RESUMO
Despite major advances in linking single genetic variants to single causal genes, the significance of genetic variation on transcript-level regulation of expression, transcript-specific functions, and relevance to human disease has been poorly investigated. Strawberry notch homolog 2 (SBNO2) is a candidate gene in a susceptibility locus with different variants associated with Crohn's disease and bone mineral density. The SBNO2 locus is also differentially methylated in Crohn's disease but the functional mechanisms are unknown. Here we show that the isoforms of SBNO2 are differentially regulated by lipopolysaccharide and IL-10. We identify Crohn's disease associated isoform quantitative trait loci that negatively regulate the expression of the noncanonical isoform 2 corresponding with the methylation signals at the isoform 2 promoter in IBD and CD. The two isoforms of SBNO2 drive differential gene networks with isoform 2 dominantly impacting antimicrobial activity in macrophages. Our data highlight the role of isoform quantitative trait loci to understand disease susceptibility and resolve underlying mechanisms of disease.
Assuntos
Doença de Crohn , Predisposição Genética para Doença , Lipopolissacarídeos , Isoformas de Proteínas , Locos de Características Quantitativas , Doença de Crohn/genética , Humanos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Interleucina-10/genética , Interleucina-10/metabolismo , Regiões Promotoras Genéticas/genética , Metilação de DNA , Macrófagos/metabolismo , Regulação da Expressão GênicaRESUMO
BACKGROUND: Mechanisms governing the diversity of CFTR gene expression throughout the body are complex. Multiple intronic and distal regulatory elements are responsible for regulating differential CFTR expression across tissues. METHODS: Drawing on published data, 18 high-priority genomic regions were identified and interrogated for CFTR-enhancer function using CRISPR/dCas9-based epigenome editing tools. Each region was evaluated by dCas9p300 and dCas9KRAB for its ability to enhance or repress CFTR expression, respectively. RESULTS: Multiple genomic regions were tested for enhancer activity using CRISPR/dCas9 epigenome editing. dCas9p300 mediates a significant increase in CFTR mRNA levels when targeted to the promoter and a region 44 kb upstream of the transcriptional start site in a CFTR-low expressing cell line. Multiple gRNAs targeting the promoter induced a robust increase in CFTR protein levels. In contrast, dCas9KRAB-mediated repression is much more robust with 10 of the 18 evaluated genomic regions inducing CFTR protein knockdown. To evaluate the therapeutic efficacy of modulating CFTR gene regulation, dCas9p300 was used to induce elevated levels of CFTR from the endogenous locus in ΔF508/ΔF508 human bronchial epithelial cells. Ussing chamber studies demonstrated a synergistic increase in ion transport in response to CRISPR-induced expression of ΔF508 CFTR mRNA along with VX809 treatment. CONCLUSIONS: CRISPR/dCas9-based epigenome-editing provides a previously unexplored tool for interrogating CFTR enhancer function. Here, we demonstrate that therapeutic interventions that increase the expression of CFTR may improve the efficacy of CFTR modulators. A better understanding CFTR regulatory mechanisms could uncover novel therapeutic interventions for the development of cystic fibrosis therapies.
Assuntos
Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas/genética , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/genética , Edição de Genes/métodos , Fibrose Cística/tratamento farmacológico , Regulador de Condutância Transmembrana em Fibrose Cística/uso terapêutico , Epigenoma , Regulação da Expressão Gênica , Células HEK293 , Humanos , RNA Guia de Cinetoplastídeos/genéticaRESUMO
Many diseases, such as diabetes, autoimmune diseases, cancer, and neurological disorders, are caused by a dysregulation of a complex interplay of genes. Genome-wide association studies have identified thousands of disease-linked polymorphisms in the human population. However, detailing the causative gene expression or functional changes underlying those associations has been elusive in many cases. Functional genomics is an emerging field of research that aims to deconvolute the link between genotype and phenotype by making use of large -omic data sets and next-generation gene and epigenome editing tools to perturb genes of interest. Here we review how functional genomic tools can be used to better understand the biological interplay between genes, improve disease modeling, and identify novel drug targets. Incorporation of functional genomic capabilities into conventional drug development pipelines is predicted to expedite the development of first-in-class therapeutics.
Assuntos
Descoberta de Drogas/tendências , Predisposição Genética para Doença , Genoma Humano/genética , Genômica/tendências , Sistemas CRISPR-Cas/genética , Epigenoma/genética , Edição de Genes , Estudo de Associação Genômica Ampla , Genótipo , Humanos , Fenótipo , RNA Interferente Pequeno/genéticaRESUMO
Over 5 million people in the United States suffer from heart failure, due to the limited ability to regenerate functional cardiac tissue. One potential therapeutic strategy is to enhance proliferation of resident cardiomyocytes. However, phenotypic screening for therapeutic agents is challenged by the limited ability of conventional markers to discriminate between cardiomyocyte proliferation and endoreplication (e.g. polyploidy and multinucleation). Here, we developed a novel assay that combines automated live-cell microscopy and image processing algorithms to discriminate between proliferation and endoreplication by quantifying changes in the number of nuclei, changes in the number of cells, binucleation, and nuclear DNA content. We applied this assay to further prioritize hits from a primary screen for DNA synthesis, identifying 30 compounds that enhance proliferation of human induced pluripotent stem cell-derived cardiomyocytes. Among the most active compounds from the phenotypic screen are clinically approved L-type calcium channel blockers from multiple chemical classes whose activities were confirmed across different sources of human induced pluripotent stem cell-derived cardiomyocytes. Identification of compounds that stimulate human cardiomyocyte proliferation may provide new therapeutic strategies for heart failure.
Assuntos
Canais de Cálcio Tipo L/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Proliferação de Células , DNA/biossíntese , Humanos , Processamento de Imagem Assistida por Computador , Fenótipo , PloidiasRESUMO
Activation and proliferation of resident cardiac progenitor cells has therapeutic potential to repair the heart after injury. However, research has been impeded by a lack of well-defined and characterized cell sources and difficulties in translation to screening platforms. Here, we describe the development, validation, and use of a 384-well phenotypic assay in primary human epicardium-derived cells (EPDCs) to identify compounds that induce proliferation while maintaining the progenitor phenotype. Using this assay, we screened 7400 structurally diverse compounds where greater than 90% are biologically annotated and known to modulate a broad range of biological targets. From the primary screen, we identified and validated hits and expanded upon the lead molecules of interest. A counterscreen was developed in human cardiac fibroblasts to filter out compounds with a general proliferative effect, after which the activity of selected molecules was confirmed across multiple EPDC donors. To further examine the mechanism of action of compounds with annotated targets, we performed knockdown experiments to understand whether a single known target was responsible for the proliferative effect, confirming results with protein expression and activity assays. Here, we were able to show that the annotated targets of compounds of interest were not responsible for the proliferative effect, which highlights potential differences in cell types and signaling pathways and possible polypharmacology. These studies demonstrate the feasibility of using relevant human primary cells in a phenotypic screen to identify compounds as novel biological tools and starting points for drug discovery projects, and we disclose the first small molecules to proliferate human primary EPDCs.
Assuntos
Proliferação de Células/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Miocárdio/citologia , Pericárdio/efeitos dos fármacos , Técnicas de Cultura de Células , Células Cultivadas , Descoberta de Drogas , Fibroblastos/citologia , Fibroblastos/metabolismo , Coração/efeitos dos fármacos , Coração/fisiologia , Humanos , Miocárdio/metabolismo , Pericárdio/citologia , Pericárdio/metabolismo , Fenótipo , Regeneração/efeitos dos fármacosRESUMO
Several progenitor cell populations have been reported to exist in hearts that play a role in cardiac turnover and/or repair. Despite the presence of cardiac stem and progenitor cells within the myocardium, functional repair of the heart after injury is inadequate. Identification of the signaling pathways involved in the expansion and differentiation of cardiac progenitor cells (CPCs) will broaden insight into the fundamental mechanisms playing a role in cardiac homeostasis and disease and might provide strategies for in vivo regenerative therapies. To understand and exploit cardiac ontogeny for drug discovery efforts, we developed an in vitro human induced pluripotent stem cell-derived CPC model system using a highly enriched population of KDR(pos)/CKIT(neg)/NKX2.5(pos) CPCs. Using this model system, these CPCs were capable of generating highly enriched cultures of cardiomyocytes under directed differentiation conditions. In order to facilitate the identification of pathways and targets involved in proliferation and differentiation of resident CPCs, we developed phenotypic screening assays. Screening paradigms for therapeutic applications require a robust, scalable, and consistent methodology. In the present study, we have demonstrated the suitability of these cells for medium to high-throughput screens to assess both proliferation and multilineage differentiation. Using this CPC model system and a small directed compound set, we identified activin-like kinase 5 (transforming growth factor-ß type 1 receptor kinase) inhibitors as novel and potent inducers of human CPC differentiation to cardiomyocytes. Significance: Cardiac disease is a leading cause of morbidity and mortality, with no treatment available that can result in functional repair. This study demonstrates how differentiation of induced pluripotent stem cells can be used to identify and isolate cell populations of interest that can translate to the adult human heart. Two separate examples of phenotypic screens are discussed, demonstrating the value of this biologically relevant and reproducible technology. In addition, this assay system was able to identify novel and potent inducers of differentiation and proliferation of induced pluripotent stem cell-derived cardiac progenitor cells.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Receptores de Fatores de Crescimento Transformadores beta/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Biomarcadores/metabolismo , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Expressão Gênica , Ensaios de Triagem em Larga Escala , Proteína Homeobox Nkx-2.5 , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Fenótipo , Plasmídeos/química , Plasmídeos/metabolismo , Cultura Primária de Células , Inibidores de Proteínas Quinases/química , Proteínas Proto-Oncogênicas c-kit/deficiência , Proteínas Proto-Oncogênicas c-kit/genética , Receptores de Fatores de Crescimento Transformadores beta/genética , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Bibliotecas de Moléculas Pequenas/química , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transfecção , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
We have previously reported the high regenerative potential of murine muscle-derived stem cells (mMDSCs) that are capable of differentiating into multiple mesodermal cell lineages, including myogenic, endothelial, chondrocytic, and osteoblastic cells. Recently, we described a putative human counterpart of mMDSCs, the myogenic endothelial cells (MECs), in adult human skeletal muscle, which efficiently repair/regenerate the injured and dystrophic skeletal muscle as well as the ischemic heart in animal disease models. Nevertheless it remained unclear whether human MECs, at the clonal level, preserve mMDSC-like chondrogenic and osteogenic potentials and classic stem cell characteristics including high proliferation and resistance to stress. Herein, we demonstrated that MECs, sorted from fresh postnatal human skeletal muscle biopsies, can be grown clonally and exhibit robust resistance to oxidative stress with no tumorigeneity. MEC clones were capable of differentiating into chondrocytes and osteoblasts under inductive conditions in vitro and participated in cartilage and bone formation in vivo. Additionally, adipogenic and angiogenic potentials of clonal MECs (cMECs) were observed. Overall, our study showed that cMECs not only display typical properties of adult stem cells but also exhibit chondrogenic and osteogenic capacities in vitro and in vivo, suggesting their potential applications in articular cartilage and bone repair/regeneration.
Assuntos
Diferenciação Celular/fisiologia , Condrogênese/fisiologia , Desenvolvimento Muscular/fisiologia , Músculo Esquelético/citologia , Músculo Esquelético/fisiologia , Osteogênese/fisiologia , Adipócitos/citologia , Adulto , Animais , Biópsia , Proliferação de Células , Células Cultivadas , Condrócitos/citologia , Endotélio/citologia , Endotélio/fisiologia , Humanos , Técnicas In Vitro , Masculino , Camundongos , Camundongos SCID , Osteoblastos/citologia , Estresse Oxidativo/fisiologia , Transplante HeterólogoRESUMO
Stem cell therapy for tissue repair is a rapidly evolving field and the factors that dictate the physiological responsiveness of stem cells remain under intense investigation. In this study we hypothesized that the mechanical loading history of muscle-derived stem cells (MDSCs) would significantly impact MDSC survival, host tissue angiogenesis, and myocardial function after MDSC transplantation into acutely infarcted myocardium. Mice with acute myocardial infarction by permanent left coronary artery ligation were injected with either nonstimulated (NS) or mechanically stimulated (MS) MDSCs. Mechanical stimulation consisted of stretching the cells with equibiaxial stretch with a magnitude of 10% and frequency of 0.5 Hz. MS cell-transplanted hearts showed improved cardiac contractility, increased numbers of host CD31+ cells, and decreased fibrosis, in the peri-infarct region, compared to the hearts treated with NS MDSCs. MS MDSCs displayed higher vascular endothelial growth factor expression than NS cells in vitro. These findings highlight an important role for cyclic mechanical loading preconditioning of donor MDSCs in optimizing MDSC transplantation for myocardial repair.
Assuntos
Infarto do Miocárdio/terapia , Transplante de Células-Tronco , Células-Tronco/citologia , Estresse Mecânico , Animais , Fenômenos Biomecânicos , Diferenciação Celular , Proliferação de Células , Sobrevivência Celular , Cicatriz/patologia , Cicatriz/fisiopatologia , Modelos Animais de Doenças , Testes de Função Cardíaca , Precondicionamento Isquêmico Miocárdico , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Infarto do Miocárdio/patologia , Infarto do Miocárdio/fisiopatologia , Miocárdio/patologia , Neovascularização Fisiológica , Estresse Oxidativo , Resultado do Tratamento , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Identification of cells that are endowed with maximum potency could be critical for the clinical success of cell-based therapies. We investigated whether cells with an enhanced efficacy for cardiac cell therapy could be enriched from adult human skeletal muscle on the basis of their adhesion properties to tissue culture flasks following tissue dissociation. Cells that adhered slowly displayed greater myogenic purity and more readily differentiated into myotubes in vitro than rapidly adhering cells (RACs). The slowly adhering cell (SAC) population also survived better than the RAC population in kinetic in vitro assays that simulate conditions of oxidative and inflammatory stress. When evaluated for the treatment of a myocardial infarction (MI), intramyocardial injection of the SACs more effectively improved echocardiographic indexes of left ventricular (LV) remodeling and contractility than the transplantation of the RACs. Immunohistological analysis revealed that hearts injected with SACs displayed a reduction in myocardial fibrosis and an increase in infarct vascularization, donor cell proliferation, and endogenous cardiomyocyte survival and proliferation in comparison with the RAC-treated hearts. In conclusion, these results suggest that adult human skeletal muscle-derived cells are inherently heterogeneous with regard to their efficacy for enhancing cardiac function after cardiac implantation, with SACs outperforming RACs.
Assuntos
Fibras Musculares Esqueléticas/transplante , Isquemia Miocárdica/terapia , Estresse Fisiológico , Adolescente , Idoso , Animais , Apoptose/genética , Adesão Celular , Diferenciação Celular , Proliferação de Células , Sobrevivência Celular/genética , Cicatriz/patologia , Perfilação da Expressão Gênica , Humanos , Imunofenotipagem , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Pessoa de Meia-Idade , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/metabolismo , Isquemia Miocárdica/metabolismo , Miocárdio/metabolismo , Miocárdio/patologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Neovascularização Fisiológica , Estresse OxidativoRESUMO
Although cellular transplantation has been shown to promote improvements in cardiac function following injury, poor cell survival following transplantation continues to limit the efficacy of this therapy. We have previously observed that transplantation of muscle-derived stem cells (MDSCs) improves cardiac function in an acute murine model of myocardial infarction to a greater extent than myoblasts. This improved regenerative capacity of MDSCs is linked to their increased level of antioxidants such as glutathione (GSH) and superoxide dismutase. In the current study, we demonstrated the pivotal role of antioxidant levels on MDSCs survival and cardiac functional recovery by either reducing the antioxidant levels with diethyl maleate or increasing antioxidant levels with N-acetylcysteine (NAC). Both the anti- and pro-oxidant treatments dramatically influenced the survival of the MDSCs in vitro. When NAC-treated MDSCs were transplanted into infarcted myocardium, we observed significantly improved cardiac function, decreased scar tissue formation, and increased numbers of CD31(+) endothelial cell structures, compared to the injection of untreated and diethyl maleate-treated cells. These results indicate that elevating the levels of antioxidants in MDSCs with NAC can significantly influence their tissue regeneration capacity.
Assuntos
Antioxidantes/metabolismo , Terapia Baseada em Transplante de Células e Tecidos/métodos , Músculo Esquelético/citologia , Células-Tronco/metabolismo , Acetilcisteína/farmacologia , Animais , Apoptose/efeitos dos fármacos , Western Blotting , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Ensaio de Imunoadsorção Enzimática , Feminino , Masculino , Maleatos/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos SCID , Infarto do Miocárdio/terapia , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
We have previously shown that populations of skeletal muscle-derived stem cells (MDSCs) exhibit sex-based differences for skeletal muscle and bone repair, with female cells demonstrating superior engrafting abilities to males in skeletal muscle while male cells differentiating more robustly toward the osteogenic and chondrogenic lineages. In this study, we tested the hypothesis that the therapeutic capacity of MDSCs transplanted into myocardium is influenced by sex of donor MDSCs or recipient. Male and female MDSCs isolated from the skeletal muscle of 3-week-old mice were transplanted into recipient male or female dystrophin-deficient (mdx) hearts or into the hearts of male SCID mice following acute myocardial infarction. In the mdx model, no difference was seen in engraftment or blood vessel formation based on donor cell or recipient sex. In the infarction model, MDSC-transplanted hearts showed higher postinfarction angiogenesis, less myocardial scar formation, and improved cardiac function compared to vehicle controls. However, sex of donor MDSCs had no significant effects on engraftment, angiogenesis, and cardiac function. VEGF expression, a potent angiogenic factor, was similar between male and female MDSCs. Our results suggest that donor MDSC or recipient sex has no significant effect on the efficiency of MDSC-triggered myocardial engraftment or regeneration following cardiac injury. The ability of the MDSCs to improve cardiac regeneration and repair through promotion of angiogenesis without differentiation into the cardiac lineage may have contributed to the lack of sex difference observed in these models.
Assuntos
Músculo Esquelético/citologia , Infarto do Miocárdio/terapia , Transplante de Células-Tronco , Animais , Linhagem da Célula , Distrofina/deficiência , Distrofina/genética , Distrofina/metabolismo , Feminino , Coração/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos SCID , Miocárdio/patologia , Fatores Sexuais , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
We document anatomic, molecular and developmental relationships between endothelial and myogenic cells within human skeletal muscle. Cells coexpressing myogenic and endothelial cell markers (CD56, CD34, CD144) were identified by immunohistochemistry and flow cytometry. These myoendothelial cells regenerate myofibers in the injured skeletal muscle of severe combined immunodeficiency mice more effectively than CD56+ myogenic progenitors. They proliferate long term, retain a normal karyotype, are not tumorigenic and survive better under oxidative stress than CD56+ myogenic cells. Clonally derived myoendothelial cells differentiate into myogenic, osteogenic and chondrogenic cells in culture. Myoendothelial cells are amenable to biotechnological handling, including purification by flow cytometry and long-term expansion in vitro, and may have potential for the treatment of human muscle disease.