RESUMO
Pioneer transcription factors (TFs) regulate cell fate by establishing transcriptionally primed and active states. However, cell fate control requires the coordination of both lineage-specific gene activation and repression of alternative-lineage programs, a process that is poorly understood. Here, we demonstrate that the pioneer TF FOXA coordinates with PRDM1 TF to recruit nucleosome remodeling and deacetylation (NuRD) complexes and Polycomb repressive complexes (PRCs), which establish highly occupied, accessible nucleosome conformation with bivalent epigenetic states, thereby preventing precocious and alternative-lineage gene expression during human endoderm differentiation. Similarly, the pioneer TF OCT4 coordinates with PRDM14 to form bivalent enhancers and repress cell differentiation programs in human pluripotent stem cells, suggesting that this may be a common and critical function of pioneer TFs. We propose that pioneer and PRDM TFs coordinate to safeguard cell fate through epigenetic repression mechanisms.
Assuntos
Nucleossomos , Fatores de Transcrição , Humanos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Nucleossomos/genética , Diferenciação Celular/genética , Proteínas do Grupo Polycomb/metabolismo , Epigênese GenéticaRESUMO
Large-scale genome sequencing is poised to provide a substantial increase in the rate of discovery of disease-associated mutations, but the functional interpretation of such mutations remains challenging. Here we show that deletions of a sequence on human chromosome 16 that we term the intestine-critical region (ICR) cause intractable congenital diarrhoea in infants1,2. Reporter assays in transgenic mice show that the ICR contains a regulatory sequence that activates transcription during the development of the gastrointestinal system. Targeted deletion of the ICR in mice caused symptoms that recapitulated the human condition. Transcriptome analysis revealed that an unannotated open reading frame (Percc1) flanks the regulatory sequence, and the expression of this gene was lost in the developing gut of mice that lacked the ICR. Percc1-knockout mice displayed phenotypes similar to those observed upon ICR deletion in mice and patients, whereas an ICR-driven Percc1 transgene was sufficient to rescue the phenotypes found in mice that lacked the ICR. Together, our results identify a gene that is critical for intestinal function and underscore the need for targeted in vivo studies to interpret the growing number of clinical genetic findings that do not affect known protein-coding genes.
Assuntos
Diarreia/congênito , Diarreia/genética , Elementos Facilitadores Genéticos/genética , Regulação da Expressão Gênica no Desenvolvimento , Genes , Intestinos/fisiologia , Deleção de Sequência/genética , Animais , Cromossomos Humanos Par 16/genética , Modelos Animais de Doenças , Feminino , Genes Reporter , Loci Gênicos/genética , Humanos , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Linhagem , Fenótipo , Ativação Transcricional , Transcriptoma/genética , Transgenes/genéticaRESUMO
Gaucher disease (GD) is caused by GBA1 mutations leading to functional deficiency of acid-ß-glucosidase (GCase). No effective treatment is available for neuronopathic GD (nGD). A subclass of neural stem and precursor cells (NPCs) expresses VLA4 (integrin α4ß1, very late antigen-4) that facilitates NPC entry into the brain following intravenous (IV) infusion. Here, the therapeutic potential of IV VLA4+NPCs was assessed for nGD using wild-type mouse green fluorescent protein (GFP)-positive multipotent induced pluripotent stem cell (iPSC)-derived VLA4+NPCs. VLA4+NPCs successfully engrafted in the nGD (4L;C*) mouse brain. GFP-positive cells differentiated into neurons, astrocytes and oligodendrocytes in the brainstem, midbrain and thalamus of the transplanted mice and significantly improved sensorimotor function and prolonged life span compared to vehicle-treated 4L;C* mice. VLA4+NPC transplantation significantly decreased levels of CD68 and glial fibrillary acidic protein, as well as TNFα mRNA levels in the brain, indicating reduced neuroinflammation. Furthermore, decreased Fluoro-Jade C and NeuroSilver staining suggested inhibition of neurodegeneration. VLA4+NPC-engrafted 4L;C* midbrains showed 35% increased GCase activity, reduced substrate [glucosylceramide (GC, -34%) and glucosylsphingosine (GS, -11%)] levels and improved mitochondrial oxygen consumption rates in comparison to vehicle-4L;C* mice. VLA4+NPC engraftment in 4L;C* brain also led to enhanced expression of neurotrophic factors that have roles in neuronal survival and the promotion of neurogenesis. This study provides evidence that iPSC-derived NPC transplantation has efficacy in an nGD mouse model and provides proof of concept for autologous NPC therapy in nGD.
Assuntos
Doença de Gaucher/metabolismo , Doença de Gaucher/terapia , Glucosilceramidase/metabolismo , Células-Tronco Pluripotentes Induzidas/fisiologia , Células-Tronco Neurais/fisiologia , Animais , Terapia Baseada em Transplante de Células e Tecidos/métodos , Células-Tronco Pluripotentes Induzidas/citologia , Infusões Intravenosas , Integrina alfa4beta1/metabolismo , Camundongos , Células-Tronco Neurais/citologia , beta-Glucosidase/metabolismoRESUMO
Gastric diseases, including peptic ulcer disease and gastric cancer, affect 10% of the world's population and are largely due to chronic Helicobacter pylori infection. Species differences in embryonic development and architecture of the adult stomach make animal models suboptimal for studying human stomach organogenesis and pathogenesis, and there is no experimental model of normal human gastric mucosa. Here we report the de novo generation of three-dimensional human gastric tissue in vitro through the directed differentiation of human pluripotent stem cells. We show that temporal manipulation of the FGF, WNT, BMP, retinoic acid and EGF signalling pathways and three-dimensional growth are sufficient to generate human gastric organoids (hGOs). Developing hGOs progressed through molecular and morphogenetic stages that were nearly identical to the developing antrum of the mouse stomach. Organoids formed primitive gastric gland- and pit-like domains, proliferative zones containing LGR5-expressing cells, surface and antral mucous cells, and a diversity of gastric endocrine cells. We used hGO cultures to identify novel signalling mechanisms that regulate early endoderm patterning and gastric endocrine cell differentiation upstream of the transcription factor NEUROG3. Using hGOs to model pathogenesis of human disease, we found that H. pylori infection resulted in rapid association of the virulence factor CagA with the c-Met receptor, activation of signalling and induction of epithelial proliferation. Together, these studies describe a new and robust in vitro system for elucidating the mechanisms underlying human stomach development and disease.
Assuntos
Infecções por Helicobacter/fisiopatologia , Modelos Biológicos , Organogênese , Organoides/citologia , Células-Tronco Pluripotentes/citologia , Estômago/citologia , Diferenciação Celular , Helicobacter pylori , Humanos , Organoides/microbiologia , Transdução de SinaisRESUMO
Studies in embryonic development have guided successful efforts to direct the differentiation of human embryonic and induced pluripotent stem cells (PSCs) into specific organ cell types in vitro. For example, human PSCs have been differentiated into monolayer cultures of liver hepatocytes and pancreatic endocrine cells that have therapeutic efficacy in animal models of liver disease and diabetes, respectively. However, the generation of complex three-dimensional organ tissues in vitro remains a major challenge for translational studies. Here we establish a robust and efficient process to direct the differentiation of human PSCs into intestinal tissue in vitro using a temporal series of growth factor manipulations to mimic embryonic intestinal development. This involved activin-induced definitive endoderm formation, FGF/Wnt-induced posterior endoderm pattering, hindgut specification and morphogenesis, and a pro-intestinal culture system to promote intestinal growth, morphogenesis and cytodifferentiation. The resulting three-dimensional intestinal 'organoids' consisted of a polarized, columnar epithelium that was patterned into villus-like structures and crypt-like proliferative zones that expressed intestinal stem cell markers. The epithelium contained functional enterocytes, as well as goblet, Paneth and enteroendocrine cells. Using this culture system as a model to study human intestinal development, we identified that the combined activity of WNT3A and FGF4 is required for hindgut specification whereas FGF4 alone is sufficient to promote hindgut morphogenesis. Our data indicate that human intestinal stem cells form de novo during development. We also determined that NEUROG3, a pro-endocrine transcription factor that is mutated in enteric anendocrinosis, is both necessary and sufficient for human enteroendocrine cell development in vitro. PSC-derived human intestinal tissue should allow for unprecedented studies of human intestinal development and disease.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Células-Tronco Embrionárias/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Intestinos/citologia , Ativinas/farmacologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Padronização Corporal/efeitos dos fármacos , Técnicas de Cultura de Células , Células Cultivadas , Meios de Cultura/química , Meios de Cultura/farmacologia , Células-Tronco Embrionárias/efeitos dos fármacos , Endoderma/citologia , Endoderma/efeitos dos fármacos , Endoderma/embriologia , Fator 4 de Crescimento de Fibroblastos/farmacologia , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Intestinos/anatomia & histologia , Intestinos/efeitos dos fármacos , Intestinos/embriologia , Microvilosidades/efeitos dos fármacos , Morfogênese/efeitos dos fármacos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Organogênese/efeitos dos fármacos , Fatores de Tempo , Proteínas Wnt/farmacologia , Proteína Wnt3 , Proteína Wnt3ARESUMO
BACKGROUND: Induced pluripotent stem cells (iPSCs) hold tremendous potential, both as a biological tool to uncover the pathophysiology of disease by creating relevant human cell models and as a source of cells for cell-based therapeutic applications. Studying the reprogramming process will also provide significant insight into tissue development. OBJECTIVE: We sought to characterize the derivation of iPSC lines from nasal epithelial cells (NECs) isolated from nasal mucosa samples of children, a highly relevant and easily accessible tissue for pediatric populations. METHODS: We performed detailed comparative analysis on the transcriptomes and methylomes of NECs, iPSCs derived from NECs (NEC-iPSCs), and embryonic stem cells (ESCs). RESULTS: NEC-iPSCs express pluripotent cell markers, can differentiate into all 3 germ layers in vivo and in vitro, and have a transcriptome and methylome remarkably similar to those of ESCs. However, residual DNA methylation marks exist, which are differentially methylated between NEC-iPSCs and ESCs. A subset of these methylation markers related to epithelium development and asthma and specific to NEC-iPSCs persisted after several passages in vitro, suggesting the retention of an epigenetic memory of their tissue of origin. Our analysis also identified novel candidate genes with dynamic gene expression and DNA methylation changes during reprogramming, which are indicative of possible roles in airway epithelium development. CONCLUSION: NECs are an excellent tissue source to generate iPSCs in pediatric asthmatic patients, and detailed characterization of the resulting iPSC lines would help us better understand the reprogramming process and retention of epigenetic memory.
Assuntos
Asma/genética , Células-Tronco Embrionárias/metabolismo , Células Epiteliais , Células-Tronco Pluripotentes Induzidas , Mucosa Nasal/citologia , Adolescente , Animais , Linhagem Celular , Células Cultivadas , Metilação de DNA , Epigenômica , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Fibroblastos , Prepúcio do Pênis/citologia , Perfilação da Expressão Gênica , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Masculino , CamundongosRESUMO
UNLABELLED: DNA repair plays a crucial role in embryonic and somatic stem cell biology and cell reprogramming. The Fanconi anemia (FA) pathway, which promotes error-free repair of DNA double-strand breaks, is required for somatic cell reprogramming to induced pluripotent stem cells (iPSC). Thus, cells from Fanconi anemia patients, which lack this critical pathway, fail to be reprogrammed to iPSC under standard conditions unless the defective FA gene is complemented. In this study, we utilized the oncogenes of high-risk human papillomavirus 16 (HPV16) to overcome the resistance of FA patient cells to reprogramming. We found that E6, but not E7, recovers FA iPSC colony formation and, furthermore, that p53 inhibition is necessary and sufficient for this activity. The iPSC colonies resulting from each of these approaches stained positive for alkaline phosphatase, NANOG, and Tra-1-60, indicating that they were fully reprogrammed into pluripotent cells. However, FA iPSC were incapable of outgrowth into stable iPSC lines regardless of p53 suppression, whereas their FA-complemented counterparts grew efficiently. Thus, we conclude that the FA pathway is required for the growth of iPSC beyond reprogramming and that p53-independent mechanisms are involved. IMPORTANCE: A novel approach is described whereby HPV oncogenes are used as tools to uncover DNA repair-related molecular mechanisms affecting somatic cell reprogramming. The findings indicate that p53-dependent mechanisms block FA cells from reprogramming but also uncover a previously unrecognized defect in FA iPSC proliferation independent of p53.
Assuntos
Reprogramação Celular/genética , Anemia de Fanconi/genética , Células-Tronco Pluripotentes Induzidas/virologia , Queratinócitos/virologia , Proteínas Oncogênicas Virais/genética , Proteínas Repressoras/genética , Proteína Supressora de Tumor p53/genética , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Antígenos de Superfície/genética , Antígenos de Superfície/metabolismo , Biomarcadores/metabolismo , Diferenciação Celular , Proliferação de Células , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Anemia de Fanconi/metabolismo , Anemia de Fanconi/patologia , Regulação da Expressão Gênica , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/patologia , Queratinócitos/metabolismo , Queratinócitos/patologia , Proteína Homeobox Nanog , Proteínas Oncogênicas Virais/metabolismo , Cultura Primária de Células , Proteoglicanas/genética , Proteoglicanas/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas Repressoras/metabolismo , Transdução Genética , Proteína Supressora de Tumor p53/antagonistas & inibidores , Proteína Supressora de Tumor p53/metabolismoRESUMO
Diverse etiologic events are associated with the development of hepatocellular carcinoma. During hepatocarcinogenesis, genetic events likely occur that subsequently cooperate with long-term exposures to further drive the progression of hepatocellular carcinoma. In this study, the frequent loss of the retinoblastoma (RB) tumor suppressor in hepatocellular carcinoma was modeled in response to diverse hepatic stresses. Loss of RB did not significantly affect the response to a steatotic stress as driven by a methionine- and choline-deficient diet. In addition, RB status did not significantly influence the response to peroxisome proliferators that can drive hepatomegaly and tumor development in rodents. However, RB loss exhibited a highly significant effect on the response to the xenobiotic1,4-Bis-[2-(3,5-dichloropyridyloxy)] benzene. Loss of RB yielded a unique proliferative response to this agent, which was distinct from both regenerative stresses and genotoxic carcinogens. Long-term exposure to 1,4-Bis-[2-(3,5-dichloropyridyloxy)] benzene yielded profound tumor development in RB-deficient livers that was principally absent in RB-sufficient tissue. These data demonstrate the context specificity of RB and the key role RB plays in the suppression of hepatocellular carcinoma driven by xenobiotic stress.
Assuntos
Carcinógenos/farmacologia , Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas Experimentais/metabolismo , Piridinas/efeitos adversos , Proteína do Retinoblastoma/metabolismo , Xenobióticos/efeitos adversos , Animais , Carcinoma Hepatocelular/induzido quimicamente , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas Experimentais/induzido quimicamente , Neoplasias Hepáticas Experimentais/genética , Neoplasias Hepáticas Experimentais/patologia , Camundongos Mutantes , Piridinas/farmacologia , Proteína do Retinoblastoma/genética , Xenobióticos/farmacologiaRESUMO
Inducible loss-of-function strategies are crucial for understanding gene function. However, creating inducible, multiple-gene knockout models is challenging and time-consuming. Here, we present a protocol for establishing a doxycycline-inducible CRISPR interference (CRISPRi) system to concurrently silence multiple genes in human induced pluripotent stem cells (hPSCs). We describe the steps for establishing host CRISPRi hPSCs, designing and cloning single-guide RNAs (sgRNAs) into a lentivirus plasmid, and establishing monoclonal CRISPRi hPSC lines transduced with sgRNAs. We also detail the procedures for selecting effective CRISPRi clones. For complete details on the use and execution of this protocol, please refer to Matsui et al.1.
RESUMO
Transcription factor (TF) gene knockout or knockdown experiments provide comprehensive downstream effects on gene regulation. However, distinguishing primary direct effects from secondary effects remains challenging. To assess the direct effect of TF binding events, we present a protocol for establishing a doxycycline (Dox)-inducible CRISPRd system in human pluripotent stem cells (hPSCs). We describe the steps for establishing CRISPRd host hPSCs, designing and preparing single-guide RNA (sgRNA) expression lentivirus vectors, generating CRISPRd hPSCs transduced with sgRNAs, and analyzing CRISPRd TF-block effects by chromatin immunoprecipitation (ChIP)-qPCR. For complete details on the use and execution of this protocol, please refer to Matsui et al.1.
RESUMO
Recent breakthroughs in human stem cell technologies have enabled the generation of 3D brain organoid platforms for modeling human neurodevelopment and disease. Here, we review advances in brain organoid development, approaches for generating whole-brain or cerebral organoids and region-specific brain organoids, and their applications in disease modeling. We present a comprehensive overview of various brain organoid generation protocols, including culture steps, media, timelines, and technical considerations associated with each protocol, and highlight the advantages and disadvantages of each protocol. We also discuss the current limitations as well as increasing sophistication of brain organoid technology, and future directions for the field. These insights provide a valuable assessment of multiple commonly used brain organoid models and main considerations for investigators who are considering implementing brain organoid technologies in their laboratories.
Assuntos
Encéfalo , Organoides , Humanos , Células-TroncoRESUMO
Mutations in GBA1, encoding the lysosomal acid ß-glucosidase (GCase), cause neuronopathic Gaucher disease (nGD) and promote Parkinson disease (PD). The mutations on GBA1 include deletion and missense mutations that are pathological and lead to GCase deficiency in Gaucher disease. Both nGD and PD lack disease-modifying treatments and are critical unmet medical needs. In this study, we evaluated a cell therapy treatment using mouse iPSC-derived neural precursor cells (NPCs) engineered to overexpress GCase (termed hGBA1-NPCs). The hGBA1-NPCs secreted GCase that was taken up by adjacent mouse Gba -/- neurons and improved GCase activity, reduced GCase substrate accumulation, and improved mitochondrial function. Short-term in vivo effects were evaluated in 9H/PS-NA mice, an nGD mouse model exhibiting neuropathology and α-synuclein aggregation, the typical PD phenotypes. Intravenously administrated hGBA1-NPCs were engrafted throughout the brain and differentiated into neural lineages. GCase activity was increased in various brain regions of treated 9H/PS-NA mice. Compared with vehicle, hGBA1-NPC-transplanted mice showed â¼50% reduction of α-synuclein aggregates in the substantia nigra, significant reduction of neuroinflammation and neurodegeneration in the regions of NPC migration, and increased expression of neurotrophic factors that support neural cell function. Together, these results support the therapeutic benefit of intravenous delivery of iPSC-derived NPCs overexpressing GCase in mitigating nGD and PD phenotypes and establish the feasibility of combined cell and gene therapy for GBA1-associated PD.
RESUMO
Most organs have tissue-resident immune cells. Human organoids lack these immune cells, which limits their utility in modeling many normal and disease processes. Here, we describe that pluripotent stem cell-derived human colonic organoids (HCOs) co-develop a diverse population of immune cells, including hemogenic endothelium (HE)-like cells and erythromyeloid progenitors that undergo stereotypical steps in differentiation, resulting in the generation of functional macrophages. HCO macrophages acquired a transcriptional signature resembling human fetal small and large intestine tissue-resident macrophages. HCO macrophages modulate cytokine secretion in response to pro- and anti-inflammatory signals and were able to phagocytose and mount a robust response to pathogenic bacteria. When transplanted into mice, HCO macrophages were maintained within the colonic organoid tissue, established a close association with the colonic epithelium, and were not displaced by the host bone-marrow-derived macrophages. These studies suggest that HE in HCOs gives rise to multipotent hematopoietic progenitors and functional tissue-resident macrophages.
Assuntos
Células-Tronco Pluripotentes , Humanos , Camundongos , Animais , Células-Tronco Hematopoéticas , Colo , Organoides , MacrófagosRESUMO
BACKGROUND & AIMS: The tumor suppressors retinoblastoma (RB) and p53 are important regulators of the cell cycle. Although human cancer cells inactivate RB and p53 by many mechanisms, the cooperative roles of these proteins in tumorigenesis are complex and tissue specific. We analyzed the cooperation of RB and p53 in liver development and pathogenesis of hepatocellular carcinoma. METHODS: Spontaneous and carcinogen-induced (diethylnitrosamine) tumorigenesis were studied in mice with liver-specific deletions of Rb and/or p53 (Rbf/f;albcre+, p53f/f;albcre+ and Rbf/f; p53f/f;albcre+ mice). Genotype, histologic, immunohistochemical, microarray, quantitative polymerase chain reaction, immunoblot, and comparative genomic hybridization analyses were performed using normal and tumor samples. Comparative microarray analyses were performed against publicly available human microarray data sets. RESULTS: Deletion of RB and p53 from livers of mice deregulated the transcriptional programs associated with human disease. These changes were not sufficient for spontaneous tumorigenesis; potent quiescence mechanisms compensated for loss of these tumor suppressors. In response to hepatocarcinogen-induced damage, distinct and cooperative roles of RB and p53 were revealed; their loss affected cell cycle control, checkpoint response, and genome stability. In damaged tissue, combined loss of RB and p53 resulted in early lesion formation, aggressive tumor progression, and gene expression signatures and histologic characteristics of advanced human hepatocellular carcinoma. CONCLUSIONS: The effects RB and p53 loss are determined by the tissue environment; cell stresses that promote aggressive disease reveal the functions of these tumor suppressors.
Assuntos
Carcinoma Hepatocelular/prevenção & controle , Neoplasias Hepáticas Experimentais/prevenção & controle , Fígado/metabolismo , Proteína do Retinoblastoma/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Carcinoma Hepatocelular/induzido quimicamente , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Ciclo Celular , Proliferação de Células , Aberrações Cromossômicas , Hibridização Genômica Comparativa , Dietilnitrosamina , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica , Instabilidade Genômica , Genótipo , Humanos , Immunoblotting , Imuno-Histoquímica , Fígado/patologia , Neoplasias Hepáticas Experimentais/induzido quimicamente , Neoplasias Hepáticas Experimentais/metabolismo , Neoplasias Hepáticas Experimentais/patologia , Camundongos , Camundongos da Linhagem 129 , Camundongos Knockout , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , Proteína do Retinoblastoma/deficiência , Proteína do Retinoblastoma/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Transcrição Gênica , Proteína Supressora de Tumor p53/deficiência , Proteína Supressora de Tumor p53/genéticaRESUMO
A major technical limitation hindering the widespread adoption of human pluripotent stem cell (hPSC)-derived gastrointestinal (GI) organoid technologies is the need for de novo hPSC differentiation and dependence on spontaneous morphogenesis to produce detached spheroids. Here, we report a method for simple, reproducible, and scalable production of small intestinal organoids (HIOs) based on the aggregation of cryopreservable hPSC-derived mid-hindgut endoderm (MHE) monolayers. MHE aggregation eliminates variability in spontaneous spheroid production and generates HIOs that are comparable to those arising spontaneously. With a minor modification to the protocol, MHE can be cryopreserved, thawed, and aggregated, facilitating HIO production without de novo hPSC differentiation. Finally, aggregation can also be used to generate antral stomach organoids and colonic organoids. This improved method removes significant barriers to the implementation and successful use of hPSC-derived GI organoid technologies and provides a framework for improved dissemination and increased scalability of GI organoid production.
Assuntos
Organoides , Células-Tronco Pluripotentes , Diferenciação Celular , Endoderma , Humanos , Intestino DelgadoRESUMO
Background & Aims: The truncating mutations in tight junction protein 2 (TJP2) cause progressive cholestasis, liver failure, and hepatocyte carcinogenesis. Due to the lack of effective model systems, there are no targeted medications for the liver pathology with TJP2 deficiency. We leveraged the technologies of patient-specific induced pluripotent stem cells (iPSC) and CRISPR genome-editing, and we aim to establish a disease model which recapitulates phenotypes of patients with TJP2 deficiency. Methods: We differentiated iPSC to hepatocyte-like cells (iHep) on the Transwell membrane in a polarized monolayer. Immunofluorescent staining of polarity markers was detected by a confocal microscope. The epithelial barrier function and bile acid transport of bile canaliculi were quantified between the two chambers of Transwell. The morphology of bile canaliculi was measured in iHep cultured in the Matrigel sandwich system using a fluorescent probe and live-confocal imaging. Results: The iHep differentiated from iPSC with TJP2 mutations exhibited intracellular inclusions of disrupted apical membrane structures, distorted canalicular networks, altered distribution of apical and basolateral markers/transporters. The directional bile acid transport of bile canaliculi was compromised in the mutant hepatocytes, resembling the disease phenotypes observed in the liver of patients. Conclusions: Our iPSC-derived in vitro hepatocyte system revealed canalicular membrane disruption in TJP2 deficient hepatocytes and demonstrated the ability to model cholestatic disease with TJP2 deficiency to serve as a platform for further pathophysiologic study and drug discovery. Lay summary: We investigated a genetic liver disease, progressive familial intrahepatic cholestasis (PFIC), which causes severe liver disease in newborns and infants due to a lack of gene called TJP2. By using cutting-edge stem cell technology and genome editing methods, we established a novel disease modeling system in cell culture experiments. Our experiments demonstrated that the lack of TJP2 induced abnormal cell polarity and disrupted bile acid transport. These findings will lead to the subsequent investigation to further understand disease mechanisms and develop an effective treatment.
RESUMO
The retinoblastoma (RB) tumor suppressor pathway is disrupted at high frequency in hepatocellular carcinoma. However, the mechanisms through which RB modulates physiological responses in the liver remain poorly defined. Despite the well established role of RB in cell cycle control, the deletion of RB had no impact on the kinetics of cell cycle entry or the restoration of quiescence during the course of liver regeneration. Although these findings indicated compensatory effects from the RB-related proteins p107 and p130, even the dual deletion of RB with p107 or p130 failed to deregulate hepatic proliferation. Furthermore, although these findings suggested a modest role for the RB-pathway in the context of proliferative control, RB loss had striking effects on response to the genotoxic hepatocarcinogen diethylnitrosamine. With diethylnitrosamine, RB deletion resulted in inappropriate cell cycle entry that facilitated secondary genetic damage and further uncoupling of DNA replication with mitotic entry. Analysis of the mechanism underlying the differential impact of RB status on liver biology revealed that, while liver regeneration is associated with the conventional induction of cyclin D1 expression, the RB-dependent cell cycle entry, occurring with diethylnitrosamine treatment, was independent of cyclin D1 levels and associated with the specific induction of E2F1. Combined, these studies demonstrate that RB loss has disparate effects on the response to unique tumorigenic stresses, which is reflective of distinct mechanisms of cell cycle entry.
Assuntos
Alquilantes/farmacologia , Carcinoma Hepatocelular/metabolismo , Ciclo Celular/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Dietilnitrosamina/farmacologia , Neoplasias Hepáticas/metabolismo , Proteína do Retinoblastoma , Animais , Carcinoma Hepatocelular/genética , Ciclo Celular/genética , Ciclina D1/genética , Ciclina D1/metabolismo , Dano ao DNA/genética , Replicação do DNA/efeitos dos fármacos , Replicação do DNA/genética , Fator de Transcrição E2F1/genética , Fator de Transcrição E2F1/metabolismo , Deleção de Genes , Neoplasias Hepáticas/genética , Camundongos , Camundongos Transgênicos , Proteína p107 Retinoblastoma-Like/genética , Proteína p107 Retinoblastoma-Like/metabolismo , Proteína p130 Retinoblastoma-Like/genética , Proteína p130 Retinoblastoma-Like/metabolismoRESUMO
BACKGROUND & AIMS: Hepatocellular carcinoma is the third leading cause of cancer mortality worldwide; current chemotherapeutic interventions for this disease are largely ineffective. The retinoblastoma tumor suppressor (RB) is functionally inactivated at relatively high frequency in hepatocellular carcinoma and hepatoma cell lines. Here, we analyzed the ability of CDK4/6 inhibition to inhibit hepatocyte proliferation and the effect of RB status on this process. METHODS: Hepatoma cell lines and xenograft models harboring RB knockdown and mice harboring liver-specific Rb deletion were used to define the role of RB function in response to CDK4/6 inhibition. RESULTS: Our study shows that CDK4/6-dependent cell cycle progression in hepatoma cells was readily arrested by inhibition of CDK4/6 by PD-0332991 or p16ink4a irrespective of RB status. Interestingly, upon CDK4/6 inhibition, p107 protein stability was dramatically increased as a function of RB loss. This engagement of compensatory mechanisms was critical for cell cycle inhibition in the absence of RB, because both the E1A oncoprotein and overexpression of E2F proteins were capable of overcoming the effect of CDK4/6 inhibition. These findings were recapitulated in xenograft models. Furthermore, to determine how these findings relate to hepatocyte proliferation in vivo, mice were exposed to carbon tetrachloride to induce liver regeneration followed by treatment with PD-0332991. This treatment significantly inhibited hepatocyte proliferation. Strikingly, this facet of PD-0332991 function was retained even in RB-deficient livers. CONCLUSIONS: These data show that CDK4/6 inhibition is a potent mediator of cytostasis and that RB loss can be readily compensated for in the context of both hepatoma cell lines and liver tissue.
Assuntos
Carcinoma Hepatocelular/metabolismo , Proliferação de Células/efeitos dos fármacos , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Quinases Ciclina-Dependentes/antagonistas & inibidores , Hepatócitos/efeitos dos fármacos , Neoplasias Hepáticas/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteína do Retinoblastoma/metabolismo , Proteínas E1A de Adenovirus/metabolismo , Animais , Tetracloreto de Carbono , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Ciclo Celular/efeitos dos fármacos , Doença Hepática Induzida por Substâncias e Drogas/genética , Doença Hepática Induzida por Substâncias e Drogas/patologia , Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Quinase 4 Dependente de Ciclina/metabolismo , Quinase 6 Dependente de Ciclina/antagonistas & inibidores , Quinase 6 Dependente de Ciclina/metabolismo , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Modelos Animais de Doenças , Fatores de Transcrição E2F/metabolismo , Técnicas de Silenciamento de Genes , Células Hep G2 , Hepatócitos/metabolismo , Hepatócitos/patologia , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Masculino , Camundongos , Camundongos Knockout , Camundongos Nus , Transplante de Neoplasias , Fosforilação , Piperazinas/farmacologia , Purinas/farmacologia , Piridinas/farmacologia , Interferência de RNA , Proteína do Retinoblastoma/deficiência , Proteína do Retinoblastoma/genética , Proteína p107 Retinoblastoma-Like/metabolismo , Roscovitina , Fatores de Tempo , TransfecçãoRESUMO
Induced Pluripotent Stem Cells (iPSCs) can be differentiated into epithelial organoids that recapitulate the relevant context for CFTR and enable testing of therapies targeting Cystic Fibrosis (CF)-causing mutant proteins. However, to date, CF-iPSC-derived organoids have only been used to study pharmacological modulation of mutant CFTR channel activity and not the activity of other disease-relevant membrane protein constituents. In the current work, we describe a high-throughput, fluorescence-based assay of CFTR channel activity in iPSC-derived intestinal organoids and describe how this method can be adapted to study other apical membrane proteins. Specifically, we show how this assay can be employed to study CFTR and ENaC channels and an electrogenic acid transporter in the same iPSC-derived intestinal tissue. This phenotypic platform promises to expand CF therapy discovery to include strategies that target multiple determinants of epithelial fluid transport.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Intestinos/metabolismo , Organoides/metabolismo , Sistemas de Transporte de Aminoácidos/metabolismo , Animais , Diferenciação Celular , Cães , Canais Epiteliais de Sódio/metabolismo , Edição de Genes , Humanos , Células Madin Darby de Rim CaninoRESUMO
PURPOSE OF REVIEW: The transplantation of insulin-producing beta-cells derived from human embryonic stem cells and induced pluripotent stem cells (collectively termed pluripotent stem cells or PSCs) holds great promise for therapy of diabetes mellitus. The purpose of this review is to summarize recent advances in this area, emphasizing the importance of studies of endocrine pancreas development in efforts to direct PSC differentiation into endocrine cells, as well as to outline the major challenges remaining before transplantation of PSC-derived beta-cells can become a reality. RECENT FINDINGS: Although several protocols to generate glucose-responsive pancreatic beta-cells in vitro have been described, the most successful approaches are those that most closely mimic embryonic development of the endocrine pancreas. Until recently, cells generated by these methods have exhibited immature pancreatic endocrine phenotypes. However, protocols that generate more functional beta-like cells have now been described. In addition, small molecules are being used to improve protocols to direct differentiation of PSCs into endoderm and pancreatic lineages. SUMMARY: Advances over the last decade suggest that generating functional beta-cells from human PSCs is achievable. However, there are aspects of beta-cell development that are not well understood and are hampering generation of PSC-derived beta-cells. In particular, the signaling pathways that instruct endocrine progenitor cells to differentiate into mature and functional beta-cells are poorly understood. Other significant obstacles remain, including the need for safe and cost-effective differentiation methods for large-scale generation of transplantation quality beta-cells, methods to prevent immune rejection of grafted tissues, and amelioration of the risks of tumorigenesis.