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.

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.

The pro-domains of neurotrophins, including BDNF, are linked to Alzheimer's disease through a toxic synergy with Aß.

Brain-derived neurotrophic factor (BDNF) has a crucial role in learning and memory by promoting neuronal survival and modulating synaptic connectivity. BDNF levels are lower in the brains of individuals with Alzheimer's disease (AD), suggesting a pathogenic involvement. The Drosophila orthologue of BDNF is the highly conserved Neurotrophin 1 (DNT1). BDNF and DNT1 have the same overall protein structure and can be cleaved, resulting in the conversion of a full-length polypeptide into separate pro- and mature-domains. While the BDNF mature-domain is neuroprotective, the role of the pro-domain is less clear. In flies and mammalian cells, we have identified a synergistic toxic interaction between the amyloid-ß peptide (Aß1-42) and the pro-domains of both DNT1 and BDNF. Specifically, we show that DNT1 pro-domain acquires a neurotoxic activity in the presence of Aß1-42. In contrast, DNT1 mature-domain is protective against Aß1-42 toxicity. Likewise, in SH-SY5Y cell culture, BDNF pro-domain is toxic only in the presence of Aß1-42. Western blots indicate that this synergistic interaction likely results from the Aß1-42-induced upregulation of the BDNF pro-domain receptor p75(NTR). The clinical relevance of these findings is underlined by a greater than thirty fold increase in the ratio of BDNF pro- to mature-domains in the brains of individuals with AD. This unbalanced BDNF pro:mature-domain ratio in patients represents a possible biomarker of AD and may offer a target for therapeutic intervention.

The Free Radical Scavenger NecroX-7 Attenuates Acute Graft-versus-Host Disease via Reciprocal Regulation of Th1/Regulatory T Cells and Inhibition of HMGB1 Release.

Graft-versus-host disease (GVHD) is a major complication associated with allogeneic hematopoietic stem cell transplantation. Despite the prominent role of the adaptive immune system, the importance of controlling the innate immune system in the pathogenesis of GVHD has recently been rediscovered. High-mobility group box 1 (HMGB1) is a crucial damage-associated molecular pattern signal that functions as a potent innate immune mediator in GVHD. In the present study, we investigated treatment of experimental GVHD through HMGB1 blockade using the compound cyclopentylamino carboxymethylthiazolylindole (NecroX)-7. Treated animals significantly attenuated GVHD-related mortality and inhibited severe tissue damage. These protective effects correlated with the decrease in HMGB1 expression and lower levels of reactive oxidative stress. Additionally, NecroX-7 inhibited the HMGB1-induced release of TNF and IL-6, as well as the expression of TLR-4 and receptor for advanced glycation end products. We also observed increased regulatory T cell numbers, which may be associated with regulation of differentiation signals independent of HMGB1. Taken together, these data indicate that NecroX-7 protects mice against lethal GVHD by reciprocal regulation of regulatory T/Th1 cells, attenuating systemic HMGB1 accumulation and inhibiting HMGB1-mediated inflammatory response. Our results indicate the possibility of a new use for a clinical drug that is effective for the treatment of GVHD.

Therapeutic potential of low-dose IL-2 in a chronic GVHD patient by in vivo expansion of regulatory T cells.

Chronic graft-versus-host disease (cGVHD) is a common complication following allogeneic hematopoietic stem cell transplantation (HSCT), which is characterized by autoimmune like inflammatory responses and reduced levels of regulatory T cells (Tregs). Recently, the use of low-dose IL-2 has been reported to selectively increase Tregs and therefore facilitate immune regulation and promote clinical improvements in cGVHD patients. In this report, we describe the case of a cGVHD patient who was treated with daily low-dose IL-2 therapy. Our observations demonstrate that low-dose IL-2 could induce significant expansion of Tregs in vivo leading to improved Treg/Th17 ratios. The patient showed moderate clinical benefits suggesting that multiple factors may be involved in the immunological responses. Therefore, while the therapeutic potential of low-dose IL-2 is promising, strategic approaches may be needed to induce a clinically significant and sustained Treg effect.

Mesenchymal stem cells for the treatment and prevention of graft-versus-host disease: experiments and practice.

Mesenchymal stem cells (MSCs) have emerged as a therapeutic approach in a range of medical fields, including regenerative medicine, cancer, autoimmune diseases, and inflammatory diseases, because of their unique properties of tissue repair and major histocompatibility complex-unmatched immunosuppression. Because both in vitro and in vivo findings demonstrate that MSCs possess potent immunoregulatory functions, there has been increasing interest in the role of MSCs in allogeneic hematopoietic stem cell transplantation, especially in the prevention and treatment of graft-versus-host disease (GVHD). GVHD is a major cause of transplantation-related mortality, and conventional immunosuppressants frequently fail to treat patients suffering from GVHD. Following Ringden's pilot study that used third-party MSCs to treat a steroid-refractory GVHD patient, MSCs have created growing interest as a therapeutic agent for GVHD. There have been further studies which demonstrated the potentials of MSC treatment in steroid-refractory GVHD, de novo GVHD, and also GVHD prevention. However, MSCs still present limitations. The need for MSCs to be "licensed" in a pro-inflammatory environment, especially in the presence of interferon gamma, allows only a narrow window for their administration. Thus, their effects have been less clear as a preventive measure before the inflammatory environment of GVHD is established and also when administered during a chronic setting where MSCs may be alternatively licensed. In this review, we focus on the immunomodulatory properties of MSCs and their effects in relation to GVHD. Given the efficacy of MSCs in murine models of GVHD and their safety in clinical trials, it is crucial that larger clinical trials are conducted and further modifications are investigated.

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.

Human umbilical cord blood-derived mesenchymal stem cell therapy promotes functional recovery of contused rat spinal cord through enhancement of endogenous cell proliferation and oligogenesis.

Numerous studies have shown the benefits of mesenchymal stem cells (MSCs) on the repair of spinal cord injury (SCI) model and on behavioral improvement, but the underlying mechanisms remain unclear. In this study, to investigate possible mechanisms by which MSCs contribute to the alleviation of neurologic deficits, we examined the potential effect of human umbilical cord blood-derived MSCs (hUCB-MSCs) on the endogenous cell proliferation and oligogenesis after SCI. SCI was injured by contusion using a weight-drop impactor and hUCB-MSCs were transplanted into the boundary zone of the injured site. Animals received a daily injection of bromodeoxyuridine (BrdU) for 7 days after treatment to identity newly synthesized cells of ependymal and periependymal cells that immunohistochemically resembled stem/progenitor cells was evident. Behavior analysis revealed that locomotor functions of hUCB-MSCs group were restored significantly and the cavity volume was smaller in the MSCs-transplanted rats compared to the control group. In MSCs-transplanted group, TUNEL-positive cells were decreased and BrdU-positive cells were significantly increased rats compared with control group. In addition, more of BrdU-positive cells expressed neural stem/progenitor cell nestin and oligo-lineage cell such as NG2, CNPase, MBP and glial fibrillary acidic protein typical of astrocytes in the MSC-transplanted rats. Thus, endogenous cell proliferation and oligogenesis contribute to MSC-promoted functional recovery following SCI.

Valproic acid downregulates the expression of MGMT and sensitizes temozolomide-resistant glioma cells.

Temozolomide (TMZ) has become a key therapeutic agent in patients with malignant gliomas; however, its survival benefit remains unsatisfactory. Valproic acid (VPA) has emerged as an anticancer drug via inhibition of histone deacetylases (HDACs), but the therapeutic advantages of a combination with VPA and TMZ remain poorly understood. The main aim of the present study was to determine whether an antitumor effect could be potentiated by a combination of VPA and TMZ, especially in TMZ-resistant cell lines. A combination of VPA and TMZ had a significantly enhanced antitumor effect in TMZ-resistant malignant glioma cells (T98 and U138). This enhanced antitumor effect correlated with VPA-mediated reduced O6-methylguanine-DNA methyltransferase (MGMT) expression, which plays an important role in cellular resistance to alkylating agents. In vitro, the combination of these drugs enhanced the apoptotic and autophagic cell death, as well as suppressed the migratory activities in TMZ-resistant cell lines. Furthermore, in vivo efficacy experiment showed that treatment of combination of VPA and TMZ significantly inhibited tumor growth compared with the monotherapy groups of mice. These results suggest that the clinical efficacy of TMZ chemotherapy in TMZ-resistant malignant glioma may be improved by combination with VPA.

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.

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.

Irradiation enhances the tumor tropism and therapeutic potential of tumor necrosis factor-related apoptosis-inducing ligand-secreting human umbilical cord blood-derived mesenchymal stem cells in glioma therapy.

Irradiation is a standard therapy for gliomas and many other cancers. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is one of the most promising candidates for cancer gene therapy. Here, we show that tumor irradiation enhances the tumor tropism of human umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) and the therapeutic effect of TRAIL delivered by UCB-MSCs. The sequential treatment with irradiation followed by TRAIL-secreting UCB-MSCs (MSC-TRAIL) synergistically enhanced apoptosis in either TRAIL-sensitive or TRAIL-resistant glioma cells by upregulating the death receptor 5 and by inducing caspase activation. Migration assays showed greater MSC migration toward irradiated glioma cells and the tumor site in glioma-bearing mice compared with unirradiated tumors. Irradiated glioma cells had increased expression of interleukin-8 (IL-8), which leads to the upregulation of the IL-8 receptor on MSCs. This upregulation, which is involved in the migratory capacity of UCB-MSCs, was confirmed by siRNA inhibition and an antibody-neutralizing assay. In vivo survival experiments in orthotopic xenografted mice showed that MSC-based TRAIL gene delivery to irradiated tumors had greater therapeutic efficacy than a single treatment. These results suggest that clinically relevant tumor irradiation increases the therapeutic efficacy of MSC-TRAIL by increasing tropism of MSCs and TRAIL-induced apoptosis, which may be a more useful strategy for cancer gene therapy.

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.

Migration of human umbilical cord blood mesenchymal stem cells mediated by stromal cell-derived factor-1/CXCR4 axis via Akt, ERK, and p38 signal transduction pathways.

Human mesenchymal stem cells (hMSCs) have been used for cell-based therapies in degenerative disease and as vehicles for delivering therapeutic genes to sites of injury and tumors. Recently, umbilical cord blood (UCB) was identified as a source for MSCs, and human UCB-derived MSCs (hUCB-MSCs) can serve as an alternative source of bone marrow-derived mesenchymal stem cells (BM-MSCs). However, migration signaling pathways required for homing and recruitment of hUCB-MSCs are not fully understood. Stromal cell-derived factor-1 (SDF-1), a ligand for the CXCR4 chemokine receptor, plays a pivotal role in mobilization and homing of stem cells and modulates different biological responses in various stem cells. In this study, expression of CXCR4 in hUCB-MSCs was studied by western blot analysis and the functional role of SDF-1 was assessed. SDF-1 induced the migration of hUCB-MSCs in a dose-dependent manner. The induced migration was inhibited by the CXCR4-specific peptide antagonist (AMD3100) and by inhibitors of phosphoinositide 3-kinase (LY294002), mitogen-activated protein kinase/extracellular signal related kinase (PD98059) and p38MAPK inhibitor (SB203580). hUCB-MSCs treated with SDF-1 displayed increased phosphorylation of Akt, ERK and p38, which were inhibited by AMD3100. Small-interfering RNA-mediated knock-down of Akt, ERK and p38 blocked SDF-1 induced hUCB-MSC migration. In addition, SDF-1-induced actin polymerization was also blocked by these inhibitors. Taken together, these results demonstrate that Akt, ERK and p38 signal transduction pathways may be involved in SDF-1-mediated migration of hUCB-MSCs.

Microporation is a valuable transfection method for efficient gene delivery into human umbilical cord blood-derived mesenchymal stem cells.

BACKGROUND: Mesenchymal stem cells (MSCs) are an attractive source of adult stem cells for therapeutic application in clinical study. Genetic modification of MSCs with beneficial genes makes them more effective for therapeutic use. However, it is difficult to transduce genes into MSCs by common transfection methods, especially nonviral methods. In this study, we applied microporation technology as a novel electroporation technique to introduce enhanced green fluorescent protein (EGFP) and brain-derived neurotropfic factor (BDNF) plasmid DNA into human umbilical cord blood-derived MSCs (hUCB-MSCs) with significant efficiency, and investigated the stem cell potentiality of engineered MSCs through their phenotypes, proliferative capacity, ability to differentiate into multiple lineages, and migration ability towards malignant glioma cells. RESULTS: Using microporation with EGFP as a reporter gene, hUCB-MSCs were transfected with higher efficiency (83%) and only minimal cell damage than when conventional liposome-based reagent (<20%) or established electroporation methods were used (30-40%). More importantly, microporation did not affect the immunophenotype of hUCB-MSCs, their proliferation activity, ability to differentiate into mesodermal and ectodermal lineages, or migration ability towards cancer cells. In addition, the BDNF gene could be successfully transfected into hUCB-MSCs, and BDNF expression remained fairly constant for the first 2 weeks in vitro and in vivo. Moreover, microporation of BDNF gene into hUCB-MSCs promoted their in vitro differentiation into neural cells. CONCLUSION: Taken together, the present data demonstrates the value of microporation as an efficient means of transfection of MSCs without changing their multiple properties. Gene delivery by microporation may enhance the feasibility of transgenic stem cell therapy.

MK886-induced apoptosis depends on the 5-LO expression level in human malignant glioma cells.

Mounting evidence suggests that lipoxygenase (LO)-catalyzed products may play a key role in the development and progression of human cancers. In this study, we analyzed the effects of a 5-LO inhibitor, which inhibits the conversion of arachidonic acid to leukotrienes, on cell proliferation and apoptosis in human malignant glioma cells, including 5-LO-expressing cells U-87MG, A172 and 5-LO non-expressing cell U373. Growth of U-87MG and A172 cells, but not that of U373 cells, was inhibited in a dose-dependent manner by treatment with MK886. Similarly, specific 5-LO silencing by small interfering RNA reduced the growth of U-87MG and A172 cells. MK886 treatment reduced 5-LO activity independently of 5-LO-activating protein (FLAP) in human malignant glioma cells. MK886 treatment also induced cell apoptosis, measured by DNA fragmentation and nuclear condensation, in U-87MG and A172 cells but there were no signs in U373 cells. Moreover, this treatment reduced ERKs phosphorylation and anti-apoptotic molecule Bcl-2 expression, and increased Bax expression in U-87MG and A172 cells. In summary, our results show there is a link between the 5-LO expression status and the extent of MK886-inhibited cell proliferation and apoptosis. Taken together, this study suggest that 5-LO is a possible target for treating patients with gliomas, and 5-LO inhibition might be potent therapy for patients with 5-LO-expressing malignant gliomas.