Lgr6-expressing functional nail stem-like cells differentiated from human-induced pluripotent stem cells.

The nail matrix containing stem cell populations produces nails and may contribute to fingertip regeneration. Nails are important tissues that maintain the functions of the hand and foot for handling objects and locomotion. Tumor chemotherapy impairs nail growth and, in many cases, loses them, although not permanently. In this report, we have achieved the successful differentiation of nail stem (NS)-like cells from human-induced pluripotent stem cells (iPSCs) via digit organoids by stepwise stimulation, tracing the molecular processes involved in limb development. Comprehensive mRNA sequencing analysis revealed that the digit organoid global gene expression profile fits human finger development. The NS-like cells expressed Lgr6 mRNA and protein and produced type-I keratin, KRT17, and type-II keratin, KRT81, which are abundant in nails. Furthermore, we succeeded in producing functional Lgr6-reporter human iPSCs. The reporter iPSC-derived Lgr6-positive cells also produced KRT17 and KRT81 proteins in the percutaneously transplanted region. To the best of our knowledge, this is the first report of NS-like cell differentiation from human iPSCs. Our differentiation method and reporter construct enable the discovery of drugs for nail repair and possibly fingertip-regenerative therapy.

An Automated Culture System for Maintaining and Differentiating Human-Induced Pluripotent Stem Cells.

Human induced pluripotent stem cells (hiPSCs) with infinite self-proliferating ability have been expected to have applications in numerous fields, including the elucidation of rare disease pathologies, the development of new medicines, and regenerative medicine aiming to restore damaged organs. Despite this, the social implementation of hiPSCs is still limited. This is partly because of the difficulty of reproducing differentiation in culture, even with advanced knowledge and sophisticated technical skills, due to the high sensitivity of iPSCs to minute environmental changes. The application of an automated culture system can solve this issue. Experiments with high reproducibility independent of a researcher's skill can be expected according to a shared procedure across various institutes. Although several automated culture systems that can maintain iPSC cultures and induce differentiation have been developed previously, these systems are heavy, large, and costly because they make use of humanized, multi-articulated robotic arms. To improve on the above issues, we developed a new system using a simple x-y-z axis slide rail system, allowing it to be more compact, lighter, and cheaper. Furthermore, the user can easily modify parameters in the new system to develop new handling tasks. Once a task is established, all the user needs to do is prepare the iPSC, supply the reagents and consumables needed for the desired task in advance, select the task number, and specify the time. We confirmed that the system could maintain iPSCs in an undifferentiated state through several passages without feeder cells and differentiate into various cell types, including cardiomyocytes, hepatocytes, neural progenitors, and keratinocytes. The system will enable highly reproducible experiments across institutions without the need for skilled researchers and will facilitate the social implementation of hiPSCs in a wider range of research fields by diminishing the obstacles for new entries.

Application of Live Mitochondria Staining in Cell-Sorting to Purify Hepatocytes Derived from Human Induced Pluripotent Stem Cells.

Human embryonic stem (ES) and induced pluripotent stem (iPS) cells have potential applications in cell-based regenerative medicine for treating severely diseased organs due to their unlimited proliferation and pluripotent properties. However, differentiating human ES/iPS cells into 100% pure target cell types is challenging due to their high sensitivity to the environment. Tumorigenesis after transplantation is caused by contaminated, proliferating, and undifferentiated cells, making high-purification technology essential for the safe realization of regenerative medicine. To mitigate the risk of tumorigenesis, a high-purification technology has been developed for human iPS cell-derived hepatocytes. The method employs FACS (fluorescence-activated cell sorting) using a combination of high mitochondrial content and the cell-surface marker ALCAM (activated leukocyte cell adhesion molecule) without genetic modification. 97% ± 0.38% (n = 5) of the purified hepatocytes using this method exhibited albumin protein expression. This article aims to provide detailed procedures for this method, as applied to the most current two-dimensional differentiation method for human iPS cells into hepatocytes.

Efficient protocol for the differentiation of kidney podocytes from induced pluripotent stem cells, involving the inhibition of mTOR.

The mechanistic/mammalian target of rapamycin (mTOR) is involved in a wide range of cellular processes. However, the role of mTOR in podocytes remains unclear. In this study, we aimed to clarify the role of mTOR in podocyte differentiation from human induced pluripotent stem cells (hiPSCs) and to establish an efficient differentiation protocol for human podocytes. We generated podocytes from hiPSCs by modifying protocol. The expression of the podocyte-specific slit membrane components nephrin and podocin was measured using PCR, western blotting, flow cytometry, and immunostaining; and the role of mTOR was evaluated using inhibitors of the mTOR pathway. Nephrin and podocin were found to be expressed in cells differentiated from hiPSCs, and their expression was increased by mTOR inhibitor treatment. S6, a downstream component of the mTOR pathway, was also found to be involved in podocyte differentiation. we evaluated its permeability to albumin, urea, and electrolytes. The induced podocytes were permeable to the small molecules, but only poorly permeable to albumin. We have shown that the mTOR pathway is involved in podocyte differentiation. Our monolayer podocyte differential protocol, using an mTOR inhibitor, provides a novel in vitro model for studies of kidney physiology and pathology.

The Induction of Parathyroid Cell Differentiation from Human Induced Pluripotent Stem Cells Promoted Via TGF-α/EGFR Signaling.

The parathyroid gland plays an essential role in mineral and bone metabolism. Cultivation of physiological human parathyroid cells has yet to be established and the method by which parathyroid cells differentiate from pluripotent stem cells remains uncertain. Therefore, it has been hard to clarify the mechanisms underlying the onset of parathyroid disorders, such as hyperparathyroidism. In this study, we developed a new method of parathyroid cell differentiation from human induced pluripotent stem (iPS) cells. Parathyroid cell differentiation occurred in accordance with embryologic development. Differentiated cells, which expressed the parathyroid hormone, adopted unique cell aggregation similar to the parathyroid gland. In addition, these differentiated cells were identified as calcium-sensing receptor (CaSR)/epithelial cell adhesion molecule (EpCAM) double-positive cells. Interestingly, stimulation with transforming growth factor-α (TGF-α), which is considered a causative molecule of parathyroid hyperplasia, increased the CaSR/EpCAM double-positive cells, but this effect was suppressed by erlotinib, which is an epidermal growth factor receptor (EGFR) inhibitor. These results suggest that TGF-α/EGFR signaling promotes parathyroid cell differentiation from iPS cells in a similar manner to parathyroid hyperplasia.

Machine learning discriminates P2X7-mediated intracellular calcium sparks in human-induced pluripotent stem cell-derived neural stem cells.

Adenosine triphosphate (ATP) is an extracellular signaling molecule that mainly affects the pathophysiological situation in the body and can be sensed by purinergic receptors, including ionotropic P2X7. Neuronal stem cells (NSCs) remain in adult neuronal tissues and can contribute to physiological processes via activation by evoked pathophysiological situations. In this study, we revealed that human-induced pluripotent stem cell-derived NSCs (iNSCs) have ATP-sensing ability primarily via the purinergic and ionotropic receptor P2X7. Next, to develop a machine learning (ML)-based screening system for food-derived neuronal effective substances and their effective doses, we collected ATP-triggered calcium responses of iNSCs pretreated with several substances and doses. Finally, we discovered that ML was performed using composite images, each containing nine waveform images, to achieve a better ML model (MLM) with higher precision. Our MLM can correctly sort subtle unidentified changes in waveforms produced by pretreated iNSCs with each substance and/or dose into the positive group, with common mRNA expression changes belonging to the gene ontology signatures.

A Novel Cell Injection Method with Minimum Invasion.

Directly injecting cells into tissues is a necessary process in cell administration and/or replacement therapy. The cell injection requires a sufficient amount of suspension solution to allow the cells to enter the tissue. The volume of the suspension solution affects the tissue, and this can cause major invasive injury as a result of the cell injection. This paper reports on a novel cell injection method, called slow injection, that aims to avoid this injury. However, pushing out the cells from the needle tip requires a sufficiently high injection speed according to Newton's law of shear force. To solve the above contradiction, a non-Newtonian fluid, such as gelatin solution, was used as the cell suspension solution in this work. Gelatins solution have temperature sensitivity, as their form changes from gel to sol at approximately 20 °C. Therefore, to maintain the cell suspension solution in the gel form, the syringe was kept cooled in this protocol; however, once the solution was injected into the body, the body temperature converted it to a sol. The interstitial tissue fluid flow can absorb excess solution. In this work, the slow injection technique allowed cardiomyocyte balls to enter the host myocardium and engraft without surrounding fibrosis. This study employed a slow injection method to inject purified and ball-formed neonatal rat cardiomyocytes into a remote area of myocardial infarction in the adult rat heart. At 2 months following the injection, the hearts of the transplanted groups showed significantly improved contractile function. Furthermore, histological analyses of the slow-injected hearts revealed seamless connections between the host and graft cardiomyocytes via intercalated disks containing gap junction connections. This method could contribute to next-generation cell therapies, particularly in cardiac regenerative medicine.

Human Induced Pluripotent Stem Cell-Derived Keratinocyte-Like Cells for Research on Protease-Activated Receptor 2 in Nonhistaminergic Cascades of Atopic Dermatitis .

Keratinocytes are the most abundant cells in the epidermis, and as part of the frontline immunologic defense system, keratinocytes function as a barrier to exogenous attacks. Protease-activated receptor 2 (PAR2) is expressed in human keratinocytes and activated in several inflammatory conditions, such as atopic dermatitis (AD). In this study, we demonstrated the differentiation of human induced pluripotent stem cell into keratinocytes by the improved, robust differentiation procedure and confirmed that human induced pluripotent stem cell-derived keratinocyte-like cells (iKera) express PAR2, which is activated by external addition of the ligand peptide and trypsin. The activation of PAR2 led to the release of calcium from intracellular calcium storage, followed by the release of the proinflammatory cytokine tumor necrosis factor α Moreover, PAR2 antagonist I-191 (CAS No. 1690172-25-8) inhibited calcium release in a dose-dependent manner. This is the first study to demonstrate that iKera expresses a functional PAR2 protein. Furthermore, our results indicate crosstalk between the PAR2- and IL-4-mediated inflammatory axes in iKera, suggesting that iKera can be used as a platform for a broad range of mechanism-targeted drug screening in AD. SIGNIFICANCE STATEMENT: This is the first study to provide evidence that human induced pluripotent stem cell-derived keratinocyte-like cells (iKera) express functional protease-activated receptor 2 (PAR2). Furthermore, this study demonstrated in iKera that the IL-4 inflammatory axis can crosstalk with the PAR2-mediated inflammatory axis in keratinocytes. To the best of our knowledge, this is the first report to indicate that iKera can be used for research and as a drug screening platform for atopic dermatitis.

Compact automated culture machine for human induced pluripotent stem cell maintenance and differentiation.

The technologies used to generate human induced pluripotent stem cell (iPSC) from somatic cells potentially enable the wide application of iPSC-derived differentiated cells in industrial research fields as a replacement for animals. However, as highly trained individuals are required to obtain reproducible results, this approach has limited social implementation. In the research field of iPSC, it is believed that documentable information is not enough for reproducing the quality of the differentiated cells. Therefore, automated culture machines for cell processing should make the starting of iPSC-using researches easier. We developed a programmable all-in-one automated culture machine, with dense and compact constitution that fits within a normal biosafety cabinet (200 mm wide, 233 mm height, and 110 mm depth). This instrument was fabricated using novel x-y-z-axes-rail-system, such as an overhead traveling crane, in a factory, which served as the main handling machinery. This machine enabled stable and efficient expansion of human iPSC under the feeder-free condition, without karyotype alterations, and simultaneously differentiated the cells into various cell types, including cardiomyocytes, hepatocytes, neural progenitors, and keratinocytes. Overall, this machine would facilitate the social implementation of human pluripotent stem cells and contribute to the accumulation of sharable knowledge for the standardization of the entire handling processes of iPSC in pharmaceutical, food, and cosmetic research.

Possible internal viral shedding and interferon production after clinical recovery from COVID-19: Case report.

A 70-year-old man underwent off-pump coronary artery bypass grafting 28 days after his recovery from coronavirus disease 2019 (COVID-19), which was confirmed by a negative polymerase chain reaction (PCR) test result for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from a nasopharyngeal swab. The PCR test result was also negative for nasopharyngeal sampling 5 days prior to the surgery. However, his redundant saphenous vein and sputum through the endotracheal tube that was taken on the operative day showed the presence of SARS-CoV-2 by PCR. Immunohistochemical analysis of Spike and Nucleoprotein of the saphenous vein showed small clusters of each antigen-positive speckle. Ultrastructural imaging of the saphenous vein showed virus-like particles. The cell-based assay suggested that the patient's serum contained a higher concentration of type-I interferons than that of healthy control sera. These observations suggest that internal viral shedding and, to some extent, innate immune responses continue after COVID-19 recovery.

Perturbation of the titin/MURF1 signaling complex is associated with hypertrophic cardiomyopathy in a fish model and in human patients.

Hypertrophic cardiomyopathy (HCM) is a hereditary disease characterized by cardiac hypertrophy with diastolic dysfunction. Gene mutations causing HCM have been found in about half of HCM patients, while the genetic etiology and pathogenesis remain unknown for many cases of HCM. To identify novel mechanisms underlying HCM pathogenesis, we generated a cardiovascular-mutant medaka fish, non-spring heart (nsh), which showed diastolic dysfunction and hypertrophic myocardium. The nsh homozygotes had fewer myofibrils, disrupted sarcomeres and expressed pathologically stiffer titin isoforms. In addition, the nsh heterozygotes showed M-line disassembly that is similar to the pathological changes found in HCM. Positional cloning revealed a missense mutation in an immunoglobulin (Ig) domain located in the M-line-A-band transition zone of titin. Screening of mutations in 96 unrelated patients with familial HCM, who had no previously implicated mutations in known sarcomeric gene candidates, identified two mutations in Ig domains close to the M-line region of titin. In vitro studies revealed that the mutations found both in medaka fish and in familial HCM increased binding of titin to muscle-specific ring finger protein 1 (MURF1) and enhanced titin degradation by ubiquitination. These findings implicate an impaired interaction between titin and MURF1 as a novel mechanism underlying the pathogenesis of HCM.

Hepatocyte-like Cells Derived from Human Pluripotent Stem Cells Can Be Enriched by a Combination of Mitochondrial Content and Activated Leukocyte Cell Adhesion Molecule.

INTRODUCTION: Non-genetic purification methods for pluripotent stem cell-derived hepatocyte-like cells are useful for liver regenerative therapy and pharmaceutical applications. METHODS: Fluorescent activated cell sorting (FACS) was used to separate cells by combining two parameters: cellular mitochondrial content evaluated by the mitochondrial membrane potential-dependent fluorescent probe (TMRM) and immunocytochemical detection of activated leukocyte cell adhesion molecule (ALCAM). This method was applied to murine fetal, human embryonic stem cell (ESC)-derived, and human induced pluripotent stem cell (iPSC)-derived cell-mixtures. Separately sorted cell fractions were evaluated by quantitative PCR, immunohistochemistry, and cytochemistry for HNF4a, AFP, and albumin mRNA and/or protein expression. RESULTS: Hepatocyte-like cells were segregated into the high TMRM signal and ALCAM-positive population. The purity of hepatocyte-like cells derived from human iPSCs was 97 ± 0.38% (n = 5). CONCLUSIONS: This hepatocyte-like cell purification method may be applicable to the quality control of cells for liver regenerative cell therapy and pharmaceutical development.

"All-in-one" in vitro selection of collagen-binding vascular endothelial growth factor.

To enhance the therapeutic effect of growth factors, a powerful strategy is to direct their localization to damaged sites. To treat skin wounds and myocardial infarction, we selected vascular endothelial growth factor (VEGF) carrying binding affinity to collagen. A simple conjugation of a reported collagen-binding sequence and VEGF did not increase the collagen-binding affinity, indicating that the molecular interaction between the two proteins abolished collagen binding activity. Here, we present a new molecular evolution strategy, "all-in-one" in vitro selection, in which a collagen-binding VEGF (CB-VEGF) was directly identified from a random library consisting of random and VEGF sequences. As expected, the selected CB-VEGFs exhibited high binding affinity to collagen and maintained the same growth enhancement activity for endothelial cells as unmodified VEGF in solution. Furthermore, the selected CB-VEGF enhanced angiogenesis at skin wounds and infarcted myocardium. This study demonstrates that "all-in-one" in vitro selection is a novel strategy for the design of functional proteins for regenerative medicine.

Glutamine Oxidation Is Indispensable for Survival of Human Pluripotent Stem Cells.

Human pluripotent stem cells (hPSCs) are uniquely dependent on aerobic glycolysis to generate ATP. However, the importance of oxidative phosphorylation (OXPHOS) has not been elucidated. Detailed amino acid profiling has revealed that glutamine is indispensable for the survival of hPSCs. Under glucose- and glutamine-depleted conditions, hPSCs quickly died due to the loss of ATP. Metabolome analyses showed that hPSCs oxidized pyruvate poorly and that glutamine was the main energy source for OXPHOS. hPSCs were unable to utilize pyruvate-derived citrate due to negligible expression of aconitase 2 (ACO2) and isocitrate dehydrogenase 2/3 (IDH2/3) and high expression of ATP-citrate lyase. Cardiomyocytes with mature mitochondria were not able to survive without glucose and glutamine, although they were able to use lactate to synthesize pyruvate and glutamate. This distinguishing feature of hPSC metabolism allows preparation of clinical-grade cell sources free of undifferentiated hPSCs, which prevents tumor formation during stem cell therapy.

Analysis of cardiomyocyte movement in the developing murine heart.

The precise assemblage of several types of cardiac precursors controls heart organogenesis. The cardiac precursors show dynamic movement during early development and then form the complicated heart structure. However, cardiomyocyte movements inside the newly organized mammalian heart remain unclear. We previously established the method of ex vivo time-lapse imaging of the murine heart to study cardiomyocyte behavior by using the Fucci (fluorescent ubiquitination-based cell cycle indicator) system, which can effectively label individual G1, S/G2/M, and G1/S-transition phase nuclei in living cardiomyocytes as red, green, and yellow, respectively. Global analysis of gene expression in Fucci green positive ventricular cardiomyocytes confirmed that cell cycle regulatory genes expressed in G1/S, S, G2/M, and M phase transitions were upregulated. Interestingly, pathway analysis revealed that many genes related to the cell cycle were significantly upregulated in the Fucci green positive ventricular cardiomyocytes, while only a small number of genes related to cell motility were upregulated. Time-lapse imaging showed that murine proliferating cardiomyocytes did not exhibit dynamic movement inside the heart, but stayed on site after entering the cell cycle.

A massive suspension culture system with metabolic purification for human pluripotent stem cell-derived cardiomyocytes.

Cardiac regenerative therapy with human pluripotent stem cells (hPSCs), such as human embryonic stem cells and induced pluripotent stem cells, has been hampered by the lack of efficient strategies for expanding functional cardiomyocytes (CMs) to clinically relevant numbers. The development of the massive suspension culture system (MSCS) has shed light on this critical issue, although it remains unclear how hPSCs could differentiate into functional CMs using a MSCS. The proliferative rate of differentiating hPSCs in the MSCS was equivalent to that in suspension cultures using nonadherent culture dishes, although the MSCS provided more homogeneous embryoid bodies (EBs), eventually reducing apoptosis. However, pluripotent markers such as Oct3/4 and Tra-1-60 were still expressed in EBs 2 weeks after differentiation, even in the MSCS. The remaining undifferentiated stem cells in such cultures could retain a strong potential for teratoma formation, which is the worst scenario for clinical applications of hPSC-derived CMs. The metabolic purification of CMs in glucose-depleted and lactate-enriched medium successfully eliminated the residual undifferentiated stem cells, resulting in a refined hPSC-derived CM population. In colony formation assays, no Tra-1-60-positive colonies appeared after purification. The nonpurified CMs in the MSCS produced teratomas at a rate of 60%. However, purified CMs never induced teratomas, and enriched CMs showed proper electrophysiological properties and calcium transients. Overall, the combination of a MSCS and metabolic selection is a highly effective and practical approach to purify and enrich massive numbers of functional CMs and provides an essential technique for cardiac regenerative therapy with hPSC-derived CMs.

Time-lapse imaging of cell cycle dynamics during development in living cardiomyocyte.

Mammalian cardiomyocytes withdraw from the cell cycle shortly after birth, although it remains unclear how cardiomyocyte cell cycles behave during development. Compared to conventional immunohistochemistry in static observation, time-lapse imaging can reveal comprehensive data in hard-to-understand biological phenomenon. However, there are no reports of an established protocol of successful time-lapse imaging in mammalian heart. Thus, it is valuable to establish a time-lapse imaging system to enable the observation of cell cycle dynamics in living murine cardiomyocytes. This study sought to establish time-lapse imaging of murine heart to study cardiomyocyte cell cycle behavior. The Fucci (fluorescent ubiquitination-based cell cycle indicator) system can effectively label individual G1, S/G2/M, and G1/S-transition phase nuclei red, green and yellow, respectively, in living mammalian cells, and could therefore be useful to visualize the real-time cell cycle transitions in living murine heart. To establish a similar system for time-lapse imaging of murine heart, we first developed an ex vivo culture system, with the culture conditions determined in terms of sample state, serum concentration, and oxygen concentration. The optimal condition (slice culture, oxygen concentration 20%, serum concentration 10%) successfully mimicked physiological cardiomyocyte proliferation in vivo. Time-lapse imaging of cardiac slices from E11.5, E14.5, E18.5, and P1 Fucci-expressing transgenic mice revealed an elongated S/G2/M phase in cardiomyocytes during development. Our time-lapse imaging of murine heart revealed a gradual elongation of the S/G2/M phase during development in living cardiomyocytes.

Distinct iPS Cells Show Different Cardiac Differentiation Efficiency.

Patient-specific induced pluripotent stem (iPS) cells can be generated by introducing transcription factors that are highly expressed in embryonic stem (ES) cells into somatic cells. This opens up new possibilities for cell transplantation-based regenerative medicine by overcoming the ethical issues and immunological problems associated with ES cells. Despite the development of various methods for the generation of iPS cells that have resulted in increased efficiency, safety, and general versatility, it remains unknown which types of iPS cells are suitable for clinical use. Therefore, the aims of the present study were to assess (1) the differentiation potential, time course, and efficiency of different types of iPS cell lines to differentiate into cardiomyocytes in vitro and (2) the properties of the iPS cell-derived cardiomyocytes. We found that high-quality iPS cells exhibited better cardiomyocyte differentiation in terms of the time course and efficiency of differentiation than low-quality iPS cells, which hardly ever differentiated into cardiomyocytes. Because of the different properties of the various iPS cell lines such as cardiac differentiation efficiency and potential safety hazards, newly established iPS cell lines must be characterized prior to their use in cardiac regenerative medicine.

Distinct metabolic flow enables large-scale purification of mouse and human pluripotent stem cell-derived cardiomyocytes.

Heart disease remains a major cause of death despite advances in medical technology. Heart-regenerative therapy that uses pluripotent stem cells (PSCs) is a potentially promising strategy for patients with heart disease, but the inability to generate highly purified cardiomyocytes in sufficient quantities has been a barrier to realizing this potential. Here, we report a nongenetic method for mass-producing cardiomyocytes from mouse and human PSC derivatives that is based on the marked biochemical differences in glucose and lactate metabolism between cardiomyocytes and noncardiomyocytes, including undifferentiated cells. We cultured PSC derivatives with glucose-depleted culture medium containing abundant lactate and found that only cardiomyocytes survived. Using this approach, we obtained cardiomyocytes of up to 99% purity that did not form tumors after transplantation. We believe that our technological method broadens the range of potential applications for purified PSC-derived cardiomyocytes and could facilitate progress toward PSC-based cardiac regenerative therapy.

Disease characterization using LQTS-specific induced pluripotent stem cells.

AIMS: Long QT syndrome (LQTS) is an inheritable and life-threatening disease; however, it is often difficult to determine disease characteristics in sporadic cases with novel mutations, and more precise analysis is necessary for the successful development of evidence-based clinical therapies. This study thus sought to better characterize ion channel cardiac disorders using induced pluripotent stem cells (iPSCs). METHODS AND RESULTS: We reprogrammed somatic cells from a patient with sporadic LQTS and from controls, and differentiated them into cardiomyocytes through embryoid body (EB) formation. Electrophysiological analysis of the LQTS-iPSC-derived EBs using a multi-electrode array (MEA) system revealed a markedly prolonged field potential duration (FPD). The IKr blocker E4031 significantly prolonged FPD in control- and LQTS-iPSC-derived EBs and induced frequent severe arrhythmia only in LQTS-iPSC-derived EBs. The IKs blocker chromanol 293B did not prolong FPD in the LQTS-iPSC-derived EBs, but significantly prolonged FPD in the control EBs, suggesting the involvement of IKs disturbance in the patient. Patch-clamp analysis and immunostaining confirmed a dominant-negative role for 1893delC in IKs channels due to a trafficking deficiency in iPSC-derived cardiomyocytes and human embryonic kidney (HEK) cells. CONCLUSIONS: This study demonstrated that iPSCs could be useful to characterize LQTS disease as well as drug responses in the LQTS patient with a novel mutation. Such analyses may in turn lead to future progress in personalized medicine.