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PIEZO1 is critical to numerous physiological processes,transducing diverse mechanical stimuli into electrical and chemical signals. Recent studies underscore the importance of visualizing endogenous PIEZO1 activity and localization to understand its functional roles. To enable physiologically and clinically relevant studies on human PIEZO1,we genetically engineered human induced pluripotent stem cells (hiPSCs) to express a HaloTag fused to endogenous PIEZO1. Combined with advanced imaging,our chemogenetic platform allows precise visualization of PIEZO1 localization dynamics in various cell types. Furthermore,the PIEZO1-HaloTag hiPSC technology facilitates the non-invasive monitoring of channel activity across diverse cell types using Ca2+-sensitive HaloTag ligands,achieving temporal resolution approaching that of patch clamp electrophysiology. Finally,we use lightsheet microscopy on hiPSC-derived neural organoids to achieve molecular scale imaging of PIEZO1 in three-dimensional tissue. Our advances establish a platform for studying PIEZO1 mechanotransduction in human systems,with potential for elucidating disease mechanisms and targeted drug screening. PIEZO1 is critical in numerous physiological processes,but monitoring its activity and localization in cells can be challenging. Here,the authors present a chemogenetic platform to visualize endogenous human PIEZO1 localization and activity in native cellular conditions,expanding the knowledge on mechanotransduction across single cells and tissue organoids. View Publication

Natural killer (NK) cells are cytotoxic immune cells that can eliminate target cells without prior stimulation. Human induced pluripotent stem cells (iPSCs) provide a robust source of NK cells for safe and effective cell-based immunotherapy against aggressive cancers. In this in vitro study,a feeder-free iPSC differentiation was performed to obtain iPSC-NK cells,and distinct maturational stages of iPSC-NK were characterized. Mature cells of CD56bright CD16bright phenotype showed upregulation of CD56,CD16,and NK cell activation markers NKG2D and NKp46 upon IL-15 exposure,while exposure to aggressive atypical teratoid/rhabdoid tumor (ATRT) cell lines enhanced NKG2D and NKp46 expression. Malignant cell exposure also increased CD107a degranulation markers and stimulated IFN-? secretion in activated NK cells. CD56bright CD16bright iPSC-NK cells showed a ratio-dependent killing of ATRT cells,and the percentage lysis of CHLA-05-ATRT was higher than that of CHLA-02-ATRT. The iPSC-NK cells were also cytotoxic against other brain,kidney,and lung cancer cell lines. Further NK maturation yielded CD56?ve CD16bright cells,which lacked activation markers even after exposure to interleukins or ATRT cells - indicating diminished cytotoxicity. Generation and characterization of different NK phenotypes from iPSCs,coupled with their promising anti-tumor activity against ATRT in vitro,offer valuable insights into potential immunotherapeutic strategies for brain tumors. Graphical abstractImage 1 Highlights•Natural killer (NK) cells were derived from human induced pluripotent stem cells (iPSCs) in the absence of feeder cells.•Various maturational subtypes of iPSC-NK cells were characterized,and the phenotypic and functional properties were studied.•iPSC-NK cells of CD56bright CD16bright phenotype expressed activation markers in response to interleukin stimuli.•iPSC-NK cells were cytotoxic toward human atypical teratoid and rhabdoid tumor (ATRT) cells and other human cancer cells.•The cytotoxicity of iPSC-NK cells against various cancer cells in vitro might be translated into an in vivo immunotherapy. View Publication

BackgroundThe human induced pluripotent stem cells (hiPSCs) can generate all the cells composing the human body,theoretically. Therefore,hiPSCs are thought to be a candidate source of stem cells for regenerative medicine. The major challenge of allogeneic hiPSC-derived cell products is their immunogenicity. The hypoimmunogenic cell strategy is allogenic cell therapy without using immune suppressants. Advances in gene engineering technology now permit the generation of hypoimmunogenic cells to avoid allogeneic immune rejection. In this study,we generated a hypoimmunogenic hiPSC (HyPSC) clone that had diminished expression of human leukocyte antigen (HLA) class Ia and class II and expressed immune checkpoint molecules and a safety switch.MethodsFirst,we generated HLA class Ia and class II double knockout (HLA class Ia/II DKO) hiPSCs. Then,a HyPSC clone was generated by introducing exogenous β-2-microglobulin (B2M),HLA-G,PD-L1,and PD-L2 genes,and the Rapamycin-activated Caspase 9 (RapaCasp9)-based suicide gene as a safety switch into the HLA class Ia/II DKO hiPSCs. The characteristics and immunogenicity of the HyPSCs and their derivatives were analyzed.ResultsWe found that the expression of HLA-G on the cell surface can be enhanced by introducing the exogenous HLA-G gene along with B2M gene into HLA class Ia/II DKO hiPSCs. The HyPSCs retained a normal karyotype and had the characteristics of pluripotent stem cells. Moreover,the HyPSCs could differentiate into cells of all three germ layer lineages including CD45+ hematopoietic progenitor cells (HPCs),functional endothelial cells,and hepatocytes. The HyPSCs-derived HPCs exhibited the ability to evade innate and adaptive immunity. Further,we demonstrated that RapaCasp9 could be used as a safety switch in vitro and in vivo.ConclusionThe HLA class Ia/II DKO hiPSCs armed with HLA-G,PD-L1,PD-L2,and RapaCasp9 molecules are a potential source of stem cells for allogeneic transplantation.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13287-024-03810-4. View Publication

The aim of this study is to establish an in vitro co-culture system to model allograft rejection using kidney organoids system derived from human induced pluripotent stem cells (hiPSCs). We co-cultured kidney organoids derived from wild-type hiPSCs with HLA-mismatched peripheral blood mononuclear cells (PBMCs) from healthy controls (HC) for 24 h. To assess allogeneic rejection modeling,we measured the expression of HLA molecules,(HLA-ABC and HLA-DR),and evaluated cellular damage in the kidney organoids. Additionally,we analyzed the distribution of T cells and their subsets within the co-cultured PBMCs. The immunosuppressive effect of tacrolimus was also evaluated in this co-culture system. Transcriptomic analysis,conducted using RNA sequencing,identified molecules associated with allogeneic rejection. When kidney organoids were co-cultured with alloreactive PBMCs for 24 h,HLA-ABC and HLA-DR expression significantly increased in kidney organoid cells. Additionally,kidney organoids showed reduced cell viability and increased apoptosis compared to syngeneic controls,as assessed by flow cytometry and Annexin V/PI staining. However,treatment with tacrolimus reduced HLA expression in a dose-dependent manner,highlighting the diminished alloimmune responses. Further analysis of PBMC subsets revealed shifts in T helper (TH) and cytotoxic T cell (TC) populations under allogeneic conditions,including increased effector TH and TC cells. Transcriptomic analysis through RNA sequencing identified 256 differentially expressed genes (DEGs),with notable immune-related pathways such as NF-kappa B and TNF signaling involved in allograft rejection. These results provide evidence that a co-culture system with allogeneic kidney organoids and PBMCs can potentially model transplant rejection in vitro.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00018-025-05867-7. View Publication

Purpose: To investigate the therapeutic potential of inducible pluripotent stem cell (hiPSC)-based vascular repair,we evaluated two vascular reparative cell populations,CD34+ cells derived from hiPSC (hiPSC-CD34+) and endothelial colony forming cells (ECFCs) derived from hiPSC (iPS-ECFCs),alone and in combination,in a type 2 diabetic (db/db) mouse model of DR. Methods: hiPSC-CD34+ cells (1 × 104) or iPSC- ECFCs (1 × 105) alone or in combination (1.1 × 105) were injected into the vitreous of immunosuppressed db/db mice with six months of established diabetes. One month post-injection,mice underwent electroretinography (ERG) and optical coherence tomography (OCT) to evaluate functional and structural retinal recovery with iPSC administration. Immunohistochemistry (IHC) was used to assess recruitment and incorporation of cells into the retinal vasculature. Retinas from the experimental groups were analyzed using Functional Proteomics via Reverse Phase Protein Array (RPPA). Results: Functional assessment via ERG demonstrated significant improvements in retinal response in the diabetic cohorts treated with either hiPSC-derived CD34+ cells or hiPSC-ECFCs. Retinal thickness,assessed by OCT,was restored to near-nondiabetic levels in mice treated with hiPSC-CD34+ cells alone and the combination group,whereas hiPSC-ECFCs alone did not significantly affect retinal thickness. One month following intravitreal injection,hiPSC-CD34+ cells were localized to perivascular regions,whereas hiPSC-ECFCs were observed to integrate directly into the retinal vasculature. RPPA analysis revealed interaction-significant changes,and this was interpreted as a combination-specific,non-additive host responses (m6A,PI3K–AKT–mTOR,glycolysis,endothelial junction pathways). Conclusions: The studies support that injection of hiPSC-CD34+ cells and hiPSC-ECFCs,both individually and in combination,showed benefit; however,iPSC combination-specific effects were identified by measurement of retinal thickness and by RPPA. View Publication

Human iPSC-derived cardiomyocytes (hiPSC-CMs) have proven invaluable for cardiac disease modeling and regeneration. Challenges with quality,inter-batch consistency,cryopreservation and scale remain,reducing experimental reproducibility and clinical translation. Here,we report a robust stirred suspension cardiac differentiation protocol,and we perform extensive morphological and functional characterization of the resulting bioreactor-differentiated iPSC-CMs (bCMs). Across multiple different iPSC lines,the protocol produces 1.2E6/mL bCMs with ~94% purity. bCMs have high viability after cryo-recovery (>90%) and predominantly ventricular identity. Compared to standard monolayer-differentiated CMs,bCMs are more reproducible across batches and have more mature functional properties. The protocol also works with magnetically stirred spinner flasks,which are more economical and scalable than bioreactors. Minor protocol modifications generate cardiac organoids fully in suspension culture. These reproducible,scalable,and resource-efficient approaches to generate iPSC-CMs and organoids will expand their applications,and our benchmark data will enable comparison to cells produced by other cardiac differentiation protocols. Subject terms: Cardiovascular biology,Induced pluripotent stem cells,Cardiovascular models View Publication

Intervertebral disc disease (IDD) is a debilitating spine condition that can be caused by intervertebral disc (IVD) damage which progresses towards IVD degeneration and dysfunction. Recently,human pluripotent stem cells (hPSCs) were recognized as a valuable resource for cell-based regenerative medicine in skeletal diseases. Therefore,adult somatic cells reprogrammed into human induced pluripotent stem cells (hiPSCs) represent an attractive cell source for the derivation of notochordal-like cells (NCs) as a first step towards the development of a regenerative therapy for IDD. Utilizing a differentiation method involving treatment with a four-factor cocktail targeting the BMP,FGF,retinoic acid,and Wnt signaling pathways,we differentiate CRISPR/Cas9-generated mCherry-reporter knock-in hiPSCs into notochordal-like cells. Comprehensive analysis of transcriptomic changes throughout the differentiation process identified regulation of histone methylation as a pivotal driver facilitating the differentiation of hiPSCs into notochordal-like cells. We further provide evidence that specific inhibition of histone demethylases KDM2A and KDM7A/B enhanced the lineage commitment of hiPSCs towards notochordal-like cells. Our results suggest that inhibition of KDMs could be leveraged to alter the epigenetic landscape of hiPSCs to control notochord-specific gene expression. Thus,our study highlights the importance of epigenetic regulators in stem cell-based regenerative approaches for the treatment of disc degeneration. View Publication

Human induced pluripotent stem cells (hiPSC) provide an attractive tool to study disease mechanisms of neurodevelopmental disorders such as schizophrenia. A pertinent problem is the development of hiPSC-based assays to discriminate schizophrenia (SZ) from autism spectrum disorder (ASD) models. Healthy control individuals as well as patients with SZ and ASD were examined by a panel of diagnostic tests. Subsequently,skin biopsies were taken for the generation,differentiation,and testing of hiPSC-derived neurons from all individuals. SZ and ASD neurons share a reduced capacity for cortical differentiation as shown by quantitative analysis of the synaptic marker PSD95 and neurite outgrowth. By contrast,pattern analysis of calcium signals turned out to discriminate among healthy control,schizophrenia,and autism samples. Schizophrenia neurons displayed decreased peak frequency accompanied by increased peak areas,while autism neurons showed a slight decrease in peak amplitudes. For further analysis of the schizophrenia phenotype,transcriptome analyses revealed a clear discrimination among schizophrenia,autism,and healthy controls based on differentially expressed genes. However,considerable differences were still evident among schizophrenia patients under inspection. For one individual with schizophrenia,expression analysis revealed deregulation of genes associated with the major histocompatibility complex class II (MHC class II) presentation pathway. Interestingly,antipsychotic treatment of healthy control neurons also increased MHC class II expression. In conclusion,transcriptome analysis combined with pattern analysis of calcium signals appeared as a tool to discriminate between SZ and ASD phenotypes in vitro. View Publication

Idiopathic pulmonary fibrosis (IPF) is a fatal disease of unknown cause that is characterized by progressive fibrotic lung remodeling. An abnormal emergence of airway epithelial-like cells within the alveolar compartments of the lung,herein termed bronchiolization,is often observed in IPF. However,the origin of this dysfunctional distal lung epithelium remains unknown due to a lack of suitable human model systems. In this study,we established a human induced pluripotent stem cell (iPSC)-derived air-liquid interface (ALI) model of alveolar epithelial type II (ATII)-like cell differentiation that allows us to investigate alveolar epithelial progenitor cell differentiation in vitro. We treated this system with an IPF-relevant cocktail (IPF-RC) to mimic the pro-fibrotic cytokine milieu present in IPF lungs. Stimulation with IPF-RC during differentiation increases secretion of IPF biomarkers and RNA sequencing (RNA-seq) of these cultures reveals significant overlap with human IPF patient data. IPF-RC treatment further impairs ATII differentiation by driving a shift toward an airway epithelial-like expression signature,providing evidence that a pro-fibrotic cytokine environment can influence the proximo-distal differentiation pattern of human lung epithelial cells. In conclusion,we show for the first time,the establishment of a human model system that recapitulates aspects of IPF-associated bronchiolization of the lung epithelium in vitro. View Publication

Human microglia are central regulators and actors in brain infections and neuro-inflammatory pathologies. However,access to such cells is limited,and studies systematically mapping the spectrum of their inflammatory states are scarce. Here,we generated microglia-like cells (MGLCs) from human induced pluripotent stem cells and characterized them as a robust,accessible model system for studying inflammatory activation. We validated lineage identity through transcriptome profiling,revealing selective upregulation of microglial signature genes and enrichment of microglia/macrophage-related gene sets. MGLCs displayed distinct morphologies and produced stimulus- and time-dependent cytokine secretion profiles upon exposure to diverse inflammatory stimuli,including pro-inflammatory cytokines (TNFα,interferon-γ) and agonists of the Toll-like receptors TLR2 (FSL-1),TLR3 (Poly(I:C)),TLR4 (lipopolysaccharide,LPS),and TLR7 (imiquimod). Transcriptome profiling and bioinformatics analysis revealed distinct activation signatures. Functional assays demonstrated stimulus-specific engagement of NFκB and JAK-STAT signaling pathways. The shared NFκB nuclear translocation response of TLR ligands and TNFα was reflected in overlapping transcriptome profiles: they shared modules (e.g.,oxidative stress response and TNFα-related signaling) identified by weighted gene co-expression network analysis. Finally,the potential consequences of microglia activation for neighboring cells were studied on the example of microglia-astrocyte crosstalk. The capacity of MGLC supernatants to stimulate astrocytes was measured by quantifying astrocytic NFκB translocation. MGLCs stimulated with FSL-1,LPS,or Poly(I:C) indirectly activated astrocytes via a strictly TNFα-dependent mechanism,highlighting the role of soluble mediators in the signal propagation. Altogether,this platform enables a dissection of microglia activation states and multi-parametric characterization of subsequent neuroinflammation. View Publication

Hypertrophic cardiomyopathy (HCM) is a common and deadly cardiac disease characterized by enlarged myocytes,increased myocardial wall thickening,and fibrosis. A majority of HCM cases are associated with mutations in the β-myosin heavy chain (MYH7) converter domain locus,which leads to varied pathophysiological and clinical manifestations. Using base-editing technology,we generated mutant human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) harboring HCM-causing myosin converter domain mutations (MYH7 c.2167C>T [R723C]; MYH6 c.2173C>T [R725C]) to define HCM pathogenesis in vitro. In this study,we integrated transcriptomic analysis with phenotypic and molecular analyses to dissect the HCM disease mechanisms using MYH6/7 myosin mutants. Our KEGG analysis of bulk RNA-sequencing data revealed significant upregulation of transcripts associated with HCM in the mutant hiPSC-CMs. Further,in-depth transcriptomic analysis using Gene-Ontology (GO-term) analysis for biological process showed upregulation of several transcripts associated with heart development and disease. Notably,our analysis showed robust upregulation of cytoskeletal transcripts,including actin-cytoskeleton networks,sarcomere components,and other structural proteins in the mutant CMs. Furthermore,cellular and nuclear morphological analysis showed that the MYH6/7 mutation induced cellular hypertrophy and increased aspect ratio compared to the isogenic control. Immunostaining experiments showed marked sarcomere disorganization with lower sarcomeric order and higher dispersion in the mutant hiPSC-CMs,highlighting the remodeling of the myofibril arrangement. Notably,the MYH6/7 mutant showed reduced cortical F-actin expression and increased central F-actin expression compared to the isogenic control,confirming the cytoskeletal remodeling and sarcomeric organization during HCM pathogenesis. These pathological changes accumulated progressively over time,underscoring the chronic and evolving nature of HCM driven by the MYH6/7 mutations. Together,our findings provide critical insights into the cellular and molecular underpinnings of MYH6/7-mutation-associated disease. These findings offer valuable insights into HCM pathogenesis,aiding in future therapies. View Publication

Rett syndrome (RTT) is a severe neurodevelopmental disorder associated with mutations in either MECP2,CDKL5 or FOXG1. The precise molecular mechanisms that lead to the pathogenesis of RTT have yet to be elucidated. We recently reported that expression of GluD1 (orphan glutamate receptor $\$-1 subunit) is increased in iPSC-derived neurons obtained from patients with mutations in either MECP2 or CDKL5. GluD1 controls synaptic differentiation and shifts the balance between excitatory and inhibitory synapses toward the latter. Thus,an increase in GluD1 might be a critical factor in the etiology of RTT by affecting the excitatory/inhibitory balance in the developing brain. To test this hypothesis,we generated iPSC-derived neurons from FOXG1(+/-) patients. We analyzed mRNA and protein levels of GluD1 together with key markers of excitatory and inhibitory synapses in these iPSC-derived neurons and in Foxg1(+/-) mouse fetal (E11.5) and adult (P70) brains. We found strong correlation between iPSC-derived neurons and fetal mouse brains,where GluD1 and inhibitory synaptic markers (GAD67 and GABA AR-$\$1) were increased,whereas the levels of a number of excitatory synaptic markers (VGLUT1,GluA1,GluN1 and PSD-95) were decreased. In adult mice,GluD1 was decreased along with all GABAergic and glutamatergic markers. Our findings further the understanding of the etiology of RTT by introducing a new pathological event occurring in the brain of FOXG1(+/-) patients during embryonic development and its time-dependent shift toward a general decrease in brain synapses. View Publication