产品说明

Current protocols generate highly pure human induced pluripotent stem cell–derived cardiomyocytes (hiPSC‐CMs) in vitro that recapitulate characteristics of mature in vivo cardiomyocytes. Yet,a risk of arrhythmias exists when hiPSC‐CMs are injected into large animal models. Thus,understanding hiPSC‐CM maturational mechanisms is crucial for clinical translation. Forkhead box (FOX) transcription factors regulate postnatal cardiomyocyte maturation through a balance between FOXO and FOXM1. We also previously demonstrated that p53 activation enhances hiPSC‐CM maturation. Here,we investigate whether p53 activation modulates the FOXO/FOXM1 balance to promote hiPSC‐CM maturation in 3‐dimensional suspension culture. Three‐dimensional cultures of hiPSC‐CMs were treated with Nutlin‐3a (p53 activator,10 μM),LOM612 (FOXO relocator,5 μM),AS1842856 (FOXO inhibitor,1 μM),or RCM‐1 (FOXM1 inhibitor,1 μM),starting 2 days after onset of beating,with dimethyl sulfoxide (0.2% vehicle) as control. P53 activation promoted hiPSC‐CM metabolic and electrophysiological maturation alongside FOXO upregulation and FOXM1 downregulation,in n=3 to 6 per group for all assays. FOXO inhibition significantly decreased expression of cardiac‐specific markers such as TNNT2. In contrast,FOXO activation or FOXM1 inhibition promoted maturational characteristics such as increased contractility,oxygen consumption,and voltage peak maximum upstroke velocity,in n=3 to 6 per group for all assays. Further,by single‐cell RNA sequencing of n=2 LOM612‐treated cells compared with dimethyl sulfoxide,LOM612‐mediated FOXO activation promoted expression of cardiac maturational pathways. We show that p53 activation promotes FOXO and suppresses FOXM1 during 3‐dimensional hiPSC‐CM maturation. These results expand our understanding of hiPSC‐CM maturational mechanisms in a clinically‐relevant 3‐dimensional culture system. View Publication

The development of haematopoiesis involves the coordinated action of numerous genes,some of which are implicated in haematological malignancies. However,the biological function of many genes remains elusive and unknown functional genes are likely to remain to be uncovered. Here,we report a previously uncharacterised gene in haematopoiesis,identified by screening mutant embryonic stem cells. The gene,‘ attenuated haematopoietic development ( Ahed )’,encodes a nuclear protein. Conditional knockout (cKO) of Ahed results in anaemia from embryonic day 14.5 onward,leading to prenatal demise. Transplantation experiments demonstrate the incapacity of Ahed -deficient haematopoietic cells to reconstitute haematopoiesis in vivo. Employing a tamoxifen-inducible cKO model,we further reveal that Ahed deletion impairs the intrinsic capacity of haematopoietic cells in adult mice. Ahed deletion affects various pathways,and published databases present cancer patients with somatic mutations in Ahed . Collectively,our findings underscore the fundamental roles of Ahed in lifelong haematopoiesis,implicating its association with malignancies. Subject terms: Lymphopoiesis,Development,Haematopoietic stem cells,Differentiation View Publication

Hypoxia-activated prodrug (HAP) is a promising candidate for highly tumor-specific chemotherapy. However,the oxygenation heterogeneity and dense extracellular matrix (ECM) of tumor,as well as the potential resistance to chemotherapy,have severely impeded the resulting overall efficacy of HAP. A HAP potentiating strategy is proposed based on ultrasound responsive nanodroplets (PTP@PLGA),which is composed of protoporphyrin (PpIX),perfluoropropane (PFP) and a typical HAP,tirapazamine (TPZ). The intense vaporization of PFP upon ultrasound irradiation can magnify the sonomechanical effect,which loosens the ECM to promote the penetration of TPZ into the deep hypoxic region. Meanwhile,the PpIX enabled sonodynamic effect can further reduce the oxygen level,thus activating the TPZ in the relatively normoxic region as well. Surprisingly,abovementioned ultrasound effect also results in the downregulation of the stemness of cancer cells,which is highly associated with drug-refractoriness. This work manifests an ideal example of ultrasound-based nanotechnology for potentiating HAP and also reveals the potential acoustic effect of intervening cancer stem-like cells. The online version contains supplementary material available at 10.1186/s12951-024-02623-0. View Publication

Mononuclear phagocytes (MNP),including macrophages and dendritic cells form an essential component of primary responses to environmental hazards and toxic exposures. This is particularly important in disease conditions such as asthma and allergic airway disease,where many different cell types are present. In this study,we differentiated CD34+ haematopoietic stem cells towards different populations of MNP in an effort to understand how different cell subtypes present in inflammatory disease microenvironments respond to the common allergen house dust mite (HDM). Using single cell mRNA sequencing,we demonstrate that macrophage subtypes MC SPP1+ and MLC MARCO+ display different patterns of gene expression after HDM challenge,noted especially for the chemokines CXCL5,CXCL8,CCL5 and CCL15. MLC CD206Hi alternatively activated macrophages displayed the greatest changes in expression,while neutrophil and monocyte populations did not respond. Further work investigated how pollutant diesel exhaust particles could modify these transcriptional responses and revealed that CXC but not CC type chemokines were further upregulated. Through the use of diesel particles with adsorbed material removed,we suggest that soluble pollutants on these particles are the active constituents responsible for the modifying effects on HDM. This study highlights that environmental exposures may influence tissue responses dependent on which MNP cell type is present,and that these should be considerations when modelling such events in vitro. Understanding the nuanced responsiveness of different immune cell types to allergen and pollutant exposure also contributes to a better understanding of how these exposures influence the development and exacerbation of human disease. View Publication

Ugale et al. demonstrate that CDC42,ERK,and mTORC1 signaling are polarized in premitotic hematopoietic stem cells and unequally segregated during asymmetric cell division. A CDC42/ERK/mTORC1 pathway maintains HSC polarity and balances symmetric and asymmetric cell division. View Publication

Precise gene editing uncovers mis-splicing of BUBR1 and CDC27 in human SF3B1-mutant HSPCs,leading to activation of mitotic checkpoint and rendering the cells sensitive to CHK1 inhibitor prexasertib. View Publication

The B cell receptor (BCR) signals together with a multi-component co-receptor complex to initiate B cell activation in response to antigen binding. Here,we take advantage of peroxidase-catalyzed proximity labeling combined with quantitative mass spectrometry to track co-receptor signaling dynamics in Raji cells from 10 s to 2 h after BCR stimulation. This approach enables tracking of 2,814 proximity-labeled proteins and 1,394 phosphosites and provides an unbiased and quantitative molecular map of proteins recruited to the vicinity of CD19,the signaling subunit of the co-receptor complex. We detail the recruitment kinetics of signaling effectors to CD19 and identify previously uncharacterized mediators of B cell activation. We show that the glutamate transporter SLC1A1 is responsible for mediating rapid metabolic reprogramming and for maintaining redox homeostasis during B cell activation. This study provides a comprehensive map of BCR signaling and a rich resource for uncovering the complex signaling networks that regulate activation. View Publication

Although platinum-based chemotherapy is the frontline regimen for colorectal cancer (CRC),drug resistance remains a major challenge affecting its therapeutic efficiency. However,there is limited research on the correlation between chemotherapy resistance and lipid metabolism,including PIK3CA mutant tumors. In this present study,we found that PIK3CA-E545K mutation attenuated cell apoptosis and increased the cell viability of CRC with L-OHP treatment in vitro and in vivo . Mechanistically,PIK3CA-E545K mutation promoted the nuclear accumulation of SREBP1,which promoted the transcription of Apolipoprotein A5 (APOA5). APOA5 activated the PPARγ signaling pathway to alleviate reactive oxygen species (ROS) production following L-OHP treatment,which contributed to cell survival of CRC cells. Moreover,APOA5 overexpression enhanced the stemness-related traits of CRC cells. Increased APOA5 expression was associated with PIK3CA mutation in tumor specimens and poor response to first-line chemotherapy,which was an independent detrimental factor for chemotherapy sensitivity in CRC patients. Taken together,this study indicated that PIK3CA-E545K mutation promoted L-OHP resistance by upregulating APOA5 transcription in CRC,which could be a potent target for improving L-OHP chemotherapeutic efficiency. Our study shed light to improve chemotherapy sensitivity through nutrient management in CRC. View Publication

Chimeric antigen receptor (CAR) T cell therapy has emerged as a powerful therapeutic approach against a range of hematologic malignancies. While the incorporation of CD28 or 4-1BB costimulatory signaling domains into CARs revolutionized immune responses,there is an exciting prospect of further enhancing CAR functionality. Here,we investigated the design of CD19 CARs enriched with distinct Toll-like receptor 4 (TLR4),myeloid differentiation primary response 88 (MyD88),or Toll/IL-1 domain-containing adaptor-inducing interferon (IFN)-β (TRIF) costimulatory domains. Screening of various designs identified several candidates with no tonic activity but with increased CD19 target cell-dependent interleukin (IL)-2 production. Human T cells transduced with the selected CAR construct exhibited augmented hIL-2 and hIFN-γ induction and cytotoxicity when cocultured with CD19-positive lymphoma and solid-tumor cell lines. RNA sequencing (RNA-seq) analysis demonstrated the upregulation of some genes involved in the innate immune response and T cell activation and proliferation. In experiments on a xenogeneic solid-tumor mice model,MyD88 and TLR4 CAR T cells exhibited prolonged remission. This study demonstrates that the integration of a truncated TLR4 signaling costimulatory domain could provide immunotherapeutic potential against both hematologic malignancies and solid tumors. View Publication

The processes that govern human haematopoietic stem cell (HSC) self-renewal and engraftment are poorly understood and challenging to recapitulate in culture to reliably expand functional HSCs 1 – 3 . Here we identify MYC target 1 (MYCT1; also known as MTLC) as a crucial human HSC regulator that moderates endocytosis and environmental sensing in HSCs. MYCT1 is selectively expressed in undifferentiated human haematopoietic stem and progenitor cells (HSPCs) and endothelial cells but becomes markedly downregulated during HSC culture. Lentivirus-mediated knockdown of MYCT1 prevented human fetal liver and cord blood (CB) HSPC expansion and engraftment. By contrast,restoring MYCT1 expression improved the expansion and engraftment of cultured CB HSPCs. Single-cell RNA sequencing of human CB HSPCs in which MYCT1 was knocked down or overexpressed revealed that MYCT1 governs important regulatory programmes and cellular properties essential for HSC stemness,such as ETS factor expression and low mitochondrial activity. MYCT1 is localized in the endosomal membrane in HSPCs and interacts with vesicle trafficking regulators and signalling machinery. MYCT1 loss in HSPCs led to excessive endocytosis and hyperactive signalling responses,whereas restoring MYCT1 expression balanced culture-induced endocytosis and dysregulated signalling. Moreover,sorting cultured CB HSPCs on the basis of lowest endocytosis rate identified HSPCs with preserved MYCT1 expression and MYCT1-regulated HSC stemness programmes. Our work identifies MYCT1-moderated endocytosis and environmental sensing as essential regulatory mechanisms required to preserve human HSC stemness. Our data also pinpoint silencing of MYCT1 as a cell-culture-induced vulnerability that compromises human HSC expansion. Subject terms: Haematopoietic stem cells,Self-renewal,Stem-cell niche,Endocytosis,Growth factor signalling View Publication

Acute myeloid leukemia (AML) is fatal in the majority of adults. Identification of new therapeutic targets and their pharmacologic modulators are needed to improve outcomes. Previous studies had shown that immunization of rabbits with normal peripheral WBCs that had been incubated with fluorodinitrobenzene elicited high titer antibodies that bound to a spectrum of human leukemias. We report that proteomic analyses of immunoaffinity-purified lysates of primary AML cells showed enrichment of scaffolding protein IQGAP1. Immunohistochemistry and gene-expression analyses confirmed IQGAP1 mRNA overexpression in various cytogenetic subtypes of primary human AML compared to normal hematopoietic cells. shRNA knockdown of IQGAP1 blocked proliferation and clonogenicity of human leukemia cell-lines. To develop small molecules targeting IQGAP1 we performed in-silico screening of 212,966 compounds,selected 4 hits targeting the IQGAP1-GRD domain,and conducted SAR of the ‘fittest hit’ to identify UR778Br,a prototypical agent targeting IQGAP1. UR778Br inhibited proliferation,induced apoptosis,resulted in G2/M arrest,and inhibited colony formation by leukemia cell-lines and primary-AML while sparing normal marrow cells. UR778Br exhibited favorable ADME/T profiles and drug-likeness to treat AML. In summary, AML shows response to IQGAP1 inhibition,and UR778Br,identified through in-silico studies,selectively targeted AML cells while sparing normal marrow. Subject terms: Cancer,Cell biology,Drug discovery,Immunology,Oncology View Publication

Adult T-cell leukemia/lymphoma (ATL) occurs after human T-cell leukemia virus type-1 (HTLV-1) infection with a long latency period exceeding several decades. This implies the presence of immune evasion mechanisms for HTLV-1-infected T cells. Although ATL cells have a CD4 + CD25 + phenotype similar to that of regulatory T cells (Tregs),they do not always possess the immunosuppressive functions of Tregs. Factors that impart effective immunosuppressive functions to HTLV-1-infected cells may exist. A previous study identified a new CD13 + Treg subpopulation with enhanced immunosuppressive activity. We,herein,describe the paired CD13 − (designated as MT-50.1) and CD13 + (MT-50.4) HTLV-1-infected T-cell lines with Treg-like phenotype,derived from the peripheral blood of a single patient with lymphoma-type ATL. The cell lines were found to be derived from HTLV-1-infected non-leukemic cells. MT-50.4 cells secreted higher levels of immunosuppressive cytokines,IL-10 and TGF-β,expressed higher levels of Foxp3,and showed stronger suppression of CD4 + CD25 − T cell proliferation than MT-50.1 cells. Furthermore,the CD13 inhibitor bestatin significantly attenuated MT-50.4 cell growth,while it did not for MT-50.1 cells. These findings suggest that CD13 expression may be involved in the increased Treg-like activity of MT-50.4 cells. Hence,MT-50.4 cells will be useful for in-depth studies of CD13 + Foxp3 + HTLV-1-infected cells. Subject terms: Cancer,Microbiology,Oncology View Publication