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Human corneal endothelial cells are derived from neural crest and because of postmitotic arrest lack competence to repair cell loss from trauma,aging,and degenerative disorders such as Fuchs endothelial corneal dystrophy (FECD). Herein,we identified a rapidly proliferating subpopulation of cells from the corneal endothelium of adult normal and FECD donors that exhibited features of neural crest-derived progenitor (NCDP) cells by showing absence of senescence with passaging,propensity to form spheres,and increased colony forming efficacy compared with the primary cells. The collective expression of stem cell-related genes SOX2,OCT4,LGR5,TP63 (p63),as well as neural crest marker genes PSIP1 (p75(NTR)),PAX3,SOX9,AP2B1 (AP-2β),and NES,generated a phenotypic footprint of endothelial NCDPs. NCDPs displayed multipotency by differentiating into microtubule-associated protein 2,β-III tubulin,and glial fibrillary acidic protein positive neurons and into p75(NTR)-positive human corneal endothelial cells that exhibited transendothelial resistance of functional endothelium. In conclusion,we found that mitotically incompetent ocular tissue cells contain adult NCDPs that exhibit a profile of transcription factors regulating multipotency and neural crest progenitor characteristics. Identification of normal NCDPs in FECD-affected endothelium holds promise for potential autologous cell therapies. View Publication

BACKGROUND Glioblastoma (GBM) is the most common and aggressive form of primary brain tumor. Although numerous postoperative therapeutic strategies have already been developed,including radiotherapy,tumors inevitably recur after several years of treatment. The coinhibitory molecule B7-H4 negatively regulates T cell immune responses and promotes immune escape. Exosomes mediate intercellular communication and initiate immune evasion in the tumor microenvironment (TME). OBJECTIVE This study aimed to determine whether B7-H4 is upregulated by radiation and loaded into exosomes,thus contributing to immunosuppression and enhancing tumor growth. METHODS Iodixanol density-gradient centrifugation and flow cytometry were used to verify exosomal B7-H4. Na{\{i}}ve T cells were differentiated into Th1 cells with or without exosomes. T cell-secreted cytokines and markers of T cell subsets were measured. Mechanistically the roles of B7-H4 and ALIX in GBM were analyzed using databases and tissue samples. Co-immunoprecipitation and pull-down assays were used to tested the direct interactions between ATM and ALIX or STAT3. In vitro ATM kinase assays western blotting and site-directed mutation were used to assess ATM-mediated STAT3 phosphorylation. Finally the contribution of exosomal B7-H4 to immunosuppression and tumor growth was investigated in vivo. RESULTS Exosomes from irradiated GBM cells decreased the anti-tumor immune response of T cell in vitro and in vivo via delivered B7-H4. Mechanistically irradiation promoted exosome biogenesis by increasing the ATM-ALIX interaction. Furthermore the ATM-phosphorylated STAT3 was found to directly binds to the B7-H4 promoter to increase its expression. Finally the radiation-induced increase in exosomal B7-H4 induced FoxP3 expression during Th1 cell differentiation via the activated STAT1 pathway. In vivo exosomal B7-H4 decreased the radiation sensitivity of GBM cells and reduced the survival of GBM mice model. CONCLUSION This study showed that radiation-enhanced exosomal B7-H4 promoted immunosuppression and tumor growth hence defining a direct link between irradiation and anti-tumor immune responses. Our results suggest that co-administration of radiotherapy with anti-B7-H4 therapy could improve local tumor control and identify exosomal B7-H4 as a potential tumor biomarker." View Publication

BACKGROUND CD73 is an ectonucleotidase producing the immunosuppressor mediator adenosine. Elevated levels of circulating CD73 in patients with cancer have been associated with disease progression and poor response to immunotherapy. Immunosuppressive pathways associated with exosomes can affect T-cell function and the therapeutic efficacy of anti-programmed cell-death protein 1 (anti-PD-1) therapy. Here,we conducted a retrospective pilot study to evaluate levels of exosomal CD73 before and early during treatment with anti-PD-1 agents in patients with melanoma and its potential contribution to affect T-cell functions and to influence the clinical outcomes of anti-PD-1 monotherapy. METHODS Exosomes were isolated by mini size exclusion chromatography from serum of patients with melanoma (n=41) receiving nivolumab or pembrolizumab monotherapy. Expression of CD73 and programmed death-ligand 1 (PD-L1) were evaluated on exosomes enriched for CD63 by on-bead flow cytometry. The CD73 AMPase activity was evaluated by mass spectrometry,also in the presence of selective inhibitors of CD73. Interferon (IFN)-$\gamma$ production and granzyme B expression were measured in CD3/28 activated T cells incubated with exosomes in presence of the CD73 substrate AMP. Levels of CD73 and PD-L1 on exosomes were correlated with therapy response. Exosomes isolated from healthy subjects were used as control. RESULTS Isolated exosomes carried CD73 on their surface,which is enzymatically active in producing adenosine. Incubation of exosomes with CD3/28 activated T cells in the presence of AMP resulted in a significant reduction of IFN-$\gamma$ release,which was reversed by the CD73 inhibitor APCP or by the selective A2A adenosine receptor antagonist ZM241385. Expression levels of exosomal CD73 from serum of patients with melanoma were not significantly different from those in healthy subjects. Early on-treatment,expression levels of both CD73 and PD-L1 on exosomes isolated from patients receiving pembrolizumab or nivolumab monotherapy were significantly increased compared with baseline. Early during therapy exosomal PD-L1 increased in responders,while exosomal CD73 resulted significantly increased in non-responders. CONCLUSIONS CD73 expressed on exosomes from serum of patients with melanoma produces adenosine and contributes to suppress T-cell functions. Early on-treatment,elevated expression levels of exosomal CD73 might affect the response to anti-PD-1 agents in patients with melanoma who failed to respond to therapy. View Publication

BackgroundTakotsubo cardiomyopathy (TTC) is marked by an acute,transient,and reversible left ventricular systolic dysfunction triggered by stress,with endothelial dysfunction being one of its pathophysiological mechanisms. However,the precise molecular mechanism underlying the interaction between endothelial cells and cardiomyocytes during TTC remains unclear. This study reveals that exosomal miRNAs derived from endothelial cells exposed to catecholamine contribute to ion channel dysfunction in the setting of TTC.MethodsHuman-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were treated with epinephrine (Epi) or exosomes (Exo) from Epi-treated human cardiac microvascular endothelial cells (HCMECs) or Exo derived from HCMECs transfected with miR-126-3p. The immunofluorescence staining,flow cytometry,qPCR,single-cell contraction,intracellular calcium transients,patch-clamp,dual luciferase reporter assay and western blot were performed for the study.ResultsModeling TTC with high doses of epinephrine (Epi) treatment in hiPSC-CMs shows suppression of depolarization velocity (Vmax),prolongation of action potential duration (APD),and induction of arrhythmic events. Exo derived from HCMECs treated with Epi (Epi-exo) mimicked or enhanced the effects of Epi. Epi exposure led to elevated levels of miR-126-3p in both HCMECs and their exosomes. Exo enriched with miR-126-3p demonstrated similar effects as Epi-exo,establishing the crucial role of miR-126-3p in the mechanism of Epi-exo. Dual luciferase reporter assay coupled with gene mutation techniques identified that miR-126-3p was found to target the regulator of G-protein signaling 3 (RGS3) gene. Western blot and qPCR analyses confirmed that miR-126-3p-mimic reduced RGS3 expression in both HCMECs and hiPSC-CMs,indicating miR-126-3p inhibits RGS3 signaling. Additionally,miR-126-3p levels were significantly higher in the serum of TTC patients compared to healthy controls and patients who had recovered from TTC.ConclusionsOur study is the first to reveal that exosomal miR-126-3p,originating from endothelial cells,contributes to ion channel dysfunction by regulating RGS3 signaling in cardiomyocytes. These findings provide new perspectives on the pathogenesis of TTC and suggest potential therapeutic targets for treatment.Graphical Abstract Supplementary InformationThe online version contains supplementary material available at 10.1186/s13287-025-04157-0. View Publication

Activated neutrophil-derived exosomes reportedly contribute to the proliferation of airway smooth muscle cells (ASMCs),thereby aggravating the airway wall remodeling during asthma; however,the specific mechanism remains unclear. Lipopolysaccharide (LPS)-EXO and si-CRNDE-EXO were extracted from the media of human neutrophils treated with LPS and LPS??+??si-CRNDE (a siRNA targets long non-coding RNA CRNDE),respectively. Human ASMCs were co-cultured with LPS-EXO or si-CRNDE-EXO,and cell viability,proliferation and migration were measured. The interplay of colorectal neoplasia differentially expressed (CRNDE),inhibitor of nuclear factor kappa B kinase subunit beta (IKK$\beta$) and nuclear receptor subfamily 2 group C member 2 (TAK1) was explored using RNA immunoprecipitation (RIP) and Co-IP assays. A mouse model of asthma was induced using ovalbumin. CRNDE was upregulated in LPS-EXO and successfully transferred from LPS-treated neutrophils to ASMCs through exosome. Mechanically,CRNDE loaded in LPS-EXO reinforced TAK1-mediated IKK$\beta$ phosphorylation,thereby activating the nuclear factor kappa B (NF-$\kappa$B) pathway. Functionally,silencing CRNDE in LPS-EXO,an IKK$\beta$ inhibitor,and an NF-$\kappa$B inhibitor all removed the upregulation of cell viability,proliferation and migration induced by LPS-EXO in ASMCs. In the end,the in vivo experiment demonstrated that CRNDE knockdown in neutrophils effectively reduced the thickness of bronchial smooth muscle in a mouse model for asthma. Activated neutrophils-derived CRNDE was transferred to ASMCs through exosomes and activated the NF-$\kappa$B pathway by enhancing IKK$\beta$ phosphorylation. The latter promoted the proliferation and migration of ASMCs and then contributed to airway remodeling in asthma. View Publication

Background: Local ischemia is the main pathological performance in osteonecrosis of the femoral head (ONFH). There is currently no effective therapy to promote angiogenesis in the femoral head. Recent studies revealed that exosomes secreted by induced pluripotent stem cell-derived mesenchymal stem cells (iPS-MSC-Exos) have great therapeutic potential in ischemic tissues,but whether they could promote angiogenesis in ONFH has not been reported,and little is known regarding the underlying mechanism. Methods: iPS-MSC-Exos were intravenously injected to a steroid-induced rat osteonecrosis model. Samples of the femoral head were obtained 3 weeks after all the injections. The effects were assessed by measuring local angiogenesis and bone loss through histological and immunohistochemical (IHC) staining,micro-CT and three-dimensional microangiography. The effects of exosomes on endothelial cells were studied through evaluations of proliferation,migration and tube-forming analyses. The expression levels of angiogenic related PI3K/Akt signaling pathway of endothelial cells were evaluated following stimulation of iPS-MSC-Exos. The promoting effects of exosomes were re-evaluated following blockade of PI3K/Akt. Results: The in vivo study revealed that administration of iPS-MSC-Exos significantly prevented bone loss,and increased microvessel density in the femoral head compared with control group. We found that iPS-MSC-Exos significantly enhanced the proliferation,migration and tube-forming capacities of endothelial cells in vitro. iPS-MSC-Exos could activate PI3K/Akt signaling pathway in endothelial cells. Moreover,the promoting effects of iPS-MSC-Exos were abolished after blockade of PI3K/Akt on endothelial cells. Conclusions: Our findings suggest that transplantation of iPS-MSC-Exos exerts a preventative effect on ONFH by promoting local angiogenesis and preventing bone loss. The promoting effect might be attributed to activation of the PI3K/Akt signaling pathway on endothelial cells. The data provide the first evidence for the potential of iPS-MSC-Exos in treating ONFH. View Publication

BACKGROUND Recently,accumulating evidence has shown that exosomes,the naturally secreted nanocarriers of cells,can exert therapeutic effects in various disease models in the absence of parent cells. However,application of exosomes in bone defect repair and regeneration has been rarely reported,and little is known regarding their underlying mechanisms. METHODS Exosomes derived from human-induced pluripotent stem cell-derived mesenchymal stem cells (hiPS-MSC-Exos) were combined with tricalcium phosphate (β-TCP) to repair critical-sized calvarial bone defects,and the efficacy was assessed by histological examination. We evaluated the in vitro effects of hiPSC-MSC-Exos on the proliferation,migration,and osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (hBMSCs) by cell-counting,scratch assays,and qRT-PCR,respectively. Gene expression profiling and bioinformatics analyses were also used to identify the underlying mechanisms in the repair. RESULTS We found that the exosome/β-TCP combination scaffolds could enhance osteogenesis as compared to pure β-TCP scaffolds. In vitro assays showed that the exosomes could release from β-TCP and could be internalized by hBMSCs. In addition,the internalization of exosomes into hBMSCs could profoundly enhance the proliferation,migration,and osteogenic differentiation of hBMSCs. Furthermore,gene expression profiling and bioinformatics analyses demonstrated that exosome/β-TCP combination scaffolds significantly altered the expression of a network of genes involved in the PI3K/Akt signaling pathway. Functional studies further confirmed that the PI3K/Akt signaling pathway was the critical mediator during the exosome-induced osteogenic responses of hBMSCs. CONCLUSIONS We propose that the exosomes can enhance the osteoinductivity of β-TCP through activating the PI3K/Akt signaling pathway of hBMSCs,which means that the exosome/β-TCP combination scaffolds possess better osteogenesis activity than pure β-TCP scaffolds. These results indicate that naturally secreted nanocarriers-exosomes can be used as a bioactive material to improve the bioactivity of the biomaterials,and that hiPS-MSC-Exos combined with β-TCP scaffolds can be potentially used for repairing bone defects. View Publication

Umbilical cord blood (UCB) is increasingly being used for human hematopoietic stem cell (HSC) transplantation in children but often requires pooling multiple cords to obtain sufficient numbers for transplantation in adults. To overcome this limitation,we have used an ex vivo two-week culture system to expand the number of hematopoietic CD34(+) cells in cord blood. To assess the in vivo function of these expanded CD34(+) cells,cultured human UCB containing 1 x 10(6) CD34(+) cells were transplanted into conditioned NOD-scid IL2rgamma(null) mice. The expanded CD34(+) cells displayed short- and long-term repopulating cell activity. The cultured human cells differentiated into myeloid,B-lymphoid,and erythroid lineages,but not T lymphocytes. Administration of human recombinant TNFalpha to recipient mice immediately prior to transplantation promoted human thymocyte and T-cell development. These T cells proliferated vigorously in response to TCR cross-linking by anti-CD3 antibody. Engrafted TNFalpha-treated mice generated antibodies in response to T-dependent and T-independent immunization,which was enhanced when mice were co-treated with the B cell cytokine BLyS. Ex vivo expanded CD34(+) human UCB cells have the capacity to generate multiple hematopoietic lineages and a functional human immune system upon transplantation into TNFalpha-treated NOD-scid IL2rgamma(null) mice. View Publication

The role of the bone marrow (BM) microenvironment in regulating the antitumor immune response in Waldenstrom macroglobulinemia (WM) remains poorly understood. Here we transcriptionally and phenotypically profiled non-malignant (CD19- CD138-) BM cells from WM patients with a focus on myeloid derived suppressive cells (MDSCs) to provide a deeper understanding of their role in WM. We found that HLA-DRlowCD11b+CD33+ MDSCs were significantly increased in WM patients as compared to normal controls,with an expansion of predominantly polymorphonuclear (PMN)-MDSCs. Single-cell immunogenomic profiling of WM MDSCs identified an immune-suppressive gene signature with upregulated inflammatory pathways associated with interferon and tumor necrosis factor (TNF) signaling. Gene signatures associated with an inflammatory and immune suppressive environment were predominately expressed in PMN-MDSCs. In vitro,WM PMN-MDSCs demonstrated robust T-cell suppression and their viability and expansion was notably enhanced by granulocyte colony stimulating factor (G-CSF) and TNFα. Furthermore,BM malignant B-cells attracted PMN-MDSCs to a greater degree than monocytic MDSCs. Collectively,these data suggest that malignant WM B cells actively recruit PMN-MDSCs which promote an immunosuppressive BM microenvironment through a direct T cell inhibition,while release of G-CSF/TNFα in the microenvironment further promotes PMN-MDSC expansion and in turn immune suppression. Targeting PMN-MDSCs may therefore represent a potential therapeutic strategy in patients with WM. View Publication

BACKGROUND Lymphoid neoplasms,including multiple myeloma (MM),non-Hodgkin lymphoma (NHL),and NK/T cell neoplasms,are a major cause of blood cancer morbidity and mortality. CD38 (cyclic ADP ribose hydrolase) is a transmembrane glycoprotein expressed on the surface of plasma cells and MM cells. The high expression of CD38 across MM and other lymphoid malignancies and its restricted expression in normal tissues make CD38 an attractive target for immunotherapy. CD38-targeting antibodies,like daratumumab,have been approved for the treatment of MM and tested against lymphoma and leukemia in multiple clinical trials. METHODS We generated chimeric antigen receptor (CAR) T cells targeting CD38 and tested its cytotoxicity against multiple CD38high and CD38low lymphoid cancer cells. We evaluated the synergistic effects of all-trans retinoic acid (ATRA) and CAR T cells or daratumumab against cancer cells and xenograft tumors. RESULTS CD38-CAR T cells dramatically inhibited the growth of CD38high MM,mantle cell lymphoma (MCL),Waldenstrom's macroglobulinemia (WM),T-cell acute lymphoblastic leukemia (T-ALL),and NK/T-cell lymphoma (NKTCL) in vitro and in mouse xenografts. ATRA elevated CD38 expression in multiple CD38low cancer cells and enhanced the anti-tumor activity of daratumumab and CD38-CAR T cells in xenograft tumors. CONCLUSIONS These findings may expand anti-CD38 immunotherapy to a broad spectrum of lymphoid malignancies and call for the incorporation of ATRA into daratumumab or other anti-CD38 immunological agents for cancer therapy. View Publication

Introduction: Pathogenic variants in the OFD1 gene are linked to a spectrum of syndromes that exhibit partial clinical overlap. Hemizygous loss-of-function variants are considered lethal in males,while heterozygous loss-of-function variants generally result in oro-facial-digital syndrome type 1. A reported phenotype,Simpson–Golabi–Behmel syndrome type 2,was published once but remains controversial,with many specialists questioning its validity and arguing about its continued listing in the OMIM database. Methods: To investigate the genetic and phenotypic characteristics of the patients,we performed clinical exome sequencing,family-based genetic analysis,X-inactivation studies,electron microscopy,and detailed clinical assessments. Results: Three patients from unrelated families carrying loss-of-function variants in the OFD1 gene were identified,emphasizing the diverse phenotypic spectrum of OFD1 -associated disorders. The first patient,a female with a heterozygous frameshift variant p.(Gln398LeufsTer2),was diagnosed with oro-facial-digital syndrome type 1. The second patient,a male with a heterozygous nonsense variant p.(Gln892Ter),presented with features resembling Simpson–Golabi–Behmel syndrome type 2,as previously reported under this diagnosis. The third patient,a male with another heterozygous nonsense variant p.(Glu879Ter),exhibited isolated primary ciliary dyskinesia without any syndromic features. Conclusions: This study contributes to the growing body of evidence on the expanding phenotypic spectrum of OFD1 -associated disorders. It underscores the need for further investigation into the molecular mechanisms underlying the diverse presentations and the necessity of re-evaluating diagnostic classifications for conditions such as SGBS2 in the context of variants in the OFD1 gene. View Publication

Recent reports have documented the differentiation of human pluripotent stem cells toward the skeletal myogenic lineage using transgene- and cell purification-free approaches. Although these protocols generate myocytes,they have not demonstrated scalability,safety,and in vivo engraftment,which are key aspects for their future clinical application. Here we recapitulate one prominent protocol,and show that it gives rise to a heterogeneous cell population containing myocytes and other cell types. Upon transplantation,the majority of human donor cells could not contribute to myofiber formation. As a proof-of-principle,we incorporated the inducible PAX7 lentiviral system into this protocol,which then enabled scalable expansion of a homogeneous population of skeletal myogenic progenitors capable of forming myofibers in vivo. Our findings demonstrate the methods for scalable expansion of PAX7(+) myogenic progenitors and their purification are critical for practical application to cell replacement treatment of muscle degenerative diseases. View Publication