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Acral melanoma (AM) is an aggressive melanoma variant that arises from palmar, plantar, and nail unit melanocytes. Compared to non-acral cutaneous melanoma (CM), AM is biologically distinct, has an equal incidence across genetic ancestries, typically presents in advanced stage disease, is less responsive to therapy, and has an overall worse prognosis. Independent analysis of published genomic and transcriptomic sequencing identified that receptor tyrosine kinase (RTK) ligands and adapter proteins are frequently amplified, translocated, and/or overexpressed in AM. To target these unique genetic changes, a zebrafish acral melanoma model was exposed to a panel of narrow and broad spectrum multi-RTK inhibitors, revealing that dual FGFR/VEGFR inhibitors decrease acral-analogous melanocyte proliferation and migration. The potent pan-FGFR/VEGFR inhibitor, Lenvatinib, uniformly induces tumor regression in AM patient-derived xenograft (PDX) tumors but only slows tumor growth in CM models. Unlike other multi-RTK inhibitors, Lenvatinib is not directly cytotoxic to dissociated AM PDX tumor cells and instead disrupts tumor architecture and vascular networks. Considering the great difficulty in establishing AM cell culture lines, these findings suggest that AM may be more sensitive to microenvironment perturbations than CM. In conclusion, dual FGFR/VEGFR inhibition may be a viable therapeutic strategy that targets the unique biology of AM.
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Oncogenes can initiate tumors only in certain cellular contexts, which is referred to as oncogenic competence. In melanoma, whether cells in the microenvironment can endow such competence remains unclear. Using a combination of zebrafish transgenesis coupled with human tissues, we demonstrate that GABAergic signaling between keratinocytes and melanocytes promotes melanoma initiation by BRAFV600E. GABA is synthesized in melanoma cells, which then acts on GABA-A receptors in keratinocytes. Electron microscopy demonstrates specialized cell-cell junctions between keratinocytes and melanoma cells, and multielectrode array analysis shows that GABA acts to inhibit electrical activity in melanoma/keratinocyte cocultures. Genetic and pharmacologic perturbation of GABA synthesis abrogates melanoma initiation in vivo. These data suggest that GABAergic signaling across the skin microenvironment regulates the ability of oncogenes to initiate melanoma. SIGNIFICANCE: This study shows evidence of GABA-mediated regulation of electrical activity between melanoma cells and keratinocytes, providing a new mechanism by which the microenvironment promotes tumor initiation. This provides insights into the role of the skin microenvironment in early melanomas while identifying GABA as a potential therapeutic target in melanoma. See related commentary by Ceol, p. 2128. This article is featured in Selected Articles from This Issue, p. 2109.
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Melanoma , Animais , Humanos , Melanoma/tratamento farmacológico , Melanoma/genética , Melanoma/patologia , Peixe-Zebra , Melanócitos/patologia , Pele , Queratinócitos , Transformação Celular Neoplásica/genética , Ácido gama-Aminobutírico , Microambiente TumoralRESUMO
The enteric nervous system (ENS) is derived from both the vagal and sacral component of the neural crest (NC). Here, we present the derivation of sacral ENS precursors from human PSCs via timed exposure to FGF, WNT, and GDF11, which enables posterior patterning and transition from posterior trunk to sacral NC identity, respectively. Using a SOX2::H2B-tdTomato/T::H2B-GFP dual reporter hPSC line, we demonstrate that both trunk and sacral NC emerge from a double-positive neuro-mesodermal progenitor (NMP). Vagal and sacral NC precursors yield distinct neuronal subtypes and migratory behaviors in vitro and in vivo. Remarkably, xenografting of both vagal and sacral NC lineages is required to rescue a mouse model of total aganglionosis, suggesting opportunities in the treatment of severe forms of Hirschsprung's disease.
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Doença de Hirschsprung , Animais , Humanos , Camundongos , Proteínas Morfogenéticas Ósseas , Modelos Animais de Doenças , Fatores de Diferenciação de Crescimento , Xenoenxertos , Histonas , Crista NeuralRESUMO
In melanoma, predicting which tumors will ultimately metastasize guides treatment decisions. Transcriptional signatures of primary tumors have been utilized to predict metastasis, but which among these are driver or passenger events remains unclear. We used data from the adjuvant AVAST-M trial to identify a predictive gene signature in localized tumors that ultimately metastasized. Using a zebrafish model of primary melanoma, we interrogated the top genes from the AVAST-M signature in vivo. This identified GRAMD1B, a cholesterol transfer protein, as a bona fide metastasis suppressor, with a majority of knockout animals rapidly developing metastasis. Mechanistically, excess free cholesterol or its metabolite 27-hydroxycholesterol promotes invasiveness via activation of an AP-1 program, which is associated with increased metastasis in humans. Our data demonstrate that the transcriptional seeds of metastasis are embedded within localized tumors, suggesting that early targeting of these programs can be used to prevent metastatic relapse. SIGNIFICANCE: We analyzed human melanoma transcriptomics data to identify a gene signature predictive of metastasis. To rapidly test clinical signatures, we built a genetic metastasis platform in adult zebrafish and identified GRAMD1B as a suppressor of melanoma metastasis. GRAMD1B-associated cholesterol overload activates an AP-1 program to promote melanoma invasion. This article is highlighted in the In This Issue feature, p. 1.
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Melanoma , Peixe-Zebra , Animais , Humanos , Peixe-Zebra/genética , Fator de Transcrição AP-1/genética , Fator de Transcrição AP-1/metabolismo , Recidiva Local de Neoplasia/genética , Melanoma/patologia , Perfilação da Expressão Gênica , Metástase Neoplásica , Regulação Neoplásica da Expressão GênicaRESUMO
In polycystic kidney disease (PKD), fluid-filled cysts arise from tubules in kidneys and other organs. Human kidney organoids can reconstitute PKD cystogenesis in a genetically specific way, but the mechanisms underlying cystogenesis remain elusive. Here we show that subjecting organoids to fluid shear stress in a PKD-on-a-chip microphysiological system promotes cyst expansion via an absorptive rather than a secretory pathway. A diffusive static condition partially substitutes for fluid flow, implicating volume and solute concentration as key mediators of this effect. Surprisingly, cyst-lining epithelia in organoids polarize outwards towards the media, arguing against a secretory mechanism. Rather, cyst formation is driven by glucose transport into lumens of outwards-facing epithelia, which can be blocked pharmacologically. In PKD mice, glucose is imported through cysts into the renal interstitium, which detaches from tubules to license expansion. Thus, absorption can mediate PKD cyst growth in human organoids, with implications for disease mechanism and potential for therapy development.
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Cistos , Doenças Renais Policísticas , Humanos , Camundongos , Animais , Doenças Renais Policísticas/genética , Doenças Renais Policísticas/metabolismo , Rim/metabolismo , Epitélio/metabolismo , Organoides/metabolismo , Cistos/metabolismoRESUMO
Transparency in glassfrogs has potential implications for human blood clotting.
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Anuros , Coagulação Sanguínea , Eritrócitos , Fígado , Animais , Humanos , Anuros/anatomia & histologia , Anuros/sangue , Anuros/fisiologia , Contagem de Eritrócitos , Fígado/fisiologia , Eritrócitos/fisiologiaRESUMO
The functions of cilia-antenna-like organelles associated with a spectrum of disease states-are poorly understood, particularly in human cells. Here we show that human pluripotent stem cells (hPSCs) edited via CRISPR to knock out the kinesin-2 subunits KIF3A or KIF3B can be used to model ciliopathy phenotypes and to reveal ciliary functions at the tissue scale. KIF3A-/- and KIF3B-/- hPSCs lacked cilia, yet remained robustly self-renewing and pluripotent. Tissues and organoids derived from these hPSCs displayed phenotypes that recapitulated defective neurogenesis and nephrogenesis, polycystic kidney disease (PKD) and other features of the ciliopathy spectrum. We also show that human cilia mediate a critical switch in hedgehog signalling during organoid differentiation, and that they constitutively release extracellular vesicles containing signalling molecules associated with ciliopathy phenotypes. The capacity of KIF3A-/- and KIF3B-/- hPSCs to reveal endogenous mechanisms underlying complex ciliary phenotypes may facilitate the discovery of candidate therapeutics.
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Ciliopatias , Células-Tronco Pluripotentes , Cílios , Ciliopatias/genética , Proteínas Hedgehog/genética , Humanos , Cinesinas/genética , FenótipoRESUMO
Oncogenic alterations to DNA are not transforming in all cellular contexts1,2. This may be due to pre-existing transcriptional programmes in the cell of origin. Here we define anatomic position as a major determinant of why cells respond to specific oncogenes. Cutaneous melanoma arises throughout the body, whereas the acral subtype arises on the palms of the hands, soles of the feet or under the nails3. We sequenced the DNA of cutaneous and acral melanomas from a large cohort of human patients and found a specific enrichment for BRAF mutations in cutaneous melanoma and enrichment for CRKL amplifications in acral melanoma. We modelled these changes in transgenic zebrafish models and found that CRKL-driven tumours formed predominantly in the fins of the fish. The fins are the evolutionary precursors to tetrapod limbs, indicating that melanocytes in these acral locations may be uniquely susceptible to CRKL. RNA profiling of these fin and limb melanocytes, when compared with body melanocytes, revealed a positional identity gene programme typified by posterior HOX13 genes. This positional gene programme synergized with CRKL to amplify insulin-like growth factor (IGF) signalling and drive tumours at acral sites. Abrogation of this CRKL-driven programme eliminated the anatomic specificity of acral melanoma. These data suggest that the anatomic position of the cell of origin endows it with a unique transcriptional state that makes it susceptible to only certain oncogenic insults.
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Melanoma , Neoplasias Cutâneas , Animais , Animais Geneticamente Modificados , Carcinogênese/genética , Pé , Mãos , Humanos , Melanoma/patologia , Unhas , Oncogenes/genética , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Transcrição Gênica , Peixe-Zebra/genética , Melanoma Maligno CutâneoRESUMO
Pathophysiological defects in water homeostasis can lead to renal failure. Likewise, common genetic disorders associated with abnormal cytoskeletal dynamics in the kidney collecting ducts and perturbed calcium and cAMP signaling in the ciliary compartment contribute to chronic kidney failure. We show that collecting ducts in mice lacking the A-Kinase anchoring protein AKAP220 exhibit enhanced development of primary cilia. Mechanistic studies reveal that AKAP220-associated protein phosphatase 1 (PP1) mediates this phenotype by promoting changes in the stability of histone deacetylase 6 (HDAC6) with concomitant defects in actin dynamics. This proceeds through a previously unrecognized adaptor function for PP1 as all ciliogenesis and cytoskeletal phenotypes are recapitulated in mIMCD3 knock-in cells expressing a phosphatase-targeting defective AKAP220-ΔPP1 mutant. Pharmacological blocking of local HDAC6 activity alters cilia development and reduces cystogenesis in kidney-on-chip and organoid models. These findings identify the AKAP220-PPI-HDAC6 pathway as a key effector in primary cilia development.
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Proteínas de Ancoragem à Quinase A/metabolismo , Cílios/metabolismo , Desacetilase 6 de Histona/metabolismo , Homeostase , Rim/metabolismo , Proteína Fosfatase 1/metabolismo , Actinas/metabolismo , Animais , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Células HEK293 , Inibidores de Histona Desacetilases/farmacologia , Humanos , Túbulos Renais Coletores , Camundongos , Organoides/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
It is now possible to direct the differentiation of human pluripotent stem cells into three-dimensional nephron-like structures called kidney organoids. Organoids contain proximal and distal tubules as well as podocytes, in addition to a variety of other lineages such as endothelial cells. Organoid technology has great potential for kidney regeneration and has already been proven to be suitable for modeling kidney disease. However, the methodologies that are used for the generation of kidney organoids require expertise and can be daunting for the inexperienced. Here, we describe in detail a well-established and relatively simple method for the generation of human kidney organoids. We include notes on technical and design considerations for these experiments, and highlight key advantages and limitations of the system.
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Técnicas de Cultura de Células/métodos , Diferenciação Celular , Organoides/fisiologia , Células-Tronco Pluripotentes/fisiologia , Técnicas de Cultura de Células/instrumentação , Humanos , Túbulos Renais/citologia , Túbulos Renais/fisiologia , Podócitos/fisiologiaRESUMO
Organoids derived from human pluripotent stem cells are a potentially powerful tool for high-throughput screening (HTS), but the complexity of organoid cultures poses a significant challenge for miniaturization and automation. Here, we present a fully automated, HTS-compatible platform for enhanced differentiation and phenotyping of human kidney organoids. The entire 21-day protocol, from plating to differentiation to analysis, can be performed automatically by liquid-handling robots, or alternatively by manual pipetting. High-content imaging analysis reveals both dose-dependent and threshold effects during organoid differentiation. Immunofluorescence and single-cell RNA sequencing identify previously undetected parietal, interstitial, and partially differentiated compartments within organoids and define conditions that greatly expand the vascular endothelium. Chemical modulation of toxicity and disease phenotypes can be quantified for safety and efficacy prediction. Screening in gene-edited organoids in this system reveals an unexpected role for myosin in polycystic kidney disease. Organoids in HTS formats thus establish an attractive platform for multidimensional phenotypic screening.
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Diferenciação Celular , Ensaios de Triagem em Larga Escala , Rim/citologia , Organoides/citologia , Fenótipo , Células-Tronco Pluripotentes/citologia , Automação , Técnicas de Cultura de Células , Humanos , Análise de Sequência de RNARESUMO
CRISPR is a nuclease guidance system that enables rapid and efficient gene editing of specific DNA sequences within genomes. We review applications of CRISPR for the study and treatment of kidney disease. CRISPR enables functional experiments in cell lines and model organisms to validate candidate genes arising from genetic studies. CRISPR has furthermore been used to establish the first models of genetic disease in human kidney organoids derived from pluripotent stem cells. These gene-edited organoids are providing new insight into the cellular mechanisms of polycystic kidney disease and nephrotic syndrome. CRISPR-engineered cell therapies are currently in clinical trials for cancers and immunologic syndromes, an approach that may be applicable to inflammatory conditions such as lupus nephritis. Use of CRISPR in large domestic species such as pigs raises the possibility of farming kidneys for transplantation to alleviate the shortage of donor organs. However, significant challenges remain, including how to effectively deliver CRISPR to kidneys and how to control gene editing events within the genome. Thorough testing of CRISPR in preclinical models will be critical to the safe and efficacious translation of this powerful young technology into therapies.
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Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Edição de Genes/métodos , Terapia Genética/tendências , Nefropatias/genética , Nefropatias/terapia , Células-Tronco Pluripotentes/transplante , Animais , Feminino , Previsões , Regulação da Expressão Gênica , Terapia Genética/métodos , Humanos , Nefropatias/epidemiologia , Transplante de Rim/métodos , Transplante de Rim/estatística & dados numéricos , Masculino , Medição de Risco , Resultado do TratamentoRESUMO
Cell manipulation is one of the most impactful applications for optical tweezers, and derived from this promise, we demonstrate a new optical tweezers system for the study of cell adhesion and organization. This method utilizes photonic-crystal-enhanced optical tweezers to manipulate cells with low laser intensities. By doing so, it enables effective cell patterning and culturing within the conditions necessary for successful differentiation and colony formation of human pluripotent stem cells. To this end, the biocompatibility of plasma-treated parylene-C for cell culturing was studied, and a thorough characterization of cellular interactive forces was performed using this system. Furthermore, this study also demonstrates construction of patterned cell arrays at arbitrary positions with micrometer-scale precision.
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Polycystic kidney disease (PKD) is a life-threatening disorder, commonly caused by defects in polycystin-1 (PC1) or polycystin-2 (PC2), in which tubular epithelia form fluid-filled cysts. A major barrier to understanding PKD is the absence of human cellular models that accurately and efficiently recapitulate cystogenesis. Previously, we have generated a genetic model of PKD using human pluripotent stem cells and derived kidney organoids. Here we show that systematic substitution of physical components can dramatically increase or decrease cyst formation, unveiling a critical role for microenvironment in PKD. Removal of adherent cues increases cystogenesis 10-fold, producing cysts phenotypically resembling PKD that expand massively to 1-centimetre diameters. Removal of stroma enables outgrowth of PKD cell lines, which exhibit defects in PC1 expression and collagen compaction. Cyclic adenosine monophosphate (cAMP), when added, induces cysts in both PKD organoids and controls. These biomaterials establish a highly efficient model of PKD cystogenesis that directly implicates the microenvironment at the earliest stages of the disease.
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Microambiente Celular , Modelos Biológicos , Organoides/metabolismo , Doenças Renais Policísticas/metabolismo , Linhagem Celular , AMP Cíclico/metabolismo , Regulação da Expressão Gênica , Humanos , Organoides/patologia , Doenças Renais Policísticas/genética , Doenças Renais Policísticas/patologia , Canais de Cátion TRPP/biossíntese , Canais de Cátion TRPP/genéticaRESUMO
A critical event during kidney organogenesis is the differentiation of podocytes, specialized epithelial cells that filter blood plasma to form urine. Podocytes derived from human pluripotent stem cells (hPSC-podocytes) have recently been generated in nephron-like kidney organoids, but the developmental stage of these cells and their capacity to reveal disease mechanisms remains unclear. Here, we show that hPSC-podocytes phenocopy mammalian podocytes at the capillary loop stage (CLS), recapitulating key features of ultrastructure, gene expression, and mutant phenotype. hPSC-podocytes in vitro progressively establish junction-rich basal membranes (nephrin+ podocin+ ZO-1+ ) and microvillus-rich apical membranes (podocalyxin+ ), similar to CLS podocytes in vivo. Ultrastructural, biophysical, and transcriptomic analysis of podocalyxin-knockout hPSCs and derived podocytes, generated using CRISPR/Cas9, reveals defects in the assembly of microvilli and lateral spaces between developing podocytes, resulting in failed junctional migration. These defects are phenocopied in CLS glomeruli of podocalyxin-deficient mice, which cannot produce urine, thereby demonstrating that podocalyxin has a conserved and essential role in mammalian podocyte maturation. Defining the maturity of hPSC-podocytes and their capacity to reveal and recapitulate pathophysiological mechanisms establishes a powerful framework for studying human kidney disease and regeneration. Stem Cells 2017;35:2366-2378.
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Organoides/metabolismo , Podócitos/metabolismo , Animais , Adesão Celular/genética , Adesão Celular/fisiologia , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Edição de Genes , Humanos , Rim/metabolismo , Rim/patologia , Glomérulos Renais/metabolismo , Glomérulos Renais/patologia , Camundongos , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Sialoglicoproteínas/genética , Sialoglicoproteínas/metabolismoRESUMO
Mucolipidosis IV is a debilitating developmental lysosomal storage disorder characterized by severe neuromotor retardation and progressive loss of vision, leading to blindness by the second decade of life. Mucolipidosis IV is caused by loss-of-function mutations in the MCOLN1 gene, which encodes the transient receptor potential channel protein mucolipin-1. Ophthalmic pathology in patients includes corneal haze and progressive retinal and optic nerve atrophy. Herein, we report ocular pathology in Mcoln1(-/-) mouse, a good phenotypic model of the disease. Early, but non-progressive, thinning of the photoreceptor layer, reduced levels of rhodopsin, disrupted rod outer segments, and widespread accumulation of the typical storage inclusion bodies were the major histological findings in the Mcoln1(-/-) retina. Electroretinograms showed significantly decreased functional response (scotopic a- and b-wave amplitudes) in the Mcoln1(-/-) mice. At the ultrastructural level, we observed formation of axonal spheroids and decreased density of axons in the optic nerve of the aged (6-month-old) Mcoln1(-/-) mice, which indicates progressive axonal degeneration. Our data suggest that mucolipin-1 plays a role in postnatal development of photoreceptors and provides a set of outcome measures that can be used for ocular therapy development for mucolipidosis IV.
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Mucolipidoses/patologia , Nervo Óptico/patologia , Distrofias Retinianas/patologia , Animais , Western Blotting , Modelos Animais de Doenças , Eletrorretinografia , Imunofluorescência , Marcação In Situ das Extremidades Cortadas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mucolipidoses/complicações , Tomografia de Coerência Óptica , Canais de Potencial de Receptor Transitório/deficiência , Canais de Potencial de Receptor Transitório/genéticaRESUMO
Nuclear hormone receptors play a major role in many important biological processes. Most nuclear hormone receptors are ubiquitously expressed and regulate processes such as metabolism, circadian function, and development. They function in these processes to maintain homeostasis through modulation of transcriptional gene networks. In this study we evaluate the effectiveness of a nuclear hormone receptor gene to modulate retinal degeneration and restore the integrity of the retina. Currently, there are no effective treatment options for retinal degenerative diseases leading to progressive and irreversible blindness. In this study we demonstrate that the nuclear hormone receptor gene Nr1d1 (Rev-Erbα) rescues Nr2e3-associated retinal degeneration in the rd7 mouse, which lacks a functional Nr2e3 gene. Mutations in human NR2E3 are associated with several retinal degenerations including enhanced S cone syndrome and retinitis pigmentosa. The rd7 mouse, lacking Nr2e3, exhibits an increase in S cones and slow, progressive retinal degeneration. A traditional genetic mapping approach previously identified candidate modifier loci. Here, we demonstrate that in vivo delivery of the candidate modifier gene, Nr1d1 rescues Nr2e3 associated retinal degeneration. We observed clinical, histological, functional, and molecular restoration of the rd7 retina. Furthermore, we demonstrate that the mechanism of rescue at the molecular and functional level is through the re-regulation of key genes within the Nr2e3-directed transcriptional network. Together, these findings reveal the potency of nuclear receptors as modulators of disease and specifically of NR1D1 as a novel therapeutic for retinal degenerations.
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Oftalmopatias Hereditárias , Terapia Genética/métodos , Membro 1 do Grupo D da Subfamília 1 de Receptores Nucleares , Receptores Nucleares Órfãos , Degeneração Retiniana , Retinose Pigmentar , Transtornos da Visão , Animais , Oftalmopatias Hereditárias/genética , Oftalmopatias Hereditárias/metabolismo , Oftalmopatias Hereditárias/patologia , Oftalmopatias Hereditárias/terapia , Humanos , Camundongos , Camundongos Transgênicos , Membro 1 do Grupo D da Subfamília 1 de Receptores Nucleares/biossíntese , Membro 1 do Grupo D da Subfamília 1 de Receptores Nucleares/genética , Receptores Nucleares Órfãos/biossíntese , Receptores Nucleares Órfãos/genética , Degeneração Retiniana/genética , Degeneração Retiniana/metabolismo , Degeneração Retiniana/patologia , Degeneração Retiniana/terapia , Retinose Pigmentar/genética , Retinose Pigmentar/metabolismo , Retinose Pigmentar/patologia , Retinose Pigmentar/terapia , Transtornos da Visão/genética , Transtornos da Visão/metabolismo , Transtornos da Visão/patologia , Transtornos da Visão/terapiaRESUMO
Eukaryotes have numerous checkpoint pathways to protect genome fidelity during normal cell division and in response to DNA damage. Through a screen for G2/M checkpoint regulators in zebrafish, we identified ticrr (for TopBP1-interacting, checkpoint, and replication regulator), a previously uncharacterized gene that is required to prevent mitotic entry after treatment with ionizing radiation. Ticrr deficiency is embryonic-lethal in the absence of exogenous DNA damage because it is essential for normal cell cycle progression. Specifically, the loss of ticrr impairs DNA replication and disrupts the S/M checkpoint, leading to premature mitotic entry and mitotic catastrophe. We show that the human TICRR ortholog associates with TopBP1, a known checkpoint protein and a core component of the DNA replication preinitiation complex (pre-IC), and that the TICRR-TopBP1 interaction is stable without chromatin and requires BRCT motifs essential for TopBP1's replication and checkpoint functions. Most importantly, we find that ticrr deficiency disrupts chromatin binding of pre-IC, but not prereplication complex, components. Taken together, our data show that TICRR acts in association with TopBP1 and plays an essential role in pre-IC formation. It remains to be determined whether Ticrr represents the vertebrate ortholog of the yeast pre-IC component Sld3, or a hitherto unknown metazoan replication and checkpoint regulator.