技术资料

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

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

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

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

The increasing scarcity of organs and the significant morbidity linked to dialysis require the development of engineered kidney tissues from human-induced pluripotent stem cells. Integrative approaches that synergize scalable kidney organoid differentiation,tissue biomanufacturing,and comprehensive assessment of their immune response and host integration are essential to accomplish this. Here,we create engineered human kidney tissues composed of organoid building blocks (OBBs) and transplant them into mice reconstituted with allogeneic human immune cells. Tissue-infiltrating human immune cells are composed of effector T cells and innate cells. This immune infiltration leads to kidney tissue injury characterized by reduced microvasculature,enhanced kidney cell apoptosis,and an inflammatory gene signature comparable to kidney organ transplant rejection in humans. Upon treatment with the immunosuppressive agent rapamycin,the induced immune response is greatly suppressed. Our model is a translational platform to study engineered kidney tissue immunogenicity and develop therapeutic targets for kidney rejection. Subject areas: Health sciences,Immunology,Bioengineering,Tissue engineering 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

Isobaric labeling-based mass spectrometry (ILMS) has been widely used to quantify,on a proteome-wide scale,the relative protein abundance in different biological conditions. However,large-scale ILMS data sets typically involve multiple runs of mass spectrometry,bringing great computational difficulty to the integration of ILMS samples. We present zMAP,a toolset that makes ILMS intensities comparable across mass spectrometry runs by modeling the associated mean-variance dependence and accordingly applying a variance stabilizing z-transformation. The practical utility of zMAP is demonstrated in several case studies involving the dynamics of cell differentiation and the heterogeneity across cancer patients. The online version contains supplementary material available at 10.1186/s13059-024-03382-9. 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

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

Hereditary spastic paraplegias are rare genetic disorders characterized by corticospinal tract impairment. Spastic paraplegia 83 (SPG83) is associated with biallelic mutations in the HPDL gene,leading to varied severities from neonatal to juvenile onset. The function of HPDL is unclear,though it is speculated to play a role in alternative coenzyme Q10 biosynthesis. Here,we report the generation of hiPS lines from primary skin fibroblasts derived from three SPG83 patients with different HPDL mutations,using episomal reprogramming. The patients’ clinical characteristics are carefully listed. The hiPS lines were meticulously characterized,demonstrating typical pluripotent characteristics through immunofluorescence assays for stemness markers (OCT4,TRA1-60,NANOG,and SSEA4) and RT-PCR for endogenous gene expression. Genetic integrity and identity were confirmed via Sanger sequencing and short tandem repeat analysis. These hiPS cells displayed typical pluripotent characteristics and were able to differentiate into neocortical neurons via a dual SMAD inhibition protocol. In addition,HPDL mutant neurons assessed via long-term culturing were able to achieve effective maturation,similarly to their wild-type counterparts. The HPDL hiPS lines we generated will provide a valuable model for studying SPG83,offering insights into its molecular mechanisms and potential for developing targeted therapies. View Publication

Mesenchymal stromal cells (MSCs) are promising candidates for cartilage repair therapy due to their self-renewal,chondrogenic,and immunomodulatory capacities. It is widely recognized that a shift from fetal bovine serum (FBS)-containing medium toward a fully chemically defined serum-free (SF) medium would be necessary for clinical applications of MSCs to eliminate issues such as xeno-contamination and batch-to-batch variation. However,there is a notable gap in the literature regarding the evaluation of the chondrogenic ability of SF-expanded MSCs (SF-MSCs). In this study,we compared the in vivo regeneration effect of FBS-MSCs and SF-MSCs in a rat osteochondral defect model and found poor cartilage repair outcomes for SF-MSCs. Consequently,a comparative analysis of FBS-MSCs and SF-MSCs expanded using two SF media,MesenCult™-ACF (ACF),and Custom StemPro™ MSC SFM XenoFree (XF) was conducted in vitro. Our results show that SF-expanded MSCs constitute variations in morphology,surface markers,senescence status,differentiation capacity,and senescence/apoptosis status. Highly proliferative MSCs supported by SF medium do not always correlate to their chondrogenic and cartilage repair ability. Prior determination of the SF medium’s ability to support the chondrogenic ability of expanded MSCs is therefore crucial when choosing an SF medium to manufacture MSCs for clinical application in cartilage repair. View Publication

Despite a record number of clinical studies investigating various anti-myeloma treatments,the 5-year survival rate for multiple myeloma (MM) patients in the US is only 55%,and almost all patients relapse. Poor patient outcomes demonstrate that myeloma cells are “born to survive” which means they can adapt and evolve following treatment. Thus,new therapeutic approaches to combat survival mechanisms and target treatment resistance are required. Importantly,Mcl-1,anti-apoptotic protein,is required for the development of MM and treatment resistance. This study looks at the possibility of KS18,a selective Mcl-1 inhibitor,to treat MM and overcome resistance. Our investigation demonstrates that KS18 effectively induces cell death in MM by dual regulatory mechanisms targeting the Mcl-1 protein at both transcriptional and post-translational levels. Specifically,KS18 suppresses Mcl-1 activation via STAT-3 pathway and promotes Mcl-1 phosphorylation/ubiquitination/proteasome-dependent protein degradation (UPS). Significantly,KS18 triggered caspase-dependent apoptosis in MM patient samples and bortezomib-resistant cells,synergizing with venetoclax to boost apoptosis. KS18 promises to overcome bortezomib and venetoclax resistance and re-sensitize myeloma cells to chemotherapy. Furthermore,the study shows the tremendous impact of KS18 in inhibiting colony formation in bortezomib-resistant cells and demonstrates significant tumor shrinkage in KS18-treated NSG mice without notable toxicity signs after 4 weeks of therapy with a single acceptable dose each week,indicating its powerful anti-neoplastic and anti-resistance characteristics. This study strongly implies that KS18 may treat MM and provide new hope to patients who are experiencing recurrence or resistance. View Publication