RESUMO
Expansion of a G4C2 repeat in the C9orf72 gene is associated with familial Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia (FTD). To investigate the underlying mechanisms of repeat instability, which occurs both somatically and intergenerationally, we created a novel mouse model of familial ALS/FTD that harbors 96 copies of G4C2 repeats at a humanized C9orf72 locus. In mouse embryonic stem cells, we observed two modes of repeat expansion. First, we noted minor increases in repeat length per expansion event, which was dependent on a mismatch repair pathway protein Msh2. Second, we found major increases in repeat length per event when a DNA double- or single-strand break (DSB/SSB) was artificially introduced proximal to the repeats, and which was dependent on the homology-directed repair (HDR) pathway. In mice, the first mode primarily drove somatic repeat expansion. Major changes in repeat length, including expansion, were observed when SSB was introduced in one-cell embryos, or intergenerationally without DSB/SSB introduction if G4C2 repeats exceeded 400 copies, although spontaneous HDR-mediated expansion has yet to be identified. These findings provide a novel strategy to model repeat expansion in a non-human genome and offer insights into the mechanism behind C9orf72 G4C2 repeat instability.
Assuntos
Proteína C9orf72 , Expansão das Repetições de DNA , Instabilidade Genômica , Animais , Humanos , Camundongos , Esclerose Lateral Amiotrófica/genética , Proteína C9orf72/genética , Modelos Animais de Doenças , Quebras de DNA de Cadeia Dupla , Expansão das Repetições de DNA/genética , Demência Frontotemporal/genética , Técnicas de Introdução de Genes , Instabilidade Genômica/genética , Proteína 2 Homóloga a MutS/genéticaRESUMO
Human breast tumors contain a breast cancer stem cell (BCSC) population with properties reminiscent of normal stem cells. We found 37 microRNAs that were differentially expressed between human BCSCs and nontumorigenic cancer cells. Three clusters, miR-200c-141, miR-200b-200a-429, and miR-183-96-182 were downregulated in human BCSCs, normal human and murine mammary stem/progenitor cells, and embryonal carcinoma cells. Expression of BMI1, a known regulator of stem cell self-renewal, was modulated by miR-200c. miR-200c inhibited the clonal expansion of breast cancer cells and suppressed the growth of embryonal carcinoma cells in vitro. Most importantly, miR-200c strongly suppressed the ability of normal mammary stem cells to form mammary ducts and tumor formation driven by human BCSCs in vivo. The coordinated downregulation of three microRNA clusters and the similar functional regulation of clonal expansion by miR-200c provide a molecular link that connects BCSCs with normal stem cells.
Assuntos
Neoplasias da Mama/genética , Mama/citologia , Perfilação da Expressão Gênica , MicroRNAs/metabolismo , Células-Tronco Neoplásicas/metabolismo , Células-Tronco/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Regulação para Baixo , Células-Tronco de Carcinoma Embrionário/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , MicroRNAs/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Complexo Repressor Polycomb 1 , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismoRESUMO
Spinal muscular atrophy (SMA), an autosomal recessive neuromuscular disease, is the leading monogenic cause of infant mortality. Homozygous loss of the gene survival of motor neuron 1 (SMN1) causes the selective degeneration of lower motor neurons and subsequent atrophy of proximal skeletal muscles. The SMN1 protein product, survival of motor neuron (SMN), is ubiquitously expressed and is a key factor in the assembly of the core splicing machinery. The molecular mechanisms by which disruption of the broad functions of SMN leads to neurodegeneration remain unclear. We used an antisense oligonucleotide (ASO)-based inducible mouse model of SMA to investigate the SMN-specific transcriptome changes associated with neurodegeneration. We found evidence of widespread intron retention, particularly of minor U12 introns, in the spinal cord of mice 30 d after SMA induction, which was then rescued by a therapeutic ASO. Intron retention was concomitant with a strong induction of the p53 pathway and DNA damage response, manifesting as γ-H2A.X positivity in neurons of the spinal cord and brain. Widespread intron retention and markers of the DNA damage response were also observed with SMN depletion in human SH-SY5Y neuroblastoma cells and human induced pluripotent stem cell-derived motor neurons. We also found that retained introns, high in GC content, served as substrates for the formation of transcriptional R-loops. We propose that defects in intron removal in SMA promote DNA damage in part through the formation of RNA:DNA hybrid structures, leading to motor neuron death.
Assuntos
Dano ao DNA , Íntrons , Atrofia Muscular Espinal/metabolismo , Proteína 1 de Sobrevivência do Neurônio Motor/genética , Proteína 1 de Sobrevivência do Neurônio Motor/metabolismo , Animais , Modelos Animais de Doenças , Humanos , Camundongos , Neurônios Motores/metabolismo , Atrofia Muscular Espinal/genética , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/metabolismo , Splicing de RNARESUMO
Marfan syndrome (MFS) is a heritable connective tissue disorder caused by mutations in the gene coding for FIBRILLIN-1 (FBN1), an extracellular matrix protein. MFS is inherited as an autosomal dominant trait and displays major manifestations in the ocular, skeletal, and cardiovascular systems. Here we report molecular and phenotypic profiles of skeletogenesis in tissues differentiated from human embryonic stem cells and induced pluripotent stem cells that carry a heritable mutation in FBN1. We demonstrate that, as a biological consequence of the activation of TGF-ß signaling, osteogenic differentiation of embryonic stem cells with a FBN1 mutation is inhibited; osteogenesis is rescued by inhibition of TGF-ß signaling. In contrast, chondrogenesis is not perturbated and occurs in a TGF-ß cell-autonomous fashion. Importantly, skeletal phenotypes observed in human embryonic stem cells carrying the monogenic FBN1 mutation (MFS cells) are faithfully phenocopied by cells differentiated from induced pluripotent-stem cells derived independently from MFS patient fibroblasts. Results indicate a unique phenotype uncovered by examination of mutant pluripotent stem cells and further demonstrate the faithful alignment of phenotypes in differentiated cells obtained from both human embryonic stem cells and induced pluripotent-stem cells, providing complementary and powerful tools to gain further insights into human molecular pathogenesis, especially of MFS.
Assuntos
Osso e Ossos/patologia , Células-Tronco Embrionárias/patologia , Células-Tronco Pluripotentes Induzidas/patologia , Síndrome de Marfan/patologia , Sequência de Bases , Osso e Ossos/metabolismo , Diferenciação Celular , Condrogênese , Células-Tronco Embrionárias/metabolismo , Fibrilina-1 , Fibrilinas , Humanos , Síndrome de Marfan/metabolismo , Proteínas dos Microfilamentos/genética , Dados de Sequência Molecular , Osteogênese , Fenótipo , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismoRESUMO
Rats were more frequently used than mice to model human disease before mouse embryonic stem cells (mESCs) revolutionized genetic engineering in mice. Rat ESCs (rESCs) were first reported over 10 years ago, yet they are not as frequently used as mESCs. CRISPR-based gene editing in zygotes is widely used in rats but is limited by the difficulty of inserting or replacing DNA sequences larger than about 10 kb. We report here the generation of germline-competent rESC lines from several rat strains. These rESC lines maintain their potential for germline transmission after serial targeting with bacterial artificial chromosome (BAC)-based targeting vectors, and CRISPR-Cas9 cutting can increase targeting efficiency. Using these methods, we have successfully replaced entire rat genes spanning up to 101 kb with the human ortholog.
Assuntos
Células-Tronco Embrionárias , Degeneração Retiniana , Humanos , Ratos , Animais , Camundongos , Edição de Genes , Engenharia Genética , Sistemas CRISPR-Cas/genéticaRESUMO
Amyotrophic lateral sclerosis is a fatal disease pathologically typified by motor and cortical neurodegeneration as well as microgliosis. The FUS P525L mutation is highly penetrant and causes ALS cases with earlier disease onset and more aggressive progression. To date, how P525L mutations may affect microglia during ALS pathogenesis had not been explored. In this study, we engineered isogenic control and P525L mutant FUS in independent human iPSC lines and differentiated them into microglia-like cells. We report that the P525L mutation causes FUS protein to mislocalize from the nucleus to cytoplasm. Homozygous P525L mutations perturb the transcriptome profile in which many differentially expressed genes are associated with microglial functions. Specifically, the dysregulation of several chemoreceptor genes leads to altered chemoreceptor-activated calcium signaling. However, other microglial functions such as phagocytosis and cytokine release are not significantly affected. Our study underscores the cell-autonomous effects of the ALS-linked FUS P525L mutation in a human microglia model.
Assuntos
Esclerose Lateral Amiotrófica , Células-Tronco Pluripotentes Induzidas , Proteína FUS de Ligação a RNA , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/patologia , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Microglia/metabolismo , Mutação , Proteína FUS de Ligação a RNA/genética , TranscriptomaRESUMO
CRISPR-based transcriptional activation is a powerful tool for functional gene interrogation; however, delivery difficulties have limited its applications in vivo. Here, we created a mouse model expressing all components of the CRISPR-Cas9 guide RNA-directed Synergistic Activation Mediator (SAM) from a single transcript that is capable of activating target genes in a tissue-specific manner. We optimized Lipid Nanoparticles and Adeno-Associated Virus guide RNA delivery approaches to achieve expression modulation of one or more genes in vivo. We utilized the SAM mouse model to generate a hypercholesteremia disease state that we could bidirectionally modulate with various guide RNAs. Additionally, we applied SAM to optimize gene expression in a humanized Transthyretin mouse model to recapitulate human expression levels. These results demonstrate that the SAM gene activation platform can facilitate in vivo research and drug discovery.
Assuntos
Sistemas CRISPR-Cas/genética , Hipercolesterolemia/genética , Lipossomos/farmacologia , Pré-Albumina/metabolismo , Ativação Transcricional/genética , Animais , Linhagem Celular , Expressão Gênica/genética , Regulação da Expressão Gênica/genética , Engenharia Genética/métodos , Células HEK293 , Humanos , Hipercolesterolemia/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Nanopartículas , Pré-Albumina/genética , RNA Guia de Cinetoplastídeos/genética , RNA Guia de Cinetoplastídeos/metabolismoRESUMO
The terminal sugars of Fc glycans can influence the Fc-dependent biological activities of monoclonal antibody therapeutics. Afucosylated N-glycans have been shown to significantly alter binding to FcγRIIIa and affect antibody-dependent cell-mediated cytotoxicity (ADCC). Therefore, in order to maintain and ensure safety and efficacy for antibodies whose predominant mechanism of action (MOA) is ADCC, afucosylation is routinely monitored and controlled within appropriate limits. However, it is unclear how the composition and levels of afucosylated N-glycans can modulate the biological activities for a recombinant antibody whose target is not a cell surface receptor, as is the case with ADCC. The impact of different types and varying levels of enriched afucosylated N-glycan species on the in vitro bioactivities is assessed for an antibody whose target is aggregated amyloid beta (Aß). While either the presence of complex biantennary or high mannose afucosylated glycoforms significantly increased FcγRIIIa binding activity compared to fucosylated glycoforms, they did not similarly increase aggregated Aß uptake activity mediated by different effector cells. These experiments suggest that afucosylated N-glycans are not critical for the in vitro phagocytic activity of a recombinant antibody whose target is aggregated Aß and uses Fc effector function as part of its MOA.
Assuntos
Peptídeos beta-Amiloides/imunologia , Anticorpos Monoclonais/farmacologia , Citotoxicidade Celular Dependente de Anticorpos/efeitos dos fármacos , Agregados Proteicos/imunologia , Receptores de IgG/imunologia , Animais , Anticorpos Monoclonais/imunologia , Células CHO , Cricetulus , Glicosilação , Humanos , Células THP-1RESUMO
The differentiation of human embryonic stem cells (hESCs) into functional hepatocytes provides a powerful in vitro model system for studying the molecular mechanisms governing liver development. Furthermore, a well-characterized renewable supply of hepatocytes differentiated from hESCs could be used for in vitro assays of drug metabolism and toxicology, screening of potential antiviral agents, and cell-based therapies to treat liver disease. In this study, we describe a protocol for the differentiation of hESCs toward hepatic cells with complex cellular morphologies. Putative hepatic cells were identified and isolated using a lentiviral vector, containing the alpha-fetoprotein promoter driving enhanced green fluorescent protein expression (AFP:eGFP). Whole-genome transcriptional profiling was performed on triplicate samples of AFP:eGFP+ and AFP:eGFP- cell populations using the recently released Affymetrix Exon Array ST 1.0 (Santa Clara, CA, http://www.affymetrix.com). Statistical analysis of the transcriptional profiles demonstrated that the AFP:eGFP+ population is highly enriched for genes characteristic of hepatic cells. These data provide a unique insight into the complex process of hepatocyte differentiation, point to signaling pathways that may be manipulated to more efficiently direct the differentiation of hESCs toward mature hepatocytes, and identify molecular markers that may be used for further dissection of hepatic cell differentiation from hESCs. Disclosure of potential conflicts of interest is found at the end of this article.
Assuntos
Células-Tronco Embrionárias/citologia , Perfilação da Expressão Gênica , Hepatócitos/citologia , Transcrição Gênica , Albuminas/metabolismo , Animais , Diferenciação Celular , Análise por Conglomerados , Vetores Genéticos , Proteínas de Fluorescência Verde/metabolismo , Hepatócitos/metabolismo , Humanos , Lentivirus/genética , Fígado/metabolismo , Camundongos , Análise de Sequência com Séries de OligonucleotídeosRESUMO
There is need in the pharmaceutical and chemical industries for high-throughput human cell-based assays for identifying hazardous chemicals, thereby reducing the overall reliance on animal studies for predicting the risk of toxic responses in humans. Despite instances of human-specific teratogens such as thalidomide, the use of human cell-teratogenicity assays has just started to be explored. Herein, a human pluripotent stem cell test (hPST) for identifying teratogens is described, benchmarking the in vitro findings to traditional preclinical toxicology teratogenicity studies and when available to teratogenic outcomes in humans. The hPST method employs a 3-day monolayer directed differentiation of human embryonic stem cells. The teratogenic risk of a compound is gauged by measuring the reduction in nuclear translocation of the transcription factor SOX17 in mesendodermal cells. Decreased nuclear SOX17 in the hPST model was strongly correlated with in vivo teratogenicity. Specifically, 71 drug-like compounds with known in vivo effects, including thalidomide, were examined in the hPST. A threshold of 5 µM demonstrated 94% accuracy (97% sensitivity and 92% specificity). Furthermore, 15 environmental toxicants with physicochemical properties distinct from small molecule pharmaceutical agents were examined and a similarly strong concordance with teratogenicity outcomes from in vivo studies was observed. Finally, to assess the suitability of the hPST for high-throughput screens, a small library of 300 kinase inhibitors was tested, demonstrating the hPST platform's utility for interrogating teratogenic mechanisms and drug safety prediction. Thus, the hPST assay is a robust predictor of teratogenicity and appears to be an improvement over existing in vitro models.
Assuntos
Ensaios de Triagem em Larga Escala , Células-Tronco Pluripotentes/efeitos dos fármacos , Teratogênicos/toxicidade , Testes de Toxicidade/métodos , Benchmarking , Biomarcadores/metabolismo , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Estudos de Viabilidade , Ensaios de Triagem em Larga Escala/normas , Humanos , Células-Tronco Pluripotentes/metabolismo , Células-Tronco Pluripotentes/patologia , Inibidores de Proteínas Quinases/toxicidade , Padrões de Referência , Reprodutibilidade dos Testes , Fatores de Transcrição SOXF/genética , Fatores de Transcrição SOXF/metabolismo , Fatores de Tempo , Testes de Toxicidade/normasRESUMO
Inhibition of spleen tyrosine kinase has attracted much attention as a mechanism for the treatment of cancers and autoimmune diseases such as asthma, rheumatoid arthritis, and systemic lupus erythematous. We report the structure-guided optimization of pyridazine amide spleen tyrosine kinase inhibitors. Early representatives of this scaffold were highly potent and selective but mutagenic in an Ames assay. An approach that led to the successful identification of nonmutagenic examples, as well as further optimization to compounds with reduced cardiovascular liabilities is described. Select pharmacokinetic and in vivo efficacy data are presented.
Assuntos
Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Piridazinas/síntese química , Piridazinas/farmacologia , Baço/enzimologia , Amidas/síntese química , Amidas/farmacologia , Animais , Biologia Computacional , Simulação por Computador , Desenho de Fármacos , Canais de Potássio Éter-A-Go-Go/efeitos dos fármacos , Humanos , Técnicas In Vitro , Camundongos , Microssomos Hepáticos/metabolismo , Modelos Moleculares , Conformação Molecular , Mutagênese/efeitos dos fármacos , Testes de Mutagenicidade , Inibidores de Proteínas Quinases/farmacocinética , Piridazinas/farmacocinética , Ratos , Baço/efeitos dos fármacos , Relação Estrutura-Atividade , Difração de Raios XRESUMO
Human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) are capable of detecting drug-induced clinical arrhythmia, Torsade de Pointes (TdP), and QT prolongation. Efforts herein employ a broad set of structurally diverse drugs to optimize the predictive algorithm for applications in discovery toxicology and cardiac safety screening. The changes in the beat rhythm and rate of a confluent monolayer of hiPS-CMs by 88 marketed and 30 internal discovery compounds were detected with real-time cellular impedance measurement and quantified by measures of arrhythmic beating (IB20, lowest concentration inducing ≥ 20% arrhythmic [irregular, atypical] beats in 3 consecutive 20-s sweeps, and predicted proarrhythmic score [PPS]-IB20) or changes in beat rate (BR20, the lowest concentration inducing a reduction in beat rate of ≥ 20% at 3 consecutive sweeps compared with the time-matched vehicle control group, and PPS-BR20). Drug-induced arrhythmic beats and reductions in beat rates are predictive of clinical arrhythmia and QT prolongation, respectively. A threshold of ≤ 10 µM for class determination results in 82% in vitro-in vivo concordance for TdP prediction and 91% sensitivity for non-TdP arrhythmia detection, or 83% and 91% if clinically efficacious plasma (effective serum therapeutic concentration [C eff]) values are incorporated. This human cardiomyocyte arrhythmic risk (hCAR) model provides greater predictivity for torsadogenicity in humans compared with either human ether-a-go-go-related gene (hERG) inhibition (75%) or QT prolongation (81%). The concordance of beat rate reductions to predict clinical QT prolongation is 86%, or 87% when C eff is considered, which is greater than a hERG signal (80%). Further, arrhythmic beats resulting from cytotoxicity were identified by a distinct arrhythmic beating pattern, which occurred after the onset of cytolethality. This hCAR assay showed increased performance over existing preclinical tools in predicting clinical QT prolongation, arrhythmia, and TdP. Thus, hiPS-CMs are a relevant cell system to improve evaluating cardiac safety liabilities of drug candidates.
Assuntos
Arritmias Cardíacas/fisiopatologia , Células-Tronco Pluripotentes Induzidas/citologia , Modelos Teóricos , Miócitos Cardíacos/citologia , Medição de Risco , Arritmias Cardíacas/patologia , Células Cultivadas , HumanosRESUMO
Jaspamide (jasplakinolide; NSC-613009) is a cyclodepsipeptide that has antitumor activity. A narrow margin of safety was observed between doses required for efficacy in mouse tumor models and doses that caused severe acute toxicity in rats and dogs. We explored the hypothesis that the observed toxicity was due to cardiotoxicity. Jaspamide was tested in a patch clamp assay to determine its effect on selected cardiac ion channels. Jaspamide (10 µM) inhibited Kv1.5 activity by 98.5%. Jaspamide also inhibited other channels including Cav1.2, Cav3.2, and HCN2; however, the Kv11.1 (hERG) channel was minimally affected. Using spontaneously contracting human cardiomyocytes derived from induced pluripotent stem cells, effects on cardiomyocyte contraction and viability were also examined. Jaspamide (30 nM to 30 µM) decreased cardiomyocyte cell indices and beat amplitude, putative measurements of cell viability and cardiac contractility, respectively. Concentration-dependent increases in rhythmic beating rate were noted at ≤ 6 h of treatment, followed by dose-dependent decreases after 6 and 72 h exposure. The toxic effects of jaspamide were compared with that of the known cardiotoxicant mitoxantrone, and confirmed by multiparameter fluorescence imaging analysis. These results support the hypothesis that the toxicity observed in rats and dogs is due to toxic effects of jaspamide on cardiomyocytes.
Assuntos
Antineoplásicos/farmacologia , Depsipeptídeos/farmacologia , Miócitos Cardíacos/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Canais Iônicos/efeitos dos fármacos , Canais Iônicos/fisiologia , Miócitos Cardíacos/fisiologiaRESUMO
We describe the discovery of several pyrrolopyrazines as potent and selective Syk inhibitors and the efforts that eventually led to the desired improvements in physicochemical properties and human whole blood potencies. Ultimately, our mouse model revealed unexpected toxicity that precluded us from further advancing this series.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Proteínas Tirosina Quinases/antagonistas & inibidores , Pirazinas/síntese química , Pirróis/síntese química , Animais , Linfócitos B/efeitos dos fármacos , Linfócitos B/enzimologia , Proteínas Sanguíneas/metabolismo , Cristalografia por Raios X , Humanos , Camundongos , Microssomos Hepáticos/metabolismo , Modelos Moleculares , Estrutura Molecular , Ligação Proteica , Pirazinas/farmacologia , Pirazinas/toxicidade , Pirróis/farmacologia , Pirróis/toxicidade , Relação Estrutura-Atividade , Quinase SykRESUMO
To gain insight into the molecular regulation of human heart development, a detailed comparison of the mRNA and miRNA transcriptomes across differentiating human-induced pluripotent stem cell (hiPSC)-derived cardiomyocytes and biopsies from fetal, adult, and hypertensive human hearts was performed. Gene ontology analysis of the mRNA expression levels of the hiPSCs differentiating into cardiomyocytes revealed 3 distinct groups of genes: pluripotent specific, transitional cardiac specification, and mature cardiomyocyte specific. Hierarchical clustering of the mRNA data revealed that the transcriptome of hiPSC cardiomyocytes largely stabilizes 20 days after initiation of differentiation. Nevertheless, analysis of cells continuously cultured for 120 days indicated that the cardiomyocytes continued to mature toward a more adult-like gene expression pattern. Analysis of cardiomyocyte-specific miRNAs (miR-1, miR-133a/b, and miR-208a/b) revealed an miRNA pattern indicative of stem cell to cardiomyocyte specification. A biostatistitical approach integrated the miRNA and mRNA expression profiles revealing a cardiomyocyte differentiation miRNA network and identified putative mRNAs targeted by multiple miRNAs. Together, these data reveal the miRNA network in human heart development and support the notion that overlapping miRNA networks re-enforce transcriptional control during developmental specification.
Assuntos
Diferenciação Celular , MicroRNAs/metabolismo , Miócitos Cardíacos/citologia , RNA Mensageiro/metabolismo , Adulto , Biomarcadores/metabolismo , Células Cultivadas , Análise por Conglomerados , Biologia Computacional/métodos , Feto/citologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Coração/crescimento & desenvolvimento , Humanos , MicroRNAs/genética , Miócitos Cardíacos/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , RNA Mensageiro/genética , Fatores de Tempo , Transcrição Gênica , TranscriptomaRESUMO
Improved in vitro systems for predicting drug-induced toxicity are needed in the pharmaceutical and biotechnology industries to decrease late-stage drug attrition. One unmet need is an early screen for cardiotoxicity, which accounts for about one third of safety-based withdrawn pharmaceuticals. Herein, the first published report of a high-throughput functional assay employing a monolayer of beating human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) is described, detailing a model that accurately detects drug-induced cardiac abnormalities. Using 96-well plates with interdigitated electrode arrays that assess impedance, the rhythmic, synchronous contractions of the iPSC-CMs were detected. Treatment of the iPSC-CMs with 28 different compounds with known cardiac effects resulted in compound-specific changes in the beat rate and/or the amplitude of the impedance measurement. Changes in impedance for the compounds tested were comparable with the results from a related technology, electric field potential assessment obtained from microelectrode arrays. Using the results from the set of compounds, an index of drug-induced arrhythmias was calculated, enabling the determination of a drug's proarrhythmic potential. This system of interrogating human cardiac function in vitro opens new opportunities for predicting cardiac toxicity and studying cardiac biology.
Assuntos
Arritmias Cardíacas/induzido quimicamente , Avaliação Pré-Clínica de Medicamentos/efeitos adversos , Miócitos Cardíacos/efeitos dos fármacos , Células-Tronco Pluripotentes/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Descoberta de Drogas/métodos , Humanos , Preparações FarmacêuticasRESUMO
The International Stem Cell Initiative analyzed 125 human embryonic stem (ES) cell lines and 11 induced pluripotent stem (iPS) cell lines, from 38 laboratories worldwide, for genetic changes occurring during culture. Most lines were analyzed at an early and late passage. Single-nucleotide polymorphism (SNP) analysis revealed that they included representatives of most major ethnic groups. Most lines remained karyotypically normal, but there was a progressive tendency to acquire changes on prolonged culture, commonly affecting chromosomes 1, 12, 17 and 20. DNA methylation patterns changed haphazardly with no link to time in culture. Structural variants, determined from the SNP arrays, also appeared sporadically. No common variants related to culture were observed on chromosomes 1, 12 and 17, but a minimal amplicon in chromosome 20q11.21, including three genes expressed in human ES cells, ID1, BCL2L1 and HM13, occurred in >20% of the lines. Of these genes, BCL2L1 is a strong candidate for driving culture adaptation of ES cells.
Assuntos
Células-Tronco Embrionárias/citologia , Crescimento/genética , Células-Tronco Pluripotentes Induzidas/citologia , Proteínas de Ligação a RNA/metabolismo , Proteína bcl-X/metabolismo , Diferenciação Celular/genética , Linhagem Celular , Cromossomos Humanos Par 20/genética , Evolução Clonal/genética , Metilação de DNA , Etnicidade/genética , Regulação da Expressão Gênica no Desenvolvimento , Variação Genética , Genótipo , Humanos , Proteína 1 Inibidora de Diferenciação/genética , Proteína 1 Inibidora de Diferenciação/metabolismo , Polimorfismo de Nucleotídeo Único , Proteínas de Ligação a RNA/genética , Seleção Genética/genética , Proteína bcl-X/genéticaRESUMO
We have studied the encapsulation of human progenitor cells into 3D PEG hydrogels. Replication-incompetent lentivirus promoter reporter vectors were found to efficiently detect the in vivo expression of human hepatic genes in hydrogel-encapsulated liver progenitor cells. Similarly, hydrogel-encapsulated cells could be efficiently infected with hepatitis C virus, and progeny infectious virus could be recovered from the media supernatants of the hydrogels. Provocatively, the diameters of these virus particles range from approximately 50 to 100 nm, while the calculated mesh size of the 8 k hydrogel is 44.6 +/- 1.7 A. To reconcile how viral particles can penetrate the hydrogels to infect the encapsulated cells, we propose that microfractures/defects of the hydrogel result in a functional pore size of up to 20 fold greater than predicted by theoretical mesh calculations. These results suggest a new model of hydrogel structure, and have exciting implications for tissue engineering and hepatitis virus studies.
Assuntos
Materiais Biocompatíveis/química , Hepacivirus/fisiologia , Hepatócitos/virologia , Hidrogéis/química , Polietilenoglicóis/química , Células-Tronco/virologia , Engenharia Tecidual/métodos , Técnicas de Cultura de Células/métodos , Linhagem Celular , Sobrevivência Celular , Simulação por Computador , Hepatócitos/citologia , Humanos , Teste de Materiais , Modelos Biológicos , Porosidade , Células-Tronco/citologiaRESUMO
To investigate the role of DNA methylation during human development, we developed Methyl-seq, a method that assays DNA methylation at more than 90,000 regions throughout the genome. Performing Methyl-seq on human embryonic stem cells (hESCs), their derivatives, and human tissues allowed us to identify several trends during hESC and in vivo liver differentiation. First, differentiation results in DNA methylation changes at a minimal number of assayed regions, both in vitro and in vivo (2%-11%). Second, in vitro hESC differentiation is characterized by both de novo methylation and demethylation, whereas in vivo fetal liver development is characterized predominantly by demethylation. Third, hESC differentiation is uniquely characterized by methylation changes specifically at H3K27me3-occupied regions, bivalent domains, and low density CpG promoters (LCPs), suggesting that these regions are more likely to be involved in transcriptional regulation during hESC differentiation. Although both H3K27me3-occupied domains and LCPs are also regions of high variability in DNA methylation state during human liver development, these regions become highly unmethylated, which is a distinct trend from that observed in hESCs. Taken together, our results indicate that hESC differentiation has a unique DNA methylation signature that may not be indicative of in vivo differentiation.
Assuntos
Metilação de DNA , Células-Tronco Embrionárias/metabolismo , Fígado/metabolismo , Sítios de Ligação , Diferenciação Celular/genética , Linhagem Celular , Células Cultivadas , Mapeamento Cromossômico , Análise por Conglomerados , Ilhas de CpG/genética , Células-Tronco Embrionárias/citologia , Perfilação da Expressão Gênica , Genoma Humano/genética , Histonas/metabolismo , Humanos , Fígado/citologia , Fígado/embriologia , Lisina/metabolismo , Metilação , Regiões Promotoras Genéticas/genética , Análise de Sequência de DNARESUMO
This study describes a cross-species functional screen of mouse gastrula cDNA libraries for components of endoderm and mesoderm specification. Pools of 96 cDNAs from arrayed mouse gastrula cDNA libraries were transcribed into mRNA and injected into either the presumptive mesoderm or the ectoderm of one-cell Xenopus laevis embryos. Injected embryos were examined at gastrula stage by in situ hybridization with endoderm or mesoderm markers. Using this approach, we screened over 700 pools or approximately 60,000 cDNAs. We identified 17 unique cDNAs that function during mesoderm and/or endoderm specification and 16 that cause general morphology changes. Identified molecules fall into eight general functional groups as follows: cell cycle components (seven), transcription factors (four), extracellular secreted molecules (seven), transmembrane receptors (one), intracellular signaling components (five), microtubule components (two), metabolism molecules (three), and unknown (four). Several of the genes we identified would not have been predicted to be involved in endoderm or mesoderm specification, highlighting the usefulness of nonbiased screening approaches. This includes Otx2, which we show is a downstream target of Xsox17beta. The speed, low cost, and high efficiency of this cross-species screen makes it an ideal method for examining cDNAs from difficult-to-obtain sources. Therefore, this approach complements the current mouse molecular genetics systems and provides a powerful means for the genome-wide examination of mammalian gene function.