An Ethanol Extract of Coptidis rhizoma Induces Apoptotic Cell Death in Induced Pluripotent Stem Cells and Suppresses Teratoma Formation.

In cell-based regenerative medicine, induced pluripotent stem cells (iPSCs) generated from reprogrammed adult somatic cells have emerged as a useful cell source due to the lack of ethical concerns and the low risk of immune rejection. To address the risk of teratoma formation, which is a safety issue in iPSC-based cell therapy, it is essential to selectively remove undifferentiated iPSCs remaining in the iPSC-derived differentiated cell product prior to in vivo transplantation. In this study, we explored whether an ethanol extract of coptidis rhizoma (ECR) exhibited anti-teratoma activity and identified the active components involved in the selective elimination of undifferentiated iPSCs. Transcriptome analysis of iPSCs confirmed that cell death-related pathways were significantly altered by ECR treatment. Our results demonstrate that ECR effectively induced apoptotic cell death and DNA damage in iPSCs, and that reactive oxygen species generation, mitochondrial damage, caspase activation, and p53 activation were involved in ECR-mediated iPSC death. However, in iPSC-derived differentiated cells (iPSC-Diff), reduced cell viability and the DNA damage response were not observed after ECR treatment. We co-cultured iPSCs and iPSC-Diff and found that ECR treatment selectively removed iPSCs, whereas iPSC-Diff remained intact. Prior to in ovo implantation, ECR treatment of a mixed cell culture of iPSCs and iPSC-Diff significantly suppressed iPSC-derived teratoma formation. Among the main components of the ECR, berberine and coptisine showed selective cytotoxicity to iPSCs but not to iPSC-Diff. Together, these results indicate the usefulness of ECRs in preparing safe and effective iPSC-based therapeutic cell products with no risk of teratoma formation.

Neuroprotective Effects of Licochalcone D in Oxidative-Stress-Induced Primitive Neural Stem Cells from Parkinson's Disease Patient-Derived iPSCs.

Parkinson's disease (PD) is one of the most common neurodegenerative diseases caused by the loss of dopaminergic neurons in the substantia nigra pars compacta. Although the etiology of PD is still unclear, the death of dopaminergic neurons during PD progression was revealed to be associated with abnormal aggregation of α-synuclein, elevation of oxidative stress, dysfunction of mitochondrial functions, and increased neuroinflammation. In this study, the effects of Licochalcone D (LCD) on MG132-induced neurotoxicity in primitive neural stem cells (pNSCs) derived from reprogrammed iPSCs were investigated. A cell viability assay showed that LCD had anti-apoptotic properties in MG132-induced oxidative-stressed pNSCs. It was confirmed that apoptosis was reduced in pNSCs treated with LCD through 7-AAD/Annexin Ⅴ staining and cleaved caspase3. These effects of LCD were mediated through an interaction with JunD and through the EGFR/AKT and JNK signaling pathways. These findings suggest that LCD could be a potential antioxidant reagent for preventing disease-related pathological phenotypes of PD.

Advances in Gene Therapy Techniques to Treat LRRK2 Gene Mutation.

Leucine-rich repeat kinase 2 (LRRK2) gene mutation is an autosomal dominant mutation associated with Parkinson's disease (PD). Among LRRK2 gene mutations, the LRRK2 G2019S mutation is frequently involved in PD onset. Currently, diverse gene correction tools such as zinc finger nucleases (ZFNs), helper-dependent adenoviral vector (HDAdV), the bacterial artificial chromosome-based homologous recombination (BAC-based HR) system, and CRISPR/Cas9-homology-directed repair (HDR) or adenine base editor (ABE) are used in genome editing. Gene correction of the LRRK2 G2019S mutation has been applied whenever new gene therapy tools emerge, being mainly applied to induced pluripotent stem cells (LRRK2 G2019S-mutant iPSCs). Here, we comprehensively introduce the principles and methods of each programmable nuclease such as ZFN, CRISPR/Cas9-HDR or ABE applied to LRRK2 G2019S, as well as those of HDAdV or BAC-based HR systems used as nonprogrammable nuclease systems.

Caffeic acid selectively eliminates teratogenic human-induced pluripotent stem cells via apoptotic cell death.

BACKGROUND: Induced pluripotent stem cells (iPSCs) generated from reprogrammed adult somatic cells are considered as a promising cell source in cell-based regenerative medicine. To avoid teratoma formation, which is a safety issue in iPSC-based cell therapy, it is important to selectively remove undifferentiated iPSCs that remain in the differentiated cell product before in vivo transplantation. Caffeic acid (CAA, 3,4-dihydroxy-cinnamic acid) is a phenolic compound synthesized from various vegetables, fruits, and herbs; it has shown various pharmacological activities against inflammation, cancer, infection, diabetes, and neurodegenerative diseases. However, the beneficial effects of CAA in iPSC-based cell therapy, such as the selective elimination of iPSCs and anti-teratoma effects, have not yet been explored. RESULTS: Here, we found that CAA induced apoptotic cell death in iPSCs; this process did not occur in iPSC-derived mesenchymal progenitor cells (MPCs) or human dermal fibroblast (hDFs). Under co-culture conditions with MPCs and hDFs, CAA treatment selectively removed iPSCs. In addition, CAA treatment in mixed cell culture with iPSCs and MPCs prior to grafting markedly suppressed iPSC-derived teratoma formation. Finally, CAA did not induce DNA damage in MPCs or hDFs. CONCLUSION: Taken together, these results suggest that CAA is effective in preparing safe iPSC-based therapeutic cells without the risk of teratoma formation and DNA damage in normal cells and iPSC-derived differentiated cells.

Differential Transcriptional Regulation of Polymorphic p53 Codon 72 in Metabolic Pathways.

p53 is a transcription factor that is activated under DNA damage stress and regulates the expression of proapoptotic genes including the expression of growth arrest genes to subsequently determine the fate of cells. To investigate the functional differences of polymorphic p53 codon 72, we constructed isogenic lines encoding each polymorphic p53 codon 72 based on induced pluripotent stem cells, which can endogenously express each polymorphic p53 protein only, encoding either the arginine 72 (R72) variant or proline 72 (P72) variant, respectively. We found that there was no significant functional difference between P72 and R72 cells in growth arrest or apoptosis as a representative function of p53. In the comprehensive analysis, the expression pattern of the common p53 target genes, including cell cycle arrest or apoptosis, was also increased regardless of the polymorphic p53 codon 72 status, whereas the expression pattern involved in metabolism was decreased and more significant in R72 than in P72 cells. This study noted that polymorphic p53 codon 72 differentially regulated the functional categories of metabolism and not the pathways that determine cell fate, such as growth arrest and apoptosis in cells exposed to genotoxic stress.

Ciliogenesis is Not Directly Regulated by LRRK2 Kinase Activity in Neurons.

Mutations in the Leucine-rich repeat kinase 2 (LRRK2) gene are the most prevalent cause of familial Parkinson's disease (PD). The increase in LRRK2 kinase activity observed in the pathogenic G2019S mutation is important for PD development. Several studies have reported that increased LRRK2 kinase activity and treatment with LRRK2 kinase inhibitors decreased and increased ciliogenesis, respectively, in mouse embryonic fibroblasts (MEFs) and retinal pigment epithelium (RPE) cells. In contrast, treatment of SH-SY5Y dopaminergic neuronal cells with PD-causing chemicals increased ciliogenesis. Because these reports were somewhat contradictory, we tested the effect of LRRK2 kinase activity on ciliogenesis in neurons. In SH-SY5Y cells, LRRK2 inhibitor treatment slightly increased ciliogenesis, but serum starvation showed no increase. In rat primary neurons, LRRK2 inhibitor treatment repeatedly showed no significant change. Little difference was observed between primary cortical neurons prepared from wild-type (WT) and G2019S+/- mice. However, a significant increase in ciliogenesis was observed in G2019S+/- compared to WT human fibroblasts, and this pattern was maintained in neural stem cells (NSCs) differentiated from the induced pluripotent stem cells (iPSCs) prepared from the same WT/G2019S fibroblast pair. NSCs differentiated from G2019S and its gene-corrected WT counterpart iPSCs were also used to test ciliogenesis in an isogenic background. The results showed no significant difference between WT and G2019S regardless of kinase inhibitor treatment and B27-deprivation-mimicking serum starvation. These results suggest that LRRK2 kinase activity may be not a direct regulator of ciliogenesis and ciliogenesis varies depending upon the cell type or genetic background.

Quantitative Proteomic Analysis of Primitive Neural Stem Cells from LRRK2 G2019S-Associated Parkinson's Disease Patient-Derived iPSCs.

Parkinson's disease (PD) is a common neurodegenerative disease, causing movement defects. The incidence of PD is constantly increasing and this disease is still incurable. Thus, understanding PD pathophysiology would be pivotal for the development of PD therapy, and various PD models have thus been already developed. Through recent advances in reprogramming techniques, a primitive neural stem cell (pNSC) derived from PD patient induced pluripotent stem cells (iPSCs) could be potentially used as a reproducible and reliable experimental system to analyze the effect of the leucine-rich repeat kinase 2 G2019S mutation (LK2GS) in neural cells. Here, we investigated the advantages of such a model system through quantitative proteomic analysis of pNSCs from normal control iPSCs and familial PD patient iPSCs harboring LK2GS. We confirmed that the expression of molecules known to be involved in PD pathogenesis, such as oxidative stress-, cell adhesion-, and cytoskeleton-related proteins, were altered in the LK2GS pNSC. In addition, we showed that down-regulation of Ku80, which was found in the proteomic analysis with LK2GS pNSCs, resulted in apoptosis induced by DNA damage response. Taken together, we suggest that pNSCs from PD iPSCs could provide a reliable and useful model system to study PD. Moreover, the highly expandable pNSC is suitable for multi-omics approaches to understand PD pathologies and discover therapeutic targets for PD.

Simplified Brain Organoids for Rapid and Robust Modeling of Brain Disease.

Although brain organoids are an innovative technique for studying human brain development and disease by replicating the structural and functional properties of the developing human brain, some limitations such as heterogeneity and long-term differentiation (over 2 months) impede their application in disease modeling and drug discovery. In this study, we established simplified brain organoids (simBOs), composed of mature neurons and astroglial cells from expandable hPSC-derived primitive neural stem cells (pNSCs). simBOs can be rapidly generated in 2 weeks and have more homogeneous properties. Transcriptome analysis revealed that three-dimensional (3D) environment of simBOs facilitates the conversion of pNSCs to mature neuronal systems compared to a two-dimensional environment in the context of neurotransmitter release, synaptic vesicle formation, ion channels, calcium signaling, axonal guidance, extracellular matrix organization, and cell cycle. This result was correlated with the translocation of YAP1 into the cytoplasm by sensing matrix stiffness on the 3D models. Furthermore, we demonstrated that simBOs could easily be specified into midbrain-like simBOs by treatment with Shh and FGF8. Midbrain-like simBOs from a Parkinson's disease patient (LRRK2 G2019S)-derived pNSCs and gene-corrected (LRRK2 WT ) control pNSCs represented disease-associated phenotypes in terms of increased LRRK2 activity, decreased dopaminergic neurons, and increased autophagy. Treatment with the LRRK2 inhibitor, PFE-360, relieved the phenotype of Parkinson's disease in midbrain-like simBOs. Taken together, these approaches could be applied to large-scale disease models and alternative drug-testing platforms.

Elimination of Teratogenic Human Induced Pluripotent Stem Cells by Bee Venom via Calcium-Calpain Pathway.

Induced pluripotent stem cells (iPSCs) are regarded as a promising option for cell-based regenerative medicine. To obtain safe and efficient iPSC-based cell products, it is necessary to selectively eliminate the residual iPSCs prior to in vivo implantation due to the risk of teratoma formation. Bee venom (BV) has long been used in traditional Chinese medicine to treat inflammatory diseases and relieve pain, and has been shown to exhibit anti-cancer, anti-mutagenic, anti-nociceptive, and radioprotective activities. However, the potential benefits of BV in iPSC therapy, particularly its anti-teratoma activity, have not been examined. In this study, we found that BV selectively induced cell death in iPSCs, but not in iPSC-derived differentiated cells (iPSCs-Diff). BV rapidly disrupted cell membrane integrity and focal adhesions, followed by induction of apoptosis and necroptosis in iPSCs. We also found that BV remarkably enhanced intracellular calcium levels, calpain activation, and reactive oxygen speciesgeneration in iPSCs. BV treatment before in ovo grafting efficiently prevented iPSC-derived teratoma formation. In contrast, no DNA damage was observed in iPSCs-Diff following BV treatment, further demonstrating the safety of BV for use with iPSCs-Diff. Taken together, these findings show that BV has potent anti-teratoma activity by eliminating residual iPSCs, and can be used for the development of effective and safe iPSC-based cell therapies.

Prunellae Spica Extract Suppresses Teratoma Formation of Pluripotent Stem Cells through p53-Mediated Apoptosis.

Induced pluripotent stem cells (iPSCs) have similar properties to embryonic stem cells in terms of indefinite self-renewal and differentiation capacity. After in vitro differentiation of iPSCs, undifferentiated iPSCs (USCs) may exist in cell therapy material and can form teratomas after in vivo transplantation. Selective elimination of residual USCs is, therefore, very important. Prunellae Spica (PS) is a traditional medicinal plant that has been shown to exert anti-cancer, antioxidant, and anti-inflammatory activities; however, its effects on iPSCs have not been previously characterized. In this study, we find that ethanol extract of PS (EPS) effectively induces apoptotic cell death of USCs through G2/M cell cycle arrest, generation of intracellular reactive oxygen species, alteration of mitochondrial membrane potentials, and caspase activation of USCs. In addition, EPS increases p53 accumulation and expression of its downstream targets. In p53 knockout (KO) iPSCs, the EPS did not induce apoptosis, indicating that EPS-mediated apoptosis of USCs was p53-dependent. In addition, EPS was not genotoxic towards iPSCs-derived differentiated cells. EPS treatment before injection efficiently prevented in ovo teratoma formation of p53 wild-type (WT) iPSCs but not p53KO iPSCs. Collectively, these results indicate that EPS has potent anti-teratoma activity and no genotoxicity to differentiated cells. It can, therefore, be used in the development of safe and efficient iPSC-based cell therapies.

Ethanol extract of Magnoliae cortex (EEMC) limits teratoma formation of pluripotent stem cells by selective elimination of undifferentiated cells through the p53-dependent mitochondrial apoptotic pathway.

BACKGROUND: Induced pluripotent stem cells (iPSCs) are regarded as the best potential cell source for cell-based regenerative medicine. To develop a safe and efficient iPSC-based cell therapy, it is very important to avoid possible teratoma formation, which can arise from undifferentiated iPSCs (USCs) remaining among differentiated cell products. Dried bark of Magnolia officinalis (Magnolia cortex, MC) has long been used in traditional medicine to treat gastrointestinal ailments and allergic diseases, and has shown have various pharmacological activities, including anti-bacterial, anti-inflammatory, and anti-cancer effects. However, its effects on iPSCs have not yet been examined. PURPOSE: In this study, we investigated the selective cytotoxic effects of ethanol extract of MC (EEMC) on undifferentiated iPSCs and elucidated the underlying apoptotic mechanisms in detail. We also investigated the inhibitory effects of EEMC on teratoma formation via in ovo experiments. RESULTS: We found that EEMC greatly reduced cell growth and induced apoptotic cell death in USCs, but not in differentiated or normal cells. EEMC caused G2/M cell cycle arrest, mitochondrial damage, and caspase activation of USCs, accompanied by p53 accumulation. In p53KO human iPSCs, EEMC had no cytotoxicity, reinforcing that EEMC-mediated apoptosis of USCs is p53-dependent. EEMC did not cause DNA damage in iPSC-derived differentiated cells. In ovo teratoma formation assay revealed that EEMC treatment before injection efficiently eliminated USCs and prevented teratoma formation. CONCLUSIONS: These results collectively indicate that EEMC has potent anti-teratoma activity, and therefore can be used for the development of safe iPSC-based therapy.

Retroelement Insertion in a CRISPR/Cas9 Editing Site in the Early Embryo Intensifies Genetic Mosaicism.

Continued CRISPR/Cas9-mediated editing activity that allows differential and asynchronous modification of alleles in successive cell generations expands allelic complexity. To understand the earliest events during CRISPR/Cas9 editing and the allelic selection among the progeny of subsequent cell divisions, we inspected in detail the genotypes of 4- and 8-cell embryos and embryonic stem cells (ESCs) after microinjection of a CRISPR toolkit into the zygotes. We found a higher editing frequency in 8-cell embryos than in 4-cell embryos, indicating that the CRISPR/Cas9 activity persisted through the 8-cell stage. Analysis of a CRISPR/Cas9 transgenic founder mouse revealed that four different alleles were present in its organs in different combinations and that its germline included three different mutant alleles, as shown by the genotypes of the pups. The indel depth, which measured the extent of indels at the sequence level within single embryos, decreased significantly as the embryos advanced to form ESCs, suggesting that exclusion of fatal indels occurred in the subsequent cell generations. Interestingly, we discovered that the CRISPR sites frequently contained introduced retroelement sequences and that this occurred preferentially with certain classes of retroelements. Therefore, in addition to CRISPR/Cas9's innate mechanism of separate, differential enzymatic modifications of alleles, the frequent retroelement insertions that occur in early mouse embryos during CRISPR/Cas9 editing further expand the allelic diversity and mosaicism in the resulting transgenic founders.

Generation of gene-corrected iPSC line, KIOMi002-A, from Parkinson's disease patient iPSC with LRRK2 G2019S mutation using BAC-based homologous recombination.

Mutations in leucine-rich repeat kinase 2 (LRRK2) gene (LRRK2 G2019S) is a representative autosomal dominant mutation that can cause Parkinson's disease (PD). A bacterial artificial chromosome-based homologous recombination (BAC-based HR) system was utilized for gene therapy of LRRK2 G2019S-mutant induced pluripotent stem cells (iPSCs) produced by reprogramming episomal vectors. The gene-corrected iPSCs retained typical pluripotency required for their spontaneous differentiation into differentiated cells. The iPSCs had a normal karyotype and were confirmed to have no off-target sites by melting curve analysis.

Comprehensive analysis of transcriptomic changes induced by low and high doses of bisphenol A in HepG2 spheroids in vitro and rat liver in vivo.

Bisphenol A (BPA), a synthetic monomer commonly included in the daily products, has a structure similar to the estrogen receptor agonist. Therefore BPA has been anticipated to interfere with the hormone metabolisms and cause diverse pathological conditions. But the effects of BPA on the genetic landscapes of liver or hepatic cells have not been fully established. Gene expressional changes induced by low- or high-dose of BPA were evaluated in 3D cultured human hepatoma cells (HepG2 spheroids) in vitro at 0, 0.5, 5 and 200 µM and liver of rats exposed to BPA at 0, 0.5 and 250 mg/kg for 90 days in vivo. Functional enrichment analysis, pathway activity measurement and network analysis were performed using BPA-responsive genes. Treatment with BPA changed a lot of gene expressions in both HepG2 spheroids and rat livers depending on doses of BPA. Functional enrichment and pathway analysis show that lipid or steroid metabolism-related functions were altered by BPA in both HepG2 spheroids and livers of rats. Lipid metabolism-related functions altered by BPA formed a large cluster encompassing lipid biosynthesis, steroid metabolic process and cholesterol regulation process. It was also observed that distribution of pathway activities was correlated between HepG2 spheroids and rat livers at low-dose of BPA. Distance distribution in protein-protein interaction network also evidenced the closeness of BPA-responsive genes to metabolism pathways which include lipid metabolism. Collectively, we demonstrated that BPA greatly influenced overall gene expression and biological functions in both human hepatoma spheroids and rat liver, in which lipid- or steroid metabolism-associated genes were significantly altered by the exposure to BPA.

K120R mutation inactivates p53 by creating an aberrant splice site leading to nonsense-mediated mRNA decay.

The point mutation that substitutes lysine with arginine at position 120 of human p53 has been characterized as a missense mutation. The K120R mutation renders the p53 protein disabled for acetylation and, as a result, defective for apoptotic function, which provides a mechanistic link between the missense mutation and tumorigenesis. However, we noticed the failures of tumorigenesis in mice with the mutation, and of the related studies to notice that it has arbitrarily reflected in amino acid change through a sequence modification (AGA) of the original tumor mutation (AGG) by codon degeneracy. Unlike this modified version, we also discovered a novel splicing site the original mutation, TP53 c.359A>G, may induce. Using a human induced pluripotent stem cell line that was engineered to be homozygous for the original mutation, we here identified that the accidental splicing site generates a defective transcript variant with a frame-shifted premature termination codon which is subjected to nonsense-mediated mRNA decay. The authentic splicing still occurs but in extremely low amounts. Taken together, this mutation causes depletion of cellular p53 via defective mRNA, suggesting a new link to tumorigenesis.

Generation of gene-corrected iPSC line from Parkinson's disease patient iPSC line with alpha-SNCA A53T mutation.

Parkinson's disease (PD) is the second most common age-related neurodegenerative disorder. PD can result from a mutation of alpha-synuclein (α-SNCA), such as α-SNCA A53T. Using episomal vectors, induced pluripotent stem cells (iPSCs) were generated from skin fibroblasts with the α-SNCA A53T mutation. A huge bacterial artificial chromosome (BAC) harboring the normal α-SNCA gene successfully corrected the α-SNCA A53T-mutant iPSCs. Melting curve analysis for allelic composition indicated that the BAC DNA was precisely targeted to the α-SNCA A53T mutation allele, without random integration. The corrected PD-iPSCs displayed the normal karyotype and pluripotency, with the capability to differentiate to any cell type.

High-efficiency generation of induced pluripotent stem cells from human foreskin fibroblast cells using the Sagunja-tang herbal formula.

BACKGROUND: Sagunja-Tang (SGT-4) is a traditional herbal formula in Korean medicine that is used to treat anti-metabolic syndrome, and has antioxidant activity. In this study, we evaluated the effects of SGT-4 on the formation efficiency of induced pluripotent stem cells (iPSCs) from human foreskin fibroblasts (HFFs) by four reprogramming transcription factors: Oct4, Sox2, KIf4, and c-Myc (OSKM). METHODS: SGT-4 contained four different herbal medicines that are composed of Ginseng Radix, Glycyrrhizae Radix et Rhizoma, Atractylodis Rhizoma Alba, and Poria Sclerotium. The composition of SGT-4 was analyzed by high-performance liquid chromatography (HPLC). HFFs were transfected with episomal vectors contained by four OSKM. Western blotting, RT-PCR, immunofluroescence, and in vitro differentiation were used to assess the pluripotency of the iPSC cells. RESULTS: SGT-4 exhibited antioxidant activity against the generation of intracellular reactive oxygen species (ROS) as well as promoted the activation of superoxide dismutase 1 (SOD1), catalase, gluthathione peroxidase 1 (GPX1), and glutathione (GSH). Moreover, the ATP level was not significantly fluctuated depending on the concentration of SGT-4 in the hiPSCs. CONCLUSION: Our results indicate that the SGT-4, herbal formula significantly increases the efficiency of human iPSC generation via the transcription factors (Oct4, Sox2, KIf4, and c-Myc).

The five elements of the cell.

Everything in the surrounding universe can be attributed into five elements. Human organs can be also linked to the five elements. Cells, the smallest unit of the human body, consist of cellular organelles as little organs. Here, we extended the concept of the five elements to a cellular level via the human organs, theoretically re-evaluating the overall association of cellular organelles in maintaining the homeostasis of cellular functions.

Normalization of human RNA-seq experiments using chimpanzee RNA as a spike-in standard.

Normalization of human RNA-seq experiments employing chimpanzee RNA as a spike-in standard is reported. Human and chimpanzee RNAs exhibit single nucleotide variations (SNVs) in average 210-bp intervals. Spike-in chimpanzee RNA would behave the same as the human counterparts during the whole NGS procedures owing to the high sequence similarity. After discrimination of species origins of the NGS reads based on SNVs, the chimpanzee reads were used to read-by-read normalize biases and variations of human reads. By this approach, as many as 10,119 transcripts were simultaneously normalized for the entire NGS procedures leading to accurate and reproducible quantification of differential gene expression. In addition, incomparable data sets from different in-process degradations or from different library preparation methods were made well comparable by the normalization. Based on these results, we expect that the normalization approaches using near neighbor genomes as internal standards could be employed as a standard protocol, which will improve both accuracy and comparability of NGS results across different sample batches, laboratories and NGS platforms.

Concurrent progress of reprogramming and gene correction to overcome therapeutic limitation of mutant ALK2-iPSC.

Fibrodysplasia ossificans progressiva (FOP) syndrome is caused by mutation of the gene ACVR1, encoding a constitutive active bone morphogenetic protein type I receptor (also called ALK2) to induce heterotopic ossification in the patient. To genetically correct it, we attempted to generate the mutant ALK2-iPSCs (mALK2-iPSCs) from FOP-human dermal fibroblasts. However, the mALK2 leads to inhibitory pluripotency maintenance, or impaired clonogenic potential after single-cell dissociation as an inevitable step, which applies gene-correction tools to induced pluripotent stem cells (iPSCs). Thus, current iPSC-based gene therapy approach reveals a limitation that is not readily applicable to iPSCs with ALK2 mutation. Here we developed a simplified one-step procedure by simultaneously introducing reprogramming and gene-editing components into human fibroblasts derived from patient with FOP syndrome, and genetically treated it. The mixtures of reprogramming and gene-editing components are composed of reprogramming episomal vectors, CRISPR/Cas9-expressing vectors and single-stranded oligodeoxynucleotide harboring normal base to correct ALK2 c.617G>A. The one-step-mediated ALK2 gene-corrected iPSCs restored global gene expression pattern, as well as mineralization to the extent of normal iPSCs. This procedure not only helps save time, labor and costs but also opens up a new paradigm that is beyond the current application of gene-editing methodologies, which is hampered by inhibitory pluripotency-maintenance requirements, or vulnerability of single-cell-dissociated iPSCs.