技术资料

Mast cells (MCs) are tissue-resident immune cells known to degranulate in response to FcεRI crosslinking or MRGPRX2 engagement. MCs are found close to nerves,but the mechanisms that regulate this privileged localization remain unclear. Here,we investigated MRGPRX2 expression patterns and specific activities in MCs. We show that MRGPRX2 expression is heterogeneous in human MC (hMC) progenitors and mature MCs. Substance P (SP) is a rapid and specific activator of MRGPRX2,and long-term supplementation of MCs with SP expands MRGPRX2-expressing cells. While high concentrations of SP induce rapid MC degranulation,low concentrations prompt immature MC chemotaxis. Lastly,we demonstrate that in inflammatory skin conditions like psoriasis,the number of MRGPRX2 + MCs is increased,and during in vitro skin reinnervation,MRGPRX2 + MCs preferentially reside in proximity to and migrate toward SP + nerve fibers (NFs). This indicates that SP-MRGPRX2 signaling defines MC positioning and relocation within tissues and promotes immune cell-NF communication. Subject areas: Immunology,Molecular biology,Cell biology View Publication

Long-term culture of primary lymphocytes and hematopoietic stem and progenitor cells (HSPCs) is pivotal to their expansion and study. Furthermore,genetic engineering of the above-mentioned primary human cells has several safety needs,including the requirement of efficient in vitro assays for unwanted tumorigenic events. In this work,we tested and optimized the Miniaturized Optically Accessible Bioreactor (MOAB) platform. The MOAB consists of a millifluidic cell culture device with three optically-accessible culture chambers. Inside the MOAB,we inserted a silk-based framework that resembles some properties of the bone marrow environment and cultivated in this device either CD4 + T lymphocytes isolated from healthy donor buffy coat or cord blood-derived hematopoietic CD34 + cells. A fraction of these cells is viable for up to 3 months. Next,we tested the capability of the MOAB to detect tumorigenic events. Serial dilutions of engineered fluorescent tumor cells were mixed with either CD4 + or CD34 + primary cells,and their growth was followed. By this approach,we successfully detected as little as 100 tumorigenic cells mixed with 100,000 primary cells. We found that non-tumorigenic primary cells colonized the silk environment,whereas tumor cells,after an adaptation phase,expanded and entered the circulation. We conclude that the millifluidic platform allows the detection of rare tumorigenic events in the long-term culture of human cells. View Publication

Induced pluripotent stem cell (iPSC)-derived mesenchymal stromal cells (iMSCs) offer a promising alternative to primary mesenchymal stromal cells (MSCs) and their derivatives,particularly extracellular vesicles (EVs),for use in advanced therapy medicinal products. In this study we evaluated the immunomodulatory and regenerative potential of iMSCs as well as iMSC-EVs,alongside primary human umbilical cord-derived mesenchymal stromal cells (hUCMSCs). Our findings demonstrate that iMSCs exhibit comparable abilities to hUCMSCs in regulating lymphocyte proliferation and inducing an anti-inflammatory phenotype in monocytes. We also observed decreased TNFα levels and increased IL-10 induction,indicating a potential mechanism for their immunomodulatory effects. Furthermore,iMSC-EVs also showed effective immunomodulation by inhibiting T cell proliferation and inducing macrophage polarization similar to their parental cells. Additionally,iMSC-EVs exhibited pro-regenerative potential akin to hUCMSC-EVs in in vitro scratch assays. Notably,priming iMSCs with pro-inflammatory cytokines significantly enhanced the immunomodulatory potential of iMSC-EVs. These results underscore the considerable promise of iMSCs and iMSCs-EVs as an alternate source for MSC-derived therapeutics,given their potent immunomodulatory and regenerative properties. The online version contains supplementary material available at 10.1038/s41598-024-75956-3. View Publication

Translational medicine provides insight into novel drugs and predicts unwanted effects. In well-characterized pathways (e.g.,cytokine-Janus kinase [JAK]-signal transducers and activators of transcription [STAT]),a variety of in vitro assessments were used to estimate selectivity of effects on different potential targets (i.e.,JAK1,JAK2,JAK3,and tyrosine kinase 2 [TYK2]). Several approved drugs were characterized as selective for the JAK family. These assessments are challenged by a lack of compounds that only inhibit one JAK family member. Deucravacitinib is a first-in-class,oral,selective,allosteric inhibitor of TYK2,a kinase required for IL-12,IL-23,and Type I interferon signaling. Unlike deucravacitinib,which selectively binds to the TYK2 regulatory domain,JAK1,2,3 inhibitors target the catalytic domain,contributing to nonselective targeting of JAK1,2,3. Cytokines associated with JAK1,2,3 signaling are required for both immune and nonimmune functions. A similar laboratory abnormality profile was observed in clinical trials using JAK1,2,3 inhibitors that has not been observed with deucravacitinib. In vitro testing of JAK1,2,3 inhibitors has relied upon assays of signal transduction,such as those measuring STAT phosphorylation,for estimates of potency and selectivity. These assay systems can be effective in estimating in vivo efficacy; however,they may not provide insight into downstream outcomes of receptor signaling,which may be more relevant for evaluating safety aspects. Assay systems assessing functional outcomes from cells may yield a more useful translational evaluation. Here,deucravacitinib was assessed for potency and selectivity versus three representatives of the JAK inhibitor class (tofacitinib,baricitinib,and upadacitinib) based on functional assays. JAK inhibitors had suppressive activity against JAK2-dependent hematopoietic colony-forming assays modeling thrombopoiesis,erythropoiesis,and myelopoiesis; however,deucravacitinib did not. Deucravacitinib had limited potency against NK cells,cytotoxic T cells,T-helper cells,and regulatory T cells activated by JAK1/JAK3-dependent common gamma chain cytokines. These data are consistent with the biologic role of JAK1,2,3 and pharmacodynamic changes in clinical laboratory abnormalities. Against TYK2-dependent cytokines,deucravacitinib selectively inhibited Type I interferon stimulation of monocytes and dendritic cells and was a more potent inhibitor than JAK inhibitors. IL-12 and IL-23 functional outputs were similarly potently inhibited by deucravacitinib. Results are consistent with deucravacitinib selectively inhibiting TYK2. View Publication

Dysregulated innate immune responses underlie multiple inflammatory diseases,but clinical translation of preclinical innate immunity research in mice is hampered by the difficulty of studying human inflammatory reactions in an in vivo context. We therefore sought to establish in vivo human inflammatory responses in NSG-QUAD mice that express four human myelopoiesis transgenes to improve engraftment of a human innate immune system. We reconstituted NSG-QUAD mice with human hematopoietic stem and progenitor cells (HSPCs),after which we evaluated human myeloid cell development and subsequent human responses to systemic and local lipopolysaccharide (LPS) challenges. NSG-QUAD mice already displayed engraftment of human monocytes,dendritic cells and granulocytes in peripheral blood,spleen and liver at 6 weeks after HSPC reconstitution,in which both classical,intermediate and non-classical monocytes were present. These huNSG-QUAD mice responded to intraperitoneal and intranasal LPS challenges with production of NF-κB-dependent human cytokines,a human type I interferon response,as well as inflammasome-mediated production of human IL-1β and IL-18. The latter were specifically abrogated by the NLRP3 inhibitor MCC950,while LPS-induced human monocyte death was not altered. Besides providing proof-of-principle for small molecule testing of human inflammatory reactions in huNSG-QUAD mice,this observation suggests that LPS-induced in vivo release of human NLRP3 inflammasome-generated cytokines occurs in a cell death-independent manner. HuNSG-QUAD mice are competent for the NF-κB,interferon and inflammasome effectors of human innate immunity,and can thus be utilized to investigate signaling mechanisms and pharmacological targeting of human inflammatory responses in an in vivo setting. View Publication

CD19CAR-T cell therapy demonstrated promising outcomes in relapsed/refractory B-cell malignancies. Nonetheless,the limited T-cell function and ineffective T-cell apheresis for therapeutic purposes are still concern in heavily pretreated patients. We investigated the feasibility of generating hematopoietic stem cell-derived T lymphocytes (HSC-T) for cancer immunotherapy. The patients’ autologous peripheral blood HSCs were enriched for CD34 + and CD3 + cells. The CD34 + cells were then cultured following three steps of lymphoid progenitor differentiation,T-cell differentiation,and T-cell maturation processes. HSC-T cells were successfully generated with robust fold expansion of 3735 times. After lymphoid progenitor differentiation,CD5 + and CD7 + cells remarkably increased (65–84 %) while CD34 + cells consequentially declined. The mature CD3 + cells were detected up to 40 % and 90 % on days 42 and 52,respectively. The majority of HSC-T population was naïve phenotype compared to CD3-T cells (73 % vs 34 %) and CD8:CD4 ratio was 2:1. The higher level of cytokine and cytotoxic granule secretion in HSC-T was observed after activation. HSC-T cells were assessed for clinical application and found that CD19CAR-transduced HSC-T cells demonstrated higher cytokine secretion and a trend of superior cytotoxicity against CD19 + target cells compared to control CAR-T cells. A chronic antigen stimulation assay revealed similar T-cell proliferation,stemness,and exhaustion phenotypes among CAR-T cell types. In conclusions,autologous HSC-T was feasible to generate with preserved T-cell efficacy. The HSC-T cells are potentially utilized as an alternative option for cellular immunotherapy. View Publication

Breast cancer is the most frequently diagnosed cancer worldwide,constituting 15% of cases in 2023. The predominant cause of breast cancer-related mortality is metastasis,and a lack of metastasis-targeted therapies perpetuates dismal outcomes for late-stage patients. By using meiotic genetics to study inherited transcriptional network regulation,we have identified,to the best of our knowledge,a new class of “essential expression-restricted” genes as potential candidates for metastasis-targeted therapeutics. Building upon previous work implicating the CCR4-NOT RNA deadenylase complex in metastasis,we demonstrate that RNA-binding proteins NANOS1,PUM2,and CPSF4 also regulate metastatic potential. Using various models and clinical data,we pinpoint Smarcd1 mRNA as a target of all three RNA-BPs. Strikingly,both high and low expression of Smarcd1 correlate with positive clinical outcomes,while intermediate expression significantly reduces the probability of survival. Applying the theory of “essential genes” from evolution,we identify 50 additional genes that require precise expression levels for metastasis to occur. Specifically,small perturbations in Smarcd1 expression significantly reduce metastasis in mouse models and alter splicing programs relevant to the ER+/HER2-enriched breast cancer. Identification subtype-specific essential expression-restricted metastasis modifiers introduces a novel class of genes that,when therapeutically “nudged” in either direction,may significantly improve late-stage breast cancer patients. Subject terms: Metastasis,Cancer genetics,Breast cancer View Publication

Human pluripotent stem cells (hPSCs) represent a powerful model system to study early developmental processes. However,lineage specification into trophectoderm (TE) and trophoblast (TB) differentiation remains poorly understood,and access to well-characterized placental cells for biomedical research is limited,largely depending on fetal tissues or cancer cell lines. Here,we developed novel strategies enabling highly efficient TE specification that generates cytotrophoblast (CTB) and multinucleated syncytiotrophoblast (STB),followed by the establishment of trophoblast stem cells (TSCs) capable of differentiating into extravillous trophoblast (EVT) and STB after long-term expansion. We confirmed stepwise and controlled induction of lineage- and cell-type-specific genes consistent with developmental biology principles and benchmarked typical features of placental cells using morphological,biochemical,genomics,epigenomics,and single-cell analyses. Charting a well-defined roadmap from hPSCs to distinct placental phenotypes provides invaluable opportunities for studying early human development,infertility,and pregnancy-associated diseases. Subject areas: Natural sciences,Biological sciences,Cell biology,Stem cells research View Publication

Despite significant progress in the prognosis of pediatric T-cell acute lymphoblastic leukemia (T-ALL) in recent decades,a notable portion of children still confronts challenges such as treatment resistance and recurrence,leading to limited options and a poor prognosis. LIM domain-binding protein 1 (LDB1) has been confirmed to exert a crucial role in various physiological and pathological processes. In our research,we aim to elucidate the underlying function and mechanisms of LDB1 within the background of T-ALL. Employing short hairpin RNA (shRNA) techniques,we delineated the functional impact of LDB1 in T-ALL cell lines. Through the application of RNA-Seq,CUT&Tag,and immunoprecipitation assays,we scrutinized master transcription factors cooperating with LDB1 and identified downstream targets under LDB1 regulation. LDB1 emerges as a critical transcription factor co-activator in cell lines derived from T-ALL. It primarily collaborates with master transcription factors (ERG,ETV6,IRF1) to cooperatively regulate the transcription of downstream target genes. Both in vitro and in vivo experiments affirm the essential fuction of LDB1 in the proliferation and survival of cell lines derived from T-ALL,with MYB identified as a significant downstream target of LDB1. To sum up,our research establishes the pivotal fuction of LDB1 in the tumorigenesis and progression of T-ALL cell lines. Mechanistic insights reveal that LDB1 cooperates with ERG,ETV6,and IRF1 to modulate the expression of downstream effector genes. Furthermore,LDB1 controls MYB through remote enhancer modulation,providing valuable mechanistic insights into its involvement in the progression of T-ALL. The online version contains supplementary material available at 10.1186/s13046-024-03199-1. View Publication

CRISPR-based genome editing of pseudogene-associated disorders,such as p47 phox -deficient chronic granulomatous disease (p47 CGD),is challenged by chromosomal rearrangements due to presence of multiple targets. We report that interactions between highly homologous sequences that are localized on the same chromosome contribute substantially to post-editing chromosomal rearrangements. We successfully employed editing approaches at the NCF1 gene and its pseudogenes,NCF1B and NCF1C,in a human cell line model of p47 CGD and in patient-derived human hematopoietic stem and progenitor cells. Upon genetic engineering,a droplet digital PCR-based method identified cells with altered copy numbers,spanning megabases from the edited loci. We attributed the high aberration frequency to the interaction between repetitive sequences and their predisposition to recombination events. Our findings emphasize the need for careful evaluation of the target-specific genomic context,such as the presence of homologous regions,whose instability can constitute a risk factor for chromosomal rearrangements upon genome editing. Subject terms: CRISPR-Cas9 genome editing,Targeted gene repair,Haematopoietic stem cells View Publication

Human pluripotent stem cells (hPSCs) have the potential to differentiate into various cell types,including pancreatic insulin-producing β cells,which are crucial for developing therapies for diabetes. However,current methods for directing hPSC differentiation towards pancreatic β-like cells are often inefficient and produce cells that do not fully resemble the native counterparts. Here,we report that highly selective tankyrase inhibitors,such as WIKI4,significantly enhances pancreatic differentiation from hPSCs. Our results show that WIKI4 promotes the formation of pancreatic progenitors that give rise to islet-like cells with improved β-like cell frequencies and glucose responsiveness compared to our standard cultures. These findings not only advance our understanding of pancreatic development,but also provide a promising new tool for generating pancreatic cells for research and potential therapeutic applications. Subject terms: Stem-cell differentiation,Organogenesis,Type 1 diabetes View Publication

Addiction is known to occur through the consumption of substances such as pharmaceuticals,illicit drugs,food,alcohol and tobacco. These addictions can be viewed as drug addiction,resulting from the ingestion of chemical substances contained in them. Multiple neural networks,including the reward system,anti‐reward/stress system and central immune system in the brain,are believed to be involved in the onset of drug addiction. Although various compound evaluations using microelectrode array (MEA) as an in vitro testing methods to evaluate neural activities have been conducted,methods for assessing addiction have not been established. In this study,we aimed to develop an in vitro method for assessing the addiction of compounds,as an alternative to animal experiments,using human iPS cell‐derived dopaminergic neurons with MEA measurements. MEA data before and after chronic exposure revealed specific changes in addictive compounds compared to non‐addictive compounds,demonstrating the ability to estimate addiction of compound. Additionally,conducting gene expression analysis on cultured samples after the tests revealed changes in the expression levels of various receptors (nicotine,dopamine and GABA) due to chronic administration of addictive compounds,suggesting the potential interpretation of these expression changes as addiction‐like responses in MEA measurements. The addiction assessment method using MEA measurements in human iPS cell‐derived dopaminergic neurons conducted in this study proves effective in evaluating addiction of compounds on human neural networks. View Publication