory mechanisms of both MSCs. In addition, UC-MSCs inhibited more T helper 17 cells and induced more regulatory T cells than BM-MSCs. UC-MSCs and BM-MSCs exhibited similar effects on attenuating graft-versus-host disease. CONCLUSION: UC-MSCs and BM-MSCs exert similar immunosuppressive effects with different mechanisms involved. These findings suggest that UC-MSCs have distinct immunoregulatory functions and may substitute BM-MBSCs in the field of cell therapy.

Evaluation of Collagen Gel-Associated Human Nasal Septum-Derived Chondrocytes As a Clinically Applicable Injectable Therapeutic Agent for Cartilage Repair.

BACKGROUND: Articular cartilage injury has a poor repair ability and limited regeneration capacity with therapy based on articular chondrocytes (ACs) implantation. Here, we validated the hypothesis that human nasal septum-derived chondrocytes (hNCs) are potent therapeutic agents for clinical use in cartilage tissue engineering using an injectable hydrogel, type I collagen (COL1). METHODS: We manufactured hNCs incorporated in clinical-grade soluble COL1 and investigated their clinical potential as agents in an articular defect model. RESULTS: The hNCs encapsulated in COL1 (hNC-collagen) were uniformly distributed throughout the collagen and showed much greater growth rate than hACs encapsulated in collagen for the 14 days of culture. Fluorescent staining of hNC-collagen showed high expression levels of chondrocyte-specific proteins under clinical conditions. Moreover, a negative mycoplasma screening result were obtained in culture of hNC-collagen. Notably, implantation of hNC-collagen increased the repair of osteochondral defects in rats compared with implantation of collagen only. Many human cells were detected within the cartilage defects. CONCLUSION: These results provide reliable evidences supporting for clinical applications of hNC-collagen in regenerative medicine for cartilage repair.

Rapid Cartilage Regeneration of Spheroids Composed of Human Nasal Septum-Derived Chondrocyte in Rat Osteochondral Defect Model.

BACKGROUND: Cell-based therapies have been studied for articular cartilage regeneration. Articular cartilage defects have little treatments because articular cartilage was limited regenerative capacity. Damaged articular cartilage is difficult to obtain a successful therapeutic effect. In additionally these articular cartilage defects often cause osteoarthritis. Chondrocyte implantation is a widely available therapy used for regeneration of articular cartilage because this tissue has poor repair capacity after injury. Human nasal septum-drived chondrocytes (hNCs) from the septum show greater proliferation ability and chondrogenic capacity than human articular chondrocytes (hACs), even across different donors with different ages. Moreover, the chondrogenic properties of hNCs can be maintained after extensive culture expansion. METHODS: In this study, 2 dimensional (2D) monolayer cultured hNCs (hNCs-2D) and 3 dimensional (3D) spheroids cultured hNCs (hNCs-3D) were examined for chondrogenic capacity in vitro by PCR and immunofluorescence staining for chondrogenic marker, cell survival during cultured and for cartilage regeneration ability in vivo in a rat osteochondral defect model. RESULTS: hNCs-3D showed higher viability and more uniform morphology than 3D spheroids cultured hACs (hACs-3D) in culture. hNCs-3D also showed greater expression levels of the chondrocyte-specific marker Type II collagen (COL2A1) and sex-determining region Y (SRY)-box 9 (SOX9) than hNCs-2D. hNCs-3D also expressed chondrogenic markers in collagen. Specially, in the osteochondral defect model, implantation of hNCs-3D led to greater chondrogenic repair of focal cartilage defects in rats than implantation of hNCs-2D. CONCLUSION: These data suggest that hNCs-3D are valuable therapeutic agents for repair and regeneration of cartilage defects.

Regulation of HMGB1 release protects chemoradiotherapy-associated mucositis.

Oral mucositis (OM) is a common complication in cancer patients undergoing anticancer treatment. Despite the clinical and economic consequences of OM, there are no drugs available for its fundamental control. Here we show that high-mobility group box 1 (HMGB1), a "danger signal" that acts as a potent innate immune mediator, plays a critical role in the pathogenesis of OM. In addition, we investigated treatment of OM through HMGB1 blockade using NecroX-7 (tetrahydropyran-4-yl)-[2-phenyl-5-(1,1-dioxo-thiomorpholin-4-yl)methyl-1Hindole-7-yl]amine). NecroX-7 ameliorated basal layer epithelial cell death and ulcer size in OM induced by chemotherapy or radiotherapy. This protective effect of NecroX-7 was mediated by inhibition of HMGB1 release and downregulation of mitochondrial oxidative stress. Additionally, NecroX-7 inhibited the HMGB1-induced release of tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1ß, and macrophage inflammatory protein (MIP)-1ß, as well as the expression of p53-upregulated modulator of apoptosis (PUMA) and the excessive inflammatory microenvironment, including nuclear factor-kB (NF-kB) pathways. In conclusion, our findings suggest that HMGB1 plays a key role in the pathogenesis of OM; therefore, blockade of HMGB1 by NecroX-7 may be a novel therapeutic strategy for OM.

Accelerated Bone Regeneration via Three-Dimensional Cell-Printed Constructs Containing Human Nasal Turbinate-Derived Stem Cells as a Clinically Applicable Therapy.

Stem cell transplantation is a promising therapeutic strategy that includes both cell therapy and tissue engineering for the treatment of many regenerative diseases; however, the efficacy and safety of stem cell therapy depend on the cell type used in therapeutic and translational applications. In this study, we validated the hypothesis that human nasal turbinate-derived mesenchymal stem cells (hTMSCs) are a potential therapeutic source of adult stem cells for clinical use in bone tissue engineering using three-dimensional (3D) cell-printing technology. hTMSCs were cultured and evaluated for clinical use according to their cell growth, cell size, and preclinical safety and were then incorporated into a multicompositional 3D bioprinting system and investigated for bone tissue regeneration in vitro and in vivo. Finally, hTMSCs were compared with human bone marrow-derived MSCs (hBMSCs), which are the most common stem cell type used in regenerative medicine. hTMSCs from three different donors showed greater and faster cell growth than hBMSCs from two different donors when cultured. The hTMSCs were smaller in size than the hBMSCs. Furthermore, the hTMSCs did not exhibit safety issues in immunodeficient mice. hTMSCs in 3D-printed constructs (3D-hTMSC) showed much greater viability, growth, and osteogenic differentiation potential in vitro than hBMSCs in 3D-printed constructs (3D-hBMSC). Likewise, 3D-hTMSC showed better cell survival and alkaline phosphatase activity and greater osteogenic protein expression than 3D-hBMSC upon subcutaneous implantation into the dorsal region of nude mice. Notably, in an orthotopic model involving implantation into a tibial defect in rats, implantation of 3D-hTMSC led to greater bone matrix formation and enhanced bone healing to a greater degree than implantation of 3D-hBMSC. The clinically reliable evidence provided by these results is underlined by the potential for rapid tissue regeneration and ambulation in bone fracture patients implanted with 3D-hTMSC.

Enhancement of Graft-Versus-Host Disease Control Efficacy by Adoptive Transfer of Type 1 Regulatory T Cells in Bone Marrow Transplant Model.

Interleukin (IL)-10-producing type 1 regulatory T (Tr1) cells, which are Foxp3-memory T lymphocytes, play important roles in peripheral immune tolerance. We investigated whether Tr1 cells exert immunoregulatory effects in a mouse model of acute graft-versus-host disease (GVHD). Mouse CD4+ T cells were induced to differentiate in vitro into Tr1 cells using vitamin D3 and dexamethasone, and these donor-derived Tr1 cells were infused on the day of bone marrow transplantation. The Tr1 cell-transferred group showed less weight-loss and a twofold higher survival rate than the GVHD group, together with markedly decreased histopathologic grades. It was associated with the expansion of CD4+IL-4+ type 2 T-helper (Th2) cells and CD4+CD25+Foxp3+ regulatory T (Treg) cells. Furthermore, Tr1 cells decreased the numbers of CD4+interferon-γ+ Th1 and CD4+IL-17+ Th17 cells. Recipient mice harbored some Foxp3+ Tregs due to adoptive transfer of Tr1 cells, together with the upregulated expression of costimulatory molecules, including cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) and inducible T-cell costimulator (ICOS); however, the Treg cells did not show the plasticity. Therefore, adoptive Tr1 cell therapy may be effective against manifestations of GVHD, exert immunomodulatory effects in a manner dependent on CTLA-4 and ICOS, and induce differentiation of the transferred Tr1 cells into Foxp3+ Treg cells.

Characteristics of Nasal Septal Cartilage-Derived Progenitor Cells during Prolonged Cultivation.

Objective To produce alternate cell sources for tissue regeneration, human nasal septal cartilage-derived progenitor cells (NSPs) were tested to identify whether these cells meet the criteria of cartilage progenitor cells. We also evaluated the effects of prolonged cultivation on the characteristics of NSPs. Study Design In vitro study. Setting Academic research laboratory. Methods NSPs were isolated from discarded human nasal septal cartilage. NSPs were cultured for 10 passages. The expression of septal progenitor cell surface markers was assessed by fluorescence-activated cell sorting. Cell proliferation was measured with a cell-counting kit. Cytokine secretion was analyzed with multiplex immunoassays. Chondrogenic differentiation of NSPs without differentiation induction was analyzed with type II collagen immunohistochemistry. Cartilage-specific protein expression was evaluated by Western blotting. Under osteo- and adipodifferentiation media, 2 lineage differentiation potentials were evaluated by histology and gene expression analysis. Results Surface epitope analysis revealed that NSPs are positive for mesenchymal stem cells markers and negative for hematopoietic cell markers. Cultured NSPs showed sufficient cell expansion and chondrogenic potential, as demonstrated by immunostaining and expression of cartilage-specific protein. IL-6, IL-8, and transforming growth factor ß were secreted by over 200 pg/mL. The osteo- and adipodifferentiation potentials of NSPs were identified by histology and specific gene expression. The aforementioned characteristics were not influenced by prolonged cultivation. Conclusion NSPs represent an initial step toward creating a cell source from surgically discarded tissue that may prove useful in cartilage regeneration.

Robust Production of Cytomegalovirus pp65-Specific T Cells Using a Fully Automated IFN-γ Cytokine Capture System.

BACKGROUND: Cytomegalovirus(CMV)-related diseases are a serious cause of morbidity and mortality following hematopoietic stem cell transplantation (HSCT). CMV-specific cytotoxic T lymphocytes (CMV-CTLs) have been reported as an alternative to antiviral drugs that provide long-term CMV-specific immunity without major side effects. However, their application has been limited by the prolonged manufacturing process required. METHODS: In this study, we applied the IFN-γ cytokine capture system (CCS) using the fully automated CliniMACS Prodigy device for rapid production of CMV-CTLs, which may be applicable in clinically urgent CMV-related diseases. Five validation runs were performed using apheresis samples from randomly selected CMV-seropositive healthy blood donors. Successive processes, including antigen stimulation, anti-IFN-γ labeling, magnetic enrichment and elution, were then performed automatically using the CliniMACS Prodigy, which took approximately 13 h. RESULTS: The original apheresis samples consisted mainly of CD45RA+ CD62L+ naïve T cells as well as 0.3% IFN-γ-secreting CD3+ T cells that showed a response to the CMV pp65 antigen (CD3+ IFN-γ+ cells). Following IFN-γ enrichment, the target fraction contained 51.3% CD3+ IFN-γ+ cells with a reduction in naïve T cells and selection of CD45RA- CD62L- and CD45RA+ CD62L- memory T cells. Furthermore, extended culture of these isolated cells revealed functional activity, including efficient proliferation, sustained antigen-specific IFN-γ secretion, and cytotoxicity against pp65-pulsed target cells. CONCLUSION: The findings reported here suggest that the IFN-γ CCS by the CliniMACS Prodigy is a simple and robust approach to produce CMV-CTLs, which may be applicable for the treatment of clinically urgent CMV-related diseases.

Combined Treatment with Methylprednisolone and Human Bone Marrow-Derived Mesenchymal Stem Cells Ameliorate Experimental Autoimmune Encephalomyelitis.

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system. Although advances have been made in the treatment of MS, such as the use of IFN-ß, glucocorticoids and stem cells, the therapeutic effects of these treatments are not sufficient. In the present study, we evaluated whether the combination of methylprednisolone (MP) and human bone marrow-derived mesenchymal stem cells (BM-MSCs) could enhance the therapeutic effectiveness in experimental autoimmune encephalomyelitis (EAE), a model for MS. EAE was induced by immunizing C57BL/6 mice with myelin oligodendrocyte glycoprotein 35-55 (MOG 35-55). The immunized mice received an intraperitoneal injection of MP (20 mg/kg), an intravenous injection of BM-MSCs (1 × 106 cells) or both on day 14 after immunization. Combination treatment significantly ameliorated the clinical symptoms, along with attenuating inflammatory infiltration and demyelination, compared to either treatment alone. Secretion of pro-inflammatory cytokines (IFN-γ, TNF-α, IL-17) was significantly reduced, and anti-inflammatory cytokines (IL-4, IL-10) was significantly increased by the combination treatment as compared to either treatment alone. Flow cytometry analysis of MOG-reactivated T cells in spleen showed that combination treatment reduced the number of CD4+CD45+ and CD8+ T cells, and increased the number of CD4+CD25+Foxp3+ regulatory T cells. Furthermore, combination treatment enhanced apoptosis in MOG-reactivated CD4+ T cells, a key cellular subset in MS pathogenesis. Combination treatment with MP and BM-MSCs provides a novel treatment protocol for enhancing therapeutic effects in MS.

Third-party regulatory T cells prevent murine acute graft-versus-host disease.

BACKGROUND/AIMS: Adoptive therapy with regulatory T (Treg) cells to prevent graft-versus-host disease (GVHD) would benefit from a strategy to improve homing to the sites of inflammation following hematopoietic stem cell transplantation (HSCT). Although donor-derived Treg cells have mainly been used in these models, third-party-derived Treg cells are a promising alternative for cell-based immunotherapy, as they can be screened for pathogens and cell activity, and banked for GVHD prevention. In this study, we explored major histocompatibility complex (MHC) disparities between Treg cells and conventional T cells in HSCT to evaluate the impact of these different cell populations on the prevention of acute GVHD, as well as survival after allogeneic transplantation. METHODS: To induce acute GVHD, lethally irradiated BALB/c (H-2d) mice were transplanted with 5 × 105 T cell-depleted bone marrow cells and 5 × 105 CD4+CD25- splenic T cells from C57BL/6 (H-2b) mice. Recipients were injected with 5 × 105 cultured donor-, host-, or third-party-derived CD4+CD25+CD62L+ Treg cells (bone marrow transplantation + day 1). RESULTS: Systemic infusion of three groups of Treg cell improved clinicopathological manifestations and survival in an acute GVHD model. Although donor-derived Treg cells were immunologically the most effective, the third-party-derived Treg cell therapy group displayed equal regulation of expansion of CD4+CD25+- Foxp3+ Treg cells and suppressive CD4+IL-17+ T-helper (Th17) cells in ex vivo assays compared with the donor- and host-derived groups. CONCLUSION: Our findings demonstrate that the use of third-party Treg cells is a viable alternative to donor-derived Treg cellular therapy in clinical settings, in which human leukocyte antigen-matched donors are not always readily available.