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Adverse cardiac outcomes in COVID-19 patients, particularly those with preexisting cardiac disease, motivate the development of human cell-based organ-on-a-chip models to recapitulate cardiac injury and dysfunction and for screening of cardioprotective therapeutics. Here, we developed a heart-on-a-chip model to study the pathogenesis of SARS-CoV-2 in healthy myocardium established from human induced pluripotent stem cell (iPSC)-derived cardiomyocytes and a cardiac dysfunction model, mimicking aspects of preexisting hypertensive disease induced by angiotensin II (Ang II). We recapitulated cytopathic features of SARS-CoV-2-induced cardiac damage, including progressively impaired contractile function and calcium handling, apoptosis, and sarcomere disarray. SARS-CoV-2 presence in Ang II-treated hearts-on-a-chip decreased contractile force with earlier onset of contractile dysfunction and profoundly enhanced inflammatory cytokines compared to SARS-CoV-2 alone. Toward the development of potential therapeutics, we evaluated the cardioprotective effects of extracellular vesicles (EVs) from human iPSC which alleviated the impairment of contractile force, decreased apoptosis, reduced the disruption of sarcomeric proteins, and enhanced beta-oxidation gene expression. Viral load was not affected by either Ang II or EV treatment. We identified MicroRNAs miR-20a-5p and miR-19a-3p as potential mediators of cardioprotective effects of these EVs.
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Angiotensina II , COVID-19 , Células Madre Pluripotentes Inducidas , Dispositivos Laboratorio en un Chip , Miocitos Cardíacos , Humanos , Angiotensina II/farmacología , Apoptosis/efectos de los fármacos , COVID-19/virología , COVID-19/metabolismo , Citocinas/metabolismo , Vesículas Extracelulares/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , MicroARNs/metabolismo , MicroARNs/genética , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/virología , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/patología , SARS-CoV-2/fisiologíaRESUMEN
Human induced pluripotent stem cells (hiPSCs) are frequently used to study disease-associated variations. We characterized transcriptional variability from a hiPSC-derived cardiomyocyte (hiPSC-CM) study of left ventricular hypertrophy (LVH) using donor samples from the HyperGEN study. Multiple hiPSC-CM differentiations over reprogramming events (iPSC generation) across 7 donors were used to assess variabilities from reprogramming, differentiation, and donor LVH status. Variability arising from pathological alterations was assessed using a cardiac stimulant applied to the hiPSC-CMs to trigger hypertrophic responses. We found that for most genes (73.3%~85.5%), technical variability was smaller than biological variability. Further, we identified and characterized lists of "noise" genes showing greater technical variability and "signal" genes showing greater biological variability. Together, they support a "genetic robustness" hypothesis of disease-modeling whereby cellular response to relevant stimuli in hiPSC-derived somatic cells from diseased donors tends to show more transcriptional variability. Our findings suggest that hiPSC-CMs can provide a valid model for cardiac hypertrophy and distinguish between technical and disease-relevant transcriptional changes.
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Many oncology drugs have been found to induce cardiotoxicity in a subset of patients, which significantly limits their clinical use and impedes the benefit of lifesaving anticancer treatments. Human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) carry donor-specific genetic information and have been proposed for exploring the interindividual difference in oncology drug-induced cardiotoxicity. Herein, we evaluated the inter- and intraindividual variability of iPSC-CM-related assays and presented a proof of concept to prospectively predict doxorubicin (DOX)-induced cardiotoxicity (DIC) using donor-specific iPSC-CMs. Our findings demonstrated that donor-specific iPSC-CMs exhibited greater line-to-line variability than the intraindividual variability in impedance cytotoxicity and transcriptome assays. The variable and dose-dependent cytotoxic responses of iPSC-CMs resembled those observed in clinical practice and largely replicated the reported mechanisms. By categorizing iPSC-CMs into resistant and sensitive cell lines based on their time- and concentration-related phenotypic responses to DOX, we found that the sensitivity of donor-specific iPSC-CMs to DOX may predict in vivo DIC risk. Furthermore, we identified a differentially expressed gene, DND microRNA-mediated repression inhibitor 1 (DND1), between the DOX-resistant and DOX-sensitive iPSC-CMs. Our results support the utilization of donor-specific iPSC-CMs in assessing interindividual differences in DIC. Further studies will encompass a large panel of donor-specific iPSC-CMs to identify potential novel molecular and genetic biomarkers for predicting DOX and other oncology drug-induced cardiotoxicity.
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Cardiotoxicidad , Doxorrubicina , Células Madre Pluripotentes Inducidas , Miocitos Cardíacos , Prueba de Estudio Conceptual , Doxorrubicina/toxicidad , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células Madre Pluripotentes Inducidas/metabolismo , Humanos , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Antibióticos Antineoplásicos/toxicidad , Relación Dosis-Respuesta a Droga , Antineoplásicos/toxicidadRESUMEN
Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) present an attractive in vitro platform to model safety and toxicity assessments-notably screening pro-arrhythmic compounds. The utility of the platform is stymied by a hiPSC-CM contractile apparatus and calcium handling mechanism akin to fetal phenotypes, evidenced by a negative force-frequency relationship. As such, hiPSC-CMs are limited in their ability to assess compounds that modulate contraction mediated by ionotropic compounds (Robertson, Tran, & George, 2013). To address this limitation, we utilize Agilent's xCELLigence Real-Time Cell Analyzer ePacer (RTCA ePacer) to enhance hiPSC-CM functional maturity. A continuous, progressive increase of electrical pacing is applied to hiPSC-CMs for up to 15 days. Contraction and viability are recorded by measurement of impedance using the RTCA ePacer. Our data confirms hiPSC-CMs inherently demonstrate a negative impedance amplitude frequency that is reversed after long-term electrical pacing. The data also indicate positive inotropic compounds increase the contractility of paced cardiomyocytes and calcium handling machinery is improved. Increased expression of genes critical to cardiomyocyte maturation further underscores the maturity of paced cells. In summary, our data suggest the application of continuous electrical pacing can functionally mature hiPSC-CMs, enhancing cellular response to positive inotropic compounds and improving calcium handling. SUMMARY: Long-term electrical stimulation of hiPSC-CM leads to functional maturation enabling predictive assessment of inotropic compounds.
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Células Madre Pluripotentes Inducidas , Miocitos Cardíacos , Humanos , Miocitos Cardíacos/fisiología , Células Madre Pluripotentes Inducidas/fisiología , Calcio/metabolismo , Diferenciación Celular , Células CultivadasRESUMEN
Background: Indices of left ventricular (LV) structure and geometry represent useful intermediate phenotypes related to LV hypertrophy (LVH), a predictor of cardiovascular (CV) disease (CVD) outcomes. Methods and Results: We conducted an exome-wide association study of LV mass (LVM) adjusted to height2.7, LV internal diastolic dimension (LVIDD), and relative wall thickness (RWT) among 1,364 participants of African ancestry (AAs) in the Hypertension Genetic Epidemiology Network (HyperGEN). Both single-variant and gene-based sequence kernel association tests were performed to examine whether common and rare coding variants contribute to variation in echocardiographic traits in AAs. We then used a data-driven procedure to prioritize and select genes for functional validation using a human induced pluripotent stem cell cardiomyocyte (hiPSC-CM) model. Three genes [myosin VIIA and Rab interacting protein (MYRIP), trafficking protein particle complex 11 (TRAPPC11), and solute carrier family 27 member 6 (SLC27A6)] were prioritized based on statistical significance, variant functional annotations, gene expression in the hiPSC-CM model, and prior biological evidence and were subsequently knocked down in the hiPSC-CM model. Expression profiling of hypertrophic gene markers in the knockdowns suggested a decrease in hypertrophic expression profiles. MYRIP knockdowns showed a significant decrease in atrial natriuretic factor (NPPA) and brain natriuretic peptide (NPPB) expression. Knockdowns of the heart long chain fatty acid (FA) transporter SLC27A6 resulted in downregulated caveolin 3 (CAV3) expression, which has been linked to hypertrophic phenotypes in animal models. Finally, TRAPPC11 knockdown was linked to deficient calcium handling. Conclusions: The three genes are biologically plausible candidates that provide new insight to hypertrophic pathways.
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Cytochrome P450 2D6 (CYP2D6) copy number (CN) variation affects the metabolism of numerous prescribed drugs. Sequence variation within primer or probe target regions of hydrolysis probe CN assays can generate false-positive calls for CN loss. Furthermore, CYP2D6-CYP2D7 hybrids and gene conversions can cause difficult to interpret discordant CN calls. The identification of haplotypes with CN variations and structural arrangements is important to predict phenotype accurately. During clinical testing with hydrolysis probe assays targeting three CYP2D6 regions (intron 2, intron 6, and exon 9), samples with haplotypes causing inconsistent CN calls were identified. To resolve these cases, next-generation sequencing and allele-specific Sanger sequencing was performed. Sequence analysis of 16 samples, all but one from subjects of African descent, identified six novel suballeles containing single-nucleotide polymorphisms, which cause false-positive calls for CN loss in introns 2 and 6. Five samples with an exon 9 CN loss contained CYP2D6/CYP2D7 hybrids (∗13 or ∗36) and one sample was found to have a novel haplotype, CYP2D6∗141. Interestingly, CYP2D6∗141 contains a CYP2D7-derived exon 9 conversion and core single-nucleotide polymorphisms that are otherwise found in CYP2D6∗17 and ∗27. Although these variants are rare, they can cause inconsistent CN calls that typically are reported as no calls or indeterminant, and thus may deprive patients, particularly those of African descent, from taking full benefit of pharmacogenetic testing.
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Citocromo P-450 CYP2D6/genética , Variaciones en el Número de Copia de ADN , Haplotipos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Reacción en Cadena de la Polimerasa/métodos , Polimorfismo Genético , Alelos , Frecuencia de los Genes , Humanos , FenotipoRESUMEN
Exosomes are an important mechanism of cell-cell interaction in the cardiovascular system, both in maintaining homeostasis and in stress response. Interindividual differences that alter content in exosomes may play a role in cardiovascular disease pathology. To study the effect of interindividual cardiomyocyte (CM) variation, we characterized exosomal content in phenotypically diverse human induced pluripotent stem cell-derived CMs (hiPSC-CMs). Cell lines were generated from six participants in the HyperGEN cohort: three with left ventricular hypertrophy (LVH) and three with normal left ventricular mass (LVM). Sequence analysis of the intracellular and exosomal RNA populations showed distinct expression pattern differences between hiPSC-CM lines derived from individuals with LVH and those with normal LVM. Functional analysis of hiPSC-endothelial cells (hiPSC-ECs) treated with exosomes from both hiPSC-CM groups showed significant variation in response, including differences in tube formation, migration, and proliferation. Overall, treatment of hiPSC-ECs with exosomes resulted in significant expression changes associated with angiogenesis and endothelial cell vasculogenesis. However, the hiPSC-ECs treated with exosomes from the LVH-affected donors exhibited significantly increased proliferation but decreased tube formation and migration, suggesting angiogenic dysregulation.NEW & NOTEWORTHY The intracellular RNA and the miRNA content in exosomes are significantly different in hiPSC-CMs derived from LVH-affected individuals compared with those from unaffected individuals. Treatment of endothelial cells with these exosomes functionally affects cellular phenotypes in a donor-specific manner. These findings provide novel insight into underlying mechanisms of hypertrophic cell signaling between different cell types. With a growing interest in stem cells and exosomes for cardiovascular therapeutic use, this also provides information important for regenerative medicine.
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Diferenciación Celular , Exosomas/metabolismo , Hipertrofia Ventricular Izquierda/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Miocitos Cardíacos/metabolismo , Neovascularización Fisiológica , Donantes de Tejidos , Adulto , Anciano , Estudios de Casos y Controles , Movimiento Celular , Proliferación Celular , Separación Celular , Células Cultivadas , Exosomas/genética , Exosomas/ultraestructura , Femenino , Regulación de la Expresión Génica , Humanos , Hipertrofia Ventricular Izquierda/genética , Hipertrofia Ventricular Izquierda/patología , Células Madre Pluripotentes Inducidas/ultraestructura , Masculino , MicroARNs/genética , MicroARNs/metabolismo , Persona de Mediana Edad , Miocitos Cardíacos/ultraestructura , Neovascularización Fisiológica/genética , Fenotipo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transducción de Señal , TranscriptomaRESUMEN
Pharmacogenetic testing increasingly is available from clinical and research laboratories. However, only a limited number of quality control and other reference materials currently are available for the complex rearrangements and rare variants that occur in the CYP2D6 gene. To address this need, the Division of Laboratory Systems, CDC-based Genetic Testing Reference Material Coordination Program, in collaboration with members of the pharmacogenetic testing and research communities and the Coriell Cell Repositories (Camden, NJ), has characterized 179 DNA samples derived from Coriell cell lines. Testing included the recharacterization of 137 genomic DNAs that were genotyped in previous Genetic Testing Reference Material Coordination Program studies and 42 additional samples that had not been characterized previously. DNA samples were distributed to volunteer testing laboratories for genotyping using a variety of commercially available and laboratory-developed tests. These publicly available samples will support the quality-assurance and quality-control programs of clinical laboratories performing CYP2D6 testing.
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Alelos , Citocromo P-450 CYP2D6/genética , Técnicas de Genotipaje/normas , Variación Genética , Haplotipos/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Colaboración Intersectorial , Reacción en Cadena de la Polimerasa Multiplex , Reacción en Cadena en Tiempo Real de la Polimerasa , Estándares de ReferenciaRESUMEN
Tissue engineering using cardiomyocytes derived from human pluripotent stem cells holds a promise to revolutionize drug discovery, but only if limitations related to cardiac chamber specification and platform versatility can be overcome. We describe here a scalable tissue-cultivation platform that is cell source agnostic and enables drug testing under electrical pacing. The plastic platform enabled on-line noninvasive recording of passive tension, active force, contractile dynamics, and Ca2+ transients, as well as endpoint assessments of action potentials and conduction velocity. By combining directed cell differentiation with electrical field conditioning, we engineered electrophysiologically distinct atrial and ventricular tissues with chamber-specific drug responses and gene expression. We report, for the first time, engineering of heteropolar cardiac tissues containing distinct atrial and ventricular ends, and we demonstrate their spatially confined responses to serotonin and ranolazine. Uniquely, electrical conditioning for up to 8 months enabled modeling of polygenic left ventricular hypertrophy starting from patient cells.
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Miocitos Cardíacos/citología , Técnicas de Cultivo de Tejidos/instrumentación , Ingeniería de Tejidos/métodos , Potenciales de Acción , Diferenciación Celular , Células Cultivadas , Fenómenos Electrofisiológicos , Humanos , Células Madre Pluripotentes Inducidas/citología , Modelos Biológicos , Miocardio/citología , Miocitos Cardíacos/metabolismo , Células Madre Pluripotentes/citología , Técnicas de Cultivo de Tejidos/métodosRESUMEN
The developmental programs that generate a broad repertoire of regulatory T cells (Treg cells) able to respond to both self antigens and non-self antigens remain unclear. Here we found that mature Treg cells were generated through two distinct developmental programs involving CD25+ Treg cell progenitors (CD25+ TregP cells) and Foxp3lo Treg cell progenitors (Foxp3lo TregP cells). CD25+ TregP cells showed higher rates of apoptosis and interacted with thymic self antigens with higher affinity than did Foxp3lo TregP cells, and had a T cell antigen receptor repertoire and transcriptome distinct from that of Foxp3lo TregP cells. The development of both CD25+ TregP cells and Foxp3lo TregP cells was controlled by distinct signaling pathways and enhancers. Transcriptomics and histocytometric data suggested that CD25+ TregP cells and Foxp3lo TregP cells arose by coopting negative-selection programs and positive-selection programs, respectively. Treg cells derived from CD25+ TregP cells, but not those derived from Foxp3lo TregP cells, prevented experimental autoimmune encephalitis. Our findings indicate that Treg cells arise through two distinct developmental programs that are both required for a comprehensive Treg cell repertoire capable of establishing immunotolerance.
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Diferenciación Celular/inmunología , Encefalomielitis Autoinmune Experimental/inmunología , Células Progenitoras Linfoides/fisiología , Linfocitos T Reguladores/fisiología , Timo/crecimiento & desarrollo , Animales , Autoantígenos/inmunología , Colitis/inmunología , Modelos Animales de Enfermedad , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Adyuvante de Freund/administración & dosificación , Adyuvante de Freund/inmunología , Humanos , Tolerancia Inmunológica/inmunología , Subunidad alfa del Receptor de Interleucina-2/metabolismo , Células Progenitoras Linfoides/trasplante , Ratones , Ratones Transgénicos , Mycobacterium tuberculosis/inmunología , Glicoproteína Mielina-Oligodendrócito/administración & dosificación , Glicoproteína Mielina-Oligodendrócito/inmunología , Fragmentos de Péptidos/administración & dosificación , Fragmentos de Péptidos/inmunología , Transducción de Señal , Organismos Libres de Patógenos Específicos , Timo/citología , Timo/inmunologíaRESUMEN
Human IL12RB1 is an autosomal gene that is essential for mycobacterial disease resistance and T cell differentiation. Using primary human tissue and PBMCs, we demonstrate that lung and T cell IL12RB1 expression is allele-biased, and the extent to which cells express one IL12RB1 allele is unaffected by activation. Furthermore following its expression the IL12RB1 pre-mRNA is processed into either IL12RB1 Isoform 1 (IL12Rß1, a positive regulator of IL12 responsiveness) or IL12RB1 Isoform 2 (a protein of heretofore unknown function). T cells choice to process pre-mRNA into Isoform 1 or Isoform 2 is controlled by intragenic competition of IL12RB1 exon 9-10 splicing with IL12RB1 exon 9b splicing, as well as an IL12RB1 exon 9b-associated polyadenylation site. Heterogeneous nuclear ribonucleoprotein H (hnRNP H) binds near the regulated polyadenylation site, but is not required for exon 9b polyadenylation. Finally, microRNA-mediated knockdown experiments demonstrated that IL12RB1 Isoform 2 promotes T cell IL12 responses. Collectively, our data support a model wherein tissue expression of human IL12RB1 is allele-biased and produces an hnRNP H-bound pre-mRNA, the processing of which generates a novel IL12 response regulator.
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Alelos , Interleucina-12/genética , Empalme del ARN , Receptores de Interleucina-12/genética , Células Cultivadas , Ribonucleoproteína Heterogénea-Nuclear Grupo F-H/metabolismo , Humanos , Interleucina-12/metabolismo , Células Jurkat , Pulmón/metabolismo , Unión Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptores de Interleucina-12/metabolismo , Linfocitos T/metabolismoRESUMEN
The precise molecular mechanism underlying the regulation of early B cell lymphopoiesis is unclear. The PLCγ signaling pathway is critical for antigen receptor-mediated lymphocyte activation, but its function in cytokine signaling is unknown. Here we show that PLCγ1/PLCγ2 double deficiency in mice blocks early B cell development at the pre-pro-B cell stage and renders B cell progenitors unresponsive to IL-7. PLCγ pathway inhibition blocks IL-7-induced activation of mTOR, but not Stat5. The PLCγ pathway activates mTOR through the DAG/PKC signaling branch, independent of the conventional Akt/TSC/Rheb signaling axis. Inhibition of PLCγ/PKC-induced mTOR activation impairs IL-7-mediated B cell development. PLCγ1/PLCγ2 double-deficient B cell progenitors have reduced expression of genes related to B cell lineage, IL-7 signaling, and cell cycle. Thus, IL-7 receptor controls early B lymphopoiesis through activation of mTOR via PLCγ/DAG/PKC signaling, not via Akt/Rheb signaling.
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Interleucina-7/inmunología , Linfopoyesis/inmunología , Fosfolipasa C gamma/deficiencia , Fosfolipasa C gamma/inmunología , Células Precursoras de Linfocitos B/citología , Serina-Treonina Quinasas TOR/metabolismo , Animales , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fosfolipasa C gamma/genética , Células Precursoras de Linfocitos B/inmunología , Factor de Transcripción STAT5/metabolismo , Transducción de Señal/inmunologíaRESUMEN
Pharmacogenetic testing is increasingly available from clinical laboratories. However, only a limited number of quality control and other reference materials are currently available to support clinical testing. To address this need, the Centers for Disease Control and Prevention-based Genetic Testing Reference Material Coordination Program, in collaboration with members of the pharmacogenetic testing community and the Coriell Cell Repositories, has characterized 137 genomic DNA samples for 28 genes commonly genotyped by pharmacogenetic testing assays (CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, CYP3A5, CYP4F2, DPYD, GSTM1, GSTP1, GSTT1, NAT1, NAT2, SLC15A2, SLC22A2, SLCO1B1, SLCO2B1, TPMT, UGT1A1, UGT2B7, UGT2B15, UGT2B17, and VKORC1). One hundred thirty-seven Coriell cell lines were selected based on ethnic diversity and partial genotype characterization from earlier testing. DNA samples were coded and distributed to volunteer testing laboratories for targeted genotyping using a number of commercially available and laboratory developed tests. Through consensus verification, we confirmed the presence of at least 108 variant pharmacogenetic alleles. These samples are also being characterized by other pharmacogenetic assays, including next-generation sequencing, which will be reported separately. Genotyping results were consistent among laboratories, with most differences in allele assignments attributed to assay design and variability in reported allele nomenclature, particularly for CYP2D6, UGT1A1, and VKORC1. These publicly available samples will help ensure the accuracy of pharmacogenetic testing.
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Proteínas Portadoras/genética , Sistema Enzimático del Citocromo P-450/genética , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos/genética , Glucuronosiltransferasa/genética , Glutatión Transferasa/genética , Farmacogenética/métodos , Secuencia de Bases , Línea Celular , Pruebas Genéticas , Genotipo , Técnicas de Genotipaje , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Polimorfismo de Nucleótido Simple/genética , Análisis de Secuencia de ADN , Estados UnidosRESUMEN
Human interleukin 12 and interleukin 23 (IL12/23) influence susceptibility or resistance to multiple diseases. However, the reasons underlying individual differences in IL12/23 sensitivity remain poorly understood. Here we report that in human peripheral blood mononuclear cells (PBMCs) and inflamed lungs, the majority of interleukin-12 receptor ß1 (IL12RB1) mRNAs contain a number of RNA-DNA differences (RDDs) that concentrate in sequences essential to IL12Rß1's binding of IL12p40, the protein subunit common to both IL-12 and IL-23. IL12RB1 RDDs comprise multiple RDD types and are detectable by next-generation sequencing and classic Sanger sequencing. As a consequence of these RDDs, the resulting IL12Rß1 proteins have an altered amino acid sequence that could not be predicted on the basis of genomic DNA sequencing alone. Importantly, the introduction of RDDs into IL12RB1 mRNAs negatively regulates IL12Rß1's binding of IL12p40 and is sensitive to activation. Collectively, these results suggest that the introduction of RDDs into an individual's IL12RB1 mRNA repertoire is a novel determinant of IL12/23 sensitivity.
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ADN/metabolismo , ARN/metabolismo , Receptores de Interleucina-12/metabolismo , Adulto , Secuencia de Bases , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Interleucina-12/metabolismo , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Pulmón/patología , Modelos Biológicos , Datos de Secuencia Molecular , Fitohemaglutininas/farmacología , Neumonía/genética , Neumonía/patología , Unión Proteica/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Interleucina-12/genética , Proteínas Recombinantes/biosíntesisRESUMEN
BACKGROUND: Whole transcriptome sequencing (RNA-seq) represents a powerful approach for whole transcriptome gene expression analysis. However, RNA-seq carries a few limitations, e.g., the requirement of a significant amount of input RNA and complications led by non-specific mapping of short reads. The Ion AmpliSeq Transcriptome Human Gene Expression Kit (AmpliSeq) was recently introduced by Life Technologies as a whole-transcriptome, targeted gene quantification kit to overcome these limitations of RNA-seq. To assess the performance of this new methodology, we performed a comprehensive comparison of AmpliSeq with RNA-seq using two well-established next-generation sequencing platforms (Illumina HiSeq and Ion Torrent Proton). We analyzed standard reference RNA samples and RNA samples obtained from human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs). RESULTS: Using published data from two standard RNA reference samples, we observed a strong concordance of log2 fold change for all genes when comparing AmpliSeq to Illumina HiSeq (Pearson's r = 0.92) and Ion Torrent Proton (Pearson's r = 0.92). We used ROC, Matthew's correlation coefficient and RMSD to determine the overall performance characteristics. All three statistical methods demonstrate AmpliSeq as a highly accurate method for differential gene expression analysis. Additionally, for genes with high abundance, AmpliSeq outperforms the two RNA-seq methods. When analyzing four closely related hiPSC-CM lines, we show that both AmpliSeq and RNA-seq capture similar global gene expression patterns consistent with known sources of variations. CONCLUSIONS: Our study indicates that AmpliSeq excels in the limiting areas of RNA-seq for gene expression quantification analysis. Thus, AmpliSeq stands as a very sensitive and cost-effective approach for very large scale gene expression analysis and mRNA marker screening with high accuracy.
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Perfilación de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Análisis de Secuencia de ARN , Transcriptoma , Análisis por Conglomerados , Biología Computacional/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Reproducibilidad de los Resultados , Análisis de Secuencia de ARN/métodosRESUMEN
Cardiac hypertrophy is an independent risk factor for cardiovascular disease and heart failure. There is increasing evidence that microRNAs (miRNAs) play an important role in the regulation of messenger RNA (mRNA) and the pathogenesis of various cardiovascular diseases. However, the ability to comprehensively study cardiac hypertrophy on a gene regulatory level is impacted by the limited availability of human cardiomyocytes. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) offer the opportunity for disease modeling. Here we utilize a previously established in vitro model of cardiac hypertrophy to interrogate the regulatory mechanism associated with the cardiac disease process. We perform miRNA sequencing and mRNA expression analysis on endothelin 1 (ET-1) stimulated hiPSC-CMs to describe associated RNA expression profiles. MicroRNA sequencing revealed over 250 known and 34 predicted novel miRNAs to be differentially expressed between ET-1 stimulated and unstimulated control hiPSC-CMs. Messenger RNA expression analysis identified 731 probe sets with significant differential expression. Computational target prediction on significant differentially expressed miRNAs and mRNAs identified nearly 2000 target pairs. A principal component analysis approach comparing the in vitro data with human myocardial biopsies detected overlapping expression changes between the in vitro samples and myocardial biopsies with Left Ventricular Hypertrophy. These results provide further insights into the complex RNA regulatory mechanism associated with cardiac hypertrophy.
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Cardiomegalia/patología , Perfilación de la Expresión Génica , Células Madre Pluripotentes Inducidas/citología , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Humanos , MicroARNs/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Análisis de Secuencia de ARNRESUMEN
"Natural" regulatory T cells (nTregs) that express the transcription factor Foxp3 and produce IL-10 are required for systemic immunological tolerance. "Induced" regulatory T cells (iTregs) are nonredundant and essential for tolerance at mucosal surfaces, yet their mechanisms of suppression and stability are unknown. We investigated the role of iTreg-produced IL-10 and iTreg fate in a treatment model of inflammatory bowel disease. Colitis was induced in Rag1(-/-) mice by the adoptive transfer of naive CD4(+) T cells carrying a nonfunctional Foxp3 allele. At the onset of weight loss, mice were treated with both iTregs and nTregs where one marked subset was selectively IL-10 deficient. Body weight assessment, histological scoring, cytokine analysis, and flow cytometry were used to monitor disease activity. Transcriptional profiling and TCR repertoire analysis were used to track cell fate. When nTregs were present but IL-10 deficient, iTreg-produced IL-10 was necessary and sufficient for the treatment of disease, and vice versa. Invariably, â¼85% of the transferred iTregs lost Foxp3 expression (ex-iTregs) but retained a portion of the iTreg transcriptome, which failed to limit their pathogenic potential upon retransfer. TCR repertoire analysis revealed no clonal relationships between iTregs and ex-iTregs, either within mice or between mice treated with the same cells. These data identify a dynamic IL-10-dependent functional reciprocity between regulatory T cell subsets that maintains mucosal tolerance. The niche supporting stable iTregs is limited and readily saturated, which promotes a large population of ex-iTregs with pathogenic potential during immunotherapy.