技术资料

Mesenchymal stem cells (MSCs) are promising for cell-based therapies targeting a wide range of diseases. However,challenges in translating MSC-based therapies to clinical applications necessitate standardized protocols following Good Manufacturing Practices (GMP) guidelines. This study aimed at developing GMP-complained protocols for FPMSCs isolation and manipulation,necessary for translational research,by (1) optimize culture of MSCs derived from an infrapatellar fat pad (FPMSC) condition through animal-free media comparison and (2) establish feasibility of MSC isolation,manufacturing and storage under GMP-compliance (GMP-FPMSC). FPMSCs from three different patients were isolated following established protocols and the efficacy of two animal component-free media formulations in the culturing media were evaluated. The impact of different media formulations on cell proliferation,purity,and potency of MSCs was evaluated through doubling time,colony forming unit assay,and percentage of MSCs,respectively. Furthermore,the isolation and expansion of GMP-FPMSCs from four additional donors were optimized and characterized at each stage according to GMP requirements. Viability and sterility were checked using Trypan Blue and Bact/Alert,respectively,while purity and identity were confirmed using Endotoxin,Mycoplasma assays,and Flow Cytometry. The study also included stability assessments post-thaw and viability assessment to determine the shelf-life of the final GMP-FPMSC product. Statistical analyses were conducted using one-way ANOVA with Tukey’s Multiple Comparisons. The study demonstrated that FPMSCs exhibited enhanced proliferation rates when cultured in MSC-Brew GMP Medium compared to standard MSC media. Cells cultured in this media showed lower doubling times across passages,indicating increased proliferation. Additionally,higher colony formation in FPMSCs cultured in MSC-Brew GMP Medium were observed,supporting enhanced potency. Data from our GMP validation,including cells from 4 different donors,showed post-thaw GMP-FPMSC maintained stem cell marker expression and all the specifications required for product release,including > 95% viability (> 70% is required) and sterility,even after extended storage (up to 180 days),demonstrating the reproducibility and potential of GMP-FPMSCs for clinical use as well as the robustness of the isolation and storage protocols. The study underscores the feasibility of FPMSCs for clinical uses under GMP conditions and emphasizes the importance of optimized culture protocols to improve cell proliferation and potency in MSC-based therapies. The online version contains supplementary material available at 10.1186/s12860-025-00539-7. View Publication

Malaria elimination faces challenges from drug resistance,stemming from mutations within the parasite’s genetic makeup. Genetic adaptations in key erythrocyte proteins offer malaria protection in endemic regions. Emulating nature’s approach,and implementing methodologies to render indispensable host proteins inactive,holds the potential to reshape antimalarial therapy. This study delves into the functional implication of the single-span membrane protein Kell ectodomain,which shares consensus sequence with the zinc endopeptidase family,possesses extracellular enzyme activity crucial for parasite invasion into host erythrocytes. Through generating Kell-null erythrocytes from an erythroid progenitor,BEL-A,we demonstrate the indispensable nature of Kell activity in P. falciparum invasion. Additionally,thiorphan,a metallo-endopeptidase inhibitor,which specifically inhibits Kell activity,inhibited Plasmodium infection at nanomolar concentrations. Interestingly,individuals in malaria-endemic regions exhibit low Kell expression and activity,indicating a plausible Plasmodium-induced evolutionary pressure. Both thiorphan and its prodrug racecadotril,demonstrated potent antimalarial activity in vivo,highlighting Kell’s protease role in invasion and proposing thiorphan as a promising host-oriented antimalarial therapeutic. Subject terms: Parasite biology,Parasite host response View Publication

Nonsense-mediated RNA decay (NMD) is a highly conserved RNA turnover pathway that influences several biological processes. Specific features in messenger RNAs (mRNAs) have been found to trigger decay by NMD,leading to the assumption that NMD sensitivity is an intrinsic quality of a given transcript. Here,we provide evidence that,instead,an overriding factor dictating NMD sensitivity is the cell environment. Using several genome-wide techniques to detect NMD-target mRNAs,we find that hundreds of mRNAs are sensitized to NMD as human embryonic stem cells progress to form neural progenitor cells. Another class of mRNAs escape from NMD during this developmental progression. We show that the differential sensitivity to NMD extends to in vivo scenarios,and that the RNA-binding protein,HNRNPL,has a role in cell type-specific NMD. We also addressed another issue in the field—whether NMD factors are core or branch-specific in their action. Surprisingly,we found that UPF3B,an NMD factor critical for the nervous system,shares only 30% of NMD-target transcripts with the core NMD factor UPF2. Together,our findings have implications for how NMD is defined and measured,how NMD acts in different biological contexts,and how different NMD branches influence human diseases. View Publication

Alzheimer’s disease (AD) is characterized by the accumulation and spread of Tau intraneuronal inclusions throughout most of the telencephalon,leaving hindbrain regions like the cerebellum and spinal cord largely spared. These neuropathological observations,along with the identification of specific vulnerable sub-populations from AD brain-derived single nuclei transcriptomics,suggest that a subset of brain regions and neuronal subtypes possess a selective vulnerability to Tau pathology. Given the inability to culture neurons from patient brains,a disease-relevant in vitro model which recapitulates these features would serve as a critical tool to validate modulators of vulnerability and resilience. Using our recently established platform for inducing endogenous Tau aggregation in human induced pluripotent stem cell (hiPSC)-derived cortical excitatory neurons via application of AD brain-derived exogenous Tau aggregates,we explored whether Tau aggregates preferentially induce aggregation in specific neuronal subtypes. We compared Tau seeding in hiPSC-derived neuron subtypes representing regional identities across the forebrain,midbrain,and hindbrain. Higher susceptibility (i.e. more Tau aggregation) was consistently observed among cortical neuron subtypes,with CTIP2-positive,somatostatin (SST)-positive cortical inhibitory neurons showing the greatest aggregation levels across hiPSC lines from multiple donors. hiPSC-neurons also delineated between the disease-specific vulnerabilities of different protein aggregates,as α-synuclein preformed fibrils showed an increased propensity to induce aggregates in midbrain dopaminergic (mDA)-like neurons,mimicking Parkinson’s disease (PD)-specific susceptibility. Aggregate uptake and degradation rates were insufficient to explain differential susceptibility. The absence of a consistent transcriptional response following aggregate seeding further indicated that intrinsic neuronal subtype-specific properties could drive susceptibility. The present data provides evidence that hiPSC-neurons exhibit features of selective neuronal vulnerability which manifest in a cell autonomous manner,suggesting that mining intrinsic (or basal) transcriptomic signatures of more vulnerable compared to more resilient hiPSC-neurons could uncover the molecular underpinnings of differential susceptibility to protein aggregation found in a variety of neurodegenerative diseases. The online version contains supplementary material available at 10.1186/s40478-025-02000-4. View Publication

The ε4 isoform of apolipoprotein E (ApoE) is the most significant genetic risk factor for Alzheimer’s disease. Glial cells are the main source of ApoE in the brain,and in microglia,the ε4 isoform of ApoE has been shown to impair mitochondrial metabolism and the uptake of lipids and Aβ42. However,whether the ε4 isoform alters autophagy or lysosomal activity in microglia in basal and inflammatory conditions is unknown. Altogether,microglia-like cells (iMGs) from eight APOE 3/3 and six APOE 4/4 human induced pluripotent stem cell (iPSC) lines were used in this study. The responses of iMGs to Aβ42,LPS and IFNγ were studied by metabolomics,proteomics,and functional assays. Here,we demonstrate that iMGs with the APOE 4/4 genotype exhibit reduced basal pinocytosis levels compared to APOE 3/3 iMGs. Inflammatory stimulation with a combination of LPS and IFNγ or Aβ42 induced PI3K/AKT/mTORC signaling pathway,increased pinocytosis,and blocked autophagic flux,leading to the accumulation of sequestosome 1 (p62) in both APOE 4/4 and APOE 3/3 iMGs. Exposure to Aβ42 furthermore caused lysosomal membrane permeabilization,which was significantly stronger in APOE 4/4 iMGs and positively correlated with the secretion of the proinflammatory chemokine IL-8. Metabolomics analysis indicated a dysregulation in amino acid metabolism,primarily L-glutamine,in APOE 4/4 iMGs. Overall,our results suggest that inflammation-induced metabolic reprogramming places lysosomes under substantial stress. Lysosomal stress is more detrimental in APOE 4/4 microglia,which exhibit endo-lysosomal defects. The online version contains supplementary material available at 10.1186/s12974-025-03470-y. View Publication

Despite the major roles of choroid plexus epithelial cells (CPECs) in brain homeostasis and repair,their developmental lineage and diversity remain undefined. In simplified differentiations from human pluripotent stem cells,derived CPECs (dCPECs) display canonical properties and dynamic motile multiciliated phenotypes that interact with Aβ uptake. Single dCPEC transcriptomes over time correlate well with human organoid and fetal CPECs,while pseudotemporal and cell cycle analyses highlight the direct CPEC origin from neuroepithelial cells. In addition,time series analyses define metabolic (type 1) and ciliogenic dCPECs (type 2) at early timepoints,followed by type 1 diversification into anabolic-secretory (type 1a) and catabolic-absorptive subtypes (type 1b) as type 2 cells contract. These temporal patterns are then confirmed in independent derivations and mapped to prenatal stages using human tissues. In addition to defining the prenatal lineage of human CPECs,these findings suggest dynamic models of ChP support for the developing human brain. Subject terms: Differentiation,Neural stem cells,Functional clustering,Cell fate and cell lineage View Publication

VEXAS syndrome is a clonal hematopoietic disorder characterized by hyperinflammation,bone marrow failure,and high mortality. The molecular hallmark of VEXAS is somatic mutations at methionine 41 (M41) in the E1 ubiquitin enzyme,UBA1. These mutations induce a protein isoform switch,but the mechanisms underlying disease pathogenesis remain unclear. Here,we developed a human cell model of VEXAS syndrome by engineering the male monocytic THP1 cell line to express the common UBA1 M41V mutation. We found that mutant UBA1 M41V cells exhibit aberrant UBA1 isoform expression,increased vacuolization,and upregulation of the unfolded protein response,recapitulating key features of VEXAS. Moreover,proteomic analyses revealed dysregulated ubiquitination and proteotoxic stress in UBA1 M41V cells,with alterations in inflammatory and stress-response pathways. Functional studies demonstrated that UBA1 M41V cells were highly sensitive to genetic or pharmacological inhibition of E1 ubiquitin enzymes. Treatment with the E1 enzyme inhibitor TAK-243 preferentially suppressed colony formation of UBA1 M41V cells as compared to WT cells. Moreover,UBA1 M41V cells exhibited greater sensitivity to TAK-243 in competition assays and showed increased apoptosis. Interestingly,TAK-243 preferentially inhibited UBA6 activity over UBA1,suggesting that UBA6 may compensate for UBA1 dysfunction in UBA1 M41V cells. Targeting UBA6 using shRNA or the UBA6-specific inhibitor phytic acid further revealed an acquired dependency on UBA6 in UBA1 M41V cells. Phytic acid selectively impaired growth and colony formation in UBA1 M41V cells while sparing WT cells,highlighting a potential therapeutic vulnerability. Together,these findings establish a novel human model of VEXAS syndrome,identify key roles for UBA1 and UBA6 in disease pathogenesis,and demonstrate that UBA6 inhibition represents a promising therapeutic strategy for selectively targeting UBA1 mutant clones. Subject terms: Haematological cancer,Cell signalling View Publication

Terminal erythropoiesis is a complex multistep process involving coordination of gene transcription and dramatic nuclear condensation,which leads to the expulsion of nuclei to generate reticulocytes. However,we lack a comprehensive understanding of the key transcriptional and epigenetic regulators involved. We used a high-throughput small molecule screen in primary CD34 + -derived human erythroblasts to identify targets that promoted terminal erythropoiesis,and further confirmed the phenotype in different differentiation systems by inhibitors and shRNAs of different BRD4 isoforms. Then we performed RNA-seq,ATAC-seq,ChIP-qPCR,Co-IP,and reanalyzed previously-published transcriptional data and mass spectrometric data to clarify how BRD4 regulates terminal erythropoiesis. We identified that inhibitors of the bromodomain protein BRD4,an epigenetic reader and transcriptional activator together with CDK9,promoted terminal erythropoiesis from hematopoietic stem/progenitor cells and embryonic stem cells,and enhanced enucleation. Combined analysis of our RNA-seq,ATAC-seq,and previously-published transcriptional data of erythroblast differentiation at different stages confirmed that BRD4 inhibition accelerates erythroblast maturation. Unexpectedly,this BRD4 function was independent of its classical CDK9 interaction and transcriptional activation. Instead,RNA-seq,ATAC-seq,and Cut&Tag upon BRD4 inhibition revealed that BRD4 regulates erythropoiesis by inhibiting the small G protein RhoB and disrupts actin reorganization. ChIP-qPCR,Co-IP,and functional studies revealed that BRD4 acts as a transcriptional repressor by interacting with the histone methyltransferase EHMT1/2. We demonstrate a non-classical role for BRD4 as a transcriptional repressor of RhoB to regulate erythroid maturation,and classical CDK9 dependent role to regulate cell proliferation of erythroblasts. Besides,we clarify RhoB’s activity and function during terminal erythropoiesis. BRD4 inhibition might be a simple method to promote in vitro blood cell production,and a candidate therapeutic target for diseases leading to dyserythropoiesis such as myelodysplastic syndromes. The online version contains supplementary material available at 10.1186/s13045-025-01721-2. View Publication

R2 elements,a class of non-long terminal repeat (non-LTR) retrotransposons,have the potential to be harnessed for transgene insertion. However,efforts to achieve this are limited by our understanding of the retrotransposon mechanisms. Here,we structurally and biochemically characterize R2 from Taeniopygia guttata (R2Tg). We show that R2Tg cleaves both strands of its ribosomal DNA target and binds a pseudoknotted RNA element within the R2 3′ UTR to initiate target-primed reverse transcription. Guided by these insights,we engineer and characterize an all-RNA system for transgene insertion. We substantially reduce the system’s size and insertion scars by eliminating unnecessary R2 sequences on the donor. We further improve the integration efficiency by chemically modifying the 5′ end of the donor RNA and optimizing delivery,creating a compact system that achieves over 80% integration efficiency in several human cell lines. This work expands the genome engineering toolbox and provides mechanistic insights that will facilitate future development of R2-mediated gene insertion tools. Subject terms: Transferases,Protein design,Genetic engineering 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

Astrocytes,a subtype of glial cells,have multiple roles in regulating neuronal development and homeostasis. In addition to the typical mammalian astrocytes,in the primate cortex,interlaminar astrocytes are located in the superficial layer and project long processes traversing multiple layers of the cerebral cortex. Previously,we described a human stem cell based chimeric mouse model where interlaminar astrocytes develop. Here,we utilized this model to study the calcium signaling properties of interlaminar astrocytes. To determine how interlaminar astrocytes could contribute to neurodevelopmental disorders,we generated a chimeric mouse model for Fragile X syndrome (FXS). We report that FXS interlaminar astrocytes exhibit hyperexcitable calcium signaling and are associated with dendritic spines with increased turnover rate. View Publication

Innovative identification technologies for hematopoietic stem cells (HSCs) have expanded the scope of stem cell biology. Clinically,the functional quality of HSCs critically influences the safety and therapeutic efficacy of stem cell therapies. However,most analytical techniques capture only a single snapshot,disregarding the temporal context. A comprehensive understanding of the temporal heterogeneity of HSCs necessitates live-cell,real-time and non-invasive analysis. Here,we developed a prediction system for HSC diversity by integrating single-HSC ex vivo expansion technology with quantitative phase imaging (QPI)-driven machine learning. By analyzing the cellular kinetics of individual HSCs,we discovered previously undetectable diversity that snapshot analysis cannot resolve. The QPI-driven algorithm quantitatively evaluates stemness at the single-cell level and leverages temporal information to significantly improve prediction accuracy. This platform advances the field from snapshot-based identification of HSCs to dynamic,time-resolved prediction of their functional quality based on past cellular kinetics. Subject terms: Haematopoietic stem cells,Stem-cell differentiation,Self-renewal,Imaging View Publication