Aberrant neurodevelopment in human iPS cell-derived models of Alexander disease.

Alexander disease (AxD) is a rare and severe neurodegenerative disorder caused by mutations in glial fibrillary acidic protein (GFAP). While the exact disease mechanism remains unknown, previous studies suggest that mutant GFAP influences many cellular processes, including cytoskeleton stability, mechanosensing, metabolism, and proteasome function. While most studies have primarily focused on GFAP-expressing astrocytes, GFAP is also expressed by radial glia and neural progenitor cells, prompting questions about the impact of GFAP mutations on central nervous system (CNS) development. In this study, we observed impaired differentiation of astrocytes and neurons in co-cultures of astrocytes and neurons, as well as in neural organoids, both generated from AxD patient-derived induced pluripotent stem (iPS) cells with a GFAPR239C mutation. Leveraging single-cell RNA sequencing (scRNA-seq), we identified distinct cell populations and transcriptomic differences between the mutant GFAP cultures and a corrected isogenic control. These findings were supported by results obtained with immunocytochemistry and proteomics. In co-cultures, the GFAPR239C mutation resulted in an increased abundance of immature cells, while in unguided neural organoids and cortical organoids, we observed altered lineage commitment and reduced abundance of astrocytes. Gene expression analysis revealed increased stress susceptibility, cytoskeletal abnormalities, and altered extracellular matrix and cell-cell communication patterns in the AxD cultures, which also exhibited higher cell death after stress. Overall, our results point to altered cell differentiation in AxD patient-derived iPS-cell models, opening new avenues for AxD research.

Induced pluripotent stem cell-derived neural stem cells promote bone formation in mice with calvarial defects.

Nerve-derived factors have attracted attention in bone regeneration therapy due to their ability to promote bone regeneration and nerve innervation. Mesenchymal stem cells transported to target sites promote osteogenesis. However, there are few reports on the effects of neural stem cells on bone regeneration. Therefore, the aim of this study was to investigate the role of neural stem cells in osteogenesis. Here, embryoid bodies (EB) or primary neurospheres (1NS) were generated using mouse induced pluripotent stem cells (iPS cells), which were then seeded onto gelatin (Gel) sponges. The seeded Gel sponges were then transplanted into mouse calvarial bone defects. We noted that 1NS-seeded Gel promoted bone regeneration and the presence of tartrate-resistant acid phosphatase (TRAP)-positive cells, whereas the EB-seeded Gel did not. RNA-sequencing of the 1NS-seeded and EB seeded Gels showed an upregulation of the transforming growth factor (TGF)-ß signaling pathway in the 1NS-seeded Gel group. Immunostaining confirmed the presence of Id3 positive cells in mice with bone defects treated with the 1NS-seeded Gel. These findings suggest that the transplantation of neural stem cells may contribute to the promotion of bone regeneration. STATEMENT OF SIGNIFICANCE: This study aimed to investigate whether neural stem cells, when seeded in Gel sponges, promoted bone regeneration. It has been well documented that bone is tightly linked with the nervous systems. Bioscaffolds comprising factors that promote innervation and bone regeneration have been investigated for use in bone therapy. However, there is limited research on the use of neural stem cells for promoting bone formation. To assess this relationship, we conducted both in vivo and in vitro assays to determine whether neural stem cells promoted bone formation. We noted that 1NS-seeded Gel sponges promoted bone formation significantly in mice with calvarial defects after 4 weeks. This study provides a novel approach of neural stem cells for bone therapy.

Unraveling MLL1-fusion Leukemia: Epigenetic Revelations from an iPS Cell Point Mutation.

Our understanding of acute leukemia pathology is heavily dependent on 11q23 chromosomal translocations involving the mixed lineage leukemia-1 (MLL1) gene, a key player in histone H3 lysine 4 (H3K4) methylation. These translocations result in MLL1-fusion (MLL1F) proteins that are thought to drive leukemogenesis. However, the mechanism behind increased H3K4 trimethylation in MLL1F-leukemic stem cells (MLL1F-LSCs), following loss of the catalytic SET domain of MLL1 (known for H3K4 mono- and dimethylation), remains unclear. In our investigation, we introduced a homozygous loss-of-function point mutation in MLL1 within human induced pluripotent stem cells. This mutation mimics the histone methylation, gene expression, and epithelial-mesenchymal transition (EMT) phenotypes of MLL1F-LSCs- without requiring a translocation or functional wild-type MLL1. The mutation caused a genome-wide redistribution of the H3K4 trimethyl mark and upregulated LSC-maintenance genes like HoxA9-A13, Meis1, and the HOTTIP long non-coding RNA (lncRNA). EMT markers such as ZEB1, SNAI2, and HIC-5 were also increased leading to enhanced cellular migration and invasiveness. These observations underscore the essential role of MLL1's enzymatic activity in restraining the cascade of epigenetic changes associated with the gene-activating H3K4 trimethylation mark, which we show may be catalyzed by mislocalized SETd1a H3K4 trimethyltransferase in the absence of MLL1's enzymatic activity. Challenging existing models, our findings imply that MLL1F-induced leukemias arise from a dominant-negative impact on MLL1's histone methyltransferase activity. We propose targeting SETd1a in precision medicine as a new therapeutic approach for MLL1-associated leukemias.

Autologous hGMSC-Derived iPS: A New Proposal for Tissue Regeneration.

The high mortality in the global population due to chronic diseases highlights the urgency to identify effective alternative therapies. Regenerative medicine provides promising new approaches for this purpose, particularly in the use of induced pluripotent stem cells (iPSCs). The aim of the work is to establish a new pluripotency cell line obtained for the first time by reprogramming human gingival mesenchymal stem cells (hGMSCs) by a non-integrating method. The hGMSC-derived iPS line characterization is performed through morphological analysis with optical and electron scanning microscopy and through the pluripotency markers expression evaluation in cytofluorimetry, immunofluorescence, and RT-PCR. To confirm the pluripotency of new hGMSC-derived iPS, the formation of embryoid bodies (EBs), as an alternative to the teratoma formation test, is studied in morphological analysis and through three germ layers' markers' expression in immunofluorescence and RT-PCR. At the end, a comparative study between parental hGMSCs and derived iPS cells is performed also for the extracellular vesicles (EVs) and their miRNA content. The new hGMSC-derived iPS line demonstrated to be pluripotent in all aspects, thus representing an innovative dynamic platform for personalized tissue regeneration.

Simultaneous electro-dynamic stimulation accelerates maturation of engineered cardiac tissues generated by human iPS cells.

Electrical and dynamic stimulation are commonly employed to enhance the maturation of engineered cardiac tissue (ECT) derived from human induced pluripotent stem cells (iPSCs), reflecting the physiological environment of the heart. While electrical stimulation mimics natural bioelectrical signals and dynamic stimulation replicates mechanical forces, the combined effects of these stimuli on ECT maturation have not been thoroughly explored. We hypothesized that simultaneous electro-dynamic stimulation would enhance ECT maturation and function more effectively than either stimulus alone. Human iPSC-derived cardiovascular cells were co-cultured with Collagen I and Matrigel for 2 weeks, followed by a comparative analysis of four groups: no stimulation, dynamic stimulation, electrical stimulation, and simultaneous electro-dynamic stimulation. The functionality of ECTs was assessed by measuring contractile capacity and calcium indicators, and histological assessments examined structural maturation. Our results demonstrated that simultaneous electro-dynamic stimulation significantly increased the CM component, elevated TNNT2 mRNA expression levels, and enhanced calcium transient capacity. Additionally, ECTs subjected to simultaneous stimulation exhibited a positive force-frequency relationship in contractility and an elevation in peak calcium flux, indicative of advanced tissue maturation. Moreover, simultaneous stimulation promoted vascular network formation within the ECTs, suggesting improved structural organization. These findings underscore the importance of simultaneous stimulation for developing effective cardiac tissue engineering strategies.

Evaluating precision medicine approaches for gene therapy in patient-specific cellular models of Bietti crystalline dystrophy.

Patient-specific induced pluripotent stem cell-derived (iPSC-derived) cell lines allow for therapies to be tailored to individual patients, increasing therapeutic precision and efficiency. Bietti crystalline dystrophy (BCD) is a rare blinding disease estimated to affect about 67,000 individuals worldwide. Here, we used iPSC-derived retinal pigment epithelium (iRPE) cells from patients with BCD to evaluate adeno-associated virus-mediated (AAV-mediated) gene augmentation therapy strategies. We found that BCD iRPE cells were vulnerable to blue light-induced oxidative stress and that cellular phenotype can be quantified using 3 robust biomarkers: reactive oxygen species (ROS), 4-hydroxy 2-nonenal (4-HNE) levels, and cell death rate. Additionally, we demonstrated that AAV-mediated gene therapy can significantly reduce light-induced cell death in BCD iRPE cells. This is the first proof-of-concept study to our knowledge to show that AAV-CYP4V2 gene therapy can be used to treat light-induced RPE damage in BCD. Furthermore, we observed significant variability in cellular phenotypes among iRPE from patients with BCD of divergent mutations, which outlined genotype-phenotype correlations in BCD patient-specific cell disease models. Our results reveal that patient-specific iRPE cells retained personalized responses to AAV-mediated gene therapy. Therefore, this approach can advance BCD therapy and set a precedent for precision medicine in other diseases, emphasizing the necessity for personalization in healthcare to accommodate individual diversity.

Robust differentiation of human pluripotent stem cells into Schwann cells.

Research into Schwann cell (SC)-related diseases has been hampered by the difficulty of obtaining human-derived SCs, which have limited proliferative capacity. This has resulted in a delay in progress in drug discovery and cell therapy targeting SCs. To overcome these limitations, we developed a robust method for inducing the differentiation of human induced pluripotent stem cells (hiPSCs) into SCs. We established hiPSC lines and successfully generated high-purity Schwann cell precursors (SCPs) from size-controlled hiPSC aggregates by precisely timed treatment with our proprietary enzyme solution. Such SCPs were successfully expanded and further differentiated into myelin basic protein (MBP) expressing SC populations when treated with an appropriate medium containing dibutyryl-cAMP (db-cAMP). These differentiated cells secreted factors that induced neurite outgrowth in vitro. Our method allows for the efficient and stable production of SCPs and SCs from hiPSCs. This robust induction and maturation method has the potential to be a valuable tool in drug discovery and cell therapy targeting SC-related diseases.

Revealing the UV response of melanocytes in xeroderma pigmentosum group A using patient-derived induced pluripotent stem cells.

BACKGROUND: Xeroderma pigmentosum (XP) is characterized by photosensitivity that causes pigmentary disorder and predisposition to skin cancers on sunlight-exposed areas due to DNA repair deficiency. Patients with XP group A (XP-A) develop freckle-like pigmented maculae and depigmented maculae within a year unless strict sun-protection is enforced. Although it is crucial to study pigment cells (melanocytes: MCs) as disease target cells, establishing MCs in primary cultures is challenging. OBJECTIVE: Elucidation of the disease pathogenesis by comparison between MCs differentiated from XP-A induced pluripotent stem cells (iPSCs) and healthy control iPSCs on the response to UV irradiation. METHODS: iPSCs were established from a XP-A fibroblasts and differentiated into MCs. Differences in gene expression profiles between XP-A-iPSC-derived melanocytes (XP-A-iMCs) and Healthy control iPSC-derived MCs (HC-iMCs) were analyzed 4 and 12 h after irradiation with 30 or 150 J/m2 of UV-B using microarray analysis. RESULTS: XP-A-iMCs expressed SOX10, MITF, and TYR, and showed melanin synthesis. Further, XP-A-iMCs showed reduced DNA repair ability. Gene expression profile between XP-A-iMCs and HC-iMCs revealed that, numerous gene probes that were specifically upregulated or downregulated in XP-A-iMCs after 150-J/m2 of UV-B irradiation did not return to basal levels. Of note that apoptotic pathways were highly upregulated at 150 J/m2 UV exposure in XP-A-iMCs, and cytokine-related pathways were upregulated even at 30 J/m2 UV exposure. CONCLUSION: We revealed for the first time that cytokine-related pathways were upregulated even at low-dose UV exposure in XP-A-iMCs. Disease-specific iPSCs are useful to elucidate the disease pathogenesis and develop treatment strategies of XP.

Transcriptional analysis of primary ciliary dyskinesia airway cells reveals a dedicated cilia glutathione pathway.

Primary ciliary dyskinesia (PCD) is a genetic condition that results in dysmotile cilia. The repercussions of cilia dysmotility and gene variants on the multiciliated cell remain poorly understood. We used single-cell RNA-Seq, proteomics, and advanced microscopy to compare primary culture epithelial cells from patients with PCD, their heterozygous mothers, and healthy individuals, and we induced pluripotent stem cells (iPScs) generated from a patient with PCD. Transcriptomic analysis revealed unique signatures in PCD airway cells compared with their mothers' cells and the cells of healthy individuals. Gene expression in heterozygous mothers' cells diverged from both control and PCD cells, marked by increased inflammatory and cellular stress signatures. Primary and iPS-derived PCD multiciliated cells had increased expression of glutathione-S-transferases GSTA2 and GSTA1, as well as NRF2 target genes, accompanied by elevated levels of reactive oxygen species (ROS). Immunogold labeling in human cilia and proteomic analysis of the ciliated organism Chlamydomonas reinhardtii demonstrated that GSTA2 localizes to motile cilia. Loss of human GSTA2 and C. reinhardtii GSTA resulted in slowed cilia motility, pointing to local cilia regulatory roles. Our findings identify cellular responses unique to PCD variants and independent of environmental stress and uncover a dedicated ciliary GSTA2 pathway essential for normal motility that may be a therapeutic target.

Patient-derived and gene-edited pluripotent stem cells lacking NPHP1 recapitulate juvenile nephronophthisis in abnormalities of primary cilia and renal cyst formation.

Juvenile nephronophthisis is an inherited renal ciliopathy with cystic kidney disease, renal fibrosis, and end-stage renal failure in children and young adults. Mutations in the NPHP1 gene encoding nephrocystin-1 protein have been identified as the most frequently responsible gene and cause the formation of cysts in the renal medulla. The molecular pathogenesis of juvenile nephronophthisis remains elusive, and no effective medicines to prevent end-stage renal failure exist even today. No human cellular models have been available yet. Here, we report a first disease model of juvenile nephronophthisis using patient-derived and gene-edited human induced pluripotent stem cells (hiPSCs) and kidney organoids derived from these hiPSCs. We established NPHP1-overexpressing hiPSCs from patient-derived hiPSCs and NPHP1-deficient hiPSCs from healthy donor hiPSCs. Comparing these series of hiPSCs, we found abnormalities in primary cilia associated with NPHP1 deficiency in hiPSCs. Kidney organoids generated from the hiPSCs lacking NPHP1 formed renal cysts frequently in suspension culture with constant rotation. This cyst formation in patient-derived kidney organoids was rescued by overexpression of NPHP1. Transcriptome analysis on these kidney organoids revealed that loss of NPHP1 caused lower expression of genes related to primary cilia in epithelial cells and higher expression of genes related to the cell cycle. These findings suggested the relationship between abnormality in primary cilia induced by NPHP1 loss and abnormal proliferative characteristics in the formation of renal cysts. These findings demonstrated that hiPSC-based systematic disease modeling of juvenile nephronophthisis contributed to elucidating the molecular pathogenesis and developing new therapies.

Deep learning for quantifying spatial patterning and formation process of early differentiated human-induced pluripotent stem cells with micropattern images.

Micropatterning is reliable method for quantifying pluripotency of human-induced pluripotent stem cells (hiPSCs) that differentiate to form a spatial pattern of sorted, ordered and nonoverlapped three germ layers on the micropattern. In this study, we propose a deep learning method to quantify spatial patterning of the germ layers in the early differentiation stage of hiPSCs using micropattern images. We propose decoding and encoding U-net structures learning labelled Hoechst (DNA-stained) hiPSC regions with corresponding Hoechst and bright-field micropattern images to segment hiPSCs on Hoechst or bright-field images. We also propose a U-net structure to extract extraembryonic regions on a micropattern, and an algorithm to compares intensities of the fluorescence images staining respective germ-layer cells and extract their regions. The proposed method thus can quantify the pluripotency of a hiPSC line with spatial patterning including cell numbers, areas and distributions of germ-layer and extraembryonic cells on a micropattern, and reveal the formation process of hiPSCs and germ layers in the early differentiation stage by segmenting live-cell bright-field images. In our assay, the cell-number accuracy achieved 86% and 85%, and the cell region accuracy 89% and 81% for segmenting Hoechst and bright-field micropattern images, respectively. Applications to micropattern images of multiple hiPSC lines, micropattern sizes, groups of markers, living and fixed cells show the proposed method can be expected to be a useful protocol and tool to quantify pluripotency of a new hiPSC line before providing it to the scientific community.

Corrigendum: ACE2 knockout hinders SARS-CoV-2 propagation in iPS cell-derived airway and alveolar epithelial cells.

[This corrects the article DOI: 10.3389/fcell.2023.1290876.].

Construction of multilayered small intestine-like tissue by reproducing interstitial flow.

Recent advances have made modeling human small intestines in vitro possible, but it remains a challenge to recapitulate fully their structural and functional characteristics. We suspected interstitial flow within the intestine, powered by circulating blood plasma during embryonic organogenesis, to be a vital factor. We aimed to construct an in vivo-like multilayered small intestinal tissue by incorporating interstitial flow into the system and, in turn, developed the micro-small intestine system by differentiating definitive endoderm and mesoderm cells from human pluripotent stem cells simultaneously on a microfluidic device capable of replicating interstitial flow. This approach enhanced cell maturation and led to the development of a three-dimensional small intestine-like tissue with villi-like epithelium and an aligned mesenchymal layer. Our micro-small intestine system not only overcomes the limitations of conventional intestine models but also offers a unique opportunity to gain insights into the detailed mechanisms underlying intestinal tissue development.

A Novel CRISPR-Cas9 Strategy to Target DYSTROPHIN Mutations Downstream of Exon 44 in Patient-Specific DMD iPSCs.

Mutations in the DMD gene cause fatal Duchenne Muscular Dystrophy (DMD). An attractive therapeutic approach is autologous cell transplantation utilizing myogenic progenitors derived from induced pluripotent stem cells (iPSCs). Given that a significant number of DMD mutations occur between exons 45 and 55, we developed a gene knock-in approach to correct any mutations downstream of exon 44. We applied this approach to two DMD patient-specific iPSC lines carrying mutations in exons 45 and 51 and confirmed mini-DYSTROPHIN (mini-DYS) protein expression in corrected myotubes by western blot and immunofluorescence staining. Transplantation of gene-edited DMD iPSC-derived myogenic progenitors into NSG/mdx4Cv mice produced donor-derived myofibers, as shown by the dual expression of human DYSTROPHIN and LAMIN A/C. These findings further provide proof-of-concept for the use of programmable nucleases for the development of autologous iPSC-based therapy for muscular dystrophies.

Folate as a potential treatment for lethal ventricular arrhythmias in TANGO2-deficiency disorder.

TANGO2-deficiency disorder (TDD) is an autosomal-recessive genetic disease caused by biallelic loss-of-function variants in the TANGO2 gene. TDD-associated cardiac arrhythmias are recalcitrant to standard antiarrhythmic medications and constitute the leading cause of death. Disease modeling for TDD has been primarily carried out using human dermal fibroblast and, more recently, in Drosophila by multiple research groups. No human cardiomyocyte system has been reported, which greatly hinders the investigation and understanding of TDD-associated arrhythmias. Here, we established potentially novel patient-derived induced pluripotent stem cell differentiated cardiomyocyte (iPSC-CM) models that recapitulate key electrophysiological abnormalities in TDD. These electrophysiological abnormalities were rescued in iPSC-CMs with either adenoviral expression of WT-TANGO2 or correction of the pathogenic variant using CRISPR editing. Our natural history study in patients with TDD suggests that the intake of multivitamin/B complex greatly diminished the risk of cardiac crises in patients with TDD. In agreement with the clinical findings, we demonstrated that high-dose folate (vitamin B9) virtually abolishes arrhythmias in TDD iPSC-CMs and that folate's effect was blocked by the dihydrofolate reductase inhibitor methotrexate, supporting the need for intracellular folate to mediate antiarrhythmic effects. In summary, data from TDD iPSC-CM models together with clinical observations support the use of B vitamins to mitigate cardiac crises in patients with TDD, providing potentially life-saving treatment strategies during life-threatening events.

Pre-clinical evaluation of clinically relevant iPS cell derived neuroepithelial stem cells as an off-the-shelf cell therapy for spinal cord injury.

Preclinical transplantations using human neuroepithelial stem (NES) cells in spinal cord injury models have exhibited promising results and demonstrated cell integration and functional improvement in transplanted animals. Previous studies have relied on the generation of research grade cell lines in continuous culture. Using fresh cells presents logistic hurdles for clinical transition regarding time and resources for maintaining high quality standards. In this study, we generated a good manufacturing practice (GMP) compliant human iPS cell line in GMP clean rooms alongside a research grade iPS cell line which was produced using standardized protocols with GMP compliant chemicals. These two iPS cell lines were differentiated into human NES cells, from which six batches of cell therapy doses were produced. The doses were cryopreserved, thawed on demand and grafted in a rat spinal cord injury model. Our findings demonstrate that NES cells can be directly grafted post-thaw with high cell viability, maintaining their cell identity and differentiation capacity. This opens the possibility of manufacturing off-the-shelf cell therapy products. Moreover, our manufacturing process yields stable cell doses with minimal batch-to-batch variability, characterized by consistent expression of identity markers as well as similar viability of cells across the two iPS cell lines. These cryopreserved cell doses exhibit sustained viability, functionality, and quality for at least 2 years. Our results provide proof of concept that cryopreserved NES cells present a viable alternative to transplanting freshly cultured cells in future cell therapies and exemplify a platform from which cell formulation can be optimized and facilitate the transition to clinical trials.

iPS cell therapy 2.0: Preparing for next-generation regenerative medicine.

This year marks the tenth anniversary of the world's first transplantation of tissue generated from induced pluripotent stem cells (iPSCs). There is now a growing number of clinical trials worldwide examining the efficacy and safety of autologous and allogeneic iPSC-derived products for treating various pathologic conditions. As we patiently wait for the results from these and future clinical trials, it is imperative to strategize for the next generation of iPSC-based therapies. This review examines the lessons learned from the development of another advanced cell therapy, chimeric antigen receptor (CAR) T cells, and the possibility of incorporating various new bioengineering technologies in development, from RNA engineering to tissue fabrication, to apply iPSCs not only as a means to achieve personalized medicine but also as designer medical applications.

Thymus transplantation as immunotherapy for the enhancement and/or correction of T cell function.

The thymus is where T cells, among the most important immune cells involved in biological defense and homeostasis, are produced and developed. The thymus plays an important role in the defense against infection and cancer as well as the prevention of autoimmune diseases. However, the thymus gland atrophies with age, which might have pathological functions, and in some circumstances, there is a congenital defect in the thymus. These can be the cause of many diseases related to the dysregulation of T cell functions. Thus, the enhancement and/or normalization of thymic function may lead to protection against and treatment of a wide variety of diseases. Therefore, thymus transplantation is considered a strong candidate for permanent treatment. The status and issues related to thymus transplantation for possible immunotherapy are discussed although it is still at an early stage of development.

Generation of allogeneic and xenogeneic functional muscle stem cells for intramuscular transplantation.

Satellite cells, the stem cells of skeletal muscle tissue, hold a remarkable regeneration capacity and therapeutic potential in regenerative medicine. However, low satellite cell yield from autologous or donor-derived muscles hinders the adoption of satellite cell transplantation for the treatment of muscle diseases, including Duchenne muscular dystrophy (DMD). To address this limitation, here we investigated whether satellite cells can be derived in allogeneic or xenogeneic animal hosts. First, injection of CRISPR/Cas9-corrected Dmdmdx mouse induced pluripotent stem cells (iPSCs) into mouse blastocysts carrying an ablation system of host satellite cells gave rise to intraspecies chimeras exclusively carrying iPSC-derived satellite cells. Furthermore, injection of genetically corrected DMD iPSCs into rat blastocysts resulted in the formation of interspecies rat-mouse chimeras harboring mouse satellite cells. Notably, iPSC-derived satellite cells or derivative myoblasts produced in intraspecies or interspecies chimeras restored dystrophin expression in DMD mice following intramuscular transplantation and contributed to the satellite cell pool. Collectively, this study demonstrates the feasibility of producing therapeutically competent stem cells across divergent animal species, raising the possibility of generating human muscle stem cells in large animals for regenerative medicine purposes.

Establishment of Induced Pancreatic Stem Cells by Yes-Associated Protein 1.

Recently, we and others generated induced tissue-specific stem/progenitor (iTS/iTP) cells. The advantages of iTS/iTP cells compared with induced pluripotent stem (iPS) cells are (1) easier generation, (2) efficient differentiation, and (3) no teratomas formation. In this study, we generated mouse induced pancreatic stem cells (iTS-P cells) by the plasmid vector expressing Yes-associated protein 1 (YAP). The iTS-P YAP9 cells expressed Foxa2 (endoderm marker) and Pdx1 (pancreatic marker) while the expressions of Oct3/4 and Nanog (marker of embryonic stem [ES] cells) in iTS-P YAP9 cells was significantly lower compared with those in ES cells. The iTS-P YAP9 cells efficiently differentiated into insulin-expressing cells compared with ES cells. The ability to generate autologous iTS cells may be applied to diverse applications of regenerative medicine.