技术资料

In bone marrow,cell numbers are balanced between production and loss. After chemotherapy,blood cell counts decrease initially but later recover as hematopoietic progenitor cells expand,although the mechanisms underlying this recovery are still unclear. We investigated the influence of red blood cells (RBCs) on hematopoietic stem cell (HSC) function during bone marrow recovery. Following chemotherapy,RBC concentrations in bone marrow peaked on day 5 posttreatment,coinciding with the recovery of hematopoiesis. Coculture of HSCs with RBCs resulted in a significant increase in hematopoiesis. Direct contact between RBCs and HSCs was essential for enhancement of hematopoiesis,and HSCs precultured with RBCs resulted in greater numbers of donor‐derived mature hematopoietic cells after transplantation. RNA‐sequencing analysis showed that Hes1 was the most significantly upregulated transcription factor in RBC coculture,and the response to RBC‐induced hematopoiesis of Hes1‐deficient HSCs was reduced. These findings imply a role of RBCs and Hes1 in the enhancement of hematopoietic recovery following bone marrow stress. View Publication

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

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

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

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

Anti-obesity medications (AOMs) have become one of the most prescribed drugs in human medicine. While AOMs are known to impact adult neurogenesis in the hypothalamus,their effects on the functional maturation of hypothalamic neurons remain unexplored. Given that AOMs target neurons in the Medial Basal Hypothalamus (MBH),which play a crucial role in regulating energy homeostasis,we hypothesized that AOMs might influence the functional maturation of these neurons,potentially rewiring the MBH. To investigate this,we exposed hypothalamic neurons derived from human induced pluripotent stem cells (hiPSCs) to Semaglutide and lipidized prolactin-releasing peptide (LiPR),two anti-obesity compounds. Contrary to our expectations,treatment with Semaglutide or LiPR during neuronal maturation did not affect the proportion of anorexigenic,Pro-opiomelanocortin-expressing (POMC+) neurons. Additionally,LiPR did not alter the morphology of POMC+ neurons or the expression of selected genes critical for the metabolism or development of anorexigenic neurons. Furthermore,LiPR did not impact the proportion of adult-generated POMC+ neurons in the mouse MBH. Taken together,these results suggest that AOMs do not influence the functional maturation of anorexigenic hypothalamic neurons. View Publication

Transplantation of engineered B cells has demonstrated efficacy in HIV disease models. B cell engineering may also be utilized for the treatment of cancer. Recent studies have highlighted that B cell activity is associated with favorable clinical outcomes in oncology. In mice,polyclonal B cells have been shown to elicit anti-cancer responses. As a potential novel cell therapy,we demonstrate that engineering B cells to target a tumor-associated antigen enhances polyclonal anti-tumor responses. We observe that engineered B cells expressing an anti-HPV B cell receptor internalize the antigen,enabling subsequent activation of oncoantigen-specific T cells. Secreted antibodies from engineered B cells form immune complexes,which are taken up by antigen-presenting cells to further promote T cell activation. Engineered B cells hold promise as novel,multi-modal cell therapies and open new avenues in solid tumor targeting. View Publication

The present study focused on the culture medium of induced pluripotent stem cells (iPSCs) prior to the use of cardiomyocytes differentiation induction medium (pre-culture medium). Seven types (Nos. 1-7) of StemFit AK03 medium (Ajinomoto) for clinical iPSCs with varying compositions were prepared as pre-culture medium. The cardiac muscle troponin T (cTnT) positivity of No. 1 (StemFit AK03 medium) was 84%,No. 3 (similar to E8 medium) was 89%,No. 2 (similar to E8 medium) was 91%,No. 5 (similar to EB Formation medium) was 95%,when using differentiation induction medium prepared with known components available for clinical cell production. The formation of cardiac tissues was assessed by evaluating the expression levels of specific markers,including cTnT,atrial natriuretic peptides (ANP),and pro-B-type natriuretic peptide (proBNP). The results demonstrated that cardiac tissue with high protein expression levels of cTnT and ANP was formed when similar to E8 medium as pre-culture medium. The online version contains supplementary material available at 10.1038/s41598-025-13259-x. View Publication

Type 1 diabetes (T1D) results from the autoimmune-mediated loss of pancreatic β-cells. Current insulin therapies offer symptomatic relief but fall short of providing a definitive cure. Islet cell transplantation,while promising,faces limitations related to donor scarcity,procedural complexities,and the necessity for long-term immunosuppression. Consequently,there is an urgent need for innovative strategies aimed at β-cell regeneration. Patient-derived induced pluripotent stem cells (iPSCs),obtained from peripheral blood mononuclear cells (PBMCs) of T1D patients,hold great potential as a source of cells for therapeutic purposes. Therefore,the differentiation of T1D-iPSCs into functional pancreatic β-cells is a critical step toward effective β-cell replacement therapy. To assess the potential of patient-derived T1D-β-like cells (differentiated from T1D-iPSCs reprogrammed from T1D-PBMCs) for restoring β-cell function in T1D. T1D-iPSCs were reprogrammed from T1D-PBMCs using an episomal vector-based approach. Pluripotency was confirmed by flow cytometry (FCM),quantitative real-time polymerase chain reaction,genomic stability analysis,and teratoma formation assays. Differentiation into pancreatic β-cells was optimized using triiodothyronine (T3),vitamin C (Vc),and an adenovirus (M3C) encoding pancreatic duodenal homeobox-1,neurogenin 3 ( Ngn3 ),and MAF bZIP transcription factor A ( MafA ). Following characterization of β-cell features by immunofluorescence,quantitative real-time polymerase chain reaction,and flow cytometry,therapeutic efficacy was assessed through blood glucose monitoring after transplantation under the renal capsule of streptozotocin-induced diabetic mice. T1D-iPSCs were successfully generated from T1D-PBMCs. These cells exhibited the hallmark characteristics of pluripotent stem cells,including appropriate morphology,differentiation potential,genomic integrity,and expression of pluripotency-associated genes. Differentiation yielded insulin-positive (insulin + ) pancreatic β-like cells that,at the mRNA level,expressed key β-cell markers such as pancreatic duodenal homeobox-1,Ngn3,MafA,NeuroD,glucagon-like peptide-1 receptor,Nkx6.1,glucose transporter 2,and Kir6.2 . Notably,the T3 + Vc group displayed the lowest Ngn3 expression (1.31 ± 0.38 vs 1.96 ± 0.25 vs 2.51 ± 0.24,P < 0.01),while the M3C + T3 + Vc group exhibited the highest MafA expression (0.49 ± 0.11 vs 0.32 ± 0.06 vs 0.29 ± 0.08,P < 0.05). Both in vitro and in vivo assessments confirmed the insulin secretion ability of the generated β-like cells; however,they did not demonstrate appropriate modulation of insulin release in response to variations in extracellular glucose concentrations. T1D-iPSCs derived from T1D-PBMCs can be differentiated into insulin + β-like cells,albeit with functional immaturity. These cells represent a potential source of seed cells for β-cell replacement therapy in T1D. View Publication

The recently identified proton-activated chloride (PAC) channel is ubiquitously expressed,and it regulates several proton-sensitive physiological and pathophysiological processes. While the PAC channel is activated by strong acids due to the binding of protons to extracellular binding sites,here,we describe the way in which weak acids inhibit the PAC channel by a mechanism involving a distinct extracellular binding site. Whole-cell patch clamp was performed on wildtype HEK293T cells,PAC-knockout HEK293 cells expressing human (h)PAC mutant constructs,and on hiPSC-derived cardiomyocytes. Proton-induced cytotoxicity was examined in HEK293T cells. Acetic acid inhibited endogenous PAC channels in HEK 293T cells in a reversible,concentration-dependent,and pH-dependent manner. The inhibition of PAC channels was also induced by lactic acid,propionic acid,itaconic acid,and β-hydroxybutyrate. Weak acids also inhibited recombinant wildtype hPAC channels and PAC-like currents in hiPSC-derived cardiomyocytes. Replacement of the extracellular arginine 93 by an alanine (hPAC–Arg93Ala) strongly reduced the inhibition by some weak acids,including arachidonic acid. Although lactic acid inhibited PAC,it did not reduce the proton-induced cytotoxicity examined in wildtype HEK 293 cells. To conclude,weak acids inhibit PAC via an extracellular mechanism involving Arg93. These data warrant further investigations into the regulation of the PAC channel by endogenous weak acids. View Publication

Arteriovenous malformations (AVMs) are congenital vascular anomalies defined by abnormal direct connections between arteries and veins due to their complex structure or endovascular approaches. Pharmacological strategies targeting the underlying molecular mechanisms are thus gaining increasing attention in an effort to determine the mechanism involved in AVM regulation. In this study,we examined 30 human tissue samples,comprising 10 vascular samples,10 human fibroblasts derived from AVM tissue,and 10 vascular samples derived from healthy individuals. The pharmacological agents thalidomide,U0126,and rapamycin were applied to the isolated endothelial cells (ECs). The pharmacological treatments reduced the proliferation of AVM ECs and downregulated miR-135b-5p,a biomarker associated with AVMs. The expression levels of angiogenesis-related genes,including VEGF,ANG2,FSTL1,and MARCKS,decreased; in comparison,CSPG4,a gene related to capillary networks,was upregulated. Following analysis of these findings,skin samples from 10 AVM patients were reprogrammed into induced pluripotent stem cells (iPSCs) to generate AVM blood vessel organoids. Treatment of these AVM blood vessel organoids with thalidomide,U0126,and rapamycin resulted in a reduction in the expression of the EC markers CD31 and α-SMA. The establishment of AVM blood vessel organoids offers a physiologically relevant in vitro model for disease characterization and drug screening. The authors of future studies should aim to refine this model using advanced techniques,such as microfluidic systems,to more efficiently replicate AVMs’ pathology and support the development of personalized therapies. View Publication