Collection of homozygous mutant mouse embryonic stem cells arising from autodiploidization during haploid gene trap mutagenesis.

Haploid mouse embryonic stem cells (ESCs), in which a single hit mutation is sufficient to produce loss-of-function phenotypes, have provided a powerful tool for forward genetic screening. This strategy, however, can be hampered by undesired autodiploidization of haploid ESCs. To overcome this obstacle, we designed a new methodology that facilitates enrichment of homozygous mutant ESC clones arising from autodiploidization during haploid gene trap mutagenesis. Haploid mouse ESCs were purified by fluorescence-activated cell sorting to maintain their haploid property and then transfected with the Tol2 transposon-based biallelically polyA-trapping (BPATrap) vector that carries an invertible G418 plus puromycin double selection cassette. G418 plus puromycin double selection enriched biallelic mutant clones that had undergone autodiploidization following a single vector insertion into the haploid genome. Using this method, we successfully generated 222 homozygous mutant ESCs from 2208 clones by excluding heterozygous ESCs and ESCs with multiple vector insertions. This relatively low efficiency of generating homozygous mutant ESCs was partially overcome by cell sorting of haploid ESCs after Tol2 BPATrap transfection. These results demonstrate the feasibility of our approach to provide an efficient platform for mutagenesis of ESCs and functional analysis of the mammalian genome.

COSMOS: accurate detection of somatic structural variations through asymmetric comparison between tumor and normal samples.

An important challenge in cancer genomics is precise detection of structural variations (SVs) by high-throughput short-read sequencing, which is hampered by the high false discovery rates of existing analysis tools. Here, we propose an accurate SV detection method named COSMOS, which compares the statistics of the mapped read pairs in tumor samples with isogenic normal control samples in a distinct asymmetric manner. COSMOS also prioritizes the candidate SVs using strand-specific read-depth information. Performance tests on modeled tumor genomes revealed that COSMOS outperformed existing methods in terms of F-measure. We also applied COSMOS to an experimental mouse cell-based model, in which SVs were induced by genome engineering and gamma-ray irradiation, followed by polymerase chain reaction-based confirmation. The precision of COSMOS was 84.5%, while the next best existing method was 70.4%. Moreover, the sensitivity of COSMOS was the highest, indicating that COSMOS has great potential for cancer genome analysis.

Report on the Conference on Transposition and Genome Engineering 2015 (TGE 2015): advancing cutting-edge genomics technology in the ancient city of Nara.

From November 17 to 20 in 2015, the Conference on Transposition and Genome Engineering 2015 (TGE 2015) was held at Nara Kasugano International Forum-IRAKA-in Nara, Japan, located at the center of Nara Park. All of the presentations were carried out at Nohgaku hall in Nara Kasugano International Forum-IRAKA. Participation totaled 148 persons (30 international, 118 domestic), who were able to engage in lively scientific discussions over the 4-day period. The guest speaker list consisted of many top-notch international researchers, an achievement for which the conference received praise from the attendees. There were 36 oral presentations including the keynote lecture (22 presentations from guest speakers, complemented with 14 selected from abstract submissions). Additionally, there were 46 poster presentations. The conference uniquely combined research mainly from two different genomics approaches: (i) transposon technology allowing random genomic integration followed by gene discovery-related phenotypes and (ii) genome editing technology with designer nuclease allowing precise modification of a gene-of-interest.

Enhancement of microhomology-mediated genomic rearrangements by transient loss of mouse Bloom syndrome helicase.

Bloom syndrome, an autosomal recessive disorder of the BLM gene, confers predisposition to a broad spectrum of early-onset cancers in multiple tissue types. Loss of genomic integrity is a primary hallmark of such human malignancies, but many studies using disease-affected specimens are limited in that they are retrospective and devoid of an appropriate experimental control. To overcome this, we devised an experimental system to recapitulate the early molecular events in genetically engineered mouse embryonic stem cells, in which cells undergoing loss of heterozygosity (LOH) can be enriched after inducible down-regulation of Blm expression, with or without site-directed DNA double-strand break (DSB) induction. Transient loss of BLM increased the rate of LOH, whose breakpoints were distributed along the chromosome. Combined with site-directed DSB induction, loss of BLM synergistically increased the rate of LOH and concentrated the breakpoints around the targeted chromosomal region. We characterized the LOH events using specifically tailored genomic tools, such as high-resolution array comparative genomic hybridization and high-density single nucleotide polymorphism genotyping, revealing that the combination of BLM suppression and DSB induction enhanced genomic rearrangements, including deletions and insertions, whose breakpoints were clustered in genomic inverted repeats and associated with junctional microhomologies. Our experimental approach successfully uncovered the detailed molecular mechanisms of as-yet-uncharacterized loss of heterozygosities and reveals the significant contribution of microhomology-mediated genomic rearrangements, which could be widely applicable to the early steps of cancer formation in general.

Simulation and estimation of gene number in a biological pathway using almost complete saturation mutagenesis screening of haploid mouse cells.

BACKGROUND: Genome-wide saturation mutagenesis and subsequent phenotype-driven screening has been central to a comprehensive understanding of complex biological processes in classical model organisms such as flies, nematodes, and plants. The degree of "saturation" (i.e., the fraction of possible target genes identified) has been shown to be a critical parameter in determining all relevant genes involved in a biological function, without prior knowledge of their products. In mammalian model systems, however, the relatively large scale and labor intensity of experiments have hampered the achievement of actual saturation mutagenesis, especially for recessive traits that require biallelic mutations to manifest detectable phenotypes. RESULTS: By exploiting the recently established haploid mouse embryonic stem cells (ESCs), we present an implementation of almost complete saturation mutagenesis in a mammalian system. The haploid ESCs were mutagenized with the chemical mutagen N-ethyl-N-nitrosourea (ENU) and processed for the screening of mutants defective in various steps of the glycosylphosphatidylinositol-anchor biosynthetic pathway. The resulting 114 independent mutant clones were characterized by a functional complementation assay, and were shown to be defective in any of 20 genes among all 22 known genes essential for this well-characterized pathway. Ten mutants were further validated by whole-exome sequencing. The predominant generation of single-nucleotide substitutions by ENU resulted in a gene mutation rate proportional to the length of the coding sequence, which facilitated the experimental design of saturation mutagenesis screening with the aid of computational simulation. CONCLUSIONS: Our study enables mammalian saturation mutagenesis to become a realistic proposition. Computational simulation, combined with a pilot mutagenesis experiment, could serve as a tool for the estimation of the number of genes essential for biological processes such as drug target pathways when a positive selection of mutants is available.

A homozygous mutant embryonic stem cell bank applicable for phenotype-driven genetic screening.

Genome-wide mutagenesis in mouse embryonic stem cells (ESCs) is a powerful tool, but the diploid nature of the mammalian genome hampers its application for recessive genetic screening. We have previously reported a method to induce homozygous mutant ESCs from heterozygous mutants by tetracycline-dependent transient disruption of the Bloom's syndrome gene. However, we could not purify homozygous mutants from a large population of heterozygous mutant cells, limiting the applications. Here we developed a strategy for rapid enrichment of homozygous mutant mouse ESCs and demonstrated its feasibility for cell-based phenotypic analysis. The method uses G418-plus-puromycin double selection to enrich for homozygotes and single-nucleotide polymorphism analysis for identification of homozygosity. We combined this simple approach with gene-trap mutagenesis to construct a homozygous mutant ESC bank with 138 mutant lines and demonstrate its use in phenotype-driven genetic screening.

Pax1 acts as a negative regulator of chondrocyte maturation.

Paired box gene 1 (Pax1) indirectly promotes the early stages of chondrogenic differentiation through induction and transactivation of Nk3 homeobox 2 (Nkx3.2), a transcriptional repressor. Later in chondrogenic differentiation, Nkx3.2 blocks chondrocyte hypertrophy by repressing Runt-related transcription factor 2 (Runx2). Here we report the inhibitory action of Pax1 on chondrocyte maturation, independently of Nkx3.2. Upon cartilage formation, Pax1 expression in the ventral sclerotome was gradually decreased except for the perichondrial region of the vertebral bodies and the intervertebral region, both of which express SRY-box containing gene 9 (Sox9). Forced expression of Pax1 in the chick forelimb resulted in the formation of shortened skeletal elements with a significant reduction of proteoglycans (PGs) accumulation in cartilage as well as a lack of the cortical bone formation and vascular invasion into the primary ossification center. Pax1-misexpressing chondrocytes exhibited aberrant cell morphology with a marked downregulation of Aggrecan (Agc1). Pax1-misexpressing cultured chondrocytes failed to accumulate cartilaginous PGs and became fibroblastic, in association with downregulation of the expression of Sox9, Nkx3.2, Indian hedgehog (Ihh), type II collagen (Col2a1), Chondromodulin-1 (Chm1), and Agc1. Accumulation of cartilaginous PGs in chondrocytes was also reduced by forced expression of Pax1 and Sox9. Thus, chondrocyte maturation driven by Sox9 is antagonized by Pax1 that is downregulated during chondrogenic differentiation.

A newly identified gene Ahed plays essential roles in murine haematopoiesis.

The development of haematopoiesis involves the coordinated action of numerous genes, some of which are implicated in haematological malignancies. However, the biological function of many genes remains elusive and unknown functional genes are likely to remain to be uncovered. Here, we report a previously uncharacterised gene in haematopoiesis, identified by screening mutant embryonic stem cells. The gene, 'attenuated haematopoietic development (Ahed)', encodes a nuclear protein. Conditional knockout (cKO) of Ahed results in anaemia from embryonic day 14.5 onward, leading to prenatal demise. Transplantation experiments demonstrate the incapacity of Ahed-deficient haematopoietic cells to reconstitute haematopoiesis in vivo. Employing a tamoxifen-inducible cKO model, we further reveal that Ahed deletion impairs the intrinsic capacity of haematopoietic cells in adult mice. Ahed deletion affects various pathways, and published databases present cancer patients with somatic mutations in Ahed. Collectively, our findings underscore the fundamental roles of Ahed in lifelong haematopoiesis, implicating its association with malignancies.

Translation of quality of life scale for pediatric patients with Fabry disease in Japan.

Introduction: Fabry disease is a rare, X-linked lysosomal storage disorder that begins in childhood with a wide variety of symptoms, including neuropathic pain, gastrointestinal abnormalities, and skin abnormalities. Despite the substantial impact of these symptoms on children's quality of life (QOL), systematic QOL analysis of Japanese pediatric Fabry disease patients has been limited. Therefore, to evaluate the QOL of Japanese pediatric Fabry disease patients using standardized and disease-specific scales, we used the Fabry-specific Pediatric Health and Pain Questionnaire (FPHPQ), which was developed by the Fabry Outcome Survey. Methods: The FPHPQ was translated in accordance with the Principles of Good Practice for the Translation and Cultural Adaptation Process for Patient-Reported Outcomes. A back-translated version was reviewed twice by the original lead author of FPHPQ to confirm the conceptual equivalence. The questionnaire was then validated by cognitive debriefing, and distributed to pediatric Fabry disease patients in Japan. Results: Questionnaire responses were obtained from eight patients. The mean scores on the FPHPQ were 11.0 (± 11.43) for heat-associated pain, 5.5 (± 4.60) for cold-associated pain, and 14.8 (± 5.97) for abdominal pain and fatigue. In addition, heat-associated pain negatively correlated with physical well-being, whereas cold-associated pain positively correlated with good friendships. Conclusion: We established the Japanese version of the FPHPQ to assess the QOL of pediatric Fabry disease patients. The internal consistency and partial criterion-related validity of the Japanese version were confirmed. Analysis of a larger number of patients should be performed in the future to further validate the outcomes of this study.

Development and validation of a disease-specific quality of life scale for adult patients with Fabry disease in Japan.

BACKGROUND: Fabry disease is a rare X-linked lysosomal storage disorder. It is associated with physical distress and social challenges that may affect adults differently compared to pediatric patients. However, there is no disease-specific quality of life (QOL) scale that can provide a detailed assessment of QOL for adults with Fabry disease. Therefore, we aimed to determine the factor structure and assess the validity of a scale that was created to assess the QOL of adult patients with Fabry disease. This study was conducted in two phases. First, scale feasibility was confirmed through a questionnaire survey of nine patients. Second, a cross-sectional questionnaire survey of patients (aged ≥ 18 years) diagnosed with Fabry disease was conducted. Item development and refinement were conducted based on guidelines for scale development. Exploratory factor analysis was used to clarify the factor structure and confirm internal consistency. As a measure of QOL, construct validity was of the scale was verified based on its correlations with the Short Form-8 (SF-8) scale. RESULTS: The newly created Adult Fabry Disease QOL (AFQOL) scale comprises 39 items that cover five factors: "neuropathic pain and abdominal symptoms," "impact on work and school," "relationship challenges," "ophthalmologic and otolaryngologic symptoms," and "cardiovascular and renal symptoms." Cronbach's alpha coefficient for all factors was above 0.8, and the AFQOL total scores were significantly correlated with the physical and mental components of the SF-8 (rs = - 0.508 and - 0.400, respectively). CONCLUSIONS: The AFQOL scale assesses physical symptoms and social difficulties experienced by adult patients with Fabry disease. A strength of the scale is its ability to assess the impact of work and relationships on patients. The scale can be useful in objectively assessing QOL for a group or for individual patients. Future research should explore further aspects of the scale's validity and reliability.

Adults with Fabry disease experience severe challenges, which adversely impact their quality of life (QOL). As it is a rare disease, non-patients lack awareness of the severity of its symptoms and the resultant social difficulties of the patients. Most instruments that measure QOL are not specific enough to address issues related to Fabry disease. Therefore, in this study, a measurement instrument known as the Adult Fabry QOL (AFQOL) scale was designed and validated. The 39-item scale covers five domains that are congruent with the symptoms of adult Fabry disease. It differs from other QOL scales as it also assesses the impact of work and personal relationships on patients' QOL and symptoms that progress in adulthood. This study has important implications for healthcare providers who treat adult patients with Fabry disease, enabling them to have a fuller picture of the unique needs of this population.

Delayed dopaminergic neuron differentiation in Lrp6 mutant mice.

Wnts are known to bind and activate multiple membrane receptors/coreceptors and to regulate dopaminergic (DA) neuron development and ventral midbrain (VM) morphogenesis. The low density lipoprotein receptor-related protein (Lrp6) is a Wnt co-receptor, yet it remains unclear whether Lrp6 is required for DA neuron development or VM morphogenesis. Lrp6 is expressed ubiquitously in the developing VM. In this study, we show that Lrp6(-/-) mice exhibit normal patterning, proliferation and cell death in the VM, but display a delay in the onset of DA precursor differentiation. A transient 50% reduction in tyrosine hydroxylase-positive DA neurons and in the expression of DA markers such as Nurr1 and Pitx3, as well as a defect in midbrain morphogenesis was detected in the mutant embryos at embryonic day 11.5. Our results, therefore, suggest a role for Lrp6 in the onset of DA neuron development in the VM as well as a role in midbrain morphogenesis.

Sleeping beauty transposase has an affinity for heterochromatin conformation.

The Sleeping Beauty (SB) transposase reconstructed from salmonid fish has high transposition activity in mammals and has been a useful tool for insertional mutagenesis and gene delivery. However, the transposition efficiency has varied significantly among studies. Our previous study demonstrated that the introduction of methylation into the SB transposon enhanced transposition, suggesting that transposition efficiency is influenced by the epigenetic status of the transposon region. Here, we examined the influence of the chromatin status on SB transposition in mouse embryonic stem cells. Heterochromatin conformation was introduced into the SB transposon by using a tetracycline-controlled transrepressor (tTR) protein, consisting of a tetracycline repressor (TetR) fused to the Kruppel-associated box (KRAB) domain of human KOX1 through tetracycline operator (tetO) sequences. The excision frequency of the SB transposon, which is the first step of the transposition event, was enhanced by approximately 100-fold. SB transposase was found to be colocalized with intense DAPI (4',6'-diamidino-2-phenylindole) staining and with the HP1 family by biochemical fractionation analyses. Furthermore, chromatin immunoprecipitation analysis revealed that SB transposase was recruited to tTR-induced heterochromatic regions. These data suggest that the high affinity of SB transposase for heterochromatin conformation leads to enhancement of SB transposition efficiency.

Differential transactivation of the upstream aggrecan enhancer regulated by PAX1/9 depends on SOX9-driven transactivation.

A previously identified enhancer 10 kb upstream of the Aggrecan (Acan) gene (UE) can drive cartilage specific reporter expression in vivo. Here, we report that the paralogous transcription factors PAX1 and PAX9 differentially drive UE, depending on the presence or absence of SOX9-driven transactivation. In the developing vertebral column, PAX1/9 expression was inversely correlated with Acan expression. Moreover, PAX1/9 was co-expressed with SOX9/5/6 in the intervertebral mesenchyme and the inner annulus fibrosus (AF), and with SOX9 in the outer AF. Significant Acan upregulation was observed during chondrification of Pax1-silenced AF cells, while, Acan was significantly downregulated by persistent expression of Pax1 in cartilage. Deletion of UE using CRISPR/Cas9 resulted in ~30% and ~40% reduction of Acan expression in cartilage and the AF, respectively. In the UE, PAX1/9 acts as weak transactivators through a PAX1/9-binding site partially overlapped with a SOX9-binding site. In the presence of SOX9, which otherwise drives robust Acan expression along with SOX5/6, PAX1/9 competes with SOX9 for occupancy of the binding site, resulting in reduced transactivation of Acan. Coimmunoprecipitation revealed the physical interaction of Pax1 with SOX9. Thus, transactivation of the UE is differentially regulated by concerted action of PAX1/9, SOX9, and SOX5/6 in a context-dependent manner.

Elimination of protein aggregates prevents premature senescence in human trisomy 21 fibroblasts.

Chromosome abnormalities induces profound alterations in gene expression, leading to various disease phenotypes. Recent studies on yeast and mammalian cells have demonstrated that aneuploidy exerts detrimental effects on organismal growth and development, regardless of the karyotype, suggesting that aneuploidy-associated stress plays an important role in disease pathogenesis. However, whether and how this effect alters cellular homeostasis and long-term features of human disease are not fully understood. Here, we aimed to investigate cellular stress responses in human trisomy syndromes, using fibroblasts and induced pluripotent stem cells (iPSCs). Dermal fibroblasts derived from patients with trisomy 21, 18 and 13 showed a severe impairment of cell proliferation and enhanced premature senescence. These phenomena were accompanied by perturbation of protein homeostasis, leading to the accumulation of protein aggregates. We found that treatment with sodium 4-phenylbutyrate (4-PBA), a chemical chaperone, decreased the protein aggregates in trisomy fibroblasts. Notably, 4-PBA treatment successfully prevented the progression of premature senescence in secondary fibroblasts derived from trisomy 21 iPSCs. Our study reveals aneuploidy-associated stress as a potential therapeutic target for human trisomies, including Down syndrome.

Chromatin states shape insertion profiles of the piggyBac, Tol2 and Sleeping Beauty transposons and murine leukemia virus.

DNA transposons and retroviruses are versatile tools in functional genomics and gene therapy. To facilitate their application, we conducted a genome-wide insertion site profiling of the piggyBac (PB), Tol2 and Sleeping Beauty (SB) transposons and the murine leukemia virus (MLV) in mouse embryonic stem cells (ESCs). PB and MLV preferred highly expressed genes, whereas Tol2 and SB preferred weakly expressed genes. However, correlations with DNase I hypersensitive sites were different for all vectors, indicating that chromatin accessibility is not the sole determinant. Therefore, we analysed various chromatin states. PB and MLV highly correlated with Cohesin, Mediator and ESC-specific transcription factors. Notably, CTCF sites were correlated with PB but not with MLV, suggesting MLV prefers smaller promoter-enhancer loops, whereas PB insertion encompasses larger chromatin loops termed topologically associating domains. Tol2 also correlated with Cohesin and CTCF. However, correlations with ESC-specific transcription factors were weaker, suggesting that Tol2 prefers transcriptionally weak chromatin loops. Consistently, Tol2 insertions were associated with bivalent histone modifications characteristic of silent and inducible loci. SB showed minimum preference to all chromatin states, suggesting the least adverse effect on adjacent genes. These results will be useful for vector selection for various applications.

A Pair of Maternal Chromosomes Derived from Meiotic Nondisjunction in Trisomy 21 Affects Nuclear Architecture and Transcriptional Regulation.

Eukaryotic genomes are organised into complex higher-order structures within the nucleus, and the three-dimensional arrangement of chromosomes is functionally important for global gene regulation. The existence of supernumerary chromosome 21 in Down syndrome may perturb the nuclear architecture at different levels, which is normally optimised to maintain the physiological balance of gene expression. However, it has not been clearly elucidated whether and how aberrant configuration of chromosomes affects gene activities. To investigate the effects of trisomy 21 on nuclear organisation and gene expression, we performed three-dimensional fluorescent imaging analysis of chromosome-edited human induced pluripotent stem cells (iPSCs), which enabled identification of the parental origin of the three copies of chromosome 21. We found that two copies of maternal chromosomes resulting from meiotic nondisjunction had a higher tendency to form an adjacent pair and were located relatively distant from the nuclear membrane, suggesting the conserved interaction between these homologous chromosomes. Transcriptional profiling of parental-origin-specific corrected disomy 21 iPSC lines indicated upregulated expression of the maternal alleles for a group of genes, which was accompanied by a fluctuating expression pattern. These results suggest the unique effects of a pair of maternal chromosomes in trisomy 21, which may contribute to the pathological phenotype.

Systematic Cellular Disease Models Reveal Synergistic Interaction of Trisomy 21 and GATA1 Mutations in Hematopoietic Abnormalities.

Chromosomal aneuploidy and specific gene mutations are recognized early hallmarks of many oncogenic processes. However, the net effect of these abnormalities has generally not been explored. We focused on transient myeloproliferative disorder (TMD) in Down syndrome, which is characteristically associated with somatic mutations in GATA1. To better understand functional interplay between trisomy 21 and GATA1 mutations in hematopoiesis, we constructed cellular disease models using human induced pluripotent stem cells (iPSCs) and genome-editing technologies. Comparative analysis of these engineered iPSCs demonstrated that trisomy 21 perturbed hematopoietic development through the enhanced production of early hematopoietic progenitors and the upregulation of mutated GATA1, resulting in the accelerated production of aberrantly differentiated cells. These effects were mediated by dosage alterations of RUNX1, ETS2, and ERG, which are located in a critical 4-Mb region of chromosome 21. Our study provides insight into the genetic synergy that contributes to multi-step leukemogenesis.

Undulated short-tail deletion mutation in the mouse ablates Pax1 and leads to ectopic activation of neighboring Nkx2-2 in domains that normally express Pax1.

Previous studies have indicated that the Undulated short-tail deletion mutation in mouse Pax1 (Pax1(Un-s)) not only ablates Pax1, but also disturbs a gene or genes nearby Pax1. However, which gene(s) is involved and how the Pax1(Un-s) phenotype is confined to the Pax1-positive tissues remain unknown. In the present study, we determined the Pax1(Un-s) deletion interval to be 125 kb and characterized genes around Pax1. We show that the Pax1(Un-s) mutation affects four physically linked genes within or near the deletion, including Pax1, Nkx2-2, and their potential antisense genes. Remarkably, Nkx2-2 is ectopically activated in the sclerotome and limb buds of Pax1(Un-s) embryos, both of which normally express Pax1. This result suggests that the Pax1(Un-s) deletion leads to an illegitimate interaction between remotely located Pax1 enhancers and the Nkx2-2 promoter by disrupting an insulation mechanism between Pax1 and Nkx2-2. Furthermore, we show that expression of Bapx1, a downstream target of Pax1, is more strongly affected in Pax1(Un-s) mutants than in Pax1-null mutants, suggesting that the ectopic expression of Nkx2-2 interferes with the Pax1-Bapx1 pathway. Taken together, we propose that a combination of a loss-of-function mutation of Pax1 and a gain-of-function mutation of Nkx2-2 is the molecular basis of the Pax1(Un-s) mutation.

When half is better than the whole: advances in haploid embryonic stem cell technology.

Recent advances in embryonic stem cell (ESC) derivation and genome editing offer efficient platforms for genetic screening. In this issue, Li et al. and Leeb et al., respectively, expand such applications by generating haploid rat ESCs for screening, mutagenesis, and CRISPR-Cas-mediated gene targeting and by developing a forward genetic screen for interrogating haploid mESCs.

Removal of reprogramming transgenes improves the tissue reconstitution potential of keratinocytes generated from human induced pluripotent stem cells.

Human induced pluripotent stem cell (hiPSC) lines have a great potential for therapeutics because customized cells and organs can be induced from such cells. Assessment of the residual reprogramming factors after the generation of hiPSC lines is required, but an ideal system has been lacking. Here, we generated hiPSC lines from normal human dermal fibroblasts with piggyBac transposon bearing reprogramming transgenes followed by removal of the transposon by the transposase. Under this condition, we compared the phenotypes of transgene-residual and -free hiPSCs of the same genetic background. The transgene-residual hiPSCs, in which the transcription levels of the reprogramming transgenes were eventually suppressed, were quite similar to the transgene-free hiPSCs in a pluripotent state. However, after differentiation into keratinocytes, clear differences were observed. Morphological, functional, and molecular analyses including single-cell gene expression profiling revealed that keratinocytes from transgene-free hiPSC lines were more similar to normal human keratinocytes than those from transgene-residual hiPSC lines, which may be partly explained by reactivation of residual transgenes upon induction of keratinocyte differentiation. These results suggest that transgene-free hiPSC lines should be chosen for therapeutic purposes.