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1.
Cell Rep Methods ; 2(10): 100300, 2022 10 24.
Artigo em Inglês | MEDLINE | ID: mdl-36313798

RESUMO

Inserting large DNA payloads (>10 kb) into specific genomic sites of mammalian cells remains challenging. Applications ranging from synthetic biology to evaluating the pathogenicity of disease-associated variants for precision medicine initiatives would greatly benefit from tools that facilitate this process. Here, we merge the strengths of different classes of site-specific recombinases and combine these with CRISPR-Cas9-mediated homologous recombination to develop a strategy for stringent site-specific replacement of genomic fragments at least 50 kb in size in human induced pluripotent stem cells (hiPSCs). We demonstrate the versatility of STRAIGHT-IN (serine and tyrosine recombinase-assisted integration of genes for high-throughput investigation) by (1) inserting various combinations of fluorescent reporters into hiPSCs to assess the excitation-contraction coupling cascade in derivative cardiomyocytes and (2) simultaneously targeting multiple variants associated with inherited cardiac arrhythmic disorders into a pool of hiPSCs. STRAIGHT-IN offers a precise approach to generate genetically matched panels of hiPSC lines efficiently and cost effectively.


Assuntos
Células-Tronco Pluripotentes Induzidas , Humanos , DNA , Recombinação Homóloga
2.
Stem Cells ; 40(7): 655-668, 2022 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-35429386

RESUMO

Electrical activity and intracellular Ca2+ transients are key features of cardiomyocytes. They can be measured using organic voltage- and Ca2+-sensitive dyes but their photostability and phototoxicity mean they are unsuitable for long-term measurements. Here, we investigated whether genetically encoded voltage and Ca2+ indicators (GEVIs and GECIs) delivered as modified mRNA (modRNA) into human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) would be accurate alternatives allowing measurements over long periods. These indicators were detected in hiPSC-CMs for up to 7 days after transfection and did not affect responses to proarrhythmic compounds. Furthermore, using the GEVI ASAP2f we observed action potential prolongation in long QT syndrome models, while the GECI jRCaMP1b facilitated the repeated evaluation of Ca2+ handling responses for various tyrosine kinase inhibitors. This study demonstrated that modRNAs encoding optogenetic constructs report cardiac physiology in hiPSC-CMs without toxicity or the need for stable integration, illustrating their value as alternatives to organic dyes or other gene delivery methods for expressing transgenes.


Assuntos
Células-Tronco Pluripotentes Induzidas , Potenciais de Ação/fisiologia , Cálcio , Corantes , Humanos , Miócitos Cardíacos , Optogenética , RNA Mensageiro/genética
3.
Methods Mol Biol ; 2454: 531-557, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-33755904

RESUMO

Advances in genome editing and our ability to derive and differentiate human induced pluripotent stem cells (hiPSCs) into a wide variety of cell types present in the body is revolutionizing how we model human diseases in vitro. Central to this has been the development of the CRISPR/Cas9 system as an inexpensive and highly efficient tool for introducing or correcting disease-associated mutations. However, the ease with which CRISPR/Cas9 enables genetic modification is a double-edged sword, with the challenge now being to introduce changes precisely to just one allele without disrupting the other.In this chapter, we describe strategies to introduce specific mutations into hiPSCs without enrichment steps. Monoallelic modification is contingent on the target activity of the guide RNA, delivery method of the CRISPR/Cas9 components and design of the oligonucleotide(s) transfected. As well as addressing these aspects, we detail high throughput culturing, freezing and screening methods to identify clonal hiPSCs with the desired nucleotide change. This set of protocols offers an efficient and ultimately time- and labor-saving approach for generating isogenic pairs of hiPSCs to detect subtle phenotypic differences caused by the disease variant.


Assuntos
Células-Tronco Pluripotentes Induzidas , Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Mutação , RNA Guia de Cinetoplastídeos/genética , RNA Guia de Cinetoplastídeos/metabolismo
4.
Front Physiol ; 12: 755642, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34992545

RESUMO

While rare mutations in ion channel genes are primarily responsible for inherited cardiac arrhythmias, common genetic variants are also an important contributor to the clinical heterogeneity observed among mutation carriers. The common single nucleotide polymorphism (SNP) KCNH2-K897T is associated with QT interval duration, but its influence on the disease phenotype in patients with long QT syndrome type 2 (LQT2) remains unclear. Human induced pluripotent stem cells (hiPSCs), coupled with advances in gene editing technologies, are proving an invaluable tool for modeling cardiac genetic diseases and identifying variants responsible for variability in disease expressivity. In this study, we have used isogenic hiPSC-derived cardiomyocytes (hiPSC-CMs) to establish the functional consequences of having the KCNH2-K897T SNP in cis- or trans-orientation with LQT2-causing missense variants either within the pore-loop domain (KCNH2A561T/WT) or tail region (KCNH2N996I/WT) of the potassium ion channel, human ether-a-go-go-related gene (hERG). When KCNH2-K897T was on the same allele (cis) as the primary mutation, the hERG channel in hiPSC-CMs exhibited faster activation and deactivation kinetics compared to their trans-oriented counterparts. Consistent with this, hiPSC-CMs with KCNH2-K897T in cis orientation had longer action and field potential durations. Furthermore, there was an increased occurrence of arrhythmic events upon pharmacological blocking of hERG. Collectively, these results indicate that the common polymorphism KCNH2-K897T differs in its influence on LQT2-causing KCNH2 mutations depending on whether it is present in cis or trans. This study corroborates hiPSC-CMs as a powerful platform to investigate the modifying effects of common genetic variants on inherited cardiac arrhythmias and aids in unraveling their contribution to the variable expressivity of these diseases.

5.
Stem Cell Reports ; 15(5): 1127-1139, 2020 11 10.
Artigo em Inglês | MEDLINE | ID: mdl-33176122

RESUMO

Mutations in KCNH2 can lead to long QT syndrome type 2. Variable disease manifestation observed with this channelopathy is associated with the location and type of mutation within the protein, complicating efforts to predict patient risk. Here, we demonstrated phenotypic differences in cardiomyocytes derived from isogenic human induced pluripotent stem cells (hiPSC-CMs) genetically edited to harbor mutations either within the pore or tail region of the ion channel. Electrophysiological analysis confirmed that the mutations prolonged repolarization of the hiPSC-CMs, with differences between the mutations evident in monolayer cultures. Blocking the hERG channel revealed that the pore-loop mutation conferred greater susceptibility to arrhythmic events. These findings showed that subtle phenotypic differences related to KCNH2 mutations could be captured by hiPSC-CMs under genetically matched conditions. Moreover, the results support hiPSC-CMs as strong candidates for evaluating the underlying severity of individual KCNH2 mutations in humans, which could facilitate patient risk stratification.


Assuntos
Canal de Potássio ERG1/metabolismo , Células-Tronco Pluripotentes Induzidas/fisiologia , Síndrome do QT Longo/metabolismo , Miócitos Cardíacos/fisiologia , Arritmias Cardíacas/induzido quimicamente , Linhagem Celular , Canal de Potássio ERG1/genética , Eletrofisiologia , Edição de Genes , Predisposição Genética para Doença , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Síndrome do QT Longo/genética , Modelos Biológicos , Mutação , Miócitos Cardíacos/efeitos dos fármacos , Técnicas de Patch-Clamp , Piperidinas/efeitos adversos , Piridinas/efeitos adversos
6.
Stem Cell Res ; 43: 101698, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31945612

RESUMO

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have emerged as a powerful platform for in vitro modelling of cardiac diseases, safety pharmacology and drug screening. All these applications require large quantities of well-characterised and standardised batches of hiPSC-CMs. Cryopreservation of hiPSC-CMs without affecting their biochemical or biophysical phenotype is essential for facilitating this, but ideally requires the cells being unchanged by the freeze-thaw procedure. We therefore compared the in vitro functional and molecular characteristics of fresh and cryopreserved hiPSC-CMs generated from multiple independent hiPSC lines. While the frozen hiPSC-CMs exhibited poorer replating than their freshly-derived counterparts, there was no difference in the proportion of cardiomyocytes retrieved from the mixed population when this was factored in, although for several lines a higher percentage of ventricular-like hiPSC-CMs were recovered following cryopreservation. Furthermore, cryopreserved hiPSC-CMs from one line exhibited longer action potential durations. These results provide evidence that cryopreservation does not compromise the in vitro molecular, physiological and mechanical properties of hiPSC-CMs, though can lead to an enrichment in ventricular myocytes. It also validates this procedure for storing hiPSC-CMs, thereby allowing the same batch of hiPSC-CMs to be used for multiple applications and evaluations.


Assuntos
Criopreservação/métodos , Ventrículos do Coração/fisiopatologia , Miócitos Cardíacos/metabolismo , Células-Tronco Pluripotentes/metabolismo , Humanos
7.
Proc Natl Acad Sci U S A ; 116(35): 17361-17370, 2019 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-31413199

RESUMO

Mutations in transcription factor p63 are associated with developmental disorders that manifest defects in stratified epithelia including the epidermis. The underlying cellular and molecular mechanism is however not yet understood. We established an epidermal commitment model using human induced pluripotent stem cells (iPSCs) and characterized differentiation defects of iPSCs derived from ectrodactyly, ectodermal dysplasia, and cleft lip/palate (EEC) syndrome patients carrying p63 mutations. Transcriptome analyses revealed stepwise cell fate transitions during epidermal commitment: Specification from multipotent simple epithelium to basal stratified epithelia and ultimately to the mature epidermal fate. Differentiation defects of EEC iPSCs caused by p63 mutations occurred during the specification switch from the simple epithelium to the basal-stratified epithelial fate. Single-cell transcriptome and pseudotime analyses of cell states identified mesodermal activation that was associated with the deviated commitment route of EEC iPSCs. Integrated analyses of differentially regulated genes and p63-dependent dynamic genomic enhancers during epidermal commitment suggest that p63 directly controls epidermal gene activation at the specification switch and has an indirect effect on mesodermal gene repression. Importantly, inhibitors of mesodermal induction enhanced epidermal commitment of EEC iPSCs. Our findings demonstrate that p63 is required for specification of stratified epithelia, and that epidermal commitment defects caused by p63 mutations can be reversed by repressing mesodermal induction. This study provides insights into disease mechanisms underlying stratified epithelial defects caused by p63 mutations and suggests potential therapeutic strategies for the disease.


Assuntos
Fenda Labial/genética , Fissura Palatina/genética , Displasia Ectodérmica/genética , Epitélio/metabolismo , Fatores de Transcrição/genética , Proteínas Supressoras de Tumor/genética , Epiderme/embriologia , Epiderme/metabolismo , Epitélio/embriologia , Expressão Gênica , Perfilação da Expressão Gênica , Humanos , Imuno-Histoquímica , Células-Tronco Pluripotentes Induzidas/metabolismo , Queratinócitos/metabolismo , Mutação , Análise de Sequência de RNA , Análise de Célula Única , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/metabolismo
8.
Dis Model Mech ; 10(9): 1039-1059, 2017 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-28883014

RESUMO

It is now a decade since human induced pluripotent stem cells (hiPSCs) were first described. The reprogramming of adult somatic cells to a pluripotent state has become a robust technology that has revolutionised our ability to study human diseases. Crucially, these cells capture all the genetic aspects of the patient from which they were derived. Combined with advances in generating the different cell types present in the human heart, this has opened up new avenues to study cardiac disease in humans and investigate novel therapeutic approaches to treat these pathologies. Here, we provide an overview of the current state of the field regarding the generation of cardiomyocytes from human pluripotent stem cells and methods to assess them functionally, an essential requirement when investigating disease and therapeutic outcomes. We critically evaluate whether treatments suggested by these in vitro models could be translated to clinical practice. Finally, we consider current shortcomings of these models and propose methods by which they could be further improved.


Assuntos
Cardiopatias/patologia , Cardiopatias/terapia , Modelos Biológicos , Células-Tronco Pluripotentes/patologia , Humanos , Modelos Cardiovasculares , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia
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