Non-integrating Sendai Virus Research Articles

Generation of iPSC line FAMRCi010-A from patient with restrictive cardiomyopathy carrying genetic variant FLNC p.Gly2011Arg

Human iPSC cell line FAMRCi010-A was generated from a patient with restrictive cardiomyopathy carrying FLNC p.Gly2011Arg genetic variant. Patient-specific peripheral blood mononuclear cells were reprogrammed using non-integrative Sendai viruses containing OCT4, SOX2, KLF4, and CMYC. FAMRCi010-A was generated and characterized through the study. The reported iPSC line could be useful tool for in vitro modeling of FLNC-associated cardiomyopathies.

A heterozygous TTN (c. 79,684C>T) mutant human induced pluripotent stem cell line (ZZUNEUi023-A) generated from a Kazakh patient with dilated cardiomyopathy

Dilated cardiomyopathy (DCM) is a nonischaemic heart muscle disease with structural and functional myocardial abnormalities. TTN truncating mutations are a common cause of DCM, occurring in ∼25% of familial cases of DCM and in 18% of sporadic cases. In this study, we generated a human induced pluripotent stem cell line ZZUNEUi023-A from peripheral blood mononuclear cells of a Kazakh DCM patient with the p. Arg26562Ter (c. 79684C>T) mutation in TTN using non-integrative Sendai virus. This cell line expressed pluripotency markers, showed normal male karyotype and could differentiate into all three germ layers in vitro.

Generation and characterization of three induced pluripotent stem cell lines (NUIGi046-A, NUIGi046-B, NUIGi046-C) from a 51-year-old healthy individual

Skin punch biopsy was donated by a healthy 51-year-old Caucasian male and the dermal fibroblasts were reprogrammed into human induced pluripotent stem cell (hiPSC) lines by using non-integrative Sendai viruses expressing OCT4, SOX2, KLF4 and c-MYC. Three iPSC lines (NUIGi046-A, NUIGi046-B, NUIGi046-C) highly expressed the pluripotent markers and were capable of differentiating into cells of endodermal, mesodermal, and ectodermal origin. These iPSCs can be offered as controls and in combination with genome-editing and three-dimensional (3D) system. They may be used for human disease modelling and drug screening.

Abstract 13778: Disease Modelling and Gene Therapy of Hereditary Hemorrhagic Telangiectasia Combined With Pulmonary Artery Hypertension

Introduction: Hereditary hemorrhagic telangiectasia combined with pulmonary arterial hypertension(HHT-PAH) caused by pathogenic variants of ACVRL1 gene progresses with poor prognosis. However, there is few treatments, thus developing novel models and innovative therapy is crucial. Combination of iPSCs with gene editing technologies may be a promising applications to illuminate the disease. Hypothesis: Gene editing is a promising therapy for HHT-PAH with ACVRL1 gene mutations. Methods: Patients with HHT-PAH who carrying ACVRL1 heterozygous c.1270C>G mutation were recruited. The reprogramming factors (Oct3/4, Sox2, c-Myc, and Klf4) were transferred into the PBMCs by using non-integrating Sendai virus. The obtained hiPSCs were passaged, individually isolated and expanded 3 weeks after reprogramming factors transduction. The mutated ACVRL1 gene was repaired by CRISPR Base Editing tools to generate ACVRL1 corrected -iPSCs line. Both the patient-specific iPSCs and ACVRL1 corrected -iPSCs were differentiated into induced endothelium. EC adhesion and survival were tested in the endothelial lines. Results: The patient-specific iPSCs showed typical morphology with high alkaline phosphatase activity and normal karyotype (46, XX). Pluripotency markers OCT4, NANOG, SOX2, and TRA-1-60 were confirmed by Immunofluorescence staining and qRT-PCR. Markers of three germ layers, TUBB3+ , αSMA+ and SOX17+ cells were found in the differentiated cell population as revealed by immunostaining in vitro . These two iPSCs cell lines formed teratomas composed of tissues derived from three germ layers in immunodeficient mice. Fluorescence-activated cell sorting analysis showed that most of patient-specific iPSCs and ACVRL1 corrected -iPSCs population expressed the mature EC surface marker CD144 or CD31, and none of cells expressed the early mesoderm marker KDR during the differentiation process. Cell adhesion and survival capacity were preserved in ACVRL1 corrected iPSC-ECs, which were similar to the control, whereas these functions were significantly impaired in patient-specific iPSC-ECs. Conclusions: This study presented an in vitro cell model for exploring the pathogenesis of HHT-PAH and gene editing therapy might be a novel strategy.

Identification of a familial cleidocranial dysplasia with a novel RUNX2 mutation and establishment of patient-derived induced pluripotent stem cells

Cleidocranial dysplasia (CCD) is an autosomal dominant hereditary disease associated with the gene RUNX2. Disease-specific induced pluripotent stem cells (iPSCs) have emerged as a useful resource to further study human hereditary diseases such as CCD. In this study, we identified a novel CCD-specific RUNX2 mutation and established iPSCs with this mutation. Biopsies were obtained from familial CCD patients and mutation analyses were performed through Sanger sequencing and next generation sequencing. CCD-specific human iPSCs (CCD-hiPSCs) were established and maintained under completely defined serum, feeder, and integration-free condition using a non-integrating replication-defective Sendai virus vector. We identified the novel mutation RUNX2_c.371C>G and successfully established CCD-hiPSCs. The CCD-hiPSCs inherited the same mutation, possessed pluripotency, and showed the ability to differentiate the three germ layers. We concluded that RUNX2_c.371C>G was likely pathogenic because our results, derived from next generation sequencing, are supported by actual clinical evidence, familial tracing, and genetic data. Thus, we concluded that hiPSCs with a novel CCD-specific RUNX2 mutation are viable as a resource for future studies on CCD.

Generation and genetic repair of two human induced pluripotent cell lines from patients with Epidermolysis Bullosa simplex and dilated cardiomyopathy associated with a heterozygous mutation in the translation initiation codon of KLHL24

Fibroblasts from two patients carrying a heterozygous mutation in the translation initiation codon (c.2 T > G) of the kelch-like protein 24 (KLHL24) gene were used to generate human induced pluripotent stem cells (hiPSCs), using non-integrating Sendai virus to deliver reprogramming factors. CRISPR-Cas9 editing was used for genetic correction of the mutation in the patient-hiPSCs. The top-predicted off-target sites were not altered. Patient and isogenic hiPSCs showed typical morphology, expressed pluripotency-associated markers, had the capacity for in vitro differentiation into the three germ layers and displayed a normal karyotype. These isogenic pairs will enable in vitro modelling of KLHL24-associated heart and skin conditions.

Reprogramming of a human induced pluripotent stem cell line from a long QT syndrome patient harboring a heterozygous mutation of c.1537C > T in SCN5A gene

Long QT syndrome (LQTS) often causes malignant arrhythmia and syncope. Mutations in the SCN5A gene are the main cause of type 3 LQTS. In this study, we generated a human induced pluripotent stem cell line ZJYYPHi001-A from a female LQTS patient harboring a heterozygous mutation in SCN5A using non-integrative Sendai virus. This cell line expressed pluripotency markers, had the normal karyotype and could differentiate into three germ layers in vitro.

Generation of induced pluripotent stem cell lines from peripheral blood mononuclear cells of three drug resistant and three drug responsive epilepsy patients

Epilepsy is a common neurological disorder characterized by seizures. Unfortunately, 30–40% of all epilepsy patients are resistant to at least two or more anti-seizure medications. Attempts to treat these patients and prevent further seizures necessitates multiple drug trials for the patient. Here we describe the generation and validation of induced pluripotent stem cell (iPSC) lines from peripheral blood mononuclear cells (PBMCs) from 3 drug responsive and 3 drug resistant patients, using a non-integrative Sendai virus vector. These lines can be used to generate 2D and 3D patient-specific human cellular models that will enable personalised drug screening and pharmacogenomic studies.

Establishment of TUSMi009-A, an induced pluripotent stem cell (iPSC) line from a 24-year-old Chinese Han patient with gliocytoma

Glioblastoma is the most common malignant primary brain tumor with poor prognosis and low long-term survival rate. Here we described the production of an iPSC line generated from peripheral blood mononuclear cells (PBMCs) of a 24-year-old female gliocytoma patient. The PBMCs were reprogrammed by the non-integrating Sendai Virus with human OKSM (OCT3/4, SOX2, KLF4, and c-MYC) transcription factors. The generated iPSCs were positive for pluripotent stem cell markers demonstrated by immunocytochemistry. Besides, they displayed a normal karyotype and had the potential to differentiate spontaneously three germ layers in vitro. Our model might be useful in studying the pathogenesis of gliocytoma patients.

Human induced pluripotent stem cell line (QBRIi013-A) derivation from a 6-year-old female diagnosed with Autism spectrum disorder (ASD) and intellectual disability (ID)

Autism spectrum disorder (ASD) is a childhood-onset neurodevelopmental disorder characterized by social interaction, behavior, and communication challenges. Here, we generated an induced pluripotent stem cell (iPSC) line, QBRIi013-A using a non-integrating Sendai virus from a 6-year-old female diagnosed with ASD and intellectual disability. The QBRIi013-A cell line was fully characterized and exhibited a pluripotency capacity and trilineage differentiation potential. Furthermore, it showed normal karyotype and genetic identity to the patient’s PBMCs. Consequently, this iPSC line provides a valuable cell model in understanding the molecular mechanism underlying the complexities of ASD pathogenesis.

Generation of a SIAISi004-A hiPSC line from PBMCs of a 74 year-old Alzheimer’s disease woman by non-integrating sendai virus mediated reprogramming

A 74-year-old Alzheimer’s Disease (AD) woman patient donated her peripheral blood mononuclear cells (PBMC). Non-integrating episomal vector system were used to reprogram their PBMCs with human OKSM (OCT3/4, KLF4, SOX2, and c-MYC) transcription factors. Immunocytochemistry and flow cytometry for pluripotency makers confirmed the pluripotency of transgene-free iPSCs. Pluripotency was confirmed by the ability of iPSCs to spontaneously differentiate three germ layers in vitro as well. The newly generated human iPSC from PBMCs of a 74 year-old Alzheimer’s disease woman can be used for studying further disease mechanisms of Alzheimer’s Disease (AD).

Generation of two human induced pluripotent stem cell lines from patients with biallelic USH2A variants

Usher syndrome 2A (USH2A) is one of the most common genes associated with Usher syndrome type II (USH2) and nonsyndromic autosomal recessive retinitis pigmentosa (arRP). Here, we describe the generation and characterization of two human induced pluripotent stem cell (hiPSC) lines from a RP patient with compound heterogeneous USH2A variants and a USH2 patient with homozygous USH2A variant. Blood samples were obtained and peripheral blood mononuclear cells (PBMCs) were reprogrammed using the non-integrative Sendai virus to generate the iPSC lines. The established hiPSC lines retained the disease-associated variants and showed normal karyotype, pluripotency and differentiation capacity.

Generation of an induced pluripotent stem cell line from a patient carrying FBN1/c.6734 G > A mutation

Mutations in FBN1 (gene encodes the matrix protein fibrillin 1), are usually associated with Marfan syndrome (MS). This syndrome contains ocular, skeletal, cardiovascular, pulmonary, cutaneous and neurological abnormalities. Here, we introduced an induced pluripotent stem cell (iPSC) line from a patient who suffered from severe cardiovascular disease and carried a c.6734G > A mutation in FBN1. Dermal fibroblasts of the patient were reprogrammed with non-integrating Sendai virus (SeV). Generated iPSC line exhibited normal karyotype, showed embryonic stem cell-like morphology, expressed pluripotency markers, and was capable of differentiating into three germ layers. This iPSC line will be a valuable tool for studying MS.

Generation of an iPSC cell line (USFi003-A) from a patient with dilated cardiomyopathy carrying a heterozygous mutation in LMNA (p.R541C)

Mutations in the gene that encodes the nuclear envelope proteins lamin A/C (LMNA) are considered to be a prominent cause of Dilated cardiomyopathy (DCM), a leading cause of heart failure and a prevalent indication for heart transplant. Here we described the generation of induced pluripotent stem cells (iPSCs) from a 53-year-old female with DCM plus progressive conduction disease who carry a heterozygous mutation in LMNA (c.1621C > T, p.R541C). PBMCs isolated from the patient were reprogrammed with Yamanaka factors KOS, KLF4, and c-MYC by the non-integrating sendai virus vector system. The obtained iPSC lines demonstrated normal karyotype and pluripotent identity.

Generation and characterization of induced pluripotent stem cells heterozygous for the Portuguese BRCA2 founder mutation

Women who inherit heterozygous mutations in the BRCA2 gene have an increased risk of developing cancer, mainly breast and ovarian tumors. A particular BRCA2 mutation (c.156_157insAlu) is exclusively found in families of Portuguese ancestry and is present in approximately 30% of all Portuguese families with hereditary breast and ovarian cancers. We report the generation and characterization of the first iPSC line from a female donor harboring the Portuguese BRCA2 founder mutation. Skin fibroblasts were reprogrammed using a non-integrative Sendai virus. These iPSCs are a valuable tool to study the origin of BRCA2-associated cancer in its earliest phases.

Generation and characterization of six human induced pluripotent stem cell lines (iPSC) from three families with AP4M1-associated hereditary spastic paraplegia (SPG50)

Biallelic loss-of-function variants in the subunits of the adaptor protein complex 4 lead to childhood-onset hereditary spastic paraplegia (AP-4-HSP): SPG47 (AP4B1), SPG50 (AP4M1), SPG51 (AP4E1), and SPG52 (AP4S1). Here, we describe the generation of induced pluripotent stem cells (iPSCs) from three AP-4-HSP patients with biallelic, loss-of-function variants in AP4M1 and their sex-matched parents (asymptomatic, heterozygous carriers). Following reprogramming using non-integrating Sendai virus, iPSCs were characterized following standard protocols including karyotyping, embryoid body formation, pluripotency marker expression and STR profiling. These first iPSC lines for SPG50 provide a valuable resource for studying this rare disease and related forms of hereditary spastic paraplegia.

Generation of an iPSC line (CRICKi001-A) from an individual with a germline SMARCA4 missense mutation and autism spectrum disorder

Germline missense mutations in the BAF swi/snf chromatin remodeling subunit SMARCA4 are associated with neurodevelopmental disorders, including Coffin Siris Syndrome (CSS). Here, we generated an induced pluripotent stem cell line from a male patient with atypical CSS features and a de novo heterozygous missense mutation in the SMARCA4 gene (c.3607C>T, p.(Arg1203Cys)). Hair root derived keratinocytes were reprogrammed using non-integrative Sendai virus vector delivery of pluripotency factors. iPSCs generated display normal morphology and molecular karyotype, express pluripotency markers and are able to differentiate into the three germ layers.

Generation of integration-free induced pluripotent stem cell lines from four pediatric ADHD patients

Human induced pluripotent stem cell (iPSC) lines have been derived from four male patients with childhood attention-deficit hyperactivity disorder (ADHD). Children and adolescents between the ages 6 and 18 suffering from ADHD were recruited for this work. Isolated keratinocytes or peripheral blood mononuclear cells from the participants were reprogrammed into iPSCs using non-integrating Sendai virus to deliver the reprogramming factors Oct3/4, Sox2, Klf4 and c-Myc.

Generation of integration-free induced pluripotent stem cells from healthy individuals

Ten human induced pluripotent stem cell (iPSC) lines have been derived from five healthy controls matched to a study including Attention-Deficit Hyperactivity Disorder patients (ADHD). Both female and male children and adolescents aged 6–18 years were recruited. Isolated keratinocyte cells from the participants were reprogrammed into iPSCs using non-integrating Sendai virus to deliver the reprogramming factors Oct3/4, Sox2, Klf4 and cMyc.

Generation of an induced pluripotent stem cell line (ZZUi020-A) from a patient with Parkinson's disease harboring the intermediate-length GGC repeat expansions in the NOTCH2NLC gene.

Here, we describe the generation of an induced pluripotent stem cell (iPSC) line, from a female patient diagnosed with Parkinson's disease (PD). The patient carries a heterozygous intermediate-length GGC repeat expansions mutation in the NOTCH2NLC gene. Skin fibroblasts were reprogrammed using the non-integrating Sendai virus technology to deliver Klf4, OCT3/4, SOX2 and c-MYC factors. The generated iPSC line (ZZUi020-A) presented with expression of common pluripotency markers, showed potential of differentiating into derivatives of the three germ layers, and displayed a normal karyotype. The clone ZZUi020-A is presented thereafter, it can be used to study the mechanisms underlying NOTCH2NLC-PD pathogenesis.