Patient-specific iPSC Lines Research Articles

PAM-flexible Engineered FnCas9 variants for robust and ultra-precise genome editing and diagnostics

The clinical success of CRISPR therapies hinges on the safety and efficacy of Cas proteins. The Cas9 from Francisella novicida (FnCas9) is highly precise, with a negligible affinity for mismatched substrates, but its low cellular targeting efficiency limits therapeutic use. Here, we rationally engineer the protein to develop enhanced FnCas9 (enFnCas9) variants and broaden their accessibility across human genomic sites by ~3.5-fold. The enFnCas9 proteins with single mismatch specificity expanded the target range of FnCas9-based CRISPR diagnostics to detect the pathogenic DNA signatures. They outperform Streptococcus pyogenes Cas9 (SpCas9) and its engineered derivatives in on-target editing efficiency, knock-in rates, and off-target specificity. enFnCas9 can be combined with extended gRNAs for robust base editing at sites which are inaccessible to PAM-constrained canonical base editors. Finally, we demonstrate an RPE65 mutation correction in a Leber congenital amaurosis 2 (LCA2) patient-specific iPSC line using enFnCas9 adenine base editor, highlighting its therapeutic utility.

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.

Abstract 3113: Modeling Varicose Vein Using Induced Pluripotent Stem Cells

Background: Varicose veins (VV) pathogenesis is multifactorial and has been attributed to both genetic and epigenetic factors. Vein wall dilation occurs concurrently or because of the venous hypertension and inflammation, further exacerbating the venous reflux. These effects are due to changes in the endothelium (ECs) and smooth muscle cells (SMCs), and their impaired cross-talk that play an important role in venous constriction. Due to limited access to ECs and SMCs from patients, the molecular mechanisms underlying vascular dysfunction in VV remain largely unknown.Induced pluripotent stem cells (iPSCs) provide a unique platform to recapitulate the disease phenotype in vitro thereby allowing unprecedented access to patient-specific cells. iPSCs derived from somatic cells possess the ability to differentiate into multiple cell types, including protein expressing ECs and SMCs. Furthermore, iPSC-derivatives are patient-specific, i.e., the encoded proteins are dictated by the genome of the original somatic donor cell. Goals: By creating iPSCs from VV patients, we can model and characterize to recapitulate the disease phenotype. With these efforts, our goal is to elucidate the underlying disease mechanisms and validate the candidate genes that are, in part responsible for VV. Methods: We have recruited 32 VV patients in our Stanford clinic. Peripheral blood mononuclear cells (PBMCs) are to be isolated. Patient-specific iPSC lines are generated from patients with VV and differentiated to ECs and SMCs. Results: Our lab has developed a robust, chemically defined monolayer differentiation protocol for iPSC-ECs that consistently yields about 80-95% CD31+ cells. Using this approach, I differentiated iPSCs from both the control and VV patients to ECs. The expression of specific cell type markers (PECAM1, NOS3, CDH5, VWF) were determined by RT-qPCR. Conclusion: Our patient-specific iPSC will become possible to model individual responses to treatment and medications and to find variants that lead to differences in outcomes. However, the clear demarcation between venous and arterial phenotypes was not assessed. Therefore, our future strategies will determine the functional and developmental differences between venous and arterial iPSC-ECs.

Generation of a pluripotent stem cell line (UMGi270-A) and a corresponding CRISPR/Cas9 modified isogenic control (UMGi270-A-1) from a patient with sudden onset dilated cardiomyopathy harboring a FLNC p.R2187P mutation

Filamin C (FLNC) is a highly important actin crosslinker and multi-adaptor protein in striated skeletal and cardiac muscle. Mutations have been linked to a range of cardiomyopathy types. Here, we generated induced pluripotent stem cells (iPSC) from a patient with dilated cardiomyopathy (DCM) harboring a new, unique heterozygous FLNC mutation p.R2187P. From this patient-specific iPSC line, a corresponding isogenic control line was created by CRISPR/Cas9 genome editing. Both, the patient-specific and isogenic-control iPSC maintained full pluripotency, genomic integrity, and in vitro differentiation capacity. All iPSC lines differentiate into iPSC-cardiomyocytes, hence providing the possibility to study the pathogenesis of FLNC-mediated DCM further.

Detection of ER Stress in iPSC-Derived Neurons Carrying the p.N370S Mutation in the GBA1 Gene.

Endoplasmic reticulum (ER) stress is involved in the pathogenesis of many human diseases, such as cancer, type 2 diabetes, kidney disease, atherosclerosis and neurodegenerative diseases, in particular Parkinson's disease (PD). Since there is currently no treatment for PD, a better understanding of the molecular mechanisms underlying its pathogenesis, including the mechanisms of the switch from adaptation in the form of unfolded protein response (UPR) to apoptosis under ER stress conditions, may help in the search for treatment methods. Genetically encoded biosensors based on fluorescent proteins are suitable tools that facilitate the study of living cells and visualization of molecular events in real time. The combination of technologies to generate patient-specific iPSC lines and genetically encoded biosensors allows the creation of cell models with new properties. Using CRISPR-Cas9-mediated homologous recombination at the AAVS1 locus of iPSC with the genetic variant p.N370S (rs76763715) in the GBA1 gene, we created a cell model designed to study the activation conditions of the IRE1-XBP1 cascade of the UPR system. The cell lines obtained have a doxycycline-dependent expression of the genetically encoded biosensor XBP1-TagRFP, possess all the properties of human pluripotent cells, and can be used to test physical conditions and chemical compounds that affect the development of ER stress, the functioning of the UPR system, and in particular, the IRE1-XBP1 cascade.

Generation of Leber congenital amaurosis, type 12 patient-specific induced pluripotent stem cell line (LVPEIi006-A), harboring a homozygous mutation in RD3

Leber congenital amaurosis (LCA) is a congenital, early onset, autosomal recessive inherited retinal disease (IRD). This report describes an LCA12 patient-specific iPSC line (LVPEIi006-A), generated by the reprogramming of dermal fibroblasts using integration-free episomal plasmids.This disease-specific iPSC model carries a homozygous point mutation in RD3, within the donor splice site at the end of exon 2 (c.296 + 1G > A). The stable line at passage 15 has displayed a normal colony morphology, expressed multiple stemness and pluripotency markers, lost all transgenes, differentiated into cell types of all three germ layers, and maintained a normal karyotype.

Generation and characterization of induced pluripotent stem cells from breast cancer patients carrying ATM mutations

We generated two induced pluripotent stem cell (iPSC) lines from peripheral blood mononuclear cells (PBMCs) of breast cancer patients carrying germline ATM mutations, a gene associated with a 7% prevalence in breast cancer. These iPSC lines displayed typical morphology, expressed pluripotency markers, maintained a stable karyotype, and retained the ability to differentiate into the three germ layers. These patient-specific iPSC lines hold great potential for mechanistic investigations and the development of drug screening strategies aimed at addressing ATM-related cancer.

Generation of two induced pluripotent stem cell lines from patients with Down syndrome

Down syndrome (DS) is caused by trisomy of Homo sapiens chromosome 21 (HSA21) and is by far the most common chromosomal disorder accompanied by neurodevelopmental disorders and congenital heart disease. Here, we generated two induced pluripotent stem cell (iPSC) lines from two patients with DS. These two lines exhibited normal morphology, trisomy 21 karyotype, pluripotency and differentiation capability into derivatives of three germ layers. The patient-specific iPSC lines arean invaluable resource in research to model DS-related cellular and molecular pathologies and test possible therapeutic strategies for DS.

Generation of patient-specific induced pluripotent stem cell lines with Type 2 Long QT Syndrome and the KCNH2 c.379C > T pathogenic variant

Type 2 Long QT Syndrome (LQT2) is a rare genetic heart rhythm disorder causing life-threatening arrhythmias. We derived induced pluripotent stem cell (iPSC) lines from two patients with LQT2, aged 18 and 6, both carrying a heterozygous missense mutation on the 3rd and 11th exons of KCNH2. The iPSC lines exhibited normal genomes, expressed pluripotent markers, and differentiated into trilineage embryonic layers. These patient-specific iPSC lines provide a valuable model to study the molecular and functional impact of the hERG channel gene mutation in LQT2 and to develop personalized therapeutic approaches for this syndrome.

Abstract P1033: Derivation Of Large Biorepository Of Human Ipsc Lines For Open Access Sharing Among Academic Investigators

Background: Human induced pluripotent stem cells (iPSCs) and their ability to differentiate into numerous cell types have revolutionized biomedical and translational research in many fields such as cardiovascular disease, development, aging, regeneration, cancer, personalized medicine, and many rare diseases. The need for obtaining iPSC lines with the associated donors’ clinical data and from patients of diverse genetic backgrounds has been greater than ever. Greenstone Biosciences Inc. (GSB) has built a curated biorepository to address this demand as an asset for the scientific community utilizing iPSCs. Methods: The GSB biobank curates clinical data, isolates peripheral blood mononuclear cells (PBMCs), reprograms and banks patient-specific iPSC lines, and performs quality assurance. Reprogramming of PBMCs to iPSCs is carried out using non-integrating Sendai virus encoding for Oct4, Sox2, Klf4, and c-Myc. We then rigorously validate all iPSCs for pluripotency using immunofluorescence of stemness markers, RT-PCR, and SNP-based virtual karyotyping along with quality control methods established as a standard protocol. Results: We have a growing repository of >1,000 iPSC lines with various disease backgrounds across demographics such as age, sex, race, and ethnicity. The diseases with genetic mutations include hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM), long QT syndrome (LQTS), arrhythmogenic right ventricular dysplasia / cardiomyopathy (ARVD/C), Brugada syndrome (BrS), Duchenne’s muscular dystrophy (DMD), Spinal muscular atrophy (SMA), and many other rare diseases and syndromes. We have also established a simple material transfer agreement (MTA) for open access by academic investigators. Conclusions: We introduce the Greenstone Biosciences iPSC Biobank, a biorepository containing >1,000 human iPSC lines with de-identified patient clinical information and genetic backgrounds. This invaluable resource will help support iPSC research among academic investigators.

Generation of two induced pluripotent stem cell lines from catecholaminergic polymorphic ventricular tachycardia patients carrying RYR2 mutations

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a congenital arrhythmic syndrome caused by the RYR2 gene encoded ryanodine receptor. Mutations on RYR2 are commonly associated with ventricular tachycardia after adrenergic stimulation, leading to lethal arrhythmias and sudden cardiac death. We generated two human induced pluripotent stem cell (iPSC) lines from CPVT affected patients carrying single missense heterozygote RYR2 mutations, c.1082 G > A and c.100 A > C. Pluripotency and differentiation capability into derivatives of three germ layers were evaluated along with karyotype stability in the report. The generated patient-specific iPSC lines provide a reliable tool to investigate the CPVT phenotype and understand underlaying mechanisms.

Simple, Fast, and Efficient Method for Derivation of Dermal Fibroblasts From Skin Biopsies.

Primary fibroblasts are a precious resource in the field of translational regenerative medicine. Dermal fibroblasts derived from human subject biopsies are being used as donor tissues for the derivation of patient-specific iPSC lines, which in turn are used for disease modeling, drug screening, tissue engineering, and cell transplantation. We developed a fast and simple protocol to grow dermal fibroblasts from skin biopsies. Using this protocol, we simply and firmly fix the biopsy piece on the surface of a tissue culture-treated plate and allow the fibroblasts to grow. This novel method eliminates any need for enzymatic digestion or mechanical dissociation of the biopsy piece. By using this newly developed protocol, we have successfully established around 100 fibroblast lines characterized by the expression of specific markers [Serpin H1 (Hsp-47), F-actin, and Vimentin]. Finally, we have used many of these fibroblast lines as donor tissues to successfully derive iPSC lines. We have developed a method that is simple, fast, convenient, efficient, and gentle on the cells to derive dermal fibroblasts from human skin biopsies. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Skin biopsy collection and fibroblast derivation Support Protocol 1: Culturing, freezing, and thawing dermal fibroblasts derived from a skin biopsy Support Protocol 2: Characterization of dermal fibroblasts by immunocytochemistry.

Generation of induced pluripotent stem cell(iPSC)line CJUHi001-A derived peripheral blood mononuclear cells of spinocerebellar ataxia type 1(SCA1) the CAG repeat mutation in ATXN1 gene

Spinocerebellar ataxia type 1 (SCA1) is an autosomal dominant neurodegenerative disorder caused by CAG repeat mutations in the ATXN1 gene. In this study, we generated an induced pluripotent stem cell line (iPSC) by using non-integrating Sendai virus (SeV) from peripheral blood mononuclear cells(PBMCs)of SCA1 patient harboring a CAG repeat mutation in the ATXN1 gene. The induced patient-specific iPSC line with a normal karyotype and expresses pluripotent markers, it also shows differentiation totipotency and tridermogenesis in vitro. It may be an excellent model for studying spinocerebellar ataxia type 1 (SCA1) in vitro and will be beneficial for studying SCA1 pathogenesis and therapeutic intervention strategies.

Efficient reprogramming of human fibroblasts using RNA reprogramming with DAPT and iDOT1L under normoxia conditions.

IntroductionHuman induced pluripotent stem cells (hiPSCs) are generated through the reprogramming of somatic cells expressing a defined set of transcription factors. The advent of autologous iPSCs has enabled the generation of patient-specific iPSC lines and is expected to contribute to the exploration of cures and causes of diseases, drug screening, and tailor-made regenerative medicines. Efficient control of hiPSC derivation is beneficial for industrial applications. However, the mechanisms underlying somatic cell reprogramming remain unknown, while reprogramming efficiency remains extremely low, especially in human cells.Methods and resultsWe previously reported that chemical inhibition of the NOTCH signaling pathway and DOT1L promoted the generation of hiPSCs from keratinocytes, but the mechanisms and effect of this double inhibition on other types of cells remain to be investigated. Here, we found that the NOTCH/DOT1L inhibition markedly increased iPSC colony generation from human fibroblast cells via mRNA reprogramming, and mesenchymal to epithelial transition (MET)-related genes are significantly expressed in the early phase of the reprogramming. We successfully derived hiPSC lines using a single-cell sorting system under efficient reprogramming conditions.ConclusionsThis user-friendly reprogramming approach paves the way for the development of hiPSC derivations in industrial applications of disease modeling and drug screening.

Lab resource: Single cell line generation and characterization of a human-derived induced pluripotent stem cell line (IGIBi005-A) from a patient with spastic paraplegia/ataxia/ALS phenotype due to the mutation of the gene Kinesin Family Member 5A (KIF5A)

Human Kinesin Family Member 5A (KIF5A) gene mutations have been identified as a putative genetic cause of amyotrophic lateral sclerosis (ALS). Disease modelling using human-induced pluripotent stem cells (HiPSCs) is the next-generation approach to studying numerous human diseases. For the current investigation, we report the generation of patient-specific KIF5A iPSC lines with a mutation at the splice site mutation (c.3020 + 3 A > T) in the intronic region. The resulting line displayed markers for pluripotency, a healthy karyotype, the ability to differentiate into three germ layers in vitro, vector clearance, the KIF5A mutation, STR-based genomic identity, and contamination-free culture.

In Vitro Drug Screening Using iPSC-Derived Cardiomyocytes of a Long QT-Syndrome Patient Carrying KCNQ1 & TRPM4 Dual Mutation: An Experimental Personalized Treatment.

Congenital long QT syndrome is a type of inherited cardiovascular disorder characterized by prolonged QT interval. Patient often suffer from syncopal episodes, electrocardiographic abnormalities and life-threatening arrhythmia. Given the complexity of the root cause of the disease, a combination of clinical diagnosis and drug screening using patient-derived cardiomyocytes represents a more effective way to identify potential cures. We identified a long QT syndrome patient carrying a heterozygous KCNQ1 c.656G>A mutation and a heterozygous TRPM4 c.479C>T mutation. Implantation of implantable cardioverter defibrillator in combination with conventional medication demonstrated limited success in ameliorating long-QT-syndrome-related symptoms. Frequent defibrillator discharge also caused deterioration of patient quality of life. Aiming to identify better therapeutic agents and treatment strategy, we established a patient-specific iPSC line carrying the dual mutations and differentiated these patient-specific iPSCs into cardiomyocytes. We discovered that both verapamil and lidocaine substantially shortened the QT interval of the long QT syndrome patient-specific cardiomyocytes. Verapamil treatment was successful in reducing defibrillator discharge frequency of the KCNQ1/TRPM4 dual mutation patient. These results suggested that verapamil and lidocaine could be alternative therapeutic agents for long QT syndrome patients that do not respond well to conventional treatments. In conclusion, our approach indicated the usefulness of the in vitro disease model based on patient-specific iPSCs in identifying pharmacological mechanisms and drug screening. The long QT patient-specific iPSC line carrying KCNQ1/TRPM4 dual mutations also represents a tool for further understanding long QT syndrome pathogenesis.

Generation of human induced pluripotent stem cell lines carrying heterozygous PLN mutation from dilated cardiomyopathy patients

Familial dilated cardiomyopathy (DCM) is among the most prevalent forms of inherited heart disease. Here, two human-induced pluripotent stem cell (iPSC) lines were generated from peripheral blood mononuclear cells (PBMCs) from DCM patients carrying different mutations in the phospholamban encoding-gene (PLN). Both iPSC lines exhibited normal morphology, karyotype, pluripotency marker expression, and differentiation into the three germ layers. These patient-specific iPSC lines serve as valuable in vitro models for DCM pathology caused by PLN mutations.

Generation of the human induced pluripotent stem cell line (IBKMOLi002-A) from PBMCs of a patient carrying the heterozygous L271H mutation of the voltage-gated calcium channel subunit Cav1.3-encoding CACNA1D gene

Congenital hyperinsulinemic hypoglycemia (HH) is the most frequent cause of persistent and recurrent hypoglycemia. Peripheral mononuclear blood cells (PBMCs) from a patient diagnosed with HH, alongside autism-spectrum-disorder (ASD), carrying a heterozygous c.812 T>A (L271H) mutation in the voltage-gated calcium channel subunit Cav1.3-encoding gene CACNA1D, were reprogrammed into induced pluripotent stem cells (iPSC). The CACNA1D L271H iPSC (IBKMOLi002-A) exhibit a normal karyotype, high expression of pluripotency-associated markers and the capacity to differentiate into cells of all three germ layers. We provide a novel patient-specific iPSC line, allowing to study HH, ASD, the associated neurodevelopmental disorder as well as CACNA1D-associated channelopathies in general.

Verification of an iPSC line (LZUi002-A) from a patient with a novel mutation in the TBL1X gene

More than 120 genes have been reported to be associated with deafness, and deletion of the TBL1X gene may cause deafness in humans. In this study, we generated an induced pluripotent stem cell (iPSC) line from dermal fibroblasts of a 34-year-old deaf person with a novel variant c.342_343insGCGGCG in the TBL1X gene. The induced patient-specific iPSC line with a normal karyotype and expressed pluripotent markers, it also shows differentiation totipotency and tridermogenesis in vivo. It may be a good model for studying hearing loss in vitro and it will benefit to the development of new therapies for deafness.

Establishment of an induced pluripotent stem cell line DHMi005-A from a healthy male proband

We generated an induced pluripotent stem cell (iPSC) line from a healthy male 29-year-old proband. Adipose fibroblasts (AFs) were reprogrammed using Sendai virus. Generated iPSCs showed typical stem cell morphology. From passage 9 on, iPSCs were free of virus. Pluripotency in the iPSCs was verified and spontaneous differentiation showed expression of all three germ layers. Karyotyping indicated no anomalies for the generated iPSCs. Many patient-specific iPSCs are generated from subcutaneous fat fibroblasts obtained during surgical procedure. The described control iPSC line was generated equally and therefore serves as an ideal control for adipose-fibroblast-based patient-specific iPSC lines in disease modeling.