RESUMO
Many growth factors and cytokines signal by binding to the extracellular domains of their receptors and driving association and transphosphorylation of the receptor intracellular tyrosine kinase domains, initiating downstream signaling cascades. To enable systematic exploration of how receptor valency and geometry affect signaling outcomes, we designed cyclic homo-oligomers with up to 8 subunits using repeat protein building blocks that can be modularly extended. By incorporating a de novo-designed fibroblast growth factor receptor (FGFR)-binding module into these scaffolds, we generated a series of synthetic signaling ligands that exhibit potent valency- and geometry-dependent Ca2+ release and mitogen-activated protein kinase (MAPK) pathway activation. The high specificity of the designed agonists reveals distinct roles for two FGFR splice variants in driving arterial endothelium and perivascular cell fates during early vascular development. Our designed modular assemblies should be broadly useful for unraveling the complexities of signaling in key developmental transitions and for developing future therapeutic applications.
Assuntos
Diferenciação Celular , Fatores de Crescimento de Fibroblastos , Receptores de Fatores de Crescimento de Fibroblastos , Transdução de Sinais , Animais , Humanos , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Fatores de Crescimento de Fibroblastos/metabolismo , Camundongos , Ligantes , Cálcio/metabolismo , Sistema de Sinalização das MAP QuinasesRESUMO
The move from reading to writing the human genome offers new opportunities to improve human health. The United States National Institutes of Health (NIH) Somatic Cell Genome Editing (SCGE) Consortium aims to accelerate the development of safer and more-effective methods to edit the genomes of disease-relevant somatic cells in patients, even in tissues that are difficult to reach. Here we discuss the consortium's plans to develop and benchmark approaches to induce and measure genome modifications, and to define downstream functional consequences of genome editing within human cells. Central to this effort is a rigorous and innovative approach that requires validation of the technology through third-party testing in small and large animals. New genome editors, delivery technologies and methods for tracking edited cells in vivo, as well as newly developed animal models and human biological systems, will be assembled-along with validated datasets-into an SCGE Toolkit, which will be disseminated widely to the biomedical research community. We visualize this toolkit-and the knowledge generated by its applications-as a means to accelerate the clinical development of new therapies for a wide range of conditions.
Assuntos
Células/metabolismo , Edição de Genes/métodos , Genoma Humano/genética , National Institutes of Health (U.S.)/organização & administração , Animais , Terapia Genética , Objetivos , Humanos , Estados UnidosRESUMO
In February 2020, the European Molecular Biology Laboratory (EMBL) and the Institute for Bioengineering of Catalonia (IBEC) joined forces to unite researchers from all over the globe to discuss emerging topics in 'Engineering Multicellular Systems'. As we review here, key themes that arose throughout the meeting included the ethics of organoids in developmental biology, bottom-up versus top-down models, tissue organizing principles, and the future of improving these systems to better mimic the natural world.
Assuntos
Bioengenharia/métodos , Biologia Sintética/métodos , Animais , Biologia do Desenvolvimento/métodos , Humanos , Organoides , Engenharia Tecidual/métodosRESUMO
Kidney organoids derived from human pluripotent stem cells constitute a novel model of disease, development, and regenerative therapy. Organoids are human, experimentally accessible, high throughput, and enable reconstitution of tissue-scale biology in a petri dish. Although gene expression patterns in organoid cells have been analyzed extensively, less is known about the functionality of these structures. Here, we review assays of physiological function in human kidney organoids, including best practices for quality control, and future applications. Tubular structures in organoids accumulate specific molecules through active transport, including dextran and organic anions, and swell with fluid in response to cAMP stimulation. When engrafted into animal models in vivo, organoids form vascularized glomerulus-like structures capable of size-selective filtration. Organoids exhibit metabolic, endocrine, injury, and infection phenotypes, although their specificity is not yet fully clear. To properly interpret organoid physiology assays, it is important to incorporate appropriate negative and positive controls, statistical methods, data presentation, molecular mechanisms, and clinical data sets. Improvements in organoid perfusion, patterning, and maturation are needed to enable branching morphogenesis, urine production, and renal replacement. Reconstituting renal physiology with kidney organoids is a new field with potential to provide fresh insights into classical phenomena.
Assuntos
Organoides , Células-Tronco Pluripotentes , Animais , Diferenciação Celular , Humanos , Rim/metabolismo , Glomérulos Renais , Morfogênese , Organoides/metabolismo , Células-Tronco Pluripotentes/metabolismoRESUMO
Rare mutations in the LRP2 gene encoding for the endocytic receptor megalin cause developmental abnormalities and kidney disease. However, the mechanisms governing the dysfunction of mutant megalin remain unclear. A new study utilizing patient-derived induced pluripotent stem cells is now putting the endolysosomal system into the spotlight, as it is proposed to play a central role in the regulation of megalin in health and disease.
Assuntos
Células-Tronco Pluripotentes Induzidas , Nefropatias , Humanos , Nefropatias/genética , Nefropatias/terapia , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , MutaçãoRESUMO
Podocytes are differentiated post-mitotic cells that cannot replace themselves after injury. Glomerular parietal epithelial cells are proposed to be podocyte progenitors. To test whether a subset of parietal epithelial cells transdifferentiate to a podocyte fate, dual reporter PEC-rtTA|LC1|tdTomato|Nphs1-FLPo|FRT-EGFP mice, named PEC-PODO, were generated. Doxycycline administration permanently labeled parietal epithelial cells with tdTomato reporter (red), and upon doxycycline removal, the parietal epithelial cells (PECs) cannot label further. Despite the presence or absence of doxycycline, podocytes cannot label with tdTomato, but are constitutively labeled with an enhanced green fluorescent protein (EGFP) reporter (green). Only activation of the Nphs1-FLPo transgene by labeled parietal epithelial cells can generate a yellow color. At day 28 of experimental focal segmental glomerulosclerosis, podocyte density was 20% lower in 20% of glomeruli. At day 56 of experimental focal segmental glomerulosclerosis, podocyte density was 18% lower in 17% of glomeruli. TdTomato+ parietal epithelial cells were restricted to Bowman's capsule in healthy mice. However, by days 28 and 56 of experimental disease, two-thirds of tdTomato+ parietal epithelial cells within glomerular tufts were yellow in color. These cells co-expressed the podocyte markers podocin, nephrin, p57 and VEGF164, but not markers of endothelial (ERG) or mesangial (Perlecan) cells. Expansion microscopy showed primary, secondary and minor processes in tdTomato+EGFP+ cells in glomerular tufts. Thus, our studies provide strong evidence that parietal epithelial cells serve as a source of new podocytes in adult mice.
Assuntos
Transdiferenciação Celular , Células Epiteliais/fisiologia , Glomerulosclerose Segmentar e Focal/patologia , Podócitos/fisiologia , Animais , Modelos Animais de Doenças , Genes Reporter/genética , Glomerulosclerose Segmentar e Focal/terapia , Humanos , Microscopia Intravital , Proteínas Luminescentes/química , Proteínas Luminescentes/genética , Proteínas de Membrana/genética , Camundongos , Camundongos Transgênicos , Microscopia de Fluorescência , Proteína Vermelha FluorescenteRESUMO
Blocking the renin-angiotensin-aldosterone system (RAAS) remains a mainstay of therapy in hypertension and glomerular diseases. With the population aging, our understanding of renin-producing cells in kidneys with advanced age is more critical than ever. Accordingly, we administered tamoxifen to Ren1cCreERxRs-tdTomato-R mice to permanently fate map cells of renin lineage (CoRL). The number of Td-tomato-labeled CoRL decreased significantly in aged mice (24 mo of age) compared with young mice (3.5 mo of age), as did renin mRNA levels. To determine whether aged CoRL responded less to RAAS blockade, enalapril and losartan were administered over 25 days following uninephrectomy in young and aged mice. The number of CoRL increased in young mice in response to enalapril and losartan. However, this was significantly lower in aged mice compared with young mice due to limited proliferation, but not recruitment. Gene expression analysis of laser-captured CoRL showed a substantial increase in mRNA levels for proapoptotic and prosenescence genes, and an increase in a major prosenescence protein on immunostaining. These results show that CoRL are lower in aged mice and do not respond to RAAS inhibition to the same extent as young mice.
Assuntos
Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Pressão Sanguínea/efeitos dos fármacos , Linhagem da Célula , Enalapril/farmacologia , Rim/efeitos dos fármacos , Losartan/farmacologia , Sistema Renina-Angiotensina/efeitos dos fármacos , Renina/metabolismo , Fatores Etários , Envelhecimento , Animais , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Senescência Celular/efeitos dos fármacos , Feminino , Genes Reporter , Rim/metabolismo , Rim/patologia , Rim/cirurgia , Proteínas Luminescentes/biossíntese , Proteínas Luminescentes/genética , Camundongos Transgênicos , Nefrectomia , Proteína Vermelha FluorescenteRESUMO
Understanding of cellular transdifferentiation is limited by the technical inability to track multiple lineages in vivo. To overcome this we developed a new tool to simultaneously fate map two distinct cell types in the kidney, and genetically test whether cells of renin lineage (CoRL) can transdifferentiate to a podocyte fate. Ren1cCreER/tdTomato/Nphs1-FLPo/FRT-EGFP mice (CoRL-PODO mice) were generated by crossing Ren1c-CreER/tdTomato CoRL reporter mice with Nphs1-FLPo/FRT-EGFP podocyte reporter mice. Following tamoxifen administration in these animals, CoRL were labeled with red fluorescence (tdTomato) and co-localized with renin. Podocytes were labeled green (enhanced green fluorescent protein) and co-localized with nephrin. Following podocyte loss by nephrotoxic antibody and subsequent enalapril-enhanced partial replacement, tdTomato-EGFP-labeled CoRL were detected as yellow-colored cells in a subset of glomerular tufts, without the use of antibodies. Co-localization with podocin indicated that these cells are podocytes, derived from CoRL origin. Thus, our novel study shows that two distinct cell types can be simultaneously labeled in the mouse kidney and provide strong genetic evidence in vivo that lost podocytes can be replaced in part by CoRL.
Assuntos
Linhagem da Célula , Rastreamento de Células/métodos , Transdiferenciação Celular , Glomerulosclerose Segmentar e Focal/metabolismo , Podócitos/metabolismo , Renina/metabolismo , Células-Tronco/metabolismo , Animais , Biomarcadores , Modelos Animais de Doenças , Feminino , Genes Reporter , Glomerulosclerose Segmentar e Focal/patologia , Proteínas Luminescentes/biossíntese , Proteínas Luminescentes/genética , Masculino , Camundongos Transgênicos , Microscopia de Fluorescência , Fenótipo , Podócitos/patologia , Renina/genética , Células-Tronco/patologiaRESUMO
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.
Assuntos
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
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.
Assuntos
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
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.
Assuntos
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
Vascular progenitor cells show promise for the treatment of microvasculature endothelial injury. We investigated the function of renal artery progenitor cells derived from radical nephrectomy patients, in animal models of acute ischemic and hyperperfusion injuries. Present in human adventitia, CD34positive/CD105negative cells were clonal and expressed transcription factors Sox2/Oct4 as well as surface markers CXCR4 (CD184)/KDR(CD309) consistent with endothelial progenitor cells. Termed renal artery-derived vascular progenitor cells (RAPC), injected cells were associated with decreased serum creatinine after ischemia/reperfusion, reduced albuminuria after hyperperfusion, and improved blood flow in both models. A small population of RAPC integrated with the renal microvasculature following either experimental injury. At a cellular level, RAPC promoted local endothelial migration in co-culture. Profiling of RAPC microRNA identified high levels of miRNA 218; also found at high levels in exosomes isolated from RAPC conditioned media after cell contact for 24 hours. After hydrogen peroxide-induced endothelial injury, RAPC exosomes harbored Robo-1 transcript; a gene known to be regulated by mir218. Such exosomes enhanced endothelial cell migration in culture in the absence of RAPC. Thus, our work shows the feasibility of pre-emptive pro-angiogenic progenitor cell procurement from a targeted patient population and potential therapeutic use in the form of autologous cell transplantation.
Assuntos
Injúria Renal Aguda/terapia , Capilares/fisiologia , Rim/patologia , Transplante de Células-Tronco/métodos , Células-Tronco/metabolismo , Cicatrização , Injúria Renal Aguda/induzido quimicamente , Animais , Antígenos CD34/metabolismo , Capilares/patologia , Movimento Celular , Técnicas de Cocultura , Creatinina/sangue , Modelos Animais de Doenças , Endoglina/metabolismo , Endotélio/citologia , Exossomos/metabolismo , Estudos de Viabilidade , Humanos , Peróxido de Hidrogênio/toxicidade , Rim/irrigação sanguínea , Camundongos , MicroRNAs/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Receptores CXCR4/metabolismo , Receptores Imunológicos/metabolismo , Artéria Renal/citologia , Transplante Autólogo/métodos , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Proteínas RoundaboutRESUMO
PURPOSE OF REVIEW: Podocytes are critical components of the nephron filtration barrier and are depleted in many kidney injuries and disease states. Terminally differentiated adult podocytes are highly specialized, postmitotic cells, raising the question of whether the body has any ability to regenerate lost podocytes. This timely question has recently been illuminated by a series of innovative studies. Here, we review recent progress on this topic of significant interest and debate. RECENT FINDINGS: The innovation of genetic labeling techniques enables fate tracing of individual podocytes, providing the strongest evidence yet that podocytes can be replaced by nearby progenitor cells. In particular, two progenitor pools have recently been identified in multiple studies: parietal epithelial cells and cells of renin lineage. These studies furthermore suggest that podocyte regeneration can be enhanced using ex-vivo or pharmacological interventions. SUMMARY: Recent studies indicate that the podocyte compartment is more dynamic than previously believed. Bidirectional exchange with neighboring cellular compartments provides a mechanism for podocyte replacement. Based on these findings, we propose a set of criteria for evaluating podocyte regeneration and suggest that restoration of podocyte number to a subsclerotic threshold be targeted as a potentially achievable clinical goal.
Assuntos
Células Epiteliais/fisiologia , Nefropatias/patologia , Podócitos/fisiologia , Regeneração/fisiologia , Células-Tronco/fisiologia , Animais , Contagem de Células , Humanos , Podócitos/patologia , Renina/metabolismo , Células-Tronco/metabolismoRESUMO
The coming decades will see enormous changes in how kidney disease is diagnosed and treated. We can only predict a small proportion of the discoveries that will catalyze these changes. It is exhilarating to imagine how such discoveries might soon translate into improved medical care for millions of people.
Assuntos
Nefropatias , Humanos , Nefropatias/terapia , Tecnologia/tendênciasRESUMO
Human pluripotent stem cells (hPSCs) can generate a diversity of cell types, but few methods have been developed to derive cells of the kidney lineage. Here, we report a highly efficient system for differentiating human embryonic stem cells and induced pluripotent stem cells (referred to collectively as hPSCs) into cells expressing markers of the intermediate mesoderm (IM) that subsequently form tubule-like structures. Treatment of hPSCs with the glycogen synthase kinase-3ß inhibitor CHIR99021 induced BRACHYURY(+)MIXL1(+) mesendoderm differentiation with nearly 100% efficiency. In the absence of additional exogenous factors, CHIR99021-induced mesendodermal cells preferentially differentiated into cells expressing markers of lateral plate mesoderm with minimal IM differentiation. However, the sequential treatment of hPSCs with CHIR99021 followed by fibroblast growth factor-2 and retinoic acid generated PAX2(+)LHX1(+) cells with 70%-80% efficiency after 3 days of differentiation. Upon growth factor withdrawal, these PAX2(+)LHX1(+) cells gave rise to apically ciliated tubular structures that coexpressed the proximal tubule markers Lotus tetragonolobus lectin, N-cadherin, and kidney-specific protein and partially integrated into embryonic kidney explant cultures. With the addition of FGF9 and activin, PAX2(+)LHX1(+) cells specifically differentiated into cells expressing SIX2, SALL1, and WT1, markers of cap mesenchyme nephron progenitor cells. Our findings demonstrate the effective role of fibroblast growth factor signaling in inducing IM differentiation in hPSCs and establish the most rapid and efficient system whereby hPSCs can be differentiated into cells with features characteristic of kidney lineage cells.
Assuntos
Diferenciação Celular/fisiologia , Túbulos Renais Proximais/citologia , Mesoderma/citologia , Células-Tronco Pluripotentes/citologia , Animais , Antineoplásicos/farmacologia , Biomarcadores/metabolismo , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Feminino , Fator 2 de Crescimento de Fibroblastos/farmacologia , Fibroblastos/citologia , Prepúcio do Pênis/citologia , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Humanos , Túbulos Renais Proximais/embriologia , Túbulos Renais Proximais/metabolismo , Proteínas com Homeodomínio LIM/metabolismo , Masculino , Mesoderma/embriologia , Mesoderma/metabolismo , Camundongos , Fator de Transcrição PAX2/metabolismo , Células-Tronco Pluripotentes/efeitos dos fármacos , Gravidez , Piridinas/farmacologia , Pirimidinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Fatores de Transcrição/metabolismo , Tretinoína/farmacologiaRESUMO
iPS cells from patients with kidney disease are a new tool with the potential to impact the future of renal care. They can be used in the laboratory to model the pathophysiology of human kidney disease, and have the potential to establish a new area of immunocompatible, on-demand renal transplantation. Critical challenges remain before the full potential of these cells can be accurately assessed. We need to understand whether the derived cell types are mature and can replace kidney function(s). To what extent can iPS cells model kidney disease in the simplified environment of cell culture? Ultimately, successful integration of these cells as autograft therapies will require demonstration of safety and efficacy equal or superior to the existing gold standards of kidney allograft transplantation and dialysis. Specific educational and infrastructural changes will be necessary if these specialized technologies are to be adopted as an accepted modalities in clinical medicine. Given these barriers, the first fruit of these labors is likely to be improved understanding of pathophysiological pathways in human IPS cell disease models, followed by drug discovery and testing. These experiments will lead naturally to improvements in differentiation and experiments in animal models testing function. The time course to achieve the desired goals remains unknown, but the ultimate hope is that new, more effective and less expensive modalities for renal replacement therapy will occur in the foreseeable future. A new standard of care for patients is anticipated that addresses limitations of currently available treatments.
Assuntos
Terapia Baseada em Transplante de Células e Tecidos/tendências , Células-Tronco Pluripotentes Induzidas/transplante , Nefropatias/terapia , Humanos , FenótipoRESUMO
Heterozygous mutations in PKD1 or PKD2, which encode polycystin-1 (PC1) and polycystin-2 (PC2), respectively, cause autosomal dominant PKD (ADPKD), whereas mutations in PKHD1, which encodes fibrocystin/polyductin (FPC), cause autosomal recessive PKD (ARPKD). However, the relationship between these proteins and the pathogenesis of PKD remains unclear. To model PKD in human cells, we established induced pluripotent stem (iPS) cell lines from fibroblasts of three ADPKD and two ARPKD patients. Genetic sequencing revealed unique heterozygous mutations in PKD1 of the parental ADPKD fibroblasts but no pathogenic mutations in PKD2. Undifferentiated PKD iPS cells, control iPS cells, and embryonic stem cells elaborated primary cilia and expressed PC1, PC2, and FPC at similar levels, and PKD and control iPS cells exhibited comparable rates of proliferation, apoptosis, and ciliogenesis. However, ADPKD iPS cells as well as somatic epithelial cells and hepatoblasts/biliary precursors differentiated from these cells expressed lower levels of PC2 at the cilium. Additional sequencing confirmed the retention of PKD1 heterozygous mutations in iPS cell lines from two patients but identified possible loss of heterozygosity in iPS cell lines from one patient. Furthermore, ectopic expression of wild-type PC1 in ADPKD iPS-derived hepatoblasts rescued ciliary PC2 protein expression levels, and overexpression of PC1 but not a carboxy-terminal truncation mutant increased ciliary PC2 expression levels in mouse kidney cells. Taken together, these results suggest that PC1 regulates ciliary PC2 protein expression levels and support the use of PKD iPS cells for investigating disease pathophysiology.
Assuntos
Células-Tronco Pluripotentes Induzidas/patologia , Rim Policístico Autossômico Dominante/genética , Rim Policístico Autossômico Recessivo/genética , Canais de Cátion TRPP/genética , Adulto , Estudos de Casos e Controles , Linhagem Celular , Feminino , Humanos , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Rim Policístico Autossômico Dominante/metabolismo , Rim Policístico Autossômico Recessivo/metabolismo , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Canais de Cátion TRPP/metabolismoRESUMO
In polycystic kidney disease (PKD), microscopic tubules expand into macroscopic cysts. Among the world's most common genetic disorders, PKD is inherited via heterozygous loss-of-function mutations but is theorized to require additional loss of function. To test this, we establish human pluripotent stem cells in allelic series representing four common nonsense mutations, using CRISPR base editing. When differentiated into kidney organoids, homozygous mutants spontaneously form cysts, whereas heterozygous mutants (original or base corrected) express no phenotype. Using these, we identify eukaryotic ribosomal selective glycosides (ERSGs) as PKD therapeutics enabling ribosomal readthrough of these same nonsense mutations. Two different ERSGs not only prevent cyst initiation but also limit growth of pre-formed cysts by partially restoring polycystin expression. Furthermore, glycosides accumulate in cyst epithelia in organoids and mice. Our findings define the human polycystin threshold as a surmountable drug target for pharmacological or gene therapy interventions, with relevance for understanding disease mechanisms and future clinical trials.
Assuntos
Cistos , Doenças Renais Policísticas , Humanos , Camundongos , Animais , Códon sem Sentido/metabolismo , Canais de Cátion TRPP/genética , Canais de Cátion TRPP/metabolismo , Doenças Renais Policísticas/genética , Doenças Renais Policísticas/terapia , Doenças Renais Policísticas/metabolismo , Rim/metabolismo , Organoides/metabolismo , Cistos/genética , Cistos/metabolismo , Glicosídeos/metabolismoRESUMO
Cell culture technology has evolved, moving from single-cell and monolayer methods to 3D models like reaggregates, spheroids, and organoids, improved with bioengineering like microfabrication and bioprinting. These advancements, termed microphysiological systems (MPSs), closely replicate tissue environments and human physiology, enhancing research and biomedical uses. However, MPS complexity introduces standardization challenges, impacting reproducibility and trust. We offer guidelines for quality management and control criteria specific to MPSs, facilitating reliable outcomes without stifling innovation. Our fit-for-purpose recommendations provide actionable advice for achieving consistent MPS performance.