搜索结果: 'gmp t cell activator'

Hypomethylating agents (HMAs) are frontline therapies for Myelodysplastic Neoplasms (MDS) and Acute Myeloid Leukemia (AML). However,acquired resistance and treatment failure are commonplace. To address this,we perform a genome-wide CRISPR-Cas9 screen in a human MDS-derived cell line,MDS-L,and identify TOPORS as a loss-of-function target that synergizes with HMAs,reducing leukemic burden and improving survival in xenograft models. We demonstrate that depletion of TOPORS mediates sensitivity to HMAs by predisposing leukemic blasts to an impaired DNA damage response (DDR) accompanied by an accumulation of SUMOylated DNMT1 in HMA-treated TOPORS-depleted cells. The combination of HMAs with targeting of TOPORS does not impair healthy hematopoiesis. While inhibitors of TOPORS are unavailable,we show that inhibition of protein SUMOylation with TAK-981 partially phenocopies HMA-sensitivity and DDR impairment. Overall,our data suggest that the combination of HMAs with inhibition of SUMOylation or TOPORS is a rational treatment option for High-Risk MDS (HR-MDS) or AML. Subject terms: Myelodysplastic syndrome,Acute myeloid leukaemia,Sumoylation View Publication

Down syndrome predisposes individuals to haematological abnormalities,such as increased number of erythrocytes and leukaemia in a process that is initiated before birth and is not entirely understood 1 – 3 . Here,to understand dysregulated haematopoiesis in Down syndrome,we integrated single-cell transcriptomics of over 1.1 million cells with chromatin accessibility and spatial transcriptomics datasets using human fetal liver and bone marrow samples from 3 fetuses with disomy and 15 fetuses with trisomy. We found that differences in gene expression in Down syndrome were dependent on both cell type and environment. Furthermore,we found multiple lines of evidence that haematopoietic stem cells (HSCs) in Down syndrome are ‘primed’ to differentiate. We subsequently established a Down syndrome-specific map linking non-coding elements to genes in disomic and trisomic HSCs using 10X multiome data. By integrating this map with genetic variants associated with blood cell counts,we discovered that trisomy restructured regulatory interactions to dysregulate enhancer activity and gene expression critical to erythroid lineage differentiation. Furthermore,as mutations in Down syndrome display a signature of oxidative stress 4,5,we validated both increased mitochondrial mass and oxidative stress in Down syndrome,and observed that these mutations preferentially fell into regulatory regions of expressed genes in HSCs. Together,our single-cell,multi-omic resource provides a high-resolution molecular map of fetal haematopoiesis in Down syndrome and indicates significant regulatory restructuring giving rise to co-occurring haematological conditions. Subject terms: Haematopoietic stem cells,Leukaemia,Haematopoiesis,Haematological diseases,Aneuploidy View Publication

The ability to generate natural killer (NK) cells from induced pluripotent stem cells (iPSCs) has given rise to new possibilities for the large-scale production of homogeneous immunotherapeutic cellular products and opened new avenues towards the creation of “off-the-shelf” cancer immunotherapies. However,the differentiation of NK cells from iPSCs remains poorly understood,particularly regarding the ontogenic landscape of iPSC-derived NK (iNK) cells produced in vitro and the influence that the differentiation strategy employed may have on the iNK profile. To investigate this question,we conducted a comparative analysis of two sets of iNK cells generated from the same iPSC line using two different protocols: (i) a short-term,clinically compatible feeder-free protocol corresponding to primitive hematopoiesis,and (ii) a lymphoid-based protocol representing the definitive hematopoietic step. Our work demonstrated that both protocols are capable of producing functional iNK cells. However,the two sets of resulting iNKs exhibited distinct phenotypes and transcriptomic profiles. The lymphoid-based differentiation approach generated iNKs with a more mature and activated profile,which demonstrated higher cytotoxicity against cancer cell lines compared to iNK cells produced under short-term feeder-free conditions suggesting that the differentiation strategy must be considered when designing iNK cell–based adoptive immunotherapies. View Publication

Liver cancer stem cells (LCSCs) are thought to drive the metastasis and recurrence,however,the heterogeneity of molecular markers of LCSCs has hindered the development of effective methods to isolate them. This study introduced an effective approach to isolate and culture LCSCs from human primary liver cancer (HPLC),leveraging mouse embryonic fibroblasts (MEFs) as feeder cells in conjunction with using defined medium. Isolated LCSCs were further characterized by multiple approaches. Transcriptome sequencing data analysis was conducted to identify highly expressed genes in LCSCs and classify different subtypes of liver cancers. Total sixteen cell strains were directly isolated from 24 tissues of three types of HPLC without sorting,seven of which could be maintained long-term culture as colony growth on MEFs,which is unique characteristics of stem cells. Even 10 of cloned cells formed the tumors in immunodeficient mice,indicating that those cloned cells were tumorgenic. The histologies and gene expression pattern of human xenografts were very similar to those of HPLC where these cloned cells were isolated. Moreover,putative markers of LCSCs were further verified to all express in cloned cells,confirming that these cells were LCSCs. These cloned LCSCs could be cryopreserved,and still maintained the feature of colony growth on MEFs after the recovery. Compared to suspension culture as conventional approach to culture LCSCs,our approach much better maintained stemness of LCSCs for a long time. To date,these cloned cells could be cultured on MEFs over 12 passages. Moreover,bioinformatics analysis of sequencing data revealed the gene expression profiles in LCSCs,and liver cancers were classified into two subtypes C1 and C2 based on genes associated with the prognosis of LCSCs. Patients of the C2 subtype,which is closely related to the extracellular matrix,were found to be sensitive to treatments such as Cisplatin,Axitinib,JAK1 inhibitors,WNT-c59,Sorafenib,and RO-3306. In summary,this effective approach offers new insights into the molecular landscape of human liver cancers,and the identification of the C2 subtype and its unique response to the treatment pave the way for the creation of more effective,personalized therapeutic strategies. The online version contains supplementary material available at 10.1186/s12967-024-05870-9. View Publication

Transplantation of engineered hematopoietic stem/progenitor cells (HSPCs) showed curative potential in patients affected by neurometabolic diseases treated in early stage. Favoring the engraftment and maturation of the engineered HSPCs in the central nervous system (CNS) could allow enhancing further the therapeutic potential of this approach. Here we unveil that HSPCs haplo-insufficient at the Cx3cr1 (Cx3cr1 −/+ ) locus are favored in central nervous system (CNS) engraftment and generation of microglia-like progeny cells (MLCs) as compared to wild type (Cx3cr1 +/+ ) HSPCs upon transplantation in mice. Based on this evidence,we have developed a CRISPR-based targeted gene addition strategy at the human CX3CR1 locus resulting in an enhanced ability of the edited human HSPCs to generate mature MLCs upon transplantation in immunodeficient mice,and in lineage specific,regulated and robust transgene expression. This approach,which benefits from the modulation of pathways involved in microglia maturation and migration in haplo-insufficient cells,may broaden the application of HSPC gene therapy to a larger spectrum of neurometabolic and neurodegenerative diseases. Subject terms: Targeted gene repair,Haematopoietic stem cells,Microglial cells View Publication

Melanoma brain metastasis is linked to dismal prognosis and low overall survival and is detected in up to 80% of patients at autopsy. Circulating tumor cells (CTC) are the smallest functional units of cancer and precursors of fatal metastasis. We previously used an unbiased multilevel approach to discover a unique ribosomal protein large/small subunit (RPL/RPS) CTC gene signature associated with melanoma brain metastasis. In this study,we hypothesized that CTC-driven melanoma brain metastasis secondary metastasis (“metastasis of metastasis” per clinical scenarios) has targeted organ specificity for the liver. We injected parallel cohorts of immunodeficient and newly developed humanized NBSGW (huNBSGW) mice with cells from CTC-derived melanoma brain metastasis to identify secondary metastatic patterns. We found the presence of a melanoma brain–liver metastasis axis in huNBSGW mice. Furthermore,RNA sequencing analysis of tissues showed a significant upregulation of the RPL/RPS CTC gene signature linked to metastatic spread to the liver. Additional RNA sequencing of CTCs from huNBSGW blood revealed extensive CTC clustering with human B cells in these mice. CTC:B-cell clusters were also upregulated in the blood of patients with primary melanoma and maintained either in CTC-driven melanoma brain metastasis or melanoma brain metastasis CTC–derived cells promoting liver metastasis. CTC-generated tumor tissues were interrogated at single-cell gene and protein expression levels (10x Genomics Xenium and HALO spatial biology platforms,respectively). Collectively,our findings suggest that heterotypic CTC:B-cell interactions can be critical at multiple stages of metastasis. This study provides important insights into the relevance of prometastatic CTC:B-cell clusters in melanoma progression,extends the importance of the CTC RPL/RPS gene signature beyond primary metastasis/melanoma brain metastasis driving targeted organ specificity for liver metastasis (“metastasis of metastasis”),and identifies new targets for clinical melanoma metastasis therapies. View Publication

Chimeric antigen receptor (CAR)-engineered T cell therapy holds promise for treating myeloid malignancies,but challenges remain in bone marrow (BM) infiltration and targeting BM-resident malignant cells. Current autologous CAR-T therapies also face manufacturing and patient selection issues,underscoring the need for off-the-shelf products. In this study,we characterize primary patient samples and identify a unique therapeutic opportunity for CAR-engineered invariant natural killer T (CAR-NKT) cells. Using stem cell gene engineering and a clinically guided culture method,we generate allogeneic CD33-directed CAR-NKT cells with high yield,purity,and robustness. In preclinical mouse models,CAR-NKT cells exhibit strong BM homing and effectively target BM-resident malignant blast cells,including CD33-low/negative leukemia stem and progenitor cells. Furthermore,CAR-NKT cells synergize with hypomethylating agents,enhancing tumor-killing efficacy. These cells also show minimal off-tumor toxicity,reduced graft-versus-host disease and cytokine release syndrome risks,and resistance to allorejection,highlighting their substantial therapeutic potential for treating myeloid malignancies. Subject terms: Cancer therapy,Immunotherapy,Leukaemia View Publication

To fully utilize the potential of human induced pluripotent stem cells (hiPSCs) for allogeneic stem cell–based therapies,efficient and scalable expansion procedures must be developed. For other adherent human cell types,the combination of microcarriers (MCs) and stirred tank bioreactors has been shown to meet these demands. In this study,a hiPSC quasi-perfusion expansion procedure based on MCs was developed at 100-mL scale in spinner flasks. Process development began by assessing various medium exchange strategies and MC coatings,indicating that the hiPSCs tolerated the gradual exchange of medium well when cultivated on Synthemax II–coated MCs. This procedure was therefore scaled-up to the 1.3-L Eppendorf BioBLU 1c stirred tank bioreactor by applying the lower limit of Zwietering’s suspension criterion ( N s 1 u ),thereby demonstrating proof-of-concept when used in combination with hiPSCs for the first time. To better understand the bioreactor and its bioengineering characteristics,computational fluid dynamics and bioengineering investigations were performed prior to hiPSC cultivation. In this manner,improved process understanding allowed an expansion factor of ≈ 26 to be achieved,yielding more than 3 × 10 9 cells within 5 days. Further quality analyses confirmed that the hiPSCs maintained their viability,identity,and differentiation potential throughout cultivation. • N s 1 u can be used as a scale-up criterion for hiPSC cultivations in MC-operated stirred bioreactors • Uniform distribution and attachment of cells to the MCs are crucial for efficient expansion • Perfusion is advantageous and supports the cultivation of hiPSCs The online version contains supplementary material available at 10.1007/s00253-024-13372-3. View Publication

Acute myeloid leukemia (AML) is characterized by the accumulation of immature myeloid cells and a differentiation block,highlighting the urgent need for novel differentiation-inducing therapies. This study evaluated Adina rubella Hance (ARH) stem as a potent differentiation inducer by systematically screening 200 plant extracts. ARH stem promoted phenotypic differentiation in AML cells. In addition to its differentiation-inducing effects,ARH stem exhibited strong antileukemic activities,such as inhibiting cell proliferation,inducing cell death,and enhancing mitochondrial reactive oxygen species (mtROS) levels,the latter of which is critical for its differentiation-promoting activity. Comparative analysis with the extracts from other parts of the plant confirmed the superior efficacy of the stem extract because of its unique chemical composition. Ultra-high-performance liquid chromatography combined with quadrupole time-of-flight mass spectrometry analysis identified Picroside III as a major active compound within the stem extract,capable of recapitulating ARH stem-induced differentiation and demonstrating significant antileukemic properties. These findings underscore the therapeutic potential of ARH stem and its active component,Picroside III,as promising agents for differentiation-based treatment strategies in AML. View Publication

Understanding the interaction between genetic and epigenetic variation remains a challenge due to confounding environmental factors. We propose that human induced Pluripotent Stem Cells (iPSCs) are an excellent model to study the relationship between genetic and epigenetic variation while controlling for environmental factors. In this study,we have created a comprehensive resource of high-quality genomic,epigenomic,and transcriptomic data from iPSC lines and three iPSC-derived cell types (neural stem cell (NSC),motor neuron,monocyte) from three healthy donors. We find that epigenetic variation is most strongly associated with genetic variation at the iPSC stage,and that relationship weakens as epigenetic variation increases in differentiated cells. Additionally,cell type is a stronger source of epigenetic variation than genetic variation. Further,we elucidate a utility of studying epigenetic variation in iPSCs and their derivatives for identifying important loci for GWAS studies and the cell types in which they may be acting. Subject terms: Epigenomics,Genomics,Transcriptomics View Publication

Persistence of drug-resistant breast cancer stem cells (brCSCs) after a chemotherapeutic regime correlates with disease recurrence and elevated mortality. Therefore,deciphering mechanisms that dictate their drug-resistant phenotype is imperative for designing targeted and more effective therapeutic strategies. The transcription factor SOX2 has been recognized as a protagonist in brCSC maintenance,and previous studies have confirmed that inhibition of SOX2 purportedly eliminated these brCSCs. However,pharmacological targeting of transcription factors like SOX2 is challenging due to their structural incongruities and intrinsic disorders in their binding interfaces. Therefore,transcriptional co-activators may serve as a feasible alternative for effectively targeting the brCSCs. Incidentally,transcriptional co-activators YAP/TAZ were found to be upregulated in CD44 + /CD24 - /ALDH + cells isolated from patient breast tumors and CSC-enriched mammospheres. Interestingly,it was observed that YAP/TAZ exhibited direct physical interaction with SOX2 and silencing YAP/TAZ attenuated SOX2 expression in mammospheres,leading to significantly reduced sphere forming efficiency and cell viability. YAP/TAZ additionally manipulated redox homeostasis and regulated mitochondrial dynamics by restraining the expression of the mitochondrial fission marker,DRP1. Furthermore,YAP/TAZ inhibition induced DRP1 expression and impaired OXPHOS,consequently inducing apoptosis in mammospheres. In order to enhance clinical relevance of the study,an FDA-approved drug verteporfin (VP),was used for pharmacological inhibition of YAP/TAZ. Surprisingly,VP administration was found to reduce tumor-initiating capacity of the mammospheres,concomitant with disrupted mitochondrial homeostasis and significantly reduced brCSC population. Therefore,VP holds immense potential for repurposing and decisively eliminating the chemoresistant brCSCs,offering a potent strategy for managing tumor recurrence effectively. Subject terms: Cancer stem cells,Cancer stem cells View Publication

Toll-interacting protein (Tollip) suppresses excessive pro-inflammatory signaling,but its function in airway epithelial responses to IL-13,a key mediator in allergic diseases,remains unclear. This study investigates Tollip knockdown (TKD) effects in primary human airway epithelial cells using single-cell RNA sequencing,providing the first single-cell analysis of TKD and the first exploring its interaction with IL-13. IL-13 treatment upregulated key genes,including SPDEF,MUC5AC,POSTN,ALOX15,and CCL26,confirming IL-13’s effects and validating our methods. IL-13 reduced TNF-α signaling and epithelial-mesenchymal transition in certain cell types,suggesting a dual role in promoting type 2 inflammation while suppressing Th1-driven inflammation. Tollip deficiency alone significantly amplified TNF-α signaling and inflammatory pathways in goblet,club,and suprabasal cells. Comparisons between TKDIL13 vs IL13 and TKD vs CTR revealed that IL-13 does not substantially alter Tollip deficiency response in most cell types,reinforcing findings in TKD vs CTR. Tollip deficiency alters the response to IL-13 in a cell-type-specific manner,strongly downregulating TNF-α signaling in goblet cells but only weakly in basal and club cells. Tollip deficiency enhances IL-13’s suppression of Th1 inflammatory responses in goblet cells. These novel insights in Tollip-IL-13 interactions offer potential therapeutic targets for asthma and related diseases. The online version contains supplementary material available at 10.1186/s13104-025-07255-7. View Publication