RESUMO
There are now many reports of human kidney organoids generated via the directed differentiation of human pluripotent stem cells (PSCs) based on an existing understanding of mammalian kidney organogenesis. Such kidney organoids potentially represent tractable tools for the study of normal human development and disease with improvements in scale, structure, and functional maturation potentially providing future options for renal regeneration. The utility of such organotypic models, however, will ultimately be determined by their developmental accuracy. While initially inferred from mouse models, recent transcriptional analyses of human fetal kidney have provided greater insight into nephrogenesis. In this review, we discuss how well human kidney organoids model the human fetal kidney and how the remaining differences challenge their utility.
Assuntos
Rim/fisiologia , Modelos Biológicos , Organoides/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Rim/citologia , Rim/embriologia , Rim/crescimento & desenvolvimento , Organoides/citologiaRESUMO
Kidney organoids differentiated from pluripotent stem cells are powerful models of kidney development and disease but are characterized by cell immaturity and off-target cell fates. Comparing the cell-specific gene regulatory landscape during organoid differentiation with human adult kidney can serve to benchmark progress in differentiation at the epigenome and transcriptome level for individual organoid cell types. Using single-cell multiome and histone modification analysis, we report more broadly open chromatin in organoid cell types compared to the human adult kidney. We infer enhancer dynamics by cis-coaccessibility analysis and validate an enhancer driving transcription of HNF1B by CRISPR interference both in cultured proximal tubule cells and also during organoid differentiation. Our approach provides an experimental framework to judge the cell-specific maturation state of human kidney organoids and shows that kidney organoids can be used to validate individual gene regulatory networks that regulate differentiation.
Assuntos
Rim , Multiômica , Humanos , Diferenciação Celular/genética , Células Cultivadas , Organoides/metabolismo , Análise de Célula ÚnicaRESUMO
The postnatal kidney is predominantly composed of nephron epithelia with the interstitial components representing a small proportion of the final organ, except in the diseased state. This is in stark contrast to the developing organ, which arises from the mesoderm and comprises an expansive stromal population with distinct regional gene expression. In many organs, the identity and ultimate function of an epithelium is tightly regulated by the surrounding stroma during development. However, although the presence of a renal stromal stem cell population has been demonstrated, the focus has been on understanding the process of nephrogenesis whereas the role of distinct stromal components during kidney morphogenesis is less clear. In this Review, we consider what is known about the role of the stroma of the developing kidney in nephrogenesis, where these cells come from as well as their heterogeneity, and reflect on how this information may improve human kidney organoid models.
Assuntos
Células-Tronco Embrionárias/metabolismo , Rim/embriologia , Animais , Diferenciação Celular , Células-Tronco Embrionárias/citologia , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Rim/citologia , Rim/metabolismo , OrganogêneseRESUMO
Since 2015, Japanese researchers have made great progress in developing a method to differentiate human pluripotent stem cells (hPSCs) into kidney organoids. Protocols have been established to produce increasingly complex three-dimensional (3D) structures, which are used as a human kidney disease model and adapted for high-throughput screening. During this period, single-cell RNA sequencing (scRNA-seq) technology was developed to perform a comprehensive analysis at the single-cell level. We have performed a comprehensive analysis using scRNA-seq to define how kidney organoids can be applied to understand kidney development and pathology. The structure of kidney organoids is complex and contains many cell types of varying maturity. Since only a few proteins and mRNAs can be identified by immunostaining and other techniques, we performed scRNA-seq, which is an unbiased technology that can comprehensively categorize all cell types present in organoids. The aim of this study is to review the problems of kidney organoids based on scRNA-seq and the efforts to address the problems and predict future applications with this powerful technique.
Assuntos
Nefropatias , Rim , Humanos , Diferenciação Celular , Nefropatias/genética , Organoides , Análise de Sequência de RNARESUMO
Human kidney tissue can now be generated via the directed differentiation of human pluripotent stem cells. This advance is anticipated to facilitate the modeling of human kidney diseases, provide platforms for nephrotoxicity screening, enable cellular therapy, and potentially generate tissue for renal replacement. All such applications will rely upon the accuracy and reliability of the model and the capacity for stem cell-derived kidney tissue to recapitulate both normal and diseased states. In this review, we discuss the models available, how well they recapitulate the human kidney, and how far we are from application of these cells for use in cellular therapies.
Assuntos
Rim/citologia , Células-Tronco/citologia , Animais , Diferenciação Celular/fisiologia , Humanos , Nefropatias/fisiopatologiaRESUMO
The aim of this study is to explore the possibility of modeling Gitelman's disease (GIT) with human-induced pluripotent stem cell (hiPSC)-derived kidney organoids and to test whether gene correction using CRISPR/Cas9 can rescue the disease phenotype of GIT. To model GIT, we used the hiPSC line CMCi002 (CMC-GIT-001), generated using PBMCs from GIT patients with SLC12A3 gene mutation. Using the CRISPR-Cas9 system, we corrected CMC-GIT-001 mutations and hence generated CMC-GIT-001corr. Both hiPSCs were differentiated into kidney organoids, and we analyzed the GIT phenotype. The number of matured kidney organoids from the CMC-GIT-001corr group was significantly higher, 3.3-fold, than that of the CMC-GIT-001 group (12.2 ± 0.7/cm2 vs. 3.7 ± 0.2/cm2, p < 0.05). In qRT-PCR, performed using harvested kidney organoids, relative sodium chloride cotransporter (NCCT) mRNA levels (normalized to each iPSC) were increased in the CMC-GIT-001corr group compared with the CMC-GIT-001 group (4.1 ± 0.8 vs. 2.5 ± 0.2, p < 0.05). Consistently, immunoblot analysis revealed increased levels of NCCT protein, in addition to other tubular proteins markers, such as LTL and ECAD, in the CMC-GIT-001corr group compared to the CMC-GIT-001 group. Furthermore, we found that increased immunoreactivity of NCCT in the CMC-GIT-001corr group was colocalized with ECAD (a distal tubule marker) using confocal microscopy. Kidney organoids from GIT patient-derived iPSC recapitulated the Gitelman's disease phenotype, and correction of SLC12A3 mutation utilizing CRISPR-Cas9 technology provided therapeutic insight.
Assuntos
Sistemas CRISPR-Cas , Células-Tronco Pluripotentes Induzidas , Humanos , Sistemas CRISPR-Cas/genética , Membro 3 da Família 12 de Carreador de Soluto , Mutação , Rim , Fenótipo , OrganoidesRESUMO
Advances in clinical diagnostics and molecular tools have improved our understanding of the genetically heterogeneous causes underlying congenital anomalies of kidney and urinary tract (CAKUT). However, despite a sharp incline of CAKUT reports in the literature within the past 2 decades, there remains a plateau in the genetic diagnostic yield that is disproportionate to the accelerated ability to generate robust genome-wide data. Explanations for this observation include (i) diverse inheritance patterns with incomplete penetrance and variable expressivity, (ii) rarity of single-gene drivers such that large sample sizes are required to meet the burden of proof, and (iii) multigene interactions that might produce either intra- (e.g., copy number variants) or inter- (e.g., effects in trans) locus effects. These challenges present an opportunity for the community to implement innovative genetic and molecular avenues to explain the missing heritability and to better elucidate the mechanisms that underscore CAKUT. Here, we review recent multidisciplinary approaches at the intersection of genetics, genomics, in vivo modeling, and in vitro systems toward refining a blueprint for overcoming the diagnostic hurdles that are pervasive in urinary tract malformation cohorts. These approaches will not only benefit clinical management by reducing age at molecular diagnosis and prompting early evaluation for comorbid features but will also serve as a springboard for therapeutic development.
Assuntos
Sistema Urinário , Anormalidades Urogenitais , Variações do Número de Cópias de DNA , Genômica , Humanos , Rim/anormalidades , Anormalidades Urogenitais/diagnóstico , Anormalidades Urogenitais/genéticaRESUMO
BACKGROUND: Podocytes are critical to maintaining the glomerular filtration barrier, and mutations in nephrotic syndrome genes are known to affect podocyte calcium signaling. However, the role of calcium signaling during podocyte development remains unknown. METHODS: We undertook live imaging of calcium signaling in developing podocytes, using zebrafish larvae and human kidney organoids. To evaluate calcium signaling during development and in response to channel blockers and genetic defects, the calcium biosensor GCaMP6s was expressed in zebrafish podocytes. We used electron microscopy to evaluate filtration barrier formation in zebrafish, and Fluo-4 to detect calcium signals in differentiating podocytes in human kidney organoids. RESULTS: Immature zebrafish podocytes (2.5 days postfertilization) generated calcium transients that correlated with interactions with forming glomerular capillaries. Calcium transients persisted until 4 days postfertilization, and were absent after glomerular barrier formation was complete. We detected similar calcium transients in maturing human organoid glomeruli, suggesting a conserved mechanism. In both models, inhibitors of SERCA or IP3 receptor calcium-release channels blocked calcium transients in podocytes, whereas lanthanum was ineffective, indicating the calcium source is from intracellular podocyte endoplasmic-reticulum stores. Calcium transients were not affected by blocking heartbeat or by blocking development of endothelium or endoderm, and they persisted in isolated glomeruli, suggesting podocyte-autonomous calcium release. Inhibition of expression of phospholipase C-γ1, but not nephrin or phospholipase C-ε1, led to significantly decreased calcium activity. Finally, blocking calcium release affected glomerular shape and podocyte foot process formation, supporting the critical role of calcium signaling in glomerular morphogenesis. CONCLUSIONS: These findings establish podocyte cell-autonomous calcium signaling as a prominent and evolutionarily conserved feature of podocyte differentiation and demonstrate its requirement for podocyte foot process formation.
RESUMO
Decades of research into the molecular and cellular regulation of kidney morphogenesis in rodent models, particularly the mouse, has provided both an atlas of the mammalian kidney and a roadmap for recreating kidney cell types with potential applications for the treatment of kidney disease. With advances in both our capacity to maintain nephron progenitors in culture, reprogram to kidney cell types and direct the differentiation of human pluripotent stem cells to kidney endpoints, renal regeneration via cellular therapy or tissue engineering may be possible. Human kidney models also have potential for disease modelling and drug screening. Such applications will rely upon the accuracy of the model at the cellular level and the capacity for stem-cell derived kidney tissue to recapitulate both normal and diseased kidney tissue. In this review, we will discuss the available cell sources, how well they model the human kidney and how far we are from application either as models or for tissue engineering.
Assuntos
Terapia Baseada em Transplante de Células e Tecidos/métodos , Rim/fisiologia , Néfrons/fisiologia , Regeneração , Insuficiência Renal Crônica/terapia , Animais , Diferenciação Celular , Humanos , Rim/citologia , Néfrons/citologia , Células-Tronco Pluripotentes/citologia , Insuficiência Renal Crônica/fisiopatologia , Engenharia Tecidual/métodosRESUMO
Despite the increasing diagnostic rate of genomic sequencing, the genetic basis of more than 50% of heritable kidney disease remains unresolved. Kidney organoids differentiated from induced pluripotent stem cells (iPSCs) of individuals affected by inherited renal disease represent a potential, but unvalidated, platform for the functional validation of novel gene variants and investigation of underlying pathogenetic mechanisms. In this study, trio whole-exome sequencing of a prospectively identified nephronophthisis (NPHP) proband and her parents identified compound-heterozygous variants in IFT140, a gene previously associated with NPHP-related ciliopathies. IFT140 plays a key role in retrograde intraflagellar transport, but the precise downstream cellular mechanisms responsible for disease presentation remain unknown. A one-step reprogramming and gene-editing protocol was used to derive both uncorrected proband iPSCs and isogenic gene-corrected iPSCs, which were differentiated to kidney organoids. Proband organoid tubules demonstrated shortened, club-shaped primary cilia, whereas gene correction rescued this phenotype. Differential expression analysis of epithelial cells isolated from organoids suggested downregulation of genes associated with apicobasal polarity, cell-cell junctions, and dynein motor assembly in proband epithelial cells. Matrigel cyst cultures confirmed a polarization defect in proband versus gene-corrected renal epithelium. As such, this study represents a "proof of concept" for using proband-derived iPSCs to model renal disease and illustrates dysfunctional cellular pathways beyond the primary cilium in the setting of IFT140 mutations, which are established for other NPHP genotypes.
Assuntos
Cílios/patologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Rim/patologia , Organoides/patologia , Sequência de Aminoácidos , Sequência de Bases , Proteínas de Transporte/química , Proteínas de Transporte/genética , Células Cultivadas , Reprogramação Celular/genética , Ataxia Cerebelar/genética , Células Epiteliais/metabolismo , Feminino , Fibroblastos/patologia , Flagelos/metabolismo , Edição de Genes , Perfilação da Expressão Gênica , Heterozigoto , Humanos , Células-Tronco Pluripotentes Induzidas/patologia , Rim/diagnóstico por imagem , Fenótipo , Estabilidade de RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reprodutibilidade dos Testes , Retinose Pigmentar/genética , Esferoides Celulares/metabolismo , Esferoides Celulares/patologia , Sequenciamento do ExomaRESUMO
Kidney organoids generated from human pluripotent stem cells (hPSCs) have drastically changed the field of stem cell research on human kidneys within a few years. They are self-organizing multicellular structures that contain nephron components such as glomeruli and renal tubules in most cases, but hPSC-derived ureteric buds, the progenitors of collecting ducts and ureters, can also form three-dimensional organoids. Today's challenges facing human kidney organoids are further maturation and anatomical integrity in order to achieve a complete model of the developing kidneys and ultimately a complete adult organ. Since chronic kidney disease (CKD) and impaired kidney function are an increasing burden on public health worldwide, there is an urgent need to develop effective treatments for various renal conditions. In this regard, hPSC-derived kidney organoids may impact medicine by providing new translational approaches. The unique ability of kidney organoids derived from disease-specific hPSCs to reproduce human diseases caused by genetic alterations may help provide the next generation of kidney disease models. Recent advances in the field of kidney organoid research have been generally accompanied by progress in developmental biology and other technological breakthroughs. In this review, we consider the current trends in kidney organoid technology, especially focusing on the relationship to the study of human kidney development, and discuss the remaining hurdles and prospects in regenerating human kidney structures beyond organoids.
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Biologia do Desenvolvimento , Rim/citologia , Organoides/citologia , Animais , Diferenciação Celular , HumanosRESUMO
Nephron formation continues throughout kidney morphogenesis in both mice and humans. Lineage tracing studies in mice identified a self-renewing Six2-expressing nephron progenitor population able to give rise to the full complement of nephrons throughout kidney morphogenesis. To investigate the origin of nephrons within human pluripotent stem cell-derived kidney organoids, we performed a similar fate-mapping analysis of the SIX2-expressing lineage in induced pluripotent stem cell (iPSC)-derived kidney organoids to explore the feasibility of investigating lineage relationships in differentiating iPSCs in vitro Using CRISPR/Cas9 gene-edited lineage reporter lines, we show that SIX2-expressing cells give rise to nephron epithelial cell types but not to presumptive ureteric epithelium. The use of an inducible (CreERT2) line revealed a declining capacity for SIX2+ cells to contribute to nephron formation over time, but retention of nephron-forming capacity if provided an exogenous WNT signal. Hence, while human iPSC-derived kidney tissue appears to maintain lineage relationships previously identified in developing mouse kidney, unlike the developing kidney in vivo, kidney organoids lack a nephron progenitor niche capable of both self-renewal and ongoing nephrogenesis.
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Mapeamento Cromossômico , Perfilação da Expressão Gênica , Genes Reporter , Néfrons/embriologia , Néfrons/metabolismo , Organogênese/genética , Biomarcadores , Sistemas CRISPR-Cas , Técnicas de Cultura de Células , Diferenciação Celular , Proteínas de Homeodomínio/genética , Humanos , Proteínas do Tecido Nervoso/genética , Organoides , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Análise de Célula ÚnicaRESUMO
Autosomal dominant polycystic kidney disease (ADPKD) is a hereditary disorder which manifests progressive renal cyst formation and leads to end-stage kidney disease. Around 85% of cases are caused by PKD1 heterozygous mutations, exhibiting relatively poorer renal outcomes than those with mutations in other causative gene PKD2. Although many disease models have been proposed for ADPKD, the pre-symptomatic pathology of the human disease remains unknown. To unveil the mechanisms of early cytogenesis, robust and genetically relevant human models are needed. Here, we report a novel ADPKD model using kidney organoids derived from disease-specific human induced pluripotent stem cells (hiPSCs). Importantly, we found that kidney organoids differentiated from gene-edited heterozygous PKD1-mutant as well as ADPKD patient-derived hiPSCs can reproduce renal cysts. Further, we demonstrated the possibility of ADPKD kidney organoids serving as drug screening platforms. This newly developed model will contribute to identifying novel therapeutic targets, extending the field of ADPKD research.
Assuntos
Células-Tronco Pluripotentes Induzidas/patologia , Rim/patologia , Modelos Biológicos , Organoides/patologia , Rim Policístico Autossômico Dominante/patologia , Sequência de Aminoácidos , Sequência de Bases , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Colforsina/farmacologia , Avaliação Pré-Clínica de Medicamentos , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Mutação/genética , Fenótipo , Canais de Cátion TRPP/química , Canais de Cátion TRPP/genéticaRESUMO
It is difficult to restore kidney function following chronic kidney damage. Although dialysis is currently used to treat patients with chronic kidney disease, it does not cure the disease, while severely restricting the patient's daily and social activities. Kidney transplantation is an alternative and curative therapy, but donor numbers remain limited. However, the generation of kidney organoids from human induced pluripotent stem cells represents an important recent advance in regenerative medicine. Kidney organoids are expected to be used for disease modeling and drug discovery, and may eventually be applicable for transplantation. In this review, we describe the current status of kidney organoids and discuss the hurdles that need to be overcome to generate transplantable artificial kidneys.
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Células-Tronco Pluripotentes Induzidas , Transplante de Rim , Rim/fisiologia , Rim/fisiopatologia , Rins Artificiais , Organoides , Animais , Humanos , Rim/irrigação sanguínea , Néfrons/fisiologia , Células-TroncoAssuntos
COVID-19 , Diabetes Mellitus , Humanos , COVID-19/complicações , Organoides , SARS-CoV-2 , Rim , Diabetes Mellitus/etiologiaRESUMO
The treatment of renal failure has changed little in decades. Organ transplantation and dialysis continue to represent the only therapeutic options available. However, decades of fundamental research into the response of the kidney to acute injury and the processes driving progression to chronic kidney disease are beginning to open doors to new options. Similarly, continued investigations into the cellular and molecular basis of normal kidney development, together with major advances in stem cell biology, are now delivering options in regenerative medicine not possible as recently as a decade ago. In this review, we will discuss advances in regenerative medicine as it may be applied to the kidney. This will cover cellular therapies focused on ameliorating injury and improving repair as well as advancements in the generation of new renal tissue from stem/progenitor cells.
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Terapia Baseada em Transplante de Células e Tecidos/métodos , Nefropatias/terapia , Rim/fisiologia , Regeneração , Transplante de Células-Tronco , Animais , Reprogramação Celular , Células-Tronco Fetais/fisiologia , Humanos , Rim/citologia , Células-Tronco Mesenquimais/fisiologia , CicatrizaçãoRESUMO
Microplastics (MPs) have emerged as a pervasive environmental pollutant of global concern. Their detection within the human placenta and fetal organs has prompted apprehension regarding the potential hazards of MPs during early organogenesis. The kidney, a vital multifunctional organ, is susceptible to damage from MPs in adulthood. However, the precise adverse effects of MP exposure on human nephrogenesis remain ambiguous due to the absence of a suitable model. Here, we explore the potential impact of MPs on early kidney development utilizing human kidney organoids in vitro. Human kidney organoids were subjected to polystyrene-MPs (PS-MPs, 1 µm) during the nephron progenitor cell (NPC) stage, a critical phase in early kidney development and patterning. We delineate the effects of PS-MPs on various stages of nephrogenesis, including NPC, renal vesicle, and comma-shaped body, through sequential examination of kidney organoids. PS-MPs were observed to adhere to the surface of cells during the NPC stage and accumulate within glomerulus-like structures within kidney organoids. Moreover, both short- and long-term exposure to PS-MPs resulted in diminished organoid size and aberrant nephron structure. PS-MP exposure heightened reactive oxygen species (ROS) production, leading to NPC apoptosis during early kidney development. Increased apoptosis, diminished cell viability, and NPC reduction likely contribute to the observed organoid size reduction under PS-MP treatment. Transcriptomic analysis at both NPC and endpoint stages revealed downregulation of Notch signaling, resulting in compromised proximal and distal tubular structures, thereby disrupting normal nephron patterning following PS-MP exposure. Our findings highlight the significant disruptive impact of PS-MPs on human kidney development, offering new insights into the mechanisms underlying PS-MP-induced nephron toxicity.