RESUMO
MEGF10 myopathy is a rare inherited muscle disease that is named after the causative gene, MEGF10. The classic phenotype, early onset myopathy, areflexia, respiratory distress and dysphagia, is severe and immediately life-threatening. There are no disease-modifying therapies. We performed a small molecule screen and follow-up studies to seek a novel therapy. A primary in vitro drug screen assessed cellular proliferation patterns in Megf10-deficient myoblasts. Secondary evaluations were performed on primary screen hits using myoblasts derived from Megf10-/- mice, induced pluripotent stem cell-derived myoblasts from MEGF10 myopathy patients, mutant Drosophila that are deficient in the homologue of MEGF10 (Drpr) and megf10 mutant zebrafish. The screen yielded two promising candidates that are both selective serotonin reuptake inhibitors (SSRIs), sertraline and escitalopram. In depth follow-up analyses demonstrated that sertraline was highly effective in alleviating abnormalities across multiple models of the disease including mouse myoblast, human myoblast, Drosophila and zebrafish models. Sertraline also restored deficiencies of Notch1 in disease models. We conclude that SSRIs show promise as potential therapeutic compounds for MEGF10 myopathy, especially sertraline. The mechanism of action may involve the Notch pathway.
Assuntos
Proteínas de Membrana/genética , Doenças Musculares/tratamento farmacológico , Mioblastos/efeitos dos fármacos , Inibidores Seletivos de Recaptação de Serotonina/uso terapêutico , Sertralina/uso terapêutico , Animais , Linhagem Celular , Movimento Celular , Proliferação de Células , Citalopram/farmacologia , Citalopram/uso terapêutico , Drosophila/efeitos dos fármacos , Drosophila/genética , Avaliação Pré-Clínica de Medicamentos , Humanos , Camundongos , Camundongos Knockout , Músculo Esquelético/metabolismo , Doenças Musculares/genética , Mutação , Mioblastos/metabolismo , Receptor Notch1/metabolismo , Inibidores Seletivos de Recaptação de Serotonina/farmacologia , Sertralina/farmacologia , Transdução de Sinais , Peixe-Zebra/genética , Peixe-Zebra/metabolismoRESUMO
Myotonic dystrophy type 1 (DM1) is caused by a CTG nucleotide repeat expansion within the 3' UTR of the Dystrophia Myotonica protein kinase gene. In this study, we explored therapeutic genome editing using CRISPR/Cas9 via targeted deletion of expanded CTG repeats and targeted insertion of polyadenylation signals in the 3' UTR upstream of the CTG repeats to eliminate toxic RNA CUG repeats. We found paired SpCas9 or SaCas9 guide RNA induced deletion of expanded CTG repeats. However, this approach incurred frequent inversion in both the mutant and normal alleles. In contrast, the insertion of polyadenylation signals in the 3' UTR upstream of the CTG repeats eliminated toxic RNA CUG repeats, which led to phenotype reversal in differentiated neural stem cells, forebrain neurons, cardiomyocytes, and skeletal muscle myofibers. We concluded that targeted insertion of polyadenylation signals in the 3' UTR is a viable approach to develop therapeutic genome editing for DM1.
Assuntos
Distrofia Miotônica/genética , Miotonina Proteína Quinase/genética , Células-Tronco Neurais/fisiologia , Expansão das Repetições de Trinucleotídeos/genética , Regiões 3' não Traduzidas , Sistemas CRISPR-Cas/genética , Diferenciação Celular/genética , Edição de Genes/métodos , Terapia Genética/métodos , Células HEK293 , Humanos , Músculo Esquelético/crescimento & desenvolvimento , Miócitos Cardíacos/fisiologia , Distrofia Miotônica/patologia , Distrofia Miotônica/terapia , Neurônios/fisiologia , Sinais de Poliadenilação na Ponta 3' do RNA/genética , RNA Guia de Cinetoplastídeos , TransfecçãoRESUMO
Recent advances in DNA sequencing technologies are revealing how human genetic variations associate with differential health risks, disease susceptibilities, and drug responses. Such information is now expected to help evaluate individual health risks, design personalized health plans and treat patients with precision. It is still challenging, however, to understand how such genetic variations cause the phenotypic alterations in pathobiologies and treatment response. Human induced pluripotent stem cell (iPSC) technologies are emerging as a promising strategy to fill the knowledge gaps between genetic association studies and underlying molecular mechanisms. Breakthroughs in genome editing technologies and continuous improvement in iPSC differentiation techniques are particularly making this research direction more realistic and practical. Pioneering studies have shown that iPSCs derived from a variety of monogenic diseases can faithfully recapitulate disease phenotypes in vitro when differentiated into disease-relevant cell types. It has been shown possible to partially recapitulate disease phenotypes, even with late onset and polygenic diseases. More recently, iPSCs have been shown to validate effects of disease and treatment-related single nucleotide polymorphisms identified through genome wide association analysis. In this review, we will discuss how iPSC research will further contribute to human health in the coming era of precision medicine. Stem Cells 2017;35:545-550.
Assuntos
Células-Tronco Pluripotentes Induzidas/citologia , Medicina de Precisão , Pesquisa com Células-Tronco , Animais , Estudo de Associação Genômica Ampla , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Fenótipo , Polimorfismo de Nucleotídeo Único/genéticaRESUMO
Induced pluripotent stem cell (iPSC) technology was originally developed in 2006. Essentially, it converts somatic cells into pluripotent stem cells by transiently expressing a few transcriptional factors. Once generated, these iPSCs can differentiate into all the cell types of our body, theoretically, which has attracted great attention for clinical research including disease pathobiology studies. Could this technology then become an additional research or diagnostic tool widely available to practicing pathologists? Here we summarize progress in iPSC research toward disease pathobiology studies, its future potential, and remaining problems from a pathologist's perspective. A particular focus will be on introducing the effort to recapitulate disease-related morphological changes through three-dimensional culture of stem cells such as organoid differentiation.
Assuntos
Células-Tronco Pluripotentes Induzidas , Patologia Clínica , Pesquisa com Células-Tronco , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/fisiologia , PatologistasRESUMO
Myotonic dystrophy type 1 (DM1) is caused by expanded Cytosine-Thymine-Guanine (CTG) repeats in the 3'-untranslated region (3' UTR) of the Dystrophia myotonica protein kinase (DMPK) gene, for which there is no effective therapy. The objective of this study is to develop genome therapy in human DM1 induced pluripotent stem (iPS) cells to eliminate mutant transcripts and reverse the phenotypes for developing autologous stem cell therapy. The general approach involves targeted insertion of polyA signals (PASs) upstream of DMPK CTG repeats, which will lead to premature termination of transcription and elimination of toxic mutant transcripts. Insertion of PASs was mediated by homologous recombination triggered by site-specific transcription activator-like effector nuclease (TALEN)-induced double-strand break. We found genome-treated DM1 iPS cells continue to maintain pluripotency. The insertion of PASs led to elimination of mutant transcripts and complete disappearance of nuclear RNA foci and reversal of aberrant splicing in linear-differentiated neural stem cells, cardiomyocytes, and teratoma tissues. In conclusion, genome therapy by insertion of PASs upstream of the expanded DMPK CTG repeats prevented the production of toxic mutant transcripts and reversal of phenotypes in DM1 iPS cells and their progeny. These genetically-treated iPS cells will have broad clinical application in developing autologous stem cell therapy for DM1.
Assuntos
Terapia Genética , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Distrofia Miotônica/genética , Distrofia Miotônica/metabolismo , Transplante de Células-Tronco , Animais , Diferenciação Celular , Núcleo Celular/metabolismo , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Modelos Animais de Doenças , Marcação de Genes , Loci Gênicos , Humanos , Camundongos , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Distrofia Miotônica/terapia , Miotonina Proteína Quinase/genética , Poli A , Ligação Proteica , Splicing de RNA , Teratoma/genética , Teratoma/metabolismo , Teratoma/patologia , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição , Transplante Autólogo , Repetições de TrinucleotídeosRESUMO
Approximately 3 years ago, we assessed how patient induced pluripotent stem cell (iPSC) research could potentially impact human pathobiology studies in the future. Since then, the field has grown considerably with numerous technical developments, and the idea of modeling diseases 'in a dish' is becoming increasingly popular in biomedical research. Likely, it is even acceptable to include patient iPSCs as one of the standard research tools for disease mechanism studies, just like knockout mice. However, as the field matures, we acknowledge there remain many practical limitations and obstacles for their genuine application to understand diseases, and accept that it has not been as straightforward to model disorders as initially proposed. A major practical challenge has been efficient direction of iPSC differentiation into desired lineages and preparation of the large numbers of specific cell types required for study. Another even larger obstacle is the limited value of in vitro outcomes, which often do not closely represent disease conditions. To overcome the latter issue, many new approaches are underway, including three-dimensional organoid cultures from iPSCs, xenotransplantation of human cells to animal models and in vitro interaction of multiple cell types derived from isogenic iPSCs. Here we summarize the areas where patient iPSC studies have provided truly valuable information beyond existing skepticism, discuss the desired technologies to overcome current limitations and include practical guidance for how to utilize the resources. Undoubtedly, these human patient cells are an asset for experimental pathology studies. The future rests on how wisely we use them.
Assuntos
Pesquisa Biomédica/métodos , Células-Tronco Pluripotentes/citologia , HumanosRESUMO
Nanog or Gata6-positive cells co-exist and are convertible within the inner cell mass of murine blastocysts and embryonic stem (ES) cells. Previous studies demonstrate fibroblast growth factor receptor 2 (FGFR2) triggers Nanog gene down-regulation and differentiation to primitive endoderm (PE); however, the underlying mechanisms responsible for reversible and fluctuating cell fate are poorly understood. Using an inducible FGFR2 dimerization system in ES cells, we demonstrate that FGFR2 activation rapidly down-regulated Nanog gene transcription through activation of the Mek pathway and subsequently differentiated ES cells into PE cells. FGFR2 rather selectively repressed the Nanog gene with minimal effect on other pluripotency genes, including Oct4 and Sox2. We determined the Nanog promoter region containing minimum Oct4/Sox2 binding sites was sufficient for this transcriptional down-regulation by FGFR2, when the reporter transgenes were integrated with insulators. Of interest, FGFR2-mediated Nanog transcriptional reduction occurred without dissociation of RNA polymerase II, p300, Oct4, Sox2, and Tet1 from the Nanog proximal promoter region and with no increase in repressive histone methylation marks or DNA methylation, implying the gene repression is in the early and transient phase. Furthermore, addition of a specific FGFR inhibitor readily reversed this Nanog repression status. These findings illustrate well how FGFR2 induces rapid but reversible Nanog repression within ES cells.
Assuntos
Células-Tronco Embrionárias/metabolismo , Proteínas de Homeodomínio/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Multimerização Proteica/fisiologia , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/metabolismo , Transcrição Gênica/fisiologia , Animais , Linhagem Celular , Metilação de DNA/fisiologia , Células-Tronco Embrionárias/citologia , Endoderma/citologia , Endoderma/embriologia , Proteínas de Homeodomínio/genética , Camundongos , Proteína Homeobox Nanog , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/genética , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismoRESUMO
Stem cell-derived ß-like cells (sBC) carry the promise of providing an abundant source of insulin-producing cells for use in cell replacement therapy for patients with diabetes, potentially allowing widespread implementation of a practical cure. To achieve their clinical promise, sBC need to function comparably with mature adult ß-cells, but as yet they display varying degrees of maturity. Indeed, detailed knowledge of the events resulting in human ß-cell maturation remains obscure. Here we show that sBC spontaneously self-enrich into discreet islet-like cap structures within in vitro cultures, independent of exogenous maturation conditions. Multiple complementary assays demonstrate that this process is accompanied by functional maturation of the self-enriched sBC (seBC); however, the seBC still contain distinct subpopulations displaying different maturation levels. Interestingly, the surface protein ENTPD3 (also known as nucleoside triphosphate diphosphohydrolase-3 [NDPTase3]) is a specific marker of the most mature seBC population and can be used for mature seBC identification and sorting. Our results illuminate critical aspects of in vitro sBC maturation and provide important insights toward developing functionally mature sBC for diabetes cell replacement therapy.
Assuntos
Adenosina Trifosfatases/metabolismo , Células-Tronco Embrionárias/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Células Secretoras de Insulina/metabolismo , Adenosina Trifosfatases/genética , Cálcio/metabolismo , DNA Mitocondrial , Regulação da Expressão Gênica , Humanos , TranscriptomaRESUMO
Type 1 diabetes (T1D) is a disease that arises due to complex immunogenetic mechanisms. Key cell-cell interactions involved in the pathogenesis of T1D are activation of autoreactive T cells by dendritic cells (DC), migration of T cells across endothelial cells (EC) lining capillary walls into the islets of Langerhans, interaction of T cells with macrophages in the islets, and killing of ß-cells by autoreactive CD8+ T cells. Overall, pathogenic cell-cell interactions are likely regulated by the individual's collection of genetic T1D-risk variants. To accurately model the role of genetics, it is essential to build systems to interrogate single candidate genes in isolation during the interactions of cells that are essential for disease development. However, obtaining single-donor matched cells relevant to T1D is a challenge. Sourcing these genetic variants from human induced pluripotent stem cells (iPSC) avoids this limitation. Herein, we have differentiated iPSC from one donor into DC, macrophages, EC, and ß-cells. Additionally, we also engineered T cell avatars from the same donor to provide an in vitro platform to study genetic influences on these critical cellular interactions. This proof of concept demonstrates the ability to derive an isogenic system from a single donor to study these relevant cell-cell interactions. Our system constitutes an interdisciplinary approach with a controlled environment that provides a proof-of-concept for future studies to determine the role of disease alleles (e.g. IFIH1, PTPN22, SH2B3, TYK2) in regulating cell-cell interactions and cell-specific contributions to the pathogenesis of T1D.
Assuntos
Linfócitos T CD8-Positivos/patologia , Diabetes Mellitus Tipo 1/patologia , Células-Tronco Pluripotentes Induzidas/patologia , Diferenciação Celular/fisiologia , Humanos , Células Secretoras de Insulina/patologia , Ilhotas Pancreáticas/patologiaRESUMO
Since it is extremely difficult to establish an animal model for human chromosomal abnormalities, induced pluripotent stem cells (iPSCs) provide a powerful alternative to study underlying mechanisms of these disorders and identify potential therapeutic interventions. In this study we established iPSCs from a young girl with a hemizygous deletion of Xq27.3-q28 who exhibited global developmental delay and intellectual disability from early in infancy. The deletion site on the X chromosome includes Fragile X Mental Retardation 1 (FMR1), the gene responsible for fragile X syndrome, which likely contributes to the patient's neurodevelopmental abnormalities. The FMR1 gene was expressed in approximately half of the iPSC clones we generated while it was absent in the other half due to the random inactivation of normal and abnormal X chromosomes. The normal or absent expression pattern of the FMR1 gene was not altered when the iPSCs were differentiated into neural progenitor cells (NPCs). Moreover, chromosome reactivating reagents such as 5-aza-2-deoxycytidine, trichostatin A, and UNC0638, were tested in an attempt to reactivate the suppressed FMR1 gene in affected iPSC-NPCs. The affected and control isogenic iPSCs developed in this study are ideal models with which to identify downstream consequences caused by the Xq27.3-q28 deletion and also to provide tools for high-throughput screening to identify compounds potentially improving the well-being of this patient population.
Assuntos
Proteína do X Frágil da Deficiência Intelectual/genética , Síndrome do Cromossomo X Frágil/tratamento farmacológico , Síndrome do Cromossomo X Frágil/genética , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Diferenciação Celular , Células Cultivadas , Pré-Escolar , Deleção Cromossômica , Cromossomos Humanos X/genética , Decitabina/farmacologia , Deficiências do Desenvolvimento/tratamento farmacológico , Deficiências do Desenvolvimento/genética , Feminino , Inibidores de Histona Desacetilases/farmacologia , Humanos , Ácidos Hidroxâmicos/farmacologia , Deficiência Intelectual/tratamento farmacológico , Deficiência Intelectual/genética , Quinazolinas/farmacologiaRESUMO
Patient-derived induced pluripotent stem cells (iPSCs) have become a promising resource for exploring genetics of complex diseases, discovering new drugs, and advancing regenerative medicine. Increasingly, laboratories are creating their own banks of iPSCs derived from diverse donors. However, there are not yet standardized guidelines for qualifying these cell lines, i.e., distinguishing between bona fide human iPSCs, somatic cells, and imperfectly reprogrammed cells. Here, we report the establishment of a panel of 30 iPSCs from CD34+ peripheral blood mononuclear cells, of which 10 were further differentiated in vitro into all three germ layers. We characterized these different cell types with commonly used pluripotent and lineage specific markers, and showed that NES, TUBB3, and OTX2 cannot be reliably used as ectoderm differentiation markers. Our work highlights the importance of marker selection in iPSC authentication, and the need for the field to establish definitive standard assays.
Assuntos
Antígenos de Diferenciação/metabolismo , Biomarcadores/metabolismo , Diferenciação Celular , Ectoderma/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Leucócitos Mononucleares/metabolismo , Células Cultivadas , Ectoderma/citologia , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Leucócitos Mononucleares/citologiaRESUMO
The IFN-stimulated gene ubiquitin-specific proteinase 18 (USP18) encodes a protein that negatively regulates T1 IFN signaling via stearic inhibition of JAK1 recruitment to the IFN-α receptor 2 subunit (IFNAR2). Here, we demonstrate that USP18 expression is induced by HIV-1 in a T1 IFN-dependent manner. Experimental depletion of USP18 by clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) gene editing results in a significant restriction of HIV-1 replication in an induced pluripotent stem cell (iPSC)-derived macrophage model. In the absence of USP18, macrophages have increased responsiveness to stimulation with T1 IFNs with prolonged phosphorylation of STAT1 and STAT2 and increased expression of IFN-stimulated genes that are key for antiviral responses. Interestingly, HIV-1 requires some signaling through the T1 IFN receptor to replicate efficiently because a neutralizing antibody that inhibits T1 IFN activity reduces HIV-1 replication rate in monocyte-derived macrophages. USP18 induction by HIV-1 tunes the IFN response to optimal levels allowing for efficient transcription from the HIV-1 LTR promoter while minimizing the T1 IFN-induced antiviral response that would otherwise restrict viral replication and spread. Finally, iPSC and CRISPR/Cas9 gene targeting offer a powerful tool to study host factors that regulate innate immune responses.
RESUMO
Virus-mediated expression of defined transcription factor (TF) genes can effectively induce cellular reprogramming. However, sustained expression of the TFs often hinders pluripotent stem cell (PSC) differentiation into specific cell types, as each TF exerts its effect on PSCs for a defined period of time during differentiation. Here, we applied a bacterial type III secretion system (T3SS)-based protein delivery tool to directly translocate TFs in the form of protein into human PSCs. This transient protein delivery technique showed high delivery efficiency for hPSCs, and it avoids potential genetic alterations caused by the introduction of transgenes. In an established cardiomyocyte de novo differentiation procedure, five transcriptional factors, namely GATA4, MEF2C, TBX5, ESRRG and MESP1 (abbreviated as GMTEM), were translocated at various time points. By detecting the expression of cardiac marker genes (Nkx2.5 and cTnT), we found that GMTEM proteins delivered on mesoderm stage of the cardiomyocytes lineage differentiation significantly enhanced both the human ESC and iPSC differentiation into cardiomyocytes, while earlier or later delivery diminished the enhancing effect. Furthermore, all of the five factors were required to enhance the cardiac differentiation. This work provides a virus-free strategy of transient transcription factors delivery for directing human stem cell fate without jeopardizing genome integrity, thus safe for biomedical applications.
Assuntos
Bactérias/genética , Reprogramação Celular , Células-Tronco Pluripotentes Induzidas/citologia , Miócitos Cardíacos/citologia , Sistemas de Secreção Tipo III/genética , Diferenciação Celular , Linhagem Celular , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Miócitos Cardíacos/metabolismo , Plasmídeos/genética , Plasmídeos/metabolismo , Pseudomonas aeruginosa/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Isocitrate dehydrogenase (IDH) 2 participates in the TCA cycle and catalyzes the conversion of isocitrate to α-ketoglutarate and NADP+ to NADPH. In the mitochondria, IDH2 also plays a key role in protecting mitochondrial components from oxidative stress by supplying NADPH to both glutathione reductase (GSR) and thioredoxin reductase 2 (TXNRD2). Here, we report that loss of Idh2 accelerates age-related hearing loss, the most common form of hearing impairment, in male mice. This was accompanied by increased oxidative DNA damage, increased apoptotic cell death, and profound loss of spiral ganglion neurons and hair cells in the cochlea of 24-month-old Idh2-/- mice. In young male mice, loss of Idh2 resulted in decreased NADPH redox state and decreased activity of TXNRD2 in the mitochondria of the inner ear. In HEI-OC1 mouse inner ear cell lines, knockdown of Idh2 resulted in a decline in cell viability and mitochondrial oxygen consumption. This was accompanied by decreased NADPH redox state and decreased activity of TXNRD2 in the mitochondria of the HEI-OC1 cells. Therefore, IDH2 functions as the principal source of NADPH for the mitochondrial thioredoxin antioxidant defense and plays an essential role in protecting hair cells and neurons against oxidative stress in the cochlea of male mice.
Assuntos
Envelhecimento/genética , Perda Auditiva/genética , Isocitrato Desidrogenase/genética , Tiorredoxina Redutase 2/genética , Envelhecimento/patologia , Animais , Apoptose/genética , Sobrevivência Celular/genética , Cóclea/metabolismo , Cóclea/patologia , Modelos Animais de Doenças , Glutationa Redutase/genética , Células Ciliadas Auditivas/metabolismo , Células Ciliadas Auditivas/patologia , Perda Auditiva/metabolismo , Perda Auditiva/patologia , Humanos , Masculino , Camundongos , Mitocôndrias/genética , Mitocôndrias/metabolismo , NADP/metabolismo , Neurônios/metabolismo , Neurônios/patologia , Estresse Oxidativo/genética , Consumo de Oxigênio/genética , Gânglio Espiral da Cóclea/metabolismo , Gânglio Espiral da Cóclea/patologiaRESUMO
At least 57 independent loci within the human genome confer varying degrees of risk for the development of type 1 diabetes (T1D). The majority of these variants are thought to contribute to overall genetic risk by modulating host innate and adaptive immune responses, ultimately resulting in a loss of immunological tolerance to ß cell antigens. Early efforts to link specific risk variants with functional alterations in host immune responses have employed animal models or genotype-selected individuals from clinical bioresource banks. While some notable genotype:phenotype associations have been described, there remains an urgent need to accelerate the discovery of causal variants and elucidate the molecular mechanisms by which susceptible alleles alter immune functions. One significant limitation has been the inability to study human T1D risk loci on an isogenic background. The advent of induced pluripotent stem cells (iPSCs) and genome-editing technologies have made it possible to address a number of these outstanding questions. Specifically, the ability to drive multiple cell fates from iPSC under isogenic conditions now facilitates the analysis of causal variants in multiple cellular lineages. Bioinformatic analyses have revealed that T1D risk genes cluster within a limited number of immune signaling pathways, yet the relevant immune cell subsets and cellular activation states in which candidate risk genes impact cellular activities remain largely unknown. In this review, we summarize the functional impact of several candidate risk variants on host immunity in T1D and present an isogenic disease-in-a-dish model system for interrogating risk variants, with the goal of expediting precision therapeutics in T1D.
RESUMO
Many breast cancer deaths result from tumors acquiring resistance to available therapies. Thus, new therapeutic agents are needed for targeting drug-resistant breast cancers. Drug-refractory breast cancers include HER2+ tumors that have acquired resistance to HER2-targeted antibodies and kinase inhibitors, and "Triple-Negative" Breast Cancers (TNBCs) that lack the therapeutic targets Estrogen Receptor, Progesterone Receptor, and HER2. A significant fraction of TNBCs overexpress the HER2 family member Epidermal Growth Factor Receptor (EGFR). Thus agents that selectively kill EGFR+ and HER2+ tumors would provide new options for breast cancer therapy. We previously identified a class of compounds we termed Disulfide bond Disrupting Agents (DDAs) that selectively kill EGFR+ and HER2+ breast cancer cells in vitro and blocked the growth of HER2+ breast tumors in an animal model. DDA-dependent cytotoxicity was found to correlate with downregulation of HER1-3 and Akt dephosphorylation. Here we demonstrate that DDAs activate the Unfolded Protein Response (UPR) and that this plays a role in their ability to kill EGFR+ and HER2+ cancer cells. The use of breast cancer cell lines ectopically expressing EGFR or HER2 and pharmacological probes of UPR revealed all three DDA responses: HER1-3 downregulation, Akt dephosphorylation, and UPR activation, contribute to DDA-mediated cytotoxicity. Significantly, EGFR overexpression potentiates each of these responses. Combination studies with DDAs suggest that they may be complementary with EGFR/HER2-specific receptor tyrosine kinase inhibitors and mTORC1 inhibitors to overcome drug resistance.
Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Animais , Antineoplásicos/síntese química , Antineoplásicos/uso terapêutico , Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Regulação para Baixo , Receptores ErbB/metabolismo , Feminino , Células HEK293 , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor ErbB-2/metabolismo , Receptor ErbB-3/metabolismo , Receptores de Estrogênio/metabolismo , Receptores de Progesterona/metabolismo , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Forced expression of defined transcriptional factors has been well documented as an effective method for cellular reprogramming or directed differentiation. However, transgene expression is not amenable for therapeutic application due to potential insertional mutagenesis. Here, we have developed a bacterial type III secretion system (T3SS)-based protein delivery tool and shown its application in directing pluripotent stem cell differentiation by a controlled delivery of transcription factors relevant to early heart development. By fusing to an N-terminal secretion sequence for T3SS-dependent injection, three transcriptional factors, namely Gata4, Mef2c, and Tbx5 (abbreviated as GMT), were translocated into murine embryonic stem cells (ESCs), where the proteins are effectively targeted to the nucleus with an average intracellular half-life of 5.5 hours. Exogenous GMT protein injection activated the cardiac program, and multiple rounds of GMT protein delivery significantly improved the efficiency of ESC differentiation into cardiomyocytes. Combination of T3SS-mediated GMT delivery and Activin A treatment showed an additive effect, resulting in on average 60% of the ESCs differentiated into cardiomyocytes. ESC derived cardiomyocytes displayed spontaneous rhythmic contractile movement as well as normal hormonal responses. This work serves as a foundation for the bacterial delivery of multiple transcription factors to direct cell fate without jeopardizing genomic integrity.
Assuntos
Diferenciação Celular/genética , Células-Tronco Embrionárias Murinas/metabolismo , Miócitos Cardíacos/metabolismo , Fatores de Transcrição/genética , Animais , Western Blotting , Linhagem Celular , Núcleo Celular/genética , Núcleo Celular/metabolismo , Fator de Transcrição GATA4/genética , Fator de Transcrição GATA4/metabolismo , Expressão Gênica , Células HeLa , Humanos , Fatores de Transcrição MEF2/genética , Fatores de Transcrição MEF2/metabolismo , Camundongos , Microscopia de Fluorescência , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo , Fatores de Tempo , Fatores de Transcrição/metabolismo , Transfecção/métodosRESUMO
The type III secretion system (T3SS) of Pseudomonas aeruginosa is a powerful tool for direct protein delivery into mammalian cells and has successfully been used to deliver various exogenous proteins into mammalian cells. In the present study, transcription activator-like effector nuclease (TALEN) proteins have been efficiently delivered using the P. aeruginosa T3SS into mouse embryonic stem cells (mESCs), human ESCs (hESCs), and human induced pluripotent stem cells (hiPSCs) for genome editing. This bacterial delivery system offers an alternative method of TALEN delivery that is highly efficient in cleavage of the chromosomal target and presumably safer by avoiding plasmid DNA introduction. We combined the method of bacterial T3SS-mediated TALEN protein injection and transfection of an oligonucleotide template to effectively generate precise genetic modifications in the stem cells. Initially, we efficiently edited a single-base in the gfp gene of a mESC line to silence green fluorescent protein (GFP) production. The resulting GFP-negative mESC was cloned from a single cell and subsequently mutated back to a GFP-positive mESC line. Using the same approach, the gfp gene was also effectively knocked out in hESCs. In addition, a defined single-base edition was effectively introduced into the X-chromosome-linked HPRT1 gene in hiPSCs, generating an in vitro model of Lesch-Nyhan syndrome. T3SS-mediated TALEN protein delivery provides a highly efficient alternative for introducing precise gene editing within pluripotent stem cells for the purpose of disease genotype-phenotype relationship studies and cellular replacement therapies.
Assuntos
Células-Tronco Embrionárias/metabolismo , Endonucleases/genética , Técnicas de Transferência de Genes , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes/metabolismo , Transativadores/genética , Animais , Diferenciação Celular/genética , Humanos , Camundongos , Plasmídeos , Pseudomonas aeruginosa/genética , Ativação Transcricional/genética , TransfecçãoRESUMO
UNLABELLED: Studies in hypertension (HTN) pharmacogenomics seek to identify genetic sources of variable antihypertensive drug response. Genetic association studies have detected single-nucleotide polymorphisms (SNPs) that link to drug responses; however, to understand mechanisms underlying how genetic traits alter drug responses, a biological interface is needed. Patient-derived induced pluripotent stem cells (iPSCs) provide a potential source for studying otherwise inaccessible tissues that may be important to antihypertensive drug response. The present study established multiple iPSC lines from an HTN pharmacogenomics cohort. We demonstrated that established HTN iPSCs can robustly and reproducibly differentiate into functional vascular smooth muscle cells (VSMCs), a cell type most relevant to vasculature tone control. Moreover, a sensitive traction force microscopy assay demonstrated that iPSC-derived VSMCs show a quantitative contractile response on physiological stimulus of endothelin-1. Furthermore, the inflammatory chemokine tumor necrosis factor α induced a typical VSMC response in iPSC-derived VSMCs. These studies pave the way for a large research initiative to decode biological significance of identified SNPs in hypertension pharmacogenomics. SIGNIFICANCE: Treatment of hypertension remains suboptimal, and a pharmacogenomics approach seeks to identify genetic biomarkers that could be used to guide treatment decisions; however, it is important to understand the biological underpinnings of genetic associations. Mouse models do not accurately recapitulate individual patient responses based on their genetics, and hypertension-relevant cells are difficult to obtain from patients. Induced pluripotent stem cell (iPSC) technology provides a great interface to bring patient cells with their genomic data into the laboratory and to study hypertensive responses. As an initial step, the present study established an iPSC bank from patients with primary hypertension and demonstrated an effective and reproducible method of generating functional vascular smooth muscle cells.
Assuntos
Diferenciação Celular , Hipertensão/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Animais , Células Cultivadas , Feminino , Humanos , Hipertensão/patologia , Células-Tronco Pluripotentes Induzidas/patologia , Masculino , Camundongos , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/patologiaRESUMO
The E2f6 transcriptional repressor is an E2F-family member essential for the silencing of a group of meiosis-specific genes in somatic tissues. Although E2f6 has been shown to associate with both polycomb repressive complexes (PRC) and the methyltransferase Dnmt3b, the cross-talk between these repressive machineries during E2f6-mediated gene silencing has not been clearly demonstrated yet. In particular, it remains largely undetermined when and how E2f6 establishes repression of meiotic genes during embryonic development. We demonstrate here that the inactivation of a group of E2f6 targeted genes, including Stag3 and Smc1ß, first occurs at the transition from mouse embryonic stem cells (ESCs) to epiblast stem cells (EpiSCs), which represent pre- and post-implantation stages, respectively. This process was accompanied by de novo methylation of their promoters. Of interest, despite a clear difference in DNA methylation status, E2f6 was similarly bound to the proximal promoter regions both in ESCs and EpiSCs. Neither E2f6 nor Dnmt3b overexpression in ESCs decreased meiotic gene expression or increased DNA methylation, indicating that additional factors are required for E2f6-mediated repression during the transition. When the SET domain of Ezh2, a core subunit of the PRC2 complex, was deleted, however, repression of Stag3 and Smc1ß during embryoid body differentiation was largely impaired, indicating that the event required the enzymatic activity of Ezh2. In addition, repression of Stag3 and Smc1ß occurred in the absence of Dnmt3b. The data presented here suggest a primary role of PRC2 in E2f6-mediated gene silencing of the meiotic genes.