HOIL-1L deficiency induces cell cycle alteration which causes immaturity of skeletal muscle and cardiomyocytes.

HOIL-1L deficiency was recently reported to be one of the causes of myopathy and dilated cardiomyopathy (DCM). However, the mechanisms by which myopathy and DCM develop have not been clearly elucidated. Here, we sought to elucidate these mechanisms using the murine myoblast cell line C2C12 and disease-specific human induced pluripotent stem cells (hiPSCs). Myotubes differentiated from HOIL-1L-KO C2C12 cells exhibited deteriorated differentiation and mitotic cell accumulation. CMs differentiated from patient-derived hiPSCs had an abnormal morphology with a larger size and were excessively multinucleated compared with CMs differentiated from control hiPSCs. Further analysis of hiPSC-derived CMs showed that HOIL-1L deficiency caused cell cycle alteration and mitotic cell accumulation. These results demonstrate that abnormal cell maturation possibly contribute to the development of myopathy and DCM. In conclusion, HOIL-1L is an important intrinsic regulator of cell cycle-related myotube and CM maturation and cell proliferation.

A de novo dominant-negative variant is associated with OTULIN-related autoinflammatory syndrome.

OTULIN-related autoinflammatory syndrome (ORAS), a severe autoinflammatory disease, is caused by biallelic pathogenic variants of OTULIN, a linear ubiquitin-specific deubiquitinating enzyme. Loss of OTULIN attenuates linear ubiquitination by inhibiting the linear ubiquitin chain assembly complex (LUBAC). Here, we report a patient who harbors two rare heterozygous variants of OTULIN (p.P152L and p.R306Q). We demonstrated accumulation of linear ubiquitin chains upon TNF stimulation and augmented TNF-induced cell death in mesenchymal stem cells differentiated from patient-derived iPS cells, which confirms that the patient has ORAS. However, although the de novo p.R306Q variant exhibits attenuated deubiquitination activity without reducing the amount of OTULIN, the deubiquitination activity of the p.P152L variant inherited from the mother was equivalent to that of the wild-type. Patient-derived MSCs in which the p.P152L variant was replaced with wild-type also exhibited augmented TNF-induced cell death and accumulation of linear chains. The finding that ORAS can be caused by a dominant-negative p.R306Q variant of OTULIN furthers our understanding of disease pathogenesis.

Selective induction of human renal interstitial progenitor-like cell lineages from iPSCs reveals development of mesangial and EPO-producing cells.

Recent regenerative studies using human pluripotent stem cells (hPSCs) have developed multiple kidney-lineage cells and organoids. However, to further form functional segments of the kidney, interactions of epithelial and interstitial cells are required. Here we describe a selective differentiation of renal interstitial progenitor-like cells (IPLCs) from human induced pluripotent stem cells (hiPSCs) by modifying our previous induction method for nephron progenitor cells (NPCs) and analyzing mouse embryonic interstitial progenitor cell (IPC) development. Our IPLCs combined with hiPSC-derived NPCs and nephric duct cells form nephrogenic niche- and mesangium-like structures in vitro. Furthermore, we successfully induce hiPSC-derived IPLCs to differentiate into mesangial and erythropoietin-producing cell lineages in vitro by screening differentiation-inducing factors and confirm that p38 MAPK, hypoxia, and VEGF signaling pathways are involved in the differentiation of mesangial-lineage cells. These findings indicate that our IPC-lineage induction method contributes to kidney regeneration and developmental research.

UK-5099, a mitochondrial pyruvate carrier inhibitor, recovers impaired neutrophil maturation caused by AK2 deficiency in human pluripotent stem cell models.

Reticular dysgenesis (RD) is a rare genetic disease caused by gene mutations in the ATP:AMP phosphotransferase, adenylate kinase 2 (AK2). Patients with RD suffer from severe combined immunodeficiency with neutrophil maturation arrest. Although hematopoietic stem cell transplantation can be a curative option, it is invasive, and complications of agranulocytosis-induced infection worsen the prognosis. Here, we report that the use of UK-5099, an inhibitor of the mitochondrial pyruvate carrier (MPC), on hemo-angiogenic progenitor cells (HAPCs) derived from AK2-deficient induced pluripotent stem cells improved neutrophil maturation. Reactive oxygen species (ROS) levels in AK2-deficient HAPCs remained unchanged throughout all experiments, implying that UK-5099 improved the phenotype without affecting ROS levels. Overall, our results suggest that the MPC is a potential therapeutic target for the treatment of neutrophil maturation defects in RD.

A disease-specific iPS cell resource for studying rare and intractable diseases.

BACKGROUND: Disease-specific induced pluripotent stem cells (iPSCs) are useful tools for pathological analysis and diagnosis of rare diseases. Given the limited available resources, banking such disease-derived iPSCs and promoting their widespread use would be a promising approach for untangling the mysteries of rare diseases. Herein, we comprehensively established iPSCs from patients with designated intractable diseases in Japan and evaluated their properties to enrich rare disease iPSC resources. METHODS: Patients with designated intractable diseases were recruited for the study and blood samples were collected after written informed consent was obtained from the patients or their guardians. From the obtained samples, iPSCs were established using the episomal method. The established iPSCs were deposited in a cell bank. RESULTS: We established 1,532 iPSC clones from 259 patients with 139 designated intractable diseases. The efficiency of iPSC establishment did not vary based on age and sex. Most iPSC clones originated from non-T and non-B hematopoietic cells. All iPSC clones expressed key transcription factors, OCT3/4 (range 0.27-1.51; mean 0.79) and NANOG (range 0.15-3.03; mean 1.00), relative to the reference 201B7 iPSC clone. CONCLUSIONS: These newly established iPSCs are readily available to the researchers and can prove to be a useful resource for research on rare intractable diseases.

ZEB2 and MEIS1 independently contribute to hematopoiesis via early hematopoietic enhancer activation.

Cell differentiation is achieved by acquiring a cell type-specific transcriptional program and epigenetic landscape. While the cell type-specific patterning of enhancers has been shown to precede cell fate decisions, it remains unclear how regulators of these enhancers are induced to initiate cell specification and how they appropriately restrict cells that differentiate. Here, using embryonic stem cell-derived hematopoietic cell differentiation cultures, we show the activation of some hematopoietic enhancers during arterialization of hemogenic endothelium, a prerequisite for hematopoiesis. We further reveal that ZEB2, a factor involved in the transcriptional regulation of arterial endothelial cells, and a hematopoietic regulator MEIS1 are independently required for activating these enhancers. Concomitantly, ZEB2 or MEIS1 deficiency impaired hematopoietic cell development. These results suggest that multiple regulators expressed from an earlier developmental stage non-redundantly contribute to the establishment of hematopoietic enhancer landscape, thereby restricting cell differentiation despite the unrestricted expression of these regulators to hematopoietic cells.

Cryptotanshinone is a candidate therapeutic agent for interstitial lung disease associated with a BRICHOS-domain mutation of SFTPC.

Interstitial lung disease (ILD) represents a large group of diseases characterized by chronic inflammation and fibrosis of the lungs, for which therapeutic options are limited. Among several causative genes of familial ILD with autosomal dominant inheritance, the mutations in the BRICHOS domain of SFTPC cause protein accumulation and endoplasmic reticulum stress by misfolding its proprotein. Through a screening system using these two phenotypes in HEK293 cells and evaluation using alveolar epithelial type 2 (AT2) cells differentiated from patient-derived induced pluripotent stem cells (iPSCs), we identified Cryptotanshinone (CPT) as a potential therapeutic agent for ILD. CPT decreased cell death induced by mutant SFTPC overexpression in A549 and HEK293 cells and ameliorated the bleomycin-induced contraction of the matrix in fibroblast-dependent alveolar organoids derived from iPSCs with SFTPC mutation. CPT and this screening strategy can apply to abnormal protein-folding-associated ILD and other protein-misfolding diseases.

Evolutionary histories of breast cancer and related clones.

Recent studies have documented frequent evolution of clones carrying common cancer mutations in apparently normal tissues, which are implicated in cancer development1-3. However, our knowledge is still missing with regard to what additional driver events take place in what order, before one or more of these clones in normal tissues ultimately evolve to cancer. Here, using phylogenetic analyses of multiple microdissected samples from both cancer and non-cancer lesions, we show unique evolutionary histories of breast cancers harbouring der(1;16), a common driver alteration found in roughly 20% of breast cancers. The approximate timing of early evolutionary events was estimated from the mutation rate measured in normal epithelial cells. In der(1;16)(+) cancers, the derivative chromosome was acquired from early puberty to late adolescence, followed by the emergence of a common ancestor by the patient's early 30s, from which both cancer and non-cancer clones evolved. Replacing the pre-existing mammary epithelium in the following years, these clones occupied a large area within the premenopausal breast tissues by the time of cancer diagnosis. Evolution of multiple independent cancer founders from the non-cancer ancestors was common, contributing to intratumour heterogeneity. The number of driver events did not correlate with histology, suggesting the role of local microenvironments and/or epigenetic driver events. A similar evolutionary pattern was also observed in another case evolving from an AKT1-mutated founder. Taken together, our findings provide new insight into how breast cancer evolves.

Tofacitinib, a suppressor of NOD2 expression, is a potential treatment for Blau syndrome.

Introduction: Blau syndrome is a rare autosomal dominant autoinflammatory granulomatous disease caused by a mutation in the NOD2 gene. It is characterized by a clinical trial of granulomatous dermatitis, arthritis, and uveitis. Tofacitinib is a pan Janus kinase (JAK) inhibitor used for treatment of Blau syndrome and idiopathic sarcoidosis. Here, we evaluated its effect on inflammatory pathways associated with Blau syndrome. The effect of tofacitinib on downstream pathways regulated by mutant NOD2 was analyzed using luciferase assays with overexpression of NOD2 mutants. Methods: The effect of tofacitinib on the upstream pathway for the induction of NOD2 expression and proinflammatory cytokine production was assessed using monocytic cell lines differentiated from Blau syndrome patient-derived induced pluripotent stem cells. Results: Tofacitinib did not suppress the increased spontaneous transcriptional activity of NF-κB by mutant NOD2. In addition, mutant NOD2 was not involved in the transcription of ISRE and GAS, which are activated by type 1 and type 2 interferons (IFN), respectively. On the other hand, IFNγ induced the expression of NOD2, which led to the production of inflammatory cytokines by an autoinflammatory mechanism only in cells with mutant NOD2. Discussion: Tofacitinib suppressed the induction of NOD2 by IFNγ, thereby inhibiting the production of pro-inflammatory cytokines. Thus, tofacitinib showed anti-inflammatory effects through suppression of NOD2 expression. The JAK inhibitor tofacitinib is a potential therapeutic agent for Blau syndrome because it suppresses the autoinflammation seen in Blau syndrome by inhibiting the expression of NOD2.

A concise in vitro model for evaluating interactions between macrophage and skeletal muscle cells during muscle regeneration.

Skeletal muscle has a highly regenerative capacity, but the detailed process is not fully understood. Several in vitro skeletal muscle regeneration models have been developed to elucidate this, all of which rely on specialized culture conditions that limit the accessibility and their application to many general experiments. Here, we established a concise in vitro skeletal muscle regeneration model using mouse primary cells. This model allows evaluation of skeletal muscle regeneration in two-dimensional culture system similar to a typical cell culture, showing a macrophage-dependent regenerative capacity, which is an important process in skeletal muscle regeneration. Based on the concept that this model could assess the contribution of macrophages of various phenotypes to skeletal muscle regeneration, we evaluated the effect of endotoxin pre-stimulation for inducing various changes in gene expression on macrophages and found that the contribution to skeletal muscle regeneration was significantly reduced. The gene expression patterns differed from those of naive macrophages, especially immediately after skeletal muscle injury, suggesting that the difference in responsiveness contributed to the difference in regenerative efficiency. Our findings provide a concise in vitro model that enables the evaluation of the contribution of individual cell types, such as macrophages and muscle stem cells, on skeletal muscle regeneration.

Involvement of mTOR pathway in neurodegeneration in NSF-related developmental and epileptic encephalopathy.

Membrane fusion is mediated by soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins. During neurotransmitter exocytosis, SNARE proteins on a synaptic vesicle and the target membrane form a complex, resulting in neurotransmitter release. N-ethylmaleimide-sensitive factor (NSF), a homohexameric ATPase, disassembles the complex, allowing individual SNARE proteins to be recycled. Recently, the association between pathogenic NSF variants and developmental and epileptic encephalopathy (DEE) was reported; however, the molecular pathomechanism of NSF-related DEE remains unclear. Here, three patients with de novo heterozygous NSF variants were presented, of which two were associated with DEE and one with a very mild phenotype. One of the DEE patients also had hypocalcemia from parathyroid hormone deficiency and neuromuscular junction impairment. Using PC12 cells, a neurosecretion model, we show that NSF with DEE-associated variants impaired the recycling of vesicular membrane proteins and vesicle enlargement in response to exocytotic stimulation. In addition, DEE-associated variants caused neurodegenerative change and defective autophagy through overactivation of the mammalian/mechanistic target of rapamycin (mTOR) pathway. Treatment with rapamycin, an mTOR inhibitor or overexpression of wild-type NSF ameliorated these phenotypes. Furthermore, neurons differentiated from patient-derived induced pluripotent stem cells showed neurite degeneration, which was also alleviated by rapamycin treatment or gene correction using genome editing. Protein structure analysis of NSF revealed that DEE-associated variants might disrupt the transmission of the conformational change of NSF monomers and consequently halt the rotation of ATP hydrolysis, indicating a dominant negative mechanism. In conclusion, this study elucidates the pathomechanism underlying NSF-related DEE and identifies a potential therapeutic approach.

SMN promotes mitochondrial metabolic maturation during myogenesis by regulating the MYOD-miRNA axis.

Spinal muscular atrophy (SMA) is a congenital neuromuscular disease caused by the mutation or deletion of the survival motor neuron 1 (SMN1) gene. Although the primary cause of progressive muscle atrophy in SMA has classically been considered the degeneration of motor neurons, recent studies have indicated a skeletal muscle-specific pathological phenotype such as impaired mitochondrial function and enhanced cell death. Here, we found that the down-regulation of SMN causes mitochondrial dysfunction and subsequent cell death in in vitro models of skeletal myogenesis with both a murine C2C12 cell line and human induced pluripotent stem cells. During myogenesis, SMN binds to the upstream genomic regions of MYOD1 and microRNA (miR)-1 and miR-206. Accordingly, the loss of SMN down-regulates these miRs, whereas supplementation of the miRs recovers the mitochondrial function, cell survival, and myotube formation of SMN-deficient C2C12, indicating the SMN-miR axis is essential for myogenic metabolic maturation. In addition, the introduction of the miRs into ex vivo muscle stem cells derived from Δ7-SMA mice caused myotube formation and muscle contraction. In conclusion, our data revealed novel transcriptional roles of SMN during myogenesis, providing an alternative muscle-oriented therapeutic strategy for SMA patients.

Human genetic defects in SRP19 and SRPRA cause severe congenital neutropenia with distinctive proteome changes.

The mechanisms of coordinated changes in proteome composition and their relevance for the differentiation of neutrophil granulocytes are not well studied. Here, we discover 2 novel human genetic defects in signal recognition particle receptor alpha (SRPRA) and SRP19, constituents of the mammalian cotranslational targeting machinery, and characterize their roles in neutrophil granulocyte differentiation. We systematically study the proteome of neutrophil granulocytes from patients with variants in the SRP genes, HAX1, and ELANE, and identify global as well as specific proteome aberrations. Using in vitro differentiation of human induced pluripotent stem cells and in vivo zebrafish models, we study the effects of SRP deficiency on neutrophil granulocyte development. In a heterologous cell-based inducible protein expression system, we validate the effects conferred by SRP dysfunction for selected proteins that we identified in our proteome screen. Thus, SRP-dependent protein processing, intracellular trafficking, and homeostasis are critically important for the differentiation of neutrophil granulocytes.

Assessment of type I interferon signatures in undifferentiated inflammatory diseases: A Japanese multicenter experience.

Purpose: Upregulation of type I interferon (IFN) signaling has been increasingly detected in inflammatory diseases. Recently, upregulation of the IFN signature has been suggested as a potential biomarker of IFN-driven inflammatory diseases. Yet, it remains unclear to what extent type I IFN is involved in the pathogenesis of undifferentiated inflammatory diseases. This study aimed to quantify the type I IFN signature in clinically undiagnosed patients and assess clinical characteristics in those with a high IFN signature. Methods: The type I IFN signature was measured in patients' whole blood cells. Clinical and biological data were collected retrospectively, and an intensive genetic analysis was performed in undiagnosed patients with a high IFN signature. Results: A total of 117 samples from 94 patients with inflammatory diseases, including 37 undiagnosed cases, were analyzed. Increased IFN signaling was observed in 19 undiagnosed patients, with 10 exhibiting clinical features commonly found in type I interferonopathies. Skin manifestations, observed in eight patients, were macroscopically and histologically similar to those found in proteasome-associated autoinflammatory syndrome. Genetic analysis identified novel mutations in the PSMB8 gene of one patient, and rare variants of unknown significance in genes linked to type I IFN signaling in four patients. A JAK inhibitor effectively treated the patient with the PSMB8 mutations. Patients with clinically quiescent idiopathic pulmonary hemosiderosis and A20 haploinsufficiency showed enhanced IFN signaling. Conclusions: Half of the patients examined in this study, with undifferentiated inflammatory diseases, clinically quiescent A20 haploinsufficiency, or idiopathic pulmonary hemosiderosis, had an elevated type I IFN signature.

iPS cells from Chediak-Higashi syndrome patients recapitulate the giant granules in myeloid cells.

BACKGROUND: Chediak-Higashi syndrome (CHS) is a congenital disease characterized by immunodeficiency, hemophagocytic lymphohistiocytosis, oculocutaneous albinism, and neurological symptoms. The presence of giant granules in peripheral blood leukocytes is an important hallmark of CHS. Here we prepared induced pluripotent stem cells (iPSCs) from CHS patients (CHS-iPSCs) and differentiated them into hematopoietic cells to model the disease phenotypes. METHODS: Fibroblasts were obtained from two CHS patients and then reprogrammed into iPSCs. The iPSCs were differentiated into myeloid cells; the size of the cytosolic granules was quantified by May-Grunwald Giemsa staining and myeloperoxidase staining. RESULTS: Two clones of iPSCs were established from each patient. The differentiation efficiency to CD33+ CD45+ myeloid cells was not significantly different in CHS-iPSCs compared with control iPSCs, but significantly larger granules were observed. CONCLUSIONS: We succeeded in reproducing a characteristic cellular phenotype, giant granules in myeloid cells, using CHS-iPSCs, demonstrating that iPSCs can be used to model the pathogenesis of CHS patients.

Recapitulation of pro-inflammatory signature of monocytes with ACVR1A mutation using FOP patient-derived iPSCs.

BACKGROUND: Fibrodysplasia ossificans progressiva (FOP) is a rare genetic disease characterized by progressive heterotopic ossification (HO) in soft tissues due to a heterozygous mutation of the ACVR1A gene (FOP-ACVR1A), which erroneously transduces the BMP signal by Activin-A. Although inflammation is known to trigger HO in FOP, the role of FOP-ACVR1A on inflammatory cells remains to be elucidated. RESULTS: We generated immortalized monocytic cell lines from FOP-iPSCs (FOP-ML) and mutation rescued iPSCs (resFOP-ML). Cell morphology was evaluated during the monocyte induction and after immortalization. Fluorescence-activated cell sorting (FACS) was performed to evaluate the cell surface markers CD14 and CD16 on MLs. MLs were stimulated with lipopolysaccharide or Activin-A and the gene expression was evaluated by quantitative PCR and microarray analysis. Histological analysis was performed for HO tissue obtained from wild type mice and FOP-ACVR1A mice which conditionally express human mutant ACVR1A gene by doxycycline administration. Without any stimulation, FOP-ML showed the pro-inflammatory signature of CD16+ monocytes with an upregulation of INHBA gene, and treatment of resFOP-ML with Activin-A induced an expression profile mimicking that of FOP-ML at baseline. Treatment of FOP-ML with Activin-A further induced the inflammatory profile with an up-regulation of inflammation-associated genes, of which some, but not all, of which were suppressed by corticosteroid. Experiments using an inhibitor for TGFß or BMP signal demonstrated that Activin-A-induced genes such as CD16 and CCL7, were regulated by both signals, indicating Activin-A transduced dual signals in FOP-ML. A comparison with resFOP-ML identified several down-regulated genes in FOP-ML including LYVE-1, which is known to suppress matrix-formation in vivo. The down-regulation of LYVE-1 in HO tissues was confirmed in FOP model mice, verifying the significance of the in vitro experiments. CONCLUSION: These results indicate that FOP-ML faithfully recapitulated the phenotype of primary monocytes of FOP and the combination with resFOP-ML is a useful tool to investigate molecular events at the initial inflammation stage of HO in FOP.

Identification of Quiescent LGR5+ Stem Cells in the Human Colon.

BACKGROUND & AIMS: In the mouse intestinal epithelium, Lgr5+ stem cells are vulnerable to injury, owing to their predominantly cycling nature, and their progenies de-differentiate to replenish the stem cell pool. However, how human colonic stem cells behave in homeostasis and during regeneration remains unknown. METHODS: Transcriptional heterogeneity among colonic epithelial cells was analyzed by means of single-cell RNA sequencing analysis of human and mouse colonic epithelial cells. To trace the fate of human colonic stem or differentiated cells, we generated LGR5-tdTomato, LGR5-iCasase9-tdTomato, LGR5-split-Cre, and KRT20-ERCreER knock-in human colon organoids via genome engineering. p27+ dormant cells were further visualized with the p27-mVenus reporter. To analyze the dynamics of human colonic stem cells in vivo, we orthotopically xenotransplanted fluorescence-labeled human colon organoids into immune-deficient mice. The cell cycle dynamics in xenograft cells were evaluated using 5-ethynyl-2'-deoxyuridine pulse-chase analysis. The clonogenic capacity of slow-cycling human stem cells or differentiated cells was analyzed in the context of homeostasis, LGR5 ablation, and 5-fluorouracil-induced mucosal injury. RESULTS: Single-cell RNA sequencing analysis illuminated the presence of nondividing LGR5+ stem cells in the human colon. Visualization and lineage tracing of slow-cycling LGR5+p27+ cells and orthotopic xenotransplantation validated their homeostatic lineage-forming capability in vivo, which was augmented by 5-FU-induced mucosal damage. Transforming growth factor-ß signaling regulated the quiescent state of LGR5+ cells. Despite the plasticity of differentiated KRT20+ cells, they did not display clonal growth after 5-FU-induced injury, suggesting that occupation of the niche environment by LGR5+p27+ cells prevented neighboring differentiated cells from de-differentiating. CONCLUSIONS: Our results highlight the quiescent nature of human LGR5+ colonic stem cells and their contribution to post-injury regeneration.

Cell-matrix interface regulates dormancy in human colon cancer stem cells.

Cancer relapse after chemotherapy remains a main cause of cancer-related death. Although the relapse is thought to result from the propagation of resident cancer stem cells1, a lack of experimental platforms that enable the prospective analysis of cancer stem cell dynamics with sufficient spatiotemporal resolution has hindered the testing of this hypothesis. Here we develop a live genetic lineage-tracing system that allows the longitudinal tracking of individual cells in xenotransplanted human colorectal cancer organoids, and identify LGR5+ cancer stem cells that exhibit a dormant behaviour in a chemo-naive state. Dormant LGR5+ cells are marked by the expression of p27, and intravital imaging provides direct evidence of the persistence of LGR5+p27+ cells during chemotherapy, followed by clonal expansion. Transcriptome analysis reveals that COL17A1-a cell-adhesion molecule that strengthens hemidesmosomes-is upregulated in dormant LGR5+p27+ cells. Organoids in which COL17A1 is knocked out lose the dormant LGR5+p27+ subpopulation and become sensitive to chemotherapy, which suggests that the cell-matrix interface has a role in the maintenance of dormancy. Chemotherapy disrupts COL17A1 and breaks the dormancy in LGR5+p27+ cells through FAK-YAP activation. Abrogation of YAP signalling prevents chemoresistant cells from exiting dormancy and delays the regrowth of tumours, highlighting the therapeutic potential of YAP inhibition in preventing cancer relapse. These results offer a viable therapeutic approach to overcome the refractoriness of human colorectal cancer to conventional chemotherapy.

StemPanTox: A fast and wide-target drug assessment system for tailor-made safety evaluations using personalized iPS cells.

An alternative model that reliably predicts human-specific toxicity is necessary because the translatability of effects on animal models for human disease is limited to context. Previously, we developed a method that accurately predicts developmental toxicity based on the gene networks of undifferentiated human embryonic stem (ES) cells. Here, we advanced this method to predict adult toxicities of 24 chemicals in six categories (neurotoxins, cardiotoxins, hepatotoxins, two types of nephrotoxins, and non-genotoxic carcinogens) and achieved high predictability (AUC = 0.90-1.00) in all categories. Moreover, we screened for an induced pluripotent stem (iPS) cell line to predict the toxicities based on the gene networks of iPS cells using transfer learning of the gene networks of ES cells, and predicted toxicities in four categories (neurotoxins, hepatotoxins, glomerular nephrotoxins, and non-genotoxic carcinogens) with high performance (AUC = 0.82-0.99). This method holds promise for tailor-made safety evaluations using personalized iPS cells.