参考文献

Renal failure due to drug nephrotoxicity or disease is frequently observed in patients. The development of in vitro models able to recapitulate kidney biology offers new possibilities to study drug toxicity or model diseases. Induced pluripotent stem cell–derived kidney organoids already show promise,but several drawbacks must be overcome to maintain them in culture,among which is the presence of non-renal cell populations such as cartilage. We modified the culture protocol and maintained kidney organoids in medium containing FGF9 for 1 additional week compared to the control protocol (Takasato). In comparison to the control,the FGF9-treated kidney organoids had reduced cartilage at day 7 + 25 and diminished chondrocyte marker expression. Importantly,the renal structures assessed by immunofluorescence were unaffected by the FGF9 treatment. This reduction of cartilage produces a higher quality kidney organoid that can be maintained longer in culture to improve their maturation for further in vivo work. Subject terms: Pluripotent stem cells,Stem-cell differentiation,Kidney View Publication

Epigenetic modulators of the histone deacetylase (HDAC) family control key biological processes and are frequently dysregulated in cancer. There is superior activity of HDAC inhibitors (HDACi) in patients with myeloproliferative neoplasms (MPNs) that carry the Janus kinase-2 point mutant JAK2 V617F . This constitutively active tyrosine kinase activates signal-transducer-and-activator-of-transcription (STAT) transcription factors to promote cell proliferation and inflammatory processes. We reveal that the inhibition of HDAC1/HDAC2 with the clinically advanced HDACi romidepsin,the experimental HDACi entinostat and MERCK60,and genetic depletion of HDAC1/HDAC2 induce apoptosis and long-term growth arrest of primary and permanent MPN cells in vitro and in vivo. This treatment spares normal hematopoietic stem cells and does not compromise blood cell differentiation. At the molecular level,HDAC1 and HDAC2 control the protein stability of SIAH2 through acetylation. Genetic knockout experiments show that SIAH2 accelerates the proteasomal degradation of JAK2 V617F in conjunction with the E2 ubiquitin-conjugating enzyme UBCH8. SIAH2 binds to the surface-exposed SIAH degron motif VLP1002 in the catalytic domain of JAK2 V617F . At the functional level,SIAH2 knockout MPN cells are significantly less sensitive to HDACi. Global RNA sequencing verifies that JAK-STAT signaling is a prime target of SIAH2. Moreover,HDAC1 is an adverse prognostic factor in patients with acute myeloid leukemia ( n = 150,p = 0.02),being a possible complication of MPNs. These insights reveal a previously unappreciated link between HDAC1/HDAC2 as key molecular targets,the still undefined regulation of cytoplasmic-to-nuclear signaling by HDACs,and how HDACi kill JAK2 V617F -positive cells from MPN patients and mice with JAK2 V617F in vitro and in vivo. Subject terms: Haematological cancer,Oncogenes,Target identification,Haematopoietic stem cells View Publication

Prime editing is a promising approach for correcting pathogenic variants,but its efficiency remains variable across genomic contexts. Here,we systematically evaluated 12 modifications of the PEmax system for correcting the CFTR F508del pathogenic variant that caused cystic fibrosis in patient-derived airway basal cells. We chose EXO1 and FEN1 nucleases to improve the original system. While all tested variants showed comparatively low efficiency in this AT-rich genomic region,4-FEN modification demonstrated significantly improved editing rates (up to 2.13 fold) compared to standard PEmax. Our results highlight two key findings: first,the persistent challenge of AT-rich target sequence correction even with optimized editors,and second,the performance of 4-FEN suggests its potential value for other genomic targets. View Publication

Fatty-acid-hydroxylase-associated neurodegeneration (FAHN) is a rare neurodegenerative disorder caused by loss-of-function mutations in the FA2H gene,leading to impaired enzymatic activity and resulting in myelin sheath instability,demyelination,and axonal degeneration. In this study,we established a human in vitro model using neurons and oligodendrocytes derived from induced pluripotent stem cells (hiPSCs) of a FAHN patient. This coculture system enabled the investigation of myelination processes and myelin integrity in a disease-relevant context. Analyses using immunofluorescence and Western blot revealed impaired expression and localisation of key myelin proteins in oligodendrocytes and cocultures. FA2H-deficient cells showed reduced myelination,shortened internodes,and disrupted formation of the nodes of Ranvier. Additionally,we identified autophagy defects—a hallmark of many neurodegenerative diseases—including reduced p62 expression,elevated LC3B levels,and impaired fusion of autophagosomes with lysosomes. This study presents a robust hiPSC-based model to study FAHN,offering new insights into the molecular pathology of the disease. Our findings suggest that FA2H mutations compromise both the structural integrity of myelin and the efficiency of the autophagic machinery,highlighting potential targets for future therapeutic interventions. View Publication

Myeloproliferative Neoplasms (MPN) are malignancies of hematopoietic stem and progenitor cells (HSPCs) that lead to the overproduction of mature blood cells. These disorders include Essential Thrombocythemia (ET),Polycythemia Vera (PV),and Primary Myelofibrosis (PMF),primarily driven by somatic mutations such as JAK2 V617F . Research indicates that mesenchymal stromal cells (MSCs) support fibrosis in PMF,though their role in ET and PV remains less clear. Furthermore,in vivo studies of ET/PV HSPCs remain a challenge due to low engraftment levels in xenograft models. We employed a 3D scaffold model to create an MPN humanized xenograft mouse model,enabling in vivo functional studies of primary MPN progenitor cells and the supportive role of human MSCs. Using this model,we first demonstrated robust hematopoietic support of healthy (HD) HSPCs by PV and ET MSCs. We then investigated the role of MSCs in sustaining JAK2 V617F mutant cells by using a CRISPR‐Cas9 editing model,along with primary PV and ET HSPCs. Our results showed consistent engraftment of CRISPR‐edited JAK2 V617F mutant HSPCs and PV and ET patient‐derived HSPCs in scaffolds seeded with HD,PV,and ET stroma,providing the first in vivo evidence that PV and ET MSCs can sustain both healthy and MPN‐associated hematopoiesis. Furthermore,HD MSCs were also capable of sustaining PV and ET HSPCs in vivo. Overall,we present the first humanized MPN xenograft model that offers valuable insights into how human BM MSCs interact with JAK2 V617F mutant clones. View Publication

Spinal cord injury (SCI) remains a significant clinical challenge and poses a dramatic threat to the life quality of patients due to limited neural regeneration and detrimental post-injury alternations in tissue microenvironment. We developed a therapeutic approach by transplanting spinal neural progenitor cells (spNPGs),derived from human induced pluripotent stem cell (iPSC)-generated neuromesodermal progenitors,into a contusive SCI model in NOD-SCID mice. Single-cell RNA sequencing mapped the in vitro differentiation of iPSC-spNPGs,confirming their specification into spinal neuronal lineages. Single-nucleus transcriptomics at 1 week post-transplantation showed that the grafted cells differentiated in vivo into motor neurons and two interneuron subtypes (V2 and dI4). Additionally,spNPGs integrated into host neural circuits,enhancing synaptic connectivity,while simultaneously modulating the injury microenvironment by shifting microglia and astrocyte polarization toward anti-inflammatory and neuroprotective phenotypes. This dual mechanism promoted axonal regrowth,remyelination,and significant sensorimotor recovery,as evidenced by improved locomotor scores. Our findings highlight the therapeutic potential of human iPSC-spNPGs in reconstructing neural networks and mitigating secondary damage,providing compelling preclinical evidence for advancing stem cell-based SCI therapies. Subject terms: Stem-cell differentiation,Spinal cord injury View Publication

Strategies targeting leukemic stem and progenitor cells (LSPCs) are needed for durable remissions in acute myeloid leukemia (AML) and high-risk myelodysplastic neoplasms (MDS). While CD123 constitutes a promising target on LSPCs and leukemic blasts,previous CD123-targeting approaches showed limited efficacy and challenging safety profiles. Here,we describe the preclinical efficacy and safety of the bispecific CD123/CD16A innate cell engager “AFM28”,demonstrating superior activity against AML and MDS patient-derived LSPCs and blasts in vitro compared to an Fc-enhanced CD123-targeting antibody,especially towards CD123 low and/or CD64 + leukemic cells. AFM28 induces autologous anti-leukemic activity in fresh AML whole blood cultures,demonstrating its potential to enhance NK cell function from AML patients. Responsiveness can be further enhanced by allogeneic NK cell addition. Anti-leukemic activity of AFM28 is confirmed in xenograft mouse models. In addition,AFM28 is well tolerated and demonstrates pharmacodynamic activity in cynomolgus monkeys. Altogether,our results indicate that AFM28 has the potential to reduce relapse-inducing residual disease and promote long-term remissions for patients with AML and MDS with a favorable safety profile. Subject terms: Cancer immunotherapy,Preclinical research,Acute myeloid leukaemia,Myelodysplastic syndrome View Publication

We have developed the HybriSeq method for single-cell RNA profiling,which utilizes in situ hybridization of multiple probes for targeted transcripts,followed by split-pool barcoding and sequencing analysis of the probes. We have shown that HybriSeq can achieve high sensitivity for RNA detection with multiple probes and profile entire transcripts without an end bias. The utility of HybriSeq is demonstrated in characterizing cell-to-cell heterogeneities of a panel of 196 genes in peripheral blood mononuclear cells and the detection of missed annotations of transcripts. Subject terms: Gene expression profiling,RNA sequencing View Publication

Cytolysin A (ClyA) is a pore‐forming protein from a strongly silenced gene in non‐pathogenic Escherichia coli,including typical commensal isolates in the intestinal microbiome of healthy mammalian hosts. Upon overproduction,ClyA‐expressing bacteria display a cytolytic phenotype. However,it remains unclear whether sublytic amounts of native ClyA play a role in commensal E. coli ‐host interactions in vivo. Here,we show that sublytic amounts of ClyA are released via outer membrane vesicles (OMVs) and affect host cells in a remarkable manner. OMVs isolated from ClyA + E. coli were internalised into cultured colon cancer cells. The OMV‐associated ClyA caused reduced levels of cancer‐activating proteins such as H3K27me3,CXCR4,STAT3 and MDM2 via the EZH2/H3K27me3/microRNA 622/CXCR4 signalling axis. Our results demonstrate that sublytic amounts of ClyA in OMVs from non‐pathogenic E. coli can influence the stability of the EZH2 protein,reducing its activity in epigenetic regulation,causing elevated level of the tumour suppressor protein p53. View Publication

The limited proliferative capacity of erythroid precursors is a major obstacle to generate sufficient in vitro-derived red blood cells for clinical purposes. While BMI1,a Polycomb Repressive Complex 1 member,is both necessary and sufficient to drive extensive proliferation of self-renewing erythroblasts,its mechanism of action remains poorly understood. Here we report that BMI1 overexpression leads to 10 billion-fold increase in self-renewal of human erythroblasts,which can terminally mature and agglutinate with typing reagent monoclonal antibodies. BMI1 and RING1B occupancy,along with repressive histone marks,are present at known BMI1 target genes,including the INK-ARF locus,consistent with altered cell cycle kinetics following BMI1 inhibition. Upregulation of BMI1 target genes with low repressive histone modifications,including key regulators of cholesterol homeostasis,along with functional studies,suggest that both cholesterol import and synthesis are essential for BMI1-associated self-renewal. We conclude that BMI1 regulates erythroid self-renewal not only through gene repression but also through gene activation and offer a strategy to expand immature erythroid precursors for eventual clinical uses. Subject terms: Self-renewal,Cell growth,Stem-cell research View Publication