Integration-free Sendai Virus Research Articles

Generation of a healthy heavy smoker patient-derived induced pluripotent stem cell line UHOMi007-A from peripheral blood mononuclear cells

Human pluripotent stem cells (hiPSC) represent a unique opportunity to model lung development and chronic bronchial diseases. We generated a hiPSC line from a highly characterized healthy heavy smoker male donor free from emphysema or tobacco related disease. Peripheral blood mononuclear cells (PBMCs) were reprogrammed using integration-free Sendai virus. The cell line had normal karyotype, expressed pluripotency hallmarks, and differentiated into the three primary germ layers.The reported UHOMi007-A iPSC line may be used as a control to model lung development, study human chronic bronchial diseases and drug testing.

Generation of human-induced pluripotent stem cell line from PBMC of healthy donor using integration-free Sendai virus technology

We developed a well-characterized human induced pluripotent stem cell (iPSC) line obtained from healthy individuals' peripheral blood mononuclear cells (PBMC). The PBMCs were primed and reprogrammed using a non-integrating sendai viral vector, and the iPSC lines demonstrated complete differentiation capacity. This line, YBLi004-A, is available and registered in the human pluripotent stem cell registry. The line's legitimacy was validated using pluripotent marker expression, in vitro differentiation into three germ layers (ectoderm, mesoderm, and endoderm), karyotyping, and STR analysis. This iPSC line could be used as a healthy control for studies involving disease-specific-iPSCs, e.g. drug toxicity and efficacy testing.

Generation of Human Induced Pluripotent Stem Cell line from PBMCs of Healthy Donors using Integration-free Sendai virus Technology

Generation of Human Induced Pluripotent Stem Cell line from PBMCs of Healthy Donors using Integration-free Sendai virus Technology

Generation of iPSC line (GLNNFi001-A) from peripheral blood mononuclear cells of a patient with macular corneal dystrophy

Macular corneal dystrophy (MCD) an autosomal recessive disorder leading to severe visual impairment is caused by mutations in the carbohydrate sulfotransferase 6 (CHST6) gene. Here, we describe generation of a iPSC line from peripheral blood mononuclear cells (PBMCs) from a 38 year old MCD patient harboring a homozygous mutation, p.V172L in the CHST6 gene. The iPSC line GLNNFi001-A was generated using an integration-free Sendai virus containing (KLF4, c-MYC, OCT4 and SOX2).

Generation and characterization of IMAGINi013-A, an induced pluripotent stem cell line generated from a healthy donor

Human pluripotent stem cells are a powerful tool to study development, to model diseases or as cellular substrates for drug screening. We generated a human induced pluripotent stem cell (hiPSC) line from a healthy control donor. Peripheral blood mononuclear cells (PBMCs) from this donor were reprogrammed using integration-free Sendai virus. This cell line had normal karyotype, expressed pluripotency hallmarks and differentiated into the three primary germ layers.

Generation of four severe early-onset chronic obstructive pulmonary disease (COPD) patient-derived induced pluripotent stem cell lines from peripheral blood mononuclear cells

Evidence highlights the concept of multiple trajectories leading to COPD. Early-life events (i.e., in utero lung development) may influence the maximally attained lung function and increase the risk to develop COPD. Human pluripotent stem cells (hiPSC) represent a unique opportunity to model lung development. We generated hiPSC lines from four highly characterized COPD patients with early onset and severe phenotype. Peripheral blood mononuclear cells (PBMCs) were reprogrammed using integration-free Sendai Virus. The cell lines had normal karyotype, expressed pluripotency hallmarks, and differentiated into the three primary germ layers.These lines offer a tool to study early-life origins of COPD.

Derivation of a human induced pluripotent stem cell (hiPSC) line (PUMCSMi001-A) from peripheral blood mononuclear cells of one healthy female donor

Human induced pluripotent stem cells (hiPSCs) are valuable models for studying the molecular pathogenesis of inherited diseases and key tools for cell therapy. We derived peripheral blood mononuclear cells (PBMCs) isolated from a 30-year-old healthy female donor using integration-free Sendai virus to generate a hiPSC line, PUMCSMi001-A, which can serve as a control for the studies of pathogenesis of cardiovascular disease and drug screening. Quality assessment confirmed that the cell line PUMCSMi001-A expressed pluripotency genes, had normal karyotype and differentiation potential into three layers in vivo.

Generation of the induced pluripotent stem cell line UHOMi002-A from peripheral blood mononuclear cells of a healthy male donor.

Human pluripotent stem cells (hiPSC) are highly valuable tools to model lung development and chronic bronchial diseases. We generated a hiPSC line from a highly characterized 40-year-old healthy male nonsmoking donor. Peripheral blood mononuclear cells (PBMCs) were reprogrammed using integration-free Sendai Virus. The cell line had normal karyotype, expressed pluripotency hallmarks, and differentiated into the three primary germ layers. The reported UHOMi002-A iPSC line may be used as a control to model lung development, study human chronic bronchial diseases and drug testing.

Generation of two human iPSC lines (MDCUi001-A and MDCUi001-B) from dermal fibroblasts of a Thai patient with X-linked osteogenesis imperfecta using integration-free Sendai virus

Two clones of human induced pluripotent stem cells (iPSCs) were generated from dermal fibroblasts isolated from a one-year-old Thai patient with X-linked osteogenesis imperfecta. The patient harbored a mutation, p.N459S, in the MBTPS2 gene. The cells were reprogrammed using an integration-free Sendai virus containing KLF4, c-MYC, OCT4 and SOX2. Both of the established iPSC lines (MDCUi001-A and MDCUi001-B) maintained normal karyotype, expressed pluripotent markers and differentiated into all three germ layers.

Establishment of CSUASOi001-A, a non-integrated induced pluripotent stem cell line from urine-derived cells of a Chinese patient carrying RS1 gene mutation

X-linked juvenile retinoschisis (XLRS) is one of the most severely affected genetic causes of irreversible retinal degeneration diseases in young males, especially school-age boys. Here, we generated induced pluripotent stem cells (iPSCs) from a Chinese 11-year-old male with clinically diagnosed XLRS. Urine sample was collected with appropriate cooperation, then isolated cells were expanded for subsequent reprogramming procedure using integration-free Sendai virus. The newly derived CSUASOi001-A iPS cell line harboring the c.304C > T mutation in the RS1 gene (p.R102W) provides a useful resource to investigate pathogenic mechanisms in XLRS.

Establishment of an induced pluripotent stem cell line (FDEENTi002-A) from a patient with Best's disease carrying c.888C > A mutation in BEST1 gene

Best's disease (BD) is an inherited retinal degenerative disease caused by mutations in BEST1 gene. A human induced pluripotent stem cell (iPSC) line has been generated with integration-free Sendai virus method from peripheral blood mononuclear cells (PBMCs) of a BD patient carrying c.888C > A mutation in BEST1 gene. This cell line may serve as a model for the study of pathogenesis of BD.

Generation of induced pluripotent stem cells (iPSCs) IRMBi002-A from an Alzheimer's disease patient carrying a D694N mutation in the APP gene

Induced pluripotent stem cells (iPSC) were generated from skin fibroblasts obtained from a 58 year-old woman suffering from Alzheimer's disease and carrying a D694N mutation on Amyloid precursor protein (APP). Fibroblasts were reprogrammed into iPSC using the integration-free Sendai Virus which allows the expression of the Yamanaka factors. Verification of their pluripotency was achieved by demonstrating the expression of pluripotency markers and their differentiation potential into the three primary germ layers. The cells have the corresponding mutation and present a normal karyotype. The reported APP-D694N iPSC line may be used to model and study human AD pathology in vitro.

Generation of induced pluripotent stem cell line-NTUHi001-A from a premature ovarian failure patient with Turner's syndrome mosaicism

Turner's syndrome (TS) is one of the main causes of premature ovarian failure (POF). However, the mechanisms underlying POF are difficult to study due to the lack of suitable disease models. Herein, we have generated a human induced pluripotent stem cell (hiPSC) line derived from the peripheral blood mononuclear cells of a female patient with Turner's syndrome mosaicism via integration-free Sendai-virus system. The hiPSCs were confirmed with a 45, X karyotype and the acquisition of pluripotency. It's likely that hiPSCs can serve as a feasible cellular model for further pathophysiological studies of POF cases, especially for those originating in TS.

Generation of induced pluripotent stem cells (IRMBi001-A) from an Alzheimer's disease patient carrying a G217D mutation in the PSEN1 gene

Induced pluripotent stem cells (iPSC) were generated from skin fibroblasts obtained from a 50 year-old patient suffering from Alzheimer's disease and carrying a G217D causal mutation on presenilin 1 (PSEN1). iPSCs were obtained following reprogramming using the integration-free Sendai Virus system which allows expression of the Yamanaka factors. Verification of their pluripotency was achieved by demonstrating the expression of pluripotency markers and their differentiation potential into the three primary germ layers. iPS cells carry the patient G217D mutation and present a normal karyotype. The reported PS1-G217D iPSC line may be used to model and study human AD pathology in vitro.

Efficient Gene Transduction and Reprogramming of Hematopoietic Cells Including T-Cells By Using a Non-Integrating Measles Virus Vector

By the ectopic expression of reprogramming genes OCT, KLF4, SOX2 and MYC (OKSM), somatic cells can be reprogrammed to induced pluripotent stem cells (iPSCs). Human iPSCs are considered a promising cell source to provide an import tool for the basic investigation and the advanced medicine including gene therapy and regenerative medicine. To establish iPSCs, integration-free Sendai virus (SV) vectors have been most widely used do far, but transduction and reprogramming of T cells without stimulation is still very challenging. On the other hand, a great success of chimeric antigen receptor T cell (CAR-T) therapies highlighted the importance of anti-cancer immunity for the cancer treatment. Particularly, many refractory patients with acute lymphoblastic leukemia and B-cell lymphoma were successfully treated with CD19-CAR-T therapies, however, some patients died before receiving the treatment due to long preparation time of CAR-Ts. Therefore, rapid production systems of CAR-Ts are desired, and for this purpose, efficient and safe gene transduction systems to T cells should be developed.In this study, we developed a new non-integrating measles virus (MV) vector-based delivery system with F deletion to eliminate cell membrane fusion-associated cytotoxicity. MV vectors transduced genes through MV receptors including CD46 and signaling lymphocyte activation molecule (CD150/SLAM). First, we examined transduction efficiencies of MV vectors and SV vectors in hematopoietic cells by using GFP expression vectors (MV-Gs and SV-Gs). Compared to SV-Gs, our MV-Gs allowed more efficient gene transfer into most hematopoietic cell type including T (3-fold) and B cells (7-fold) (Fig. 1). Furthermore, at the same multiplicity of infection (MOI) of viral transduction, MV-Gs induced less apoptosis in T cell subset compared to SV-Gs (Fig. 2) due to the slower kinetics of viral RNA amplification in the transduced cells 24 h ,48 h and 72 h post transduction.Those results encouraged us to examined if MV vectors are more potent than SV vectors in iPSC generation from unstimulated T cells. To address this question, we developed MV vectors harboring four reprogramming genes (MV-OKSMGs) and compared with SV vectors harboring these genes (SV-OKSMGs). As expected, with the MV-OKSMGs, we could generate high-quality iPSCs with the similar morphology, pluripotency markers, karyotype and differentiation capacity as human embryonic stem cells. Upon the less cytotoxicity, iPSC generation efficiency of MV-OKSMGs was much higher than that of SV-OKSMGs for unstimulated T cells (0.47 ± 0.25% vs 0.008 ± 0.009%).Considering the safe history of MV vaccine, carrying capabilities of multiple genes, more flexible receptors and higher transduction efficiency for resting T cells, our exclusive MV vector would be a potential gene transfer system for iPSC generation and lymphocyte-based-immunotherapies such as CAR-T therapies. [Display omitted] DisclosuresLiao:neopharma Japan Co. Ltd: Research Funding; TAKARA BIO, INC.: Research Funding; Shinnihonseiyaku Co., Ltd: Research Funding. Soda:Shinnihonseiyaku Co., Ltd: Research Funding; neopharma Japan Co. Ltd: Research Funding; TAKARA BIO, INC.: Research Funding. Sugawara:neopharma Japan Co. Ltd: Research Funding; Shinnihonseiyaku Co., Ltd: Research Funding; TAKARA BIO, INC.: Research Funding. Miura:neopharma Japan Co. Ltd: Research Funding; Shinnihonseiyaku Co., Ltd: Research Funding; TAKARA BIO, INC.: Research Funding. Tahara:TAKARA BIO, INC.: Research Funding. Takishima:neopharma Japan Co. Ltd: Research Funding; TAKARA BIO, INC.: Research Funding; Shinnihonseiyaku Co., Ltd: Research Funding. Hirose:TAKARA BIO, INC.: Research Funding; Shinnihonseiyaku Co., Ltd: Research Funding; neopharma Japan Co. Ltd: Research Funding. Hijikata:Shinnihonseiyaku Co., Ltd: Research Funding; neopharma Japan Co. Ltd: Research Funding; TAKARA BIO, INC.: Research Funding. Miyamoto:Shinnihonseiyaku Co., Ltd: Research Funding; TAKARA BIO, INC.: Research Funding; neopharma Japan Co. Ltd: Research Funding. Takeda:TAKARA BIO, INC.: Research Funding. Tani:neopharma Japan Co. Ltd: Research Funding; Oncolys BioPharma Inc.: Equity Ownership; SymBio Pharmaceuticals Limited: Equity Ownership; TAKARA BIO, INC.: Research Funding; Shinnihonseiyaku Co., Ltd: Research Funding.

Differentiation of Sendai Virus-Reprogrammed iPSC into β Cells, Compared with Human Pancreatic Islets and Immortalized β Cell Line

Background: New sources of insulin-secreting cells are strongly in demand for treatment of diabetes. Induced pluripotent stem cells (iPSCs) have the potential to generate insulin-producing cells (iβ). However, the gene expression profile and secretory function of iβ still need to be validated in comparison with native β cells. Methods: Two clones of human iPSCs, reprogrammed from adult fibroblasts through integration-free Sendai virus, were differentiated into iβ and compared with donor pancreatic islets and EndoC-βH1, an immortalized human β cell line. Results: Both clones of iPSCs differentiated into insulin+ cells with high efficiency (up to 20%). iβ were negative for pluripotency markers (Oct4, Sox2, Ssea4) and positive for Pdx1, Nkx6.1, Chromogranin A, PC1/3, insulin, glucagon and somatostatin. iβ basally secreted C-peptide, glucagon and ghrelin and released insulin in response either to increasing concentration of glucose or a depolarizing stimulus. The comparison revealed that iβ are remarkably similar to donor derived islets in terms of gene and protein expression profile and similar level of heterogeneity. The ability of iβ to respond to glucose instead was more related to that of EndoC-βH1. Discussion: We demonstrated that insulin-producing cells generated from iPSCs recapitulate fundamental gene expression profiles and secretory function of native human β cells.

Abstract 256: Precision Medicine in Cardiac Channelopathy: Integrating Genome Editing and Induced Pluripotent Stem Cells to Decipher Variant of Unknown Significance

Background: The long QT syndrome (LQTS) is an arrhythmogenic disorder of QT interval prolongation that predisposes patients to life-threatening ventricular arrhythmias such as Torsade de pointes (TdP) and sudden cardiac death (SCD). Clinical genetic testing has emerged as the standard of care to identify genetic variants in patients suspected of having LQTS. However, these results are often confounded by the discovery of variants of unknown significance (VUS), for which there is insufficient evidence of pathogenicity. Objectives: To demonstrate that genome editing of patient-specific induced pluripotent stem cells (iPSCs) can be a valuable approach to delineate the pathogenicity of VUS in cardiac channelopathy. Methods and Results: Peripheral blood mononuclear cells (PBMCs) were isolated from a carrier with a novel missense variant (T983I) in the KCNH2 (LQT2) gene and an unrelated healthy control subject. iPSCs were generated using an integration-free Sendai virus and differentiated to iPSC-derived cardiomyocytes (iPSC-CMs). Whole-cell patch clamp recordings revealed significant prolongation of the action-potential duration (APD) and reduced rapidly activating delayed rectifier K + current ( I Kr ) density in VUS iPSC-CMs compared to healthy control iPSC-CMs. ICA-105574, a potent I Kr activator, enhanced I Kr magnitude and restored normal APD in VUS iPSC-CMs. Notably, VUS iPSC-CMs exhibited greater propensity to proarrhythmia than healthy control cells in response to high-risk torsadogenic drugs (dofetilide, ibutilide, and azimilide), suggesting a compromised repolarization reserve. Finally, the selective correction of the causal variant in iPSC-CMs using CRISPR/Cas9 gene editing (isogenic control) normalized the aberrant cellular phenotype, whereas the introduction of the homozygous variant in healthy control cells recapitulated hallmark features of the LQTS disorder. Conclusions: Our results suggest that the KCNH2 T983I VUS may be classified as potentially pathogenic.

Genome Editing of Induced Pluripotent Stem Cells to Decipher Cardiac Channelopathy Variant

BackgroundThe long QT syndrome (LQTS) is an arrhythmogenic disorder of QT interval prolongation that predisposes patients to life-threatening ventricular arrhythmias such as Torsades de pointes and sudden cardiac death. Clinical genetic testing has emerged as the standard of care to identify genetic variants in patients suspected of having LQTS. However, these results are often confounded by the discovery of variants of uncertain significance (VUS), for which there is insufficient evidence of pathogenicity. ObjectivesThe purpose of this study was to demonstrate that genome editing of patient-specific induced pluripotent stem cells (iPSCs) can be a valuable approach to delineate the pathogenicity of VUS in cardiac channelopathy. MethodsPeripheral blood mononuclear cells were isolated from a carrier with a novel missense variant (T983I) in the KCNH2 (LQT2) gene and an unrelated healthy control subject. iPSCs were generated using an integration-free Sendai virus and differentiated to iPSC-derived cardiomyocytes (CMs). ResultsWhole-cell patch clamp recordings revealed significant prolongation of the action potential duration (APD) and reduced rapidly activating delayed rectifier K+ current (IKr) density in VUS iPSC-CMs compared with healthy control iPSC-CMs. ICA-105574, a potent IKr activator, enhanced IKr magnitude and restored normal action potential duration in VUS iPSC-CMs. Notably, VUS iPSC-CMs exhibited greater propensity to proarrhythmia than healthy control cells in response to high-risk torsadogenic drugs (dofetilide, ibutilide, and azimilide), suggesting a compromised repolarization reserve. Finally, the selective correction of the causal variant in iPSC-CMs using CRISPR/Cas9 gene editing (isogenic control) normalized the aberrant cellular phenotype, whereas the introduction of the homozygous variant in healthy control cells recapitulated hallmark features of the LQTS disorder. ConclusionsThe results suggest that the KCNH2T983I VUS may be classified as potentially pathogenic.

Generation of induced pluripotent stem cells from arrhythmogenic right ventricular disease patient

To establish the induced pluripotent stem cells (iPSCs) from patients with arrhythmogenic right ventricular cardiomyopathy (ARVC). The fibroblasts were isolated from ARVC patient and DNA mutation sites were confirmed. We reprogrammed the patient fibroblasts to pluripotency by sendaiviral transduction with defined pluripotency factors. Then, we evaluated the pluripotency of ARVC-iPSCs by performing the immunofluorescence analysis, real-time PCR and 3 germ layer formation assay. We also induced the ARVC-iPSCs into cardiac specific differentiation by regulating Wnt signaling pathway. Apart from alkaline phosphatase positive staining, iPSCs derived from ARVC patients expressed pluripotent related genes and embryonic stem cell marker OCT4, SSEA4 and TRA-1-81. Embryonic body formation in vitro and teratoma test in vivo demonstrated that ARVC-iPSCs could differentiate into all three germ layers. After in vitro cardiac differentiation, ARVC-iPSC could be successfully induced to spontaneously beating mass with cTNT staining positive. Induced pluripotent stem cell was successfully established by integration free sendai virus from dermal fibroblast of patients with ARVC. It would provide the valuable experimental model to study the molecular mechanism of ARVC.

Patient-Specific and Genome-Edited Induced Pluripotent Stem Cell–Derived Cardiomyocytes Elucidate Single-Cell Phenotype of Brugada Syndrome

BackgroundBrugada syndrome (BrS), a disorder associated with characteristic electrocardiogram precordial ST-segment elevation, predisposes afflicted patients to ventricular fibrillation and sudden cardiac death. Despite marked achievements in outlining the organ level pathophysiology of the disorder, the understanding of human cellular phenotype has lagged due to a lack of adequate human cellular models of the disorder. ObjectivesThe objective of this study was to examine single cell mechanism of Brugada syndrome using induced pluripotent stem cell–derived cardiomyocytes (iPSC-CMs). MethodsThis study recruited 2 patients with type 1 BrS carrying 2 different sodium voltage-gated channel alpha subunit 5 variants as well as 2 healthy control subjects. We generated iPSCs from their skin fibroblasts by using integration-free Sendai virus. We used directed differentiation to create purified populations of iPSC-CMs. ResultsBrS iPSC-CMs showed reductions in inward sodium current density and reduced maximal upstroke velocity of action potential compared with healthy control iPSC-CMs. Furthermore, BrS iPSC-CMs demonstrated increased burden of triggered activity, abnormal calcium (Ca2+) transients, and beating interval variation. Correction of the causative variant by genome editing was performed, and resultant iPSC-CMs showed resolution of triggered activity and abnormal Ca2+ transients. Gene expression profiling of iPSC-CMs showed clustering of BrS compared with control subjects. Furthermore, BrS iPSC-CM gene expression correlated with gene expression from BrS human cardiac tissue gene expression. ConclusionsPatient-specific iPSC-CMs were able to recapitulate single-cell phenotype features of BrS, including blunted inward sodium current, increased triggered activity, and abnormal Ca2+ handling. This novel human cellular model creates future opportunities to further elucidate the cellular disease mechanism and identify novel therapeutic targets.