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AIMS: Long QT syndrome type 2 (LQTS2) is associated with inherited variants in the cardiac human ether-à-go-go-related gene (hERG) K+ channel. However, the pathogenicity of hERG channel gene variants is often uncertain. Using CRISPR-Cas9 gene-edited hiPSC-derived cardiomyocytes (hiPSC-CMs), we investigated the pathogenic mechanism underlying the LQTS-associated hERG R56Q variant and its phenotypic rescue by using the Type 1 hERG activator, RPR260243. METHODS AND RESULTS: The above approaches enable characterization of the unclear causative mechanism of arrhythmia in the R56Q variant (an N-terminal PAS domain mutation that primarily accelerates channel deactivation) and translational investigation of the potential for targeted pharmacologic manipulation of hERG deactivation. Using perforated patch clamp electrophysiology of single hiPSC-CMs, programmed electrical stimulation showed that the hERG R56Q variant does not significantly alter the mean action potential duration (APD90). However, the R56Q variant increases the beat-to-beat variability in APD90 during pacing at constant cycle lengths, enhances the variance of APD90 during rate transitions, and increases the incidence of 2:1 block. During paired S1-S2 stimulations measuring electrical restitution properties, the R56Q variant was also found to increase the variability in rise time and duration of the response to premature stimulations. Application of the hERG channel activator, RPR260243, reduces the APD variance in hERG R56Q hiPSC-CMs, reduces the variability in responses to premature stimulations, and increases the post-repolarization refractoriness. CONCLUSION: Based on our findings, we propose that the hERG R56Q variant leads to heterogeneous APD dynamics, which could result in spatial dispersion of repolarization and increased risk for re-entry without significantly affecting the average APD90. Furthermore, our data highlight the antiarrhythmic potential of targeted slowing of hERG deactivation gating, which we demonstrate increases protection against premature action potentials and reduces electrical heterogeneity in hiPSC-CMs.
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Canais de Potássio Éter-A-Go-Go , Síndrome do QT Longo , Humanos , Canais de Potássio Éter-A-Go-Go/genética , Síndrome do QT Longo/genética , Arritmias Cardíacas/genética , Arritmias Cardíacas/prevenção & controle , Miócitos Cardíacos , Potenciais de Ação , Éteres , Canal de Potássio ERG1/genéticaRESUMO
Modeling severe acute respiratory syndrome, Coronavirus 2 (SARS-CoV-2) infection in stem cell-derived organoids has helped in our understanding of the molecular pathogenesis of COVID-19 disease due to their resemblance to actual human tissues or organs. Over the past decade, organoid 3-dimensional (3D) cultures have represented a new perspective and considerable advancement over traditional in vitro 2-dimensional (2D) cell cultures. COVID-19 disease causes lung injury and multi-organ failure leading to death, especially in older patients. There is an urgent need for physiological models to study SARS-CoV-2 infection during the pandemic. Human stem cell-derived organoids can provide insight into understanding the SARS-CoV-2 cell entry molecular mechanism. Identifying such complexities will help to develop the best preventive drug targets.
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COVID-19 , SARS-CoV-2 , Humanos , Idoso , Células-Tronco , Técnicas de Cultura de Células , OrganoidesRESUMO
Lymphangioleiomyomatosis (LAM) is a rare disease involving cystic lung destruction by invasive LAM cells. These cells harbor loss-of-function mutations in TSC2, conferring hyperactive mTORC1 signaling. Here, tissue engineering tools are employed to model LAM and identify new therapeutic candidates. Biomimetic hydrogel culture of LAM cells is found to recapitulate the molecular and phenotypic characteristics of human disease more faithfully than culture on plastic. A 3D drug screen is conducted, identifying histone deacetylase (HDAC) inhibitors as anti-invasive agents that are also selectively cytotoxic toward TSC2-/- cells. The anti-invasive effects of HDAC inhibitors are independent of genotype, while selective cell death is mTORC1-dependent and mediated by apoptosis. Genotype-selective cytotoxicity is seen exclusively in hydrogel culture due to potentiated differential mTORC1 signaling, a feature that is abrogated in cell culture on plastic. Importantly, HDAC inhibitors block invasion and selectively eradicate LAM cells in vivo in zebrafish xenografts. These findings demonstrate that tissue-engineered disease modeling exposes a physiologically relevant therapeutic vulnerability that would be otherwise missed by conventional culture on plastic. This work substantiates HDAC inhibitors as possible therapeutic candidates for the treatment of patients with LAM and requires further study.
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Neoplasias Pulmonares , Linfangioleiomiomatose , Animais , Humanos , Linfangioleiomiomatose/tratamento farmacológico , Linfangioleiomiomatose/genética , Linfangioleiomiomatose/metabolismo , Neoplasias Pulmonares/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Inibidores de Histona Desacetilases/uso terapêutico , Engenharia Tecidual , Peixe-Zebra , Alvo Mecanístico do Complexo 1 de RapamicinaRESUMO
The Nobel Prize-winning discovery that human somatic cells can be readily reprogrammed into pluripotent cells has revolutionized our potential to understand the human brain. The rapid technological progression of this field has made it possible to easily obtain human neural cells and even intact tissues, offering invaluable resources to model human brain development. In this chapter, we present a brief history of hPSC-based approaches to study brain development and then, provide new insights into neurological diseases, focusing on those driven by aberrant cell death. Furthermore, we will shed light on the latest technologies and highlight the methods that researchers can use to employ established hPSC approaches in their research. Our intention is to demonstrate that hPSC-based modeling is a technical approach accessible to all researchers who seek a deeper understanding of the human brain.
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Células-Tronco Pluripotentes Induzidas , Doenças do Sistema Nervoso , Células-Tronco Pluripotentes , Encéfalo , Humanos , Doenças do Sistema Nervoso/metabolismo , Células-Tronco Pluripotentes/metabolismoRESUMO
The brain is our most complex and least understood organ. Animal models have long been the most versatile tools available to dissect brain form and function; however, the human brain is highly distinct from that of standard model organisms. In addition to existing models, access to human brain cells and tissues is essential to reach new frontiers in our understanding of the human brain and how to intervene therapeutically in the face of disease or injury. In this review, we discuss current and developing culture models of human neural tissue, outlining advantages over animal models and key challenges that remain to be overcome. Our principal focus is on advances in engineering neural cells and tissue constructs from human pluripotent stem cells (PSCs), though primary human cell and slice culture are also discussed. By highlighting studies that combine animal models and human neural cell culture techniques, we endeavor to demonstrate that clever use of these orthogonal model systems produces more reproducible, physiological, and clinically relevant data than either approach alone. We provide examples across a range of topics in neuroscience research including brain development, injury, and cancer, neurodegenerative diseases, and psychiatric conditions. Finally, as testing of PSC-derived neurons for cell replacement therapy progresses, we touch on the advancements that are needed to make this a clinical mainstay.
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Regulation of stem cell fate is best understood at the level of gene and protein regulatory networks, though it is now clear that multiple cellular organelles also have critical impacts. A growing appreciation for the functional interconnectedness of organelles suggests that an orchestration of integrated biological networks functions to drive stem cell fate decisions and regulate metabolism. Metabolic signaling itself has emerged as an integral regulator of cell fate including the determination of identity, activation state, survival, and differentiation potential of many developmental, adult, disease, and cancer-associated stem cell populations and their progeny. As the primary adenosine triphosphate-generating organelles, mitochondria are well-known regulators of stem cell fate decisions, yet it is now becoming apparent that additional organelles such as the lysosome are important players in mediating these dynamic decisions. In this review, we will focus on the emerging role of organelles, in particular lysosomes, in the reprogramming of both metabolic networks and stem cell fate decisions, especially those that impact the determination of cell identity. We will discuss the inter-organelle interactions, cell signaling pathways, and transcriptional regulatory mechanisms with which lysosomes engage and how these activities impact metabolic signaling. We will further review recent data that position lysosomes as critical regulators of cell identity determination programs and discuss the known or putative biological mechanisms. Finally, we will briefly highlight the potential impact of elucidating mechanisms by which lysosomes regulate stem cell identity on our understanding of disease pathogenesis, as well as the development of refined regenerative medicine, biomarker, and therapeutic strategies.
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We previously reported that the cell cycle-related cyclin-dependent kinase 4-retinoblastoma (RB) transcriptional corepressor pathway is essential for stroke-induced cell death both in vitro and in vivo However, how this signaling pathway induces cell death is unclear. Previously, we found that the cyclin-dependent kinase 4 pathway activates the pro-apoptotic transcriptional co-regulator Cited2 in vitro after DNA damage. In the present study, we report that Cited2 protein expression is also dramatically increased following stroke/ischemic insult. Critically, utilizing conditional knockout mice, we show that Cited2 is required for neuronal cell death, both in culture and in mice after ischemic insult. Importantly, determining the mechanism by which Cited2 levels are regulated, we found that E2F transcription factor (E2F) family members participate in Cited2 regulation. First, E2F1 expression induced Cited2 transcription, and E2F1 deficiency reduced Cited2 expression. Moreover, determining the potential E2F-binding regions on the Cited2 gene regulatory sequence by ChIP analysis, we provide evidence that E2F1/4 proteins bind to this DNA region. A luciferase reporter assay to probe the functional outcomes of this interaction revealed that E2F1 activates and E2F4 inhibits Cited2 transcription. Moreover, we identified the functional binding motif for E2F1 in the Cited2 gene promoter by demonstrating that mutation of this site dramatically reduces E2F1-mediated Cited2 transcription. Finally, E2F1 and E2F4 regulated Cited2 expression in neurons after stroke-related insults. Taken together, these results indicate that the E2F-Cited2 regulatory pathway is critically involved in stroke injury.
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Fator de Transcrição E2F1/metabolismo , Fator de Transcrição E2F4/metabolismo , Regulação da Expressão Gênica , Neurônios/metabolismo , Proteínas Repressoras/biossíntese , Acidente Vascular Cerebral/metabolismo , Transativadores/biossíntese , Motivos de Aminoácidos , Animais , Morte Celular , Fator de Transcrição E2F1/genética , Fator de Transcrição E2F4/genética , Camundongos , Camundongos Transgênicos , Neurônios/patologia , Proteínas Repressoras/genética , Acidente Vascular Cerebral/genética , Acidente Vascular Cerebral/patologia , Transativadores/genéticaRESUMO
Cell behavior is highly dependent upon microenvironment. Thus, to identify drugs targeting metastatic cancer, screens need to be performed in tissue mimetic substrates that allow cell invasion and matrix remodeling. A novel biomimetic 3D hydrogel platform that enables quantitative analysis of cell invasion and viability at the individual cell level is developed using automated data acquisition methods with an invasive lung disease (lymphangioleiomyomatosis, LAM) characterized by hyperactive mammalian target of rapamycin complex 1 (mTORC1) signaling as a model. To test the lung-mimetic hydrogel platform, a kinase inhibitor screen is performed using tuberous sclerosis complex 2 (TSC2) hypomorphic cells, identifying Cdk2 inhibition as a putative LAM therapeutic. The 3D hydrogels mimic the native niche, enable multiple modes of invasion, and delineate phenotypic differences between healthy and diseased cells, all of which are critical to effective drug screens of highly invasive diseases including lung cancer.
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Movimento Celular/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos/instrumentação , Hidrogéis , Neoplasias Pulmonares/tratamento farmacológico , Modelos Biológicos , Animais , Antineoplásicos/farmacologia , Automação Laboratorial , Materiais Biomiméticos , Movimento Celular/fisiologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/metabolismo , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Pulmão/patologia , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Teste de Materiais , Fosfotransferases/antagonistas & inibidores , Ratos , Proteína 2 do Complexo Esclerose Tuberosa/metabolismoRESUMO
The tumor suppressor PTEN is frequently inactivated in glioblastoma. PTEN-L is a long form of PTEN produced by translation from an alternate upstream start codon. Unlike PTEN, PTEN-L has a signal sequence and a tract of six arginine residues that allow PTEN-L to be secreted from cells and be taken up by neighboring cells. This suggests that PTEN-L could be used as a therapeutic to restore PTEN activity. However, effective delivery of therapeutic proteins to treat CNS cancers such as glioblastoma is challenging. One method under evaluation is cell-mediated therapy, where cells with tumor-homing abilities such as neural stem cells are genetically modified to express a therapeutic protein. Here, we have developed a version of PTEN-L that is engineered for enhanced cell-mediated delivery. This was accomplished by replacement of the native leader sequence of PTEN-L with a leader sequence from human light-chain immunoglobulin G (IgG). This version of PTEN-L showed increased secretion and an increased ability to transfer to neighboring cells. Neural stem cells derived from human fibroblasts could be modified to express this version of PTEN-L and were able to deliver catalytically active light-chain leader PTEN-L (lclPTEN-L) to neighboring glioblastoma cells.
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Lymphangioleiomyomatosis (LAM) is a progressive destructive neoplasm of the lung associated with inactivating mutations in the TSC1 or TSC2 tumor suppressor genes. Cell or animal models that accurately reflect the pathology of LAM have been challenging to develop. Here, we generated a robust human cell model of LAM by reprogramming TSC2 mutation-bearing fibroblasts from a patient with both tuberous sclerosis complex (TSC) and LAM (TSC-LAM) into induced pluripotent stem cells (iPSC), followed by selection of cells that resemble those found in LAM tumors by unbiased in vivo differentiation. We established expandable cell lines under smooth muscle cell (SMC) growth conditions that retained a patient-specific genomic TSC2+/- mutation and recapitulated the molecular and functional characteristics of pulmonary LAM cells. These include multiple indicators of hyperactive mTORC1 signaling, presence of specific neural crest and SMC markers, expression of VEGF-D and female sex hormone receptors, reduced autophagy, and metabolic reprogramming. Intriguingly, the LAM-like features of these cells suggest that haploinsufficiency at the TSC2 locus contributes to LAM pathology, and demonstrated that iPSC reprogramming and SMC lineage differentiation of somatic patient cells with germline mutations was a viable approach to generate LAM-like cells. The patient-derived SMC lines we have developed thus represent a novel cellular model of LAM that can advance our understanding of disease pathogenesis and develop therapeutic strategies against LAM. Cancer Res; 77(20); 5491-502. ©2017 AACR.
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Linfangioleiomiomatose/genética , Linfangioleiomiomatose/patologia , Miócitos de Músculo Liso/fisiologia , Células-Tronco Pluripotentes/fisiologia , Animais , Proliferação de Células/fisiologia , Feminino , Haploinsuficiência , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , Células-Tronco Pluripotentes/metabolismo , Células-Tronco Pluripotentes/patologiaRESUMO
Over the last decade significant advances have been made toward reprogramming the fate of somatic cells, typically by overexpression of cell lineage-determinant transcription factors. As key regulators of cell fate, the SOX family of transcription factors has emerged as potent drivers of direct somatic cell reprogramming into multiple lineages, in some cases as the sole overexpressed factor. The vast capacity of SOX factors, especially those of the SOXB1, E and F subclasses, to reprogram cell fate is enlightening our understanding of organismal development, cancer and disease, and offers tremendous potential for regenerative medicine and cell-based therapies. Understanding the molecular mechanisms through which SOX factors reprogram cell fate is essential to optimize the development of novel somatic cell transdifferentiation strategies.
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Diferenciação Celular/genética , Transdiferenciação Celular/genética , Reprogramação Celular/genética , Animais , Humanos , Medicina Regenerativa , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXE/genética , Fatores de Transcrição SOXF/genéticaRESUMO
Cell cycle dynamics has emerged as a key regulator of stem cell fate decisions. In particular, differentiation decisions are associated with the G1 phase, and recent evidence suggests that self-renewal is actively regulated outside of G1. The mechanisms underlying these phenomena are largely unknown, but direct control of gene regulatory programs by the cell cycle machinery is heavily implicated. A recent study sheds important mechanistic insight by demonstrating that in human embryonic stem cells (hESCs) the Cyclin-dependent kinase CDK2 controls a wide-spread epigenetic program that drives transcription at differentiation-related gene promoters specifically in G1. Here, we discuss this finding and explore whether similar mechanisms are likely to function in multipotent stem cells. The implications of this discovery toward our understanding of stem cell-related disease are discussed, and we postulate novel mechanisms that position the cell cycle as a regulator of cell fate gene networks at epigenetic, transcriptional and post-transcriptional levels.
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Quinase 2 Dependente de Ciclina/genética , Células-Tronco Embrionárias/metabolismo , Epigênese Genética , Fase G1/genética , Células-Tronco Neurais/metabolismo , Células-Tronco Pluripotentes/metabolismo , Diferenciação Celular , Quinase 2 Dependente de Ciclina/metabolismo , Células-Tronco Embrionárias/citologia , Redes Reguladoras de Genes , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Proteína Homeobox Nanog/genética , Proteína Homeobox Nanog/metabolismo , Células-Tronco Neurais/citologia , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Células-Tronco Pluripotentes/citologia , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Proteínas Smad/genética , Proteínas Smad/metabolismo , Transcrição GênicaRESUMO
E2F transcription factors and their regulatory partners, the pocket proteins (PPs), have emerged as essential regulators of stem cell fate control in a number of lineages. In mammals, this role extends from both pluripotent stem cells to those encompassing all embryonic germ layers, as well as extra-embryonic lineages. E2F/PP-mediated regulation of stem cell decisions is highly evolutionarily conserved, and is likely a pivotal biological mechanism underlying stem cell homeostasis. This has immense implications for organismal development, tissue maintenance, and regeneration. In this article, we discuss the roles of E2F factors and PPs in stem cell populations, focusing on mammalian systems. We discuss emerging findings that position the E2F and PP families as widespread and dynamic epigenetic regulators of cell fate decisions. Additionally, we focus on the ever expanding landscape of E2F/PP target genes, and explore the possibility that E2Fs are not simply regulators of general 'multi-purpose' cell fate genes but can execute tissue- and cell type-specific gene regulatory programs.
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Lymphangioleiomyomatosis (LAM) is a rare neoplastic disease, best characterized by the formation of proliferative nodules that express smooth muscle and melanocytic antigens within the lung parenchyma, leading to progressive destruction of lung tissue and function. The pathological basis of LAM is associated with Tuberous Sclerosis Complex (TSC), a multi-system disorder marked by low-grade tumors in the brain, kidneys, heart, eyes, lung and skin, arising from inherited or spontaneous germ-line mutations in either of the TSC1 or TSC2 genes. LAM can develop either in a patient with TSC (TSC-LAM) or spontaneously (S-LAM), and it is clear that the majority of LAM lesions of both forms are characterized by an inactivating mutation in either TSC1 or TSC2, as in TSC. Despite this genetic commonality, there is considerable heterogeneity in the tumor spectrum of TSC and LAM patients, the basis for which is currently unknown. There is extensive clinical evidence to suggest that the cell of origin for LAM, as well as many of the TSC-associated tumors, is a neural crest cell, a highly migratory cell type with extensive multi-lineage potential. Here we explore the hypothesis that the types of tumors that develop and the tissues that are affected in TSC and LAM are dictated by the developmental timing of TSC gene mutations, which determines the identities of the affected cell types and the size of downstream populations that acquire a mutation. We further discuss the evidence to support a neural crest origin for LAM and TSC tumors, and propose approaches for generating humanized models of TSC and LAM that will allow cell of origin theories to be experimentally tested. Identifying the cell of origin and developing appropriate humanized models is necessary to truly understand LAM and TSC pathology and to establish effective and long-lasting therapeutic approaches for these patients.
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Structural analysis of both the MDM2-p53 protein-protein interaction and several small molecules bound to MDM2 led to the design and synthesis of tetrasubstituted morpholinone 10, an MDM2 inhibitor with a biochemical IC50 of 1.0 µM. The cocrystal structure of 10 with MDM2 inspired two independent optimization strategies and resulted in the discovery of morpholinones 16 and 27 possessing distinct binding modes. Both analogues were potent MDM2 inhibitors in biochemical and cellular assays, and morpholinone 27 (IC50 = 0.10 µM) also displayed suitable PK profile for in vivo animal experiments. A pharmacodynamic (PD) experiment in mice implanted with human SJSA-1 tumors showed p21(WAF1) mRNA induction (2.7-fold over vehicle) upon oral dosing of 27 at 300 mg/kg.
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Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Proteína Supressora de Tumor p53/antagonistas & inibidores , Animais , Dicroísmo Circular , Cristalografia , Cristalografia por Raios X , Desenho de Fármacos , Feminino , Humanos , Indicadores e Reagentes , Camundongos , Camundongos Nus , Modelos Moleculares , Morfolinas/síntese química , Morfolinas/farmacologia , Piperidinas/síntese química , Piperidinas/farmacologia , Estereoisomerismo , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The mechanisms through which cell-cycle control and cell-fate decisions are coordinated in proliferating stem cell populations are largely unknown. Here, we show that E2f3 isoforms, which control cell-cycle progression in cooperation with the retinoblastoma protein (pRb), have critical effects during developmental and adult neurogenesis. Loss of either E2f3 isoform disrupts Sox2 gene regulation and the balance between precursor maintenance and differentiation in the developing cortex. Both isoforms target the Sox2 locus to maintain baseline levels of Sox2 expression but antagonistically regulate Sox2 levels to instruct fate choices. E2f3-mediated regulation of Sox2 and precursor cell fate extends to the adult brain, where E2f3a loss results in defects in hippocampal neurogenesis and memory formation. Our results demonstrate a mechanism by which E2f3a and E2f3b differentially regulate Sox2 dosage in neural precursors, a finding that may have broad implications for the regulation of diverse stem cell populations.
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Ciclo Celular , Fator de Transcrição E2F3/metabolismo , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Fatores de Transcrição SOXB1/genética , Envelhecimento/metabolismo , Animais , Sequência de Bases , Contagem de Células , Ciclo Celular/genética , Linhagem da Célula/genética , Proliferação de Células , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Regulação da Expressão Gênica , Células HEK293 , Humanos , Camundongos , Modelos Biológicos , Dados de Sequência Molecular , Neurogênese , Regiões Promotoras Genéticas/genética , Isoformas de Proteínas/metabolismo , Fatores de Transcrição SOXB1/metabolismoRESUMO
During brain morphogenesis, the mechanisms through which the cell cycle machinery integrates with differentiation signals remain elusive. Here we show that the Rb/E2F pathway regulates key aspects of differentiation and migration through direct control of the Dlx1 and Dlx2 homeodomain proteins, required for interneuron specification. Rb deficiency results in a dramatic reduction of Dlx1 and Dlx2 gene expression manifested by loss of interneuron subtypes and severe migration defects in the mouse brain. The Rb/E2F pathway modulates Dlx1/Dlx2 regulation through direct interaction with a Dlx forebrain-specific enhancer, I12b, and the Dlx1/Dlx2 proximal promoter regions, through repressor E2F sites both in vitro and in vivo. In the absence of Rb, we demonstrate that repressor E2Fs inhibit Dlx transcription at the Dlx1/Dlx2 promoters and Dlx1/2-I12b enhancer to suppress differentiation. Our findings support a model whereby the cell cycle machinery not only controls cell division but also modulates neuronal differentiation and migration through direct regulation of the Dlx1/Dlx2 bigene cluster during embryonic development.