Pulmonary fibrosis model using micro-CT analyzable human PSC-derived alveolar organoids containing alveolar macrophage-like cells.

Human lung organoids (hLOs) are useful for disease modelling and drug screening. However, a lack of immune cells in hLOs limits the recapitulation of in vivo cellular physiology. Here, we generated hLOs containing alveolar macrophage (AMφ)-like cells derived from pluripotent stem cells (PSC). To bridge hLOs with advanced human lung high-resolution X-ray computed tomography (CT), we acquired quantitative micro-CT images. Three hLO types were observed during differentiation. Among them, alveolar hLOs highly expressed not only lung epithelial cell markers but also AMφ-specific markers. Furthermore, CD68+ AMφ-like cells were spatially organized on the luminal epithelial surface of alveolar hLOs. Bleomycin-treated alveolar hLOs showed upregulated expression of fibrosis-related markers and extracellular matrix deposits in the alveolar sacs. Alveolar hLOs also showed structural alterations such as excessive tissue fraction under bleomycin treatment. Therefore, we suggest that micro-CT analyzable PSC-derived alveolar hLOs are a promising in vitro model to predict lung toxicity manifestations, including fibrosis.

Amine-modified nanoplastics promote the procoagulant activation of isolated human red blood cells and thrombus formation in rats.

BACKGROUND: Microplastics (MPs) and nanoplastics (NPs) formed from decomposed plastic are increasing environmental threats. Although MPs and NPs exposed through various routes enter the systemic circulation, the potential toxicity of those is largely unknown. We investigated whether polystyrene NPs (PS-NPs) promote the coagulation activity of red blood cells (RBCs). RESULTS: We tested several types of PS-NPs using human RBCs and found that amine-modified 100 nm PS-NPs were the most potent. We measured the uptake of PS-NPs using flow cytometry and confocal microscopy. Electron microscopy revealed morphological changes of RBCs by PS-NPs. PS-NPs induced the externalization of phosphatidylserine, generation of microvesicles in RBCs, and perturbations in the intracellular microenvironment. PS-NPs increased the activity of scramblases responsible for phospholipid translocation in RBCs. PS-NPs modulated the functional interaction to adjacent tissues and coagulation cascade, enhancing RBC adhesion and thrombin generation. Our observations in human RBCs were consistent with those in isolated rat RBCs, showing no inter-species differences. In rat venous thrombosis models, the intravenous administration of PS-NPs enhanced thrombus formation. CONCLUSION: Amine-modified PS-NPs induce the prothrombotic activation of RBCs causing thrombus formation. We believe that our study will contribute to understanding the potential toxicity of amine-modified polystyrene particles in blood cells and cardiovascular systems.

Polyhexamethylene guanidine phosphate increases stress granule formation in human 3D lung organoids under respiratory syncytial virus infection.

Polyhexamethylene guanidine phosphate (PHMG-p), a humidifier disinfectant, is known to cause lung toxicity, including inflammation and pulmonary fibrosis. In this study, we aimed to investigate the effect of PHMG-p on human lung tissue models (2D epithelial cells and 3D organoids) under conditions of oxidative stress and viral infection. The effect of PHMG-p was studied by evaluating the formation of stress granules (SGs), which play a pivotal role in cellular adaptation to various stress conditions. Under oxidative stress and respiratory syncytial virus (RSV) infection, exposure to PHMG-p remarkably increased eIF2α phosphorylation, which is essential for SG-related signalling, and significantly increased SG formation. Furthermore, PHMG-p induced fibrotic gene expression and caused cell death due to severe DNA damage, which was further increased under oxidative stress and RSV infection, indicating that PHMG-p induces severe lung toxicity under stress conditions. Taken together, toxicity evaluation under various stressful conditions is necessary to accurately predict potential lung toxicity of chemicals affecting the respiratory tract.

Live-cell screening platform using human-induced pluripotent stem cells expressing fluorescence-tagged cytochrome P450 1A1.

Human-induced pluripotent stem cells (hiPSCs) are invaluable sources for drug screening and toxicity tests because of their differentiation potential and proliferative capacity. Recently, the CRISPR-Cas9-mediated homologous recombination system has enabled reporter knock-ins at desired loci in hiPSCs, and here, we generated a hiPSC reporter line expressing mCherry-tagged cytochrome P450 1A1 (CYP1A1), which can be utilized to screen for the modulators of aryl hydrocarbon receptor (AHR) in live cells. CYP1A1-mCherry hiPSCs exhibited typical characteristics of pluripotent stem cells such as marker expression, differentiation potential, and normal karyotype. After differentiation into hepatocyte-like cells (HLCs), CYP1A1-mCherry fusion protein was expressed and localized at the endoplasmic reticulum, and induced by AHR agonists. We obtained 23 hits modulating CYP1A1 expression from high-content screening with 241 hepatotoxicity chemicals and nuclear receptor ligands, and identified three upregulating chemicals and two downregulating compounds. Responses of hiPSC-HLCs against an AHR agonist were more similar to human primary hepatocytes than of HepG2 hepatocellular carcinoma cells. This platform has the advantages of live-cell screening without sacrificing cells (unlike previously available CYP1A1 reporter cell lines), as well as an indefinite supply of cells, and can be utilized in a wide range of screening related to AHR- and CYP1A1-associated diseases in desired cell types.

LDPC Coded Massive MIMO Systems.

We design a coded massive multiple-input multiple-output (MIMO) system using low-density parity-check (LDPC) codes and iterative joint detection and decoding (JDD) algorithm employing a low complexity detection. We introduce the factor graph representation of the LDPC coded massive MIMO system, based on which the message updating rule in the JDD is defined. We devise a tool for analyzing extrinsic information transfer (EXIT) characteristics of messages flowing in the JDD and the three-dimensional (3-D) EXIT chart provides a visualization of the JDD behavior. Based on the proposed 3-D EXIT analysis, we design jointly the degree distribution of irregular LDPC codes and the JDD strategy for the coded massive MIMO system. The JDD strategy was determined to achieve a higher error correction capability with a given amount of computational complexity. It was observed that the coded massive MIMO system equipped with the proposed LDPC codes and the proposed JDD strategy has lower bit error rate than conventional LDPC coded massive MIMO systems.

Prediction of hepatotoxicity for drugs using human pluripotent stem cell-derived hepatocytes.

Drug-induced liver toxicity is a main reason for withdrawals of new drugs in late clinical phases and post-launch of the drugs. Thus, hepatotoxicity screening of drug candidates in pre-clinical stage is important for reducing drug attrition rates during the clinical development process. Here, we show commercially available hepatocytes that could be used for early toxicity evaluation of drug candidates. From our hepatic differentiation technology, we obtained highly pure (≥98%) hepatocytes from human embryonic stem cells (hESCs) having mature phenotypes and similar gene expression profiles with those of primary human tissues. Furthermore, we optimized 96-well culture condition of hESC-derived hepatocytes suitable for toxicity tests in vitro. To this end, we demonstrated the efficacy of our optimized hepatocyte model for predicting hepatotoxicity against the Chinese herbal medicines and showed that toxicity patterns from our hepatocyte model was similar to those of human primary cultured hepatocytes. We conclude that toxicity test using our hepatocyte model could be a good alternative cell source for pre-clinical study to predict potential hepatotoxicity in drug discovery industries.

Hepatic esterase activity is increased in hepatocyte-like cells derived from human embryonic stem cells using a 3D culture system.

OBJECTIVES: The aim of the study is to generate a spherical three-dimensional (3D) aggregate of hepatocyte-like cells (HLCs) differentiated from human embryonic stem cells and to investigate the effect of the 3D environment on hepatic maturation and drug metabolism. RESULTS: Quantitative real-time PCR analysis indicated that gene expression of mature hepatocyte markers, drug-metabolizing enzymes, and hepatic transporters was significantly higher in HLCs cultured in the 3D system than in those cultured in a two-dimensional system (p < 0.001). Moreover, hepatocyte-specific functions, including albumin secretion and bile canaliculi formation, were increased in HLCs cultured in the 3D system. In particular, 3D spheroidal culture increased expression of CES1 and BCHE, which encode hepatic esterases (p < 0.001). The enhanced activities of these hepatic esterases were confirmed by the cholinesterase activity assay and the increased susceptibility of HLCs to oseltamivir, which is metabolized by CES1. CONCLUSIONS: 3D spheroidal culture enhances the maturation and drug metabolism of stem cell-derived HLCs, and this may help to optimize hepatic differentiation protocols for hepatotoxicity testing.

Two zinc-aminoclays' in-vitro cytotoxicity assessment in HeLa cells and in-vivo embryotoxicity assay in zebrafish.

Two zinc-aminoclays [ZnACs] with functionalized primary amines [(-CH2)3NH2] were prepared by a simple sol-gel reaction using cationic metal precursors of ZnCl2 and Zn(NO3)2 with 3-aminopropyl triethoxysilane [APTES] under ambient conditions. Due to the facile interaction of heavy metals with primary amine sites and Zn-related intrinsic antimicrobial activity, toxicity assays of ZnACs nanoparticles (NPs) prior to their environmental and human-health applications are essential. However, such reports remain rare. Thus, in the present study, a cell viability assay of in-vitro HeLa cells comparing ZnCl2, Zn(NO3)2 salts, and ZnO (~50nm average diameter) NPs was performed. Interestingly, compared with the ZnCl2, and Zn(NO3)2 salts, and ZnO NPs (18.73/18.12/51.49µg/mL and 18.12/15.19/46.10µg/mL of IC50 values for 24 and 48h), the two ZnACs NPs exhibited the highest toxicity (IC50 values of 21.18/18.36µg/mL and 18.37/17.09µg/mL for 24 and 48h, respectively), whose concentrations were calculated on Zn elemental composition. This might be due to the enhanced bioavailability and uptake into cells of ZnAC NPs themselves and their positively charged hydrophilicity by reactive oxygen species (ROS) generation, particularly as ZnACs exist in cationic NP's form, not in released Zn2+ ionic form (i.e., dissolved nanometal). However, in an in-vivo embryotoxicity assay in zebrafish, ZnACs and ZnO NPs showed toxic effects at 50-100µg/mL (corresponding to 37.88-75.76 of Zn wt% µg/mL). The hatching rate (%) of zebrafish was lowest for the ZnO NPs, particularly where ZnAC-[(NO3)2] is slightly more toxic than ZnAC-[Cl2]. These results are all very pertinent to the issue of ZnACs' potential applications in the environmental and biomedical fields.

Nonylphenol-induced apoptotic cell death in mouse TM4 Sertoli cells via the generation of reactive oxygen species and activation of the ERK signaling pathway.

Nonylphenol (NP), a representative endocrine disruptor, interferes with reproductive function in aquatic organisms and animals. Although many previous studies have focused on apoptotic cell death by NP, the fundamental mechanism of NP on apoptosis remains poorly understood. Here, we investigated the molecular mechanism on NP-induced apoptotic cell death in mouse TM4 Sertoli cells. To evaluate NP treatment on cell viability, formazan and lactate dehydrogenase (LDH) assays were performed. Results indicate that NP reduced cell viability and increased the release of LDH in dose- and time-dependent manners. The reduction of cell viability by NP treatment appeared to involve necrosis as well as apoptosis based on nuclear fragmentation, an increase in the sub G1 population, and the detection of poly(ADP ribose) polymerase and caspase-3 cleavage. Additionally, the anti-apoptotic protein Bcl-2 diminished, whereas the pro-apoptotic protein Bax increased in a time-dependent manner. Note that NP-induced apoptotic cell death was enhanced by the generation of reactive oxygen species (ROS) and activation of extracellular signal-regulated kinase (ERK) signaling. Pretreatment with N-acetylcysteine, an antioxidant, attenuated NP-induced apoptotic cell death. Moreover, NP caused a transient activation of the MAPK pathway. In particular, NP-induced cell death was significantly suppressed by U0126, a specific inhibitor of ERK. Taken together, our results suggest that NP induces apoptosis in mouse TM4 Sertoli cells via ROS generation and ERK activation.

Prediction of Acute Hepatotoxicity With Human Pluripotent Stem Cell-Derived Hepatic Organoids.

Recent development of hepatic organoids (HOs) derived from human pluripotent stem cells (hPSCs) provides an alternative in vitro model that can mimic the human liver detoxification pathway for drug safety assessment. By recapitulating the high level of maturity and drug-metabolizing capacity of the liver in a three-dimensional organoid culture, HOs may allow researchers to assess drug toxicity and metabolism more accurately than animal models or hepatocellular carcinoma cells. Although this promising potential has contributed to the development of various protocols, only a few protocols are available to generate functional HOs with guaranteed CYP450 enzymatic activity, the key feature driving toxic responses during drug metabolism. Based on previously published protocols, we describe an optimized culture method that can substantially increase the expression and activity of CYP450s, in particular CYP3A4, CYP2C9, and CYP2C19, in HOs. To generate mass-produced and highly reproducible HOs required as models for toxicity evaluation, we first generated hepatic endodermal organoids (HEOs) from hPSCs capable of in vitro proliferation and cryopreservation. The stepwise protocol includes generating HEOs as well as efficient methods to enhance CYP450 expression and activity in terminally differentiated HOs. Furthermore, we present a simple protocol for the assessment of HO cytotoxicity, one of the hallmarks of drug-induced acute hepatotoxicity. The protocols are relatively straightforward and can be successfully used by laboratories with basic experience in culturing hPSCs. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Generation of hepatic endodermal organoids from human pluripotent stem cells Basic Protocol 2: Expansion and cryopreservation of hepatic endodermal organoids Basic Protocol 3: Differentiation of hepatic organoids from hepatic endodermal organoids Basic Protocol 4: Evaluation of hepatotoxicity using hepatic organoids Support Protocol: Human pluripotent stem cell culture.

hiPSC-derived macrophages improve drug sensitivity and selectivity in a macrophage-incorporating organoid culture model.

Accurate simulation of different cell type interactions is crucial for physiological and precisein vitrodrug testing. Human tissue-resident macrophages are critical for modulating disease conditions and drug-induced injuries in various tissues; however, their limited availability has hindered their use inin vitromodeling. Therefore, this study aimed to create macrophage-containing organoid co-culture models by directly incorporating human-induced pluripotent stem cell (hiPSC)-derived pre-macrophages into organoid and scaffold cell models. The fully differentiated cells in these organoids exhibited functional characteristics of tissue-resident macrophages with enriched pan-macrophage markers and the potential for M1/M2 subtype specialization upon cytokine stimulation. In a hepatic organoid model, the integrated macrophages replicated typical intrinsic properties, including cytokine release, polarization, and phagocytosis, and the co-culture model was more responsive to drug-induced liver injury than a macrophage-free model. Furthermore, alveolar organoid models containing these hiPSC-derived macrophages also showed increased drug and chemical sensitivity to pulmonary toxicants. Moreover, 3D adipocyte scaffold models incorporating macrophages effectively simulated in vivo insulin resistance observed in adipose tissue and showed improved insulin sensitivity on exposure to anti-diabetic drugs. Overall, the findings demonstrated that incorporating hiPSC-derived macrophages into organoid culture models resulted in more physiological and sensitivein vitrodrug evaluation and screening systems.

Micellar ordered structure effects on high-resolution CE-SSCP using Pluronic triblock copolymer blends.

Pluronic F108 block copolymers have shown a great promise to achieve the desirable high resolution in the conformation-sensitive separation of ssDNA using CE-SSCP. However, fundamental understanding of the structures and properties of Pluronic matrix affecting the resolution is still limited. Unlike conventional gel-forming homopolymers, Pluronic F108 block copolymers are amphiphilic macromolecules consisting of poly(ethylene oxide)-b-poly(propylene oxide)-b-poly(ethylene oxide) triblock copolymers, which are capable of forming a highly ordered micellar structure in aqueous solution. In this study, we have performed a series of experiments by blending different types of Pluronic polymers to control the formation of micelles and to study the correlation between separation and rheological characteristics of Pluronic gels affecting the resolution of CE-SSCP. Our experiments have been specifically designed to elucidate how the micellar structure affects the resolution of CE-SSCP upon altering the size uniformity and constituent homogeneity of the micelles. Our results suggest that uniformly sized micelle packing is the primary structural feature of Pluronic gel matrix for the high-resolution separation, while the size and constituent of the micelle themselves need to be considered as secondary factors.

Generation of a PDGFRB-mCherry knock-in reporter human induced pluripotent stem cell line (KITi001-A-1), using CRISPR/Cas9 nuclease.

PDGFRB encodes platelet-derived growth factor receptor beta (PDGFR-ß), a cell surface tyrosine kinase receptor for members of the platelet-derived growth factor family. It is required for the normal development of the vascular and nervous systems and rearrangement of the actin cytoskeleton. PDGFR-ß plays an essential role in early liver diseases, including liver fibrosis. Here, we generated a human induced pluripotent stem cell (iPSC) line, KITi001-A-1, using CRISPR/Cas9. This reporter iPSC line and its derivatives are useful for tracing PDGFR-ß-expressing cells and for screening for liver fibrosis-inducing compounds.

Establishment of a human embryonic stem cell line, WAe009-A-99, with constitutive expression of the dCas9-p300 fusion protein.

CRISPR/Cas9-based transcriptional regulation systems can induce the site-specific activation or repression of endogenous genes. p300 is a transcriptional co-activator that functions as a histone acetyltransferase that regulates gene transcription via chromatin remodeling. Here, we generated a human embryonic stem cell line stably expressing catalytically dead Cas9 (dCas9) fused to the catalytic core domain of human p300 via lentiviral transduction. This cell line can be used for locus-specific histone acetylation in combination with guide RNAs, and is a valuable tool for gene regulation in stem cell research.

Butylparaben promotes phosphatidylserine exposure and procoagulant activity of human red blood cells via increase of intracellular calcium levels.

Parabens are widely used as preservatives, added to products commonly used by humans, and to which individuals are exposed orally or dermally. Once absorbed into the body, parabens move into the bloodstream and travel through the systemic circulation. We investigated the potential impact of parabens on the enhanced generation of thrombin by red blood cells (RBCs), which are the principal cellular components of blood. We tested the effects of methylparaben (MeP), ethylparaben (EtP), propylparaben (PrP), butylparaben (BuP), and p-hydroxybenzoic acid on freshly isolated human RBCs. BuP and simultaneous exposure to BuP and PrP significantly increased phosphatidylserine (PS) externalization to the outer membranes of RBCs. PS externalization by BuP was found to be mediated by increasing intracellular Ca2+ levels in RBCs. The morphological changes in BuP-treated RBCs were observed under an electron microscope. The BuP-exposed RBCs showed increased thrombin generation and adhesion to endothelial cells. Additionally, the externalization of PS exposure and thrombin generation in BuP-treated RBCs were more susceptible to high shear stress, which mimics blood turbulence under pathological conditions. Collectively, we observed that BuP induced morphological and functional changes in RBCs, especially under high shear stress, suggesting that BuP may contribute to the thrombotic risk via procoagulant activity in RBCs.

Generation of multilineage liver organoids with luminal vasculature and bile ducts from human pluripotent stem cells via modulation of Notch signaling.

BACKGROUND: The generation of liver organoids recapitulating parenchymal and non-parenchymal cell interplay is essential for the precise in vitro modeling of liver diseases. Although different types of multilineage liver organoids (mLOs) have been generated from human pluripotent stem cells (hPSCs), the assembly and concurrent differentiation of multiple cell types in individual mLOs remain a major challenge. Particularly, most studies focused on the vascularization of mLOs in host tissue after transplantation in vivo. However, relatively little information is available on the in vitro formation of luminal vasculature in mLOs themselves. METHODS: The mLOs with luminal blood vessels and bile ducts were generated by assembling hepatic endoderm, hepatic stellate cell-like cells (HscLCs), and endothelial cells derived entirely from hPSCs using 96-well ultra-low attachment plates. We analyzed the effect of HscLC incorporation and Notch signaling modulation on the formation of both bile ducts and vasculature in mLOs using immunofluorescence staining, qRT-PCR, ELISA, and live-perfusion imaging. The potential use of the mLOs in fibrosis modeling was evaluated by histological and gene expression analyses after treatment with pro-fibrotic cytokines. RESULTS: We found that hPSC-derived HscLCs are crucial for generating functional microvasculature in mLOs. HscLC incorporation and subsequent vascularization substantially reduced apoptotic cell death and promoted the survival and growth of mLOs with microvessels. In particular, precise modulation of Notch signaling during a specific time window in organoid differentiation was critical for generating both bile ducts and vasculature. Live-cell imaging, a series of confocal scans, and electron microscopy demonstrated that blood vessels were well distributed inside mLOs and had perfusable lumens in vitro. In addition, exposure of mLOs to pro-fibrotic cytokines induced early fibrosis-associated events, including upregulation of genes associated with fibrotic induction and endothelial cell activation (i.e., collagen I, α-SMA, and ICAM) together with destruction of tissue architecture and organoid shrinkage. CONCLUSION: Our results demonstrate that mLOs can reproduce parenchymal and non-parenchymal cell interactions and suggest that their application can advance the precise modeling of liver diseases in vitro.

Gene expression analysis of toxicological pathways in TM3 leydig cell lines treated with Ethane dimethanesulfonate.

Ethane dimethanesulfonate (EDS), a well-known alkylating agent, selectively destroys Leydig cells. To clarify the molecular pathways underlying EDS action on Leydig cells, we analyzed gene expression profiles of an EDS-treated TM3 Leydig cell line. In this study, we analyzed the representative canonical pathways and toxicity pathways/gene lists using the Ingenuity Pathways Analysis program. In TM3 cells, 677 and 6756 genes were identified as being up- or downregulated after 3 and 24 h EDS treatments, respectively, (>1.3-fold changes, p < 0.05). Toxicological pathway analysis revealed that expression of genes related to Nrf2-mediated oxidative stress response showed remarkable changes in early or later stage of EDS-treated TM3 cells. Several genes related to steroidogenesis and apoptosis were also differentially expressed at 24 h in EDS-treated TM3 cells. Overall, toxicological pathway analysis using gene expression profiling showed that oxidative stress might be an important factor in cell death in TM3 cells affected by EDS treatment.

Inflammatory response in rat lungs with recurrent exposure to welding fumes: a transcriptomic approach.

As chronic exposure to welding fumes causes pulmonary diseases, such as pneumoconiosis, public concern has increased regarding continued exposure to these hazardous gases in the workplace. In a previous study, the inflammatory response to welding fume exposure was analysed in rat lungs in the case of recurrent exposure and recovery periods. Thus using lung samples, well-annotated by histological observation and biochemical analysis, this study examines the gene expression profiles to identify phenotype-anchored genes corresponding to lung inflammation and the repair phenomenon after recurrent welding fume exposure. Seven genes (Mmp12, Cd5l, LOC50101, LOC69183, Spp1, and Slc26a4) were found to be significantly up-regulated according to the severity of the lung injury. In addition, the transcription and translation of Trem2, which was up-regulated in response to the repair process, were validated using a real-time polymerase chain reaction, Western blotting, and immunohistochemistry. The differentially expressed genes in the exposure and recovery groups were also classified using k-means and hierarchical clustering, plus their toxicological function and canonical pathways were further analysed using Ingenuity Pathways Analysis Software. As a result, this comprehensive and integrative analysis of the transcriptional changes that occur during repeated exposure provides important information on the inflammation and repair processes after welding-fume-induced lung injury.

Particulate matter exposure exacerbates cellular damage by increasing stress granule formation in respiratory syncytial virus-infected human lung organoids.

Exposure to atmospheric particulate matter (PM) increases morbidity and mortality in respiratory diseases by causing various adverse health effects; however, the effects of PM exposure on cellular stress under virus-infected conditions remain unclear. The effects of PM under 10 µm (PM10) and diesel PM (DPM) on respiratory syncytial virus (RSV) infection were investigated in human two-dimensional lung epithelial cells and human three-dimensional lung organoids mimicking the lung tissue. We evaluated the formation of stress granules, which are important in cellular adaptation to various stress conditions. Furthermore, we investigated the effects of repeated exposure to PM10 and DPM on DNA damage and cell death during viral infection. PM10 and DPM did not cause stress granule formation in the absence of RSV infection but drastically increased stress granule formation and signal transduction during RSV infection in human lung epithelial cells and human lung organoids. Further, repeated exposure to PM10 and DPM caused cell death by severely damaging DNA under RSV infection conditions. Thus, PM10 and DPM induce severe lung toxicity under stress conditions, such as viral infection, suggesting that the effects of PMs under various stressful conditions should be examined to accurately predict the lung toxicity of PM.

Therapeutic correction of hemophilia A using 2D endothelial cells and multicellular 3D organoids derived from CRISPR/Cas9-engineered patient iPSCs.

The bleeding disorder hemophilia A (HA) is caused by a single-gene (F8) defect and its clinical symptom can be substantially improved by a small increase in the plasma coagulation factor VIII (FVIII) level. In this study, we used F8-defective human induced pluripotent stem cells from an HA patient (F8d-HA hiPSCs) and F8-corrected (F8c) HA hiPSCs produced by CRISPR/Cas9 genome engineering of F8d-HA hiPSCs. We obtained a highly enriched population of CD157+ cells from CRISPR/Cas9-edited F8c-HA hiPSCs. These cells exhibited multiple cellular and functional phenotypes of endothelial cells (ECs) with significant levels of FVIII activity, which was not observed in F8d-HA hiPSC-ECs. After transplantation, the engineered F8c-HA hiPSC-ECs dramatically changed bleeding episodes in HA animals and restored plasma FVIII activity. Notably, grafting a high dose of ECs substantially reduced the bleeding time during multiple consecutive bleeding challenges in HA mice, demonstrating a robust hemostatic effect (90% survival). Furthermore, the engrafted ECs survived more than 3 months in HA mice and reversed bleeding phenotypes against lethal wounding challenges. We also produced F8c-HA hiPSC-derived 3D liver organoids by assembling three different cell types in microwell devices and confirmed its therapeutic effect in HA animals. Our data demonstrate that the combination of genome-engineering and iPSC technologies represents a novel modality that allows autologous cell-mediated gene therapy for treating HA.