参考文献

The growth factor and small molecule protocol are the two primary approaches for generating human induced pluripotent stem cell-derived hepatocyte-like cells (iPSC-HLCs). We compared the efficacy of the growth factor and small molecule protocols across fifteen different human iPSC lines. Morphological assessment,relative quantification of gene expression,protein expression and proteomic studies were carried out. HLCs derived from the growth factor protocol displayed mature hepatocyte morphological features including a raised,polygonal shape with well-defined refractile borders,granular cytoplasm with lipid droplets and/or vacuoles with multiple spherical nuclei or a large centrally located nucleus; significantly elevated hepatocyte gene and protein expression including AFP,HNF4A,ALBUMIN,and proteomic and metabolic features that are more aligned with a mature phenotype. HLCs derived from the small molecule protocol showed a dedifferentiated,proliferative phenotype that is more akin to liver tumor-derived cell lines. These experimental results suggest that HLCs derived from growth factors are better suited for studies of metabolism,biotransformation,and viral infection. View Publication

The clinical application of T cells engineered with chimeric antigen receptors (CARs) for solid tumors is challenging. A major reason for this involves tumor immune evasion mechanisms,including the high expression of immune checkpoint molecules,such as the programmed death 1 (PD-1) ligands PD-L1 and PD-L2. The inducible expression of PD-L1 in tumors has been observed after CAR-T-cell infusion,even in tumors natively not expressing PD-L1. Furthermore,numerous types of pediatric cancer do not have suitable targets for CAR-T-cell therapy. Therefore,the present study aimed to develop novel CAR-T cells that target PD-L1 and PD-L2,and to evaluate their efficacy against pediatric solid tumors. A novel CAR harboring the immunoglobulin V-set domain of the human PD-1 receptor as an antigen binding site (PD-1 CAR-T) was developed without using a single-chain variable fragment. PD-1 CAR-T cells were successfully manufactured by adding an anti-PD-1 antibody,nivolumab,to the ex vivo expansion culture to prevent fratricide during the manufacturing process due to the inducible expression of PD-L1 in activated human T cells. The expression of PD-L1 (and PD-L2 to a lesser extent) was revealed to be highly upregulated in various pediatric solid tumor cells,which displayed no or very low expression initially,on in vitro exposure to interferon-γ and/or tumor necrosis factor-α,which are cytokines secreted by tumor-infiltrating T cells. Furthermore,PD-1 CAR-T cells exhibited strong cytotoxic activity against pediatric solid tumor cells expressing PD-L1 and PD-L2. Conversely,the effect of PD-1 CAR-T cells was significantly attenuated against PD-L1-positive cells coexpressing CD80,suggesting that the toxicity of PD-1 CAR-T cells to normal immune cells,including antigen presenting cells,can be minimized. In conclusion,PD-1 ligands are promising therapeutic targets for pediatric solid tumors. PD-1 CAR-T cells,either alone or in combination with CAR-T cells with other targets,represent a potential treatment option for solid tumors. View Publication

In search for broad-spectrum antivirals,we discover a small molecule inhibitor,RMC-113,that potently suppresses the replication of multiple RNA viruses including SARS-CoV-2 in human lung organoids. We demonstrate selective inhibition of the lipid kinases PIP4K2C and PIKfyve by RMC-113 and target engagement by its clickable analog. Lipidomics analysis reveals alteration of SARS-CoV-2-induced phosphoinositide signature by RMC-113 and links its antiviral effect with functional PIP4K2C and PIKfyve inhibition. We identify PIP4K2C’s roles in SARS-CoV-2 entry,RNA replication,and assembly/egress,validating it as a druggable antiviral target. Integrating proteomics,single-cell transcriptomics,and functional assays,reveals that PIP4K2C binds SARS-CoV-2 nonstructural protein 6 and regulates virus-induced autophagic flux impairment. Promoting viral protein degradation by reversing autophagic flux impairment is a mechanism of antiviral action of RMC-113. These findings reveal virus-induced autophagy regulation via PIP4K2C,an understudied kinase,and propose dual PIP4K2C and PIKfyve inhibition as a candidate strategy to combat emerging viruses. Subject terms: SARS-CoV-2,Target identification,Autophagy View Publication

Huntington’s disease (HD) is a neurodegenerative disorder caused by the expansion of CAG repeats in the HTT gene,which results in a long polyglutamine tract in the huntingtin protein (HTT). One of the earliest key molecular mechanisms underlying HD pathogenesis is transcriptional dysregulation,which is already present in the developing brain. In this study,we searched for networks of deregulated RNAs crucial for initial transcriptional changes in HD- and HTT-deficient neuronal cells. RNA-seq (including small RNAs) was used to analyze a set of isogenic human neural stem cells. The results were validated using additional methods,rescue experiments,and in the medium spiny neuron-like cells. We observed numerous changes in gene expression and substantial dysregulation of miRNA expression in HD and HTT -knockout ( HTT -KO) cell lines. The overlapping set of genes upregulated in both HD and HTT -KO cells was enriched in genes associated with DNA binding and the regulation of transcription. We observed substantial upregulation of the following transcription factors: TWIST1,SIX1,TBX1,TBX15,MSX2,MEOX2 and FOXD1 . Moreover,we identified miRNAs that were consistently deregulated in HD and HTT -KO cells,including miR-214,miR-199,and miR-9. These miRNAs may function in the network that regulates TWIST1 and HTT expression via a regulatory feed-forward loop in HD. On the basis of overlapping changes in the mRNA and miRNA profiles of HD and HTT -KO cell lines,we propose that transcriptional deregulation in HD at early neuronal stages is largely caused by a deficiency of properly functioning HTT rather than a typical gain-of-function mechanism. The online version contains supplementary material available at 10.1186/s13578-025-01443-5. View Publication

Genome‐wide association studies have implicated complement in Alzheimer's disease (AD). The CR1*2 variant of complement receptor 1 (CR1; CD35),confers increased AD risk. We confirmed CR1 expression on glial cells; however,how CR1 variants influence AD risk remains unclear. Induced pluripotent stem cell‐derived microglia and astrocytes were generated from donors homozygous for the common CR1 variants (CR1*1/CR1*1;CR1*2/CR1*2). CR1 expression was quantified and phagocytic activity assessed using diverse targets ( Escherichia coli bioparticles,amyloid β aggregates,and synaptoneurosomes),with or without serum opsonization. Expression of CR1*1 was significantly higher than CR1*2 on glial lines. Phagocytosis for all targets was markedly enhanced following serum opsonization,attenuated by Factor I‐depletion,demonstrating CR1 requirement for C3b processing. CR1*2‐expressing glia showed significantly enhanced phagocytosis of all opsonized targets compared to CR1*1‐expressing cells. CR1 is critical for glial phagocytosis of opsonized targets. CR1*2,despite lower expression,enhances glial phagocytosis,providing mechanistic explanation of increased AD risk. Induced pluripotent stem cell (iPSC)‐derived glia from individuals expressing the Alzheimer's disease (AD) risk variant complement receptor (CR) 1*2 exhibit lower CR1 expression compared to those from donors expressing the non‐risk form CR1*1. The iPSC‐derived glia from individuals expressing the AD risk variant CR1*2 exhibit enhanced phagocytic activity for opsonized bacterial particles,amyloid‐β aggregates and human synaptoneurosomes compared to those from donors expressing the non‐risk form CR1*1. We suggest that expression of the CR1*2 variant confers risk of AD by enhancing the phagocytic capacity of glia for opsonized targets. View Publication

Immunosenescence describes the gradual remodeling of immune responses,leading to disturbed immune homeostasis and increased susceptibility of older adults for infections,neoplasia and autoimmunity. Decline in cellular immunity is associated with intrinsic changes in the T cell compartment,but can be further pushed by age-related changes in cells regulating T cell immunity. Myeloid-derived suppressor cells (MDSCs) are potent inhibitors of T cell activation and function,whose induction requires chronic inflammation. Since aging is associated with low grade inflammation (inflammaging) and increased myelopoiesis,age-induced changes in MDSC induction and function in relation to T cell immunity were analyzed. MDSC numbers and functions were compared between “healthy” young and old adults,who were negatively diagnosed for severe acute and chronic diseases known to induce MDSC accumulation. MDSCs were either isolated from peripheral blood or generated in vitro from blood-derived CD14 cells. Aging was associated with significantly increased MDSC numbers in the monocytic- (M-) and polymorphonuclear (PMN-) MDSC subpopulations. MDSCs could be induced more efficiently from CD14 cells of old donors and these MDSCs inhibited CD3/28-induced T cell proliferation significantly better than MDSCs induced from young donors. Serum factors of old donors supported MDSC induction comparable to serum factors from young donors,but increased immunosuppressive activity of MDSCs was only achieved by serum from old donors. Elevated immunosuppressive activity of MDSCs from old donors was associated with major metabolic changes and increased intracellular levels of neutral and oxidized lipids known to promote immunosuppressive functions. Independent of age,MDSC-mediated suppression of T cell proliferation required direct MDSC– T cell contact. Besides their increased ability to inhibit activation-induced T cell proliferation,MDSCs from old donors strongly shift the immune response towards Th2 immunity and might thereby further contribute to impaired cell-mediated immunity during aging. These results indicate that immunosenescence of innate immunity comprises accumulation and functional changes in the MDSC compartment,which directly impacts T cell functions and contribute to age-associated impaired T cell immunity. Targeting MDSCs during aging might help to maintain functional T cell responses and increase the chance of healthy aging. The online version contains supplementary material available at 10.1186/s12979-025-00524-w. View Publication

Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm characterized by the uncontrolled proliferation of white blood cells. Tyrosine kinase inhibitors (TKIs) are the standard treatment; however,resistance to BCR::ABL1 mutations remains challenging. WEE1,a checkpoint kinase involved in mitosis and DNA repair,is a potential therapeutic target for CML treatment. Ponatinib-resistant CML cells were screened to identify candidates for overcoming drug resistance. The efficacy of the ABL TKI asciminib and the WEE1 inhibitor MK-1775 was evaluated using proliferation and colony formation assays. Public database analysis ( GSE100026 ) assessed WEE1/PKMYT1 expression in CML. In vitro screening identified MK-1775 as a promising therapeutic candidate. WEE1/PKMYT1 expression was elevated in CML cells compared to healthy cells. Both asciminib and MK-1775 inhibited CML cell proliferation after 72 h,with enhanced cytotoxicity when combined. Co-treatment reduced colony formation and induced G2/M arrest,whereas an increase in the sub-G1 cell population indicated apoptosis. Furthermore,the combination treatment disrupted the mitochondrial membrane potential. The combination of asciminib and WEE1 inhibition demonstrated greater efficacy than either drug alone,suggesting a novel therapeutic strategy for treating CML. These findings provide insights into overcoming TKI resistance and highlight a promising approach for future clinical applications. The online version contains supplementary material available at 10.1007/s12672-025-03036-7. View Publication

JAK (Janus Kinase) inhibitors,such as ruxolitinib,were introduced a decade ago for treatment of myeloproliferative neoplasms (MPN). To evaluate ruxolitinib’s impact on MPN clonal evolution,we interrogate a myelofibrosis patient cohort with longitudinal molecular evaluation and discover that ruxolitinib is associated with clonal outgrowth of RAS pathway mutations. Single-cell DNA sequencing combined with ex vivo treatment of RAS mutated CD34 + primary patient cells,demonstrates that ruxolitinib induces RAS clonal selection both in a JAK/STAT wild-type and hyper-activated context. RAS mutations are associated with decreased transformation-free and overall survival only in patients treated with ruxolitinib. In vitro and in vivo competition assays demonstrate increased cellular fitness of RAS- mutated cells under ruxolitinib or JAK2 knock-down,consistent with an on-target effect. MAPK pathway activation is associated with JAK2 downregulation resulting in enhanced oncogenic potential of RAS mutations. Our results prompt screening for pre-existing RAS mutations in JAK inhibitor treated patients with MPN. Subject terms: Myeloproliferative disease,Cancer therapeutic resistance,Tumour heterogeneity,Cancer genetics View Publication

Duplication 15q (dup15q) syndrome is a leading genetic cause of autism spectrum disorder,offering a key model for studying autism-related mechanisms. Using single-cell and single-nucleus RNA sequencing of cortical organoids from dup15q patient-derived iPSCs and post-mortem brain samples,we identify increased glycolysis,disrupted layer-specific marker expression,and aberrant morphology in deep-layer neurons during fetal-stage organoid development. In adolescent-adult postmortem brains,upper-layer neurons exhibit heightened transcriptional burden related to synaptic signaling,a pattern shared with idiopathic autism. Using spatial transcriptomics,we confirm these cell-type-specific disruptions in brain tissue. By gene co-expression network analysis,we reveal disease-associated modules that are well preserved between postmortem and organoid samples,suggesting metabolic dysregulation that may lead to altered neuron projection,synaptic dysfunction,and neuron hyperexcitability in dup15q syndrome. Subject terms: Autism spectrum disorders,Autism spectrum disorders,Disease model View Publication

Neuroblastoma (NB) is the most common extracranial solid tumor in children characterized by poor immune infiltration and resistance to adaptive immunity,contributing to its limited response to immunotherapy. A key mechanism underlying immune evasion in cancer is autophagy,a cellular process that plays many roles in cancer by supporting tumor survival and regulating immune interactions. In this study,we investigate the impact of autophagy inhibition on NB tumor growth,immune modulation,and the efficacy of immunotherapy. Using both murine and human NB cell lines,we demonstrate that genetic and pharmacological inhibition of autophagy significantly reduces 3D spheroid growth and upregulates major histocompatibility complex class I (MHC-I) expression. In vivo studies further confirm that targeting autophagy suppresses tumor progression and promotes immune infiltration into the tumor. Notably,we observe a significant increase in CD8 + T cell recruitment and activation,suggesting that autophagy inhibition reshapes the immune landscape of NB,rendering it more susceptible to immune-mediated clearance. Crucially,autophagy inhibition also sensitizes NB cells to T cell-mediated cytotoxicity and enhances the therapeutic efficacy of GD2.CAR T-cell therapy. In vitro co-culture assays reveal increased CAR T cell-mediated tumor killing upon autophagy blockade,while in vivo models show prolonged tumor control and improved survival in treated mice compared to CAR T-cell therapy alone. These findings highlight autophagy as a key regulator of immune evasion in NB and suggest that its inhibition could serve as a promising therapeutic strategy to enhance immune recognition and improve the efficacy of immunotherapy. The online version contains supplementary material available at 10.1186/s13046-025-03453-0. View Publication