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• W2887161891 abstract "Development of physiologically relevant cellular models with strong translatability to human pathophysiology is critical for identification and validation of novel therapeutic targets. Herein we describe a detailed protocol for generation of an advanced 3-dimensional kidney cellular model using induced pluripotent stem cells, where differentiation and maturation of kidney progenitors and podocytes can be monitored in live cells due to CRISPR/Cas9-mediated fluorescent tagging of kidney lineage markers (SIX2 and NPHS1). Utilizing these cell lines, we have refined the previously published procedures to generate a new, higher throughput protocol suitable for drug discovery. Using paraffin-embedded sectioning and whole-mount immunostaining, we demonstrated that organoids grown in suspension culture express key markers of kidney biology (WT1, ECAD, LTL, nephrin) and vasculature (CD31) within renal cortical structures with microvilli, tight junctions and podocyte foot processes visualized by electron microscopy. Additionally, the organoids resemble the adult kidney transcriptomics profile, thereby strengthening the translatability of our in vitro model. Thus, development of human nephron-like structures in vitro fills a major gap in our ability to assess the effect of potential treatment on key kidney structures, opening up a wide range of possibilities to improve clinical translation. Development of physiologically relevant cellular models with strong translatability to human pathophysiology is critical for identification and validation of novel therapeutic targets. Herein we describe a detailed protocol for generation of an advanced 3-dimensional kidney cellular model using induced pluripotent stem cells, where differentiation and maturation of kidney progenitors and podocytes can be monitored in live cells due to CRISPR/Cas9-mediated fluorescent tagging of kidney lineage markers (SIX2 and NPHS1). Utilizing these cell lines, we have refined the previously published procedures to generate a new, higher throughput protocol suitable for drug discovery. Using paraffin-embedded sectioning and whole-mount immunostaining, we demonstrated that organoids grown in suspension culture express key markers of kidney biology (WT1, ECAD, LTL, nephrin) and vasculature (CD31) within renal cortical structures with microvilli, tight junctions and podocyte foot processes visualized by electron microscopy. Additionally, the organoids resemble the adult kidney transcriptomics profile, thereby strengthening the translatability of our in vitro model. Thus, development of human nephron-like structures in vitro fills a major gap in our ability to assess the effect of potential treatment on key kidney structures, opening up a wide range of possibilities to improve clinical translation. see commentary on page 1040 see commentary on page 1040 Disease intervention using small molecules has been central to health care for many years. In recent years, larger molecules ranging from RNA-based approaches to peptides and proteins are all showing potential to meet a wide range of medical needs. Each modality has its strengths and weaknesses, but all require relevant cellular models for target validation and toxicity prediction. Human primary cells and patient-derived cells are in many cases most desirable to faithfully recapitulate a disease state in a dish; however, these cells often have limited availability, may lose their physiological characteristics in culture, and can rarely be expanded to the numbers needed for in vitro assays and large-scale screening purposes. The induced pluripotent stem cell (iPSC) technology has made it possible to access and modify cells of a wide variety of origins that previously have been difficult or nearly impossible to study. Within the field of nephrology, some recent advances have been made in creating 3-dimensional (3D) organoid cultures,1Ciampi O. Iacone R. Longaretti L. et al.Generation of functional podocytes from human induced pluripotent stem cells.Stem Cell Res. 2016; 17: 130-139Crossref PubMed Scopus (52) Google Scholar, 2Freedman B.S. Brooks C.R. Lam A.Q. et al.Modelling kidney disease with CRISPR-mutant kidney organoids derived from human pluripotent epiblast spheroids.Nat Commun. 2015; 6: 8715Crossref PubMed Scopus (444) Google Scholar, 3Morizane R. Lam A.Q. Freedman B.S. et al.Nephron organoids derived from human pluripotent stem cells model kidney development and injury.Nat Biotechnol. 2015; 33: 1193-1200Crossref PubMed Scopus (509) Google Scholar, 4Sharmin S. Taguchi A. Kaku Y. et al.Human induced pluripotent stem cell-derived podocytes mature into vascularized glomeruli upon experimental transplantation.J Am Soc Nephrol. 2016; 27: 1778-1791Crossref PubMed Scopus (141) Google Scholar, 5Takasato M. Er P.X. Chiu H.S. et al.Kidney organoids from human iPS cells contain multiple lineages and model human nephrogenesis.Nature. 2015; 526: 564-568Crossref PubMed Scopus (879) Google Scholar but there are still limited functional tools for drug discovery available. Approximately 10% of the population worldwide is affected by chronic kidney disease (CKD), a number expected to rise due to increased prevalence of risk factors such as diabetes, hypertension, obesity, and an aging population. Unfortunately, CKD is one of the areas where there is a great unmet need for innovative pharmacological therapies. Technologies for ex vivo nephrogenesis would not only create great advances for early-stage drug discovery and toxicity studies but could also potentially enable therapeutic replacement of damaged kidney tissue in the future when these complicated anatomical structures can be functionally recreated in vitro. The nephron is the basic structural and functional unit of the kidney, where glomerular filtration and tubular reabsorption occur to maintain body homeostasis. The filtering unit of the nephron, the glomerulus, has a highly specialized barrier made up of podocytes forming slit diaphragms, endothelial cells covered by the glycocalyx, and between them the basement membrane. This permselective barrier restricts molecules filtered into the urinary space mainly depending on size and charge.6Haraldsson B. Nystrom J. Deen W.M. Properties of the glomerular barrier and mechanisms of proteinuria.Physiol Rev. 2008; 88: 451-487Crossref PubMed Scopus (608) Google Scholar, 7Perico L. Conti S. Benigni A. et al.Podocyte-actin dynamics in health and disease.Nat Rev Nephrol. 2016; 12: 692-710Crossref PubMed Scopus (117) Google Scholar In addition, mesangial cells reside between the capillary loops.8Schlondorff D. Banas B. The mesangial cell revisited: no cell is an island.J Am Soc Nephrol. 2009; 20: 1179-1187Crossref PubMed Scopus (302) Google Scholar Glomerular disease is one of the major causes of chronic and end-stage renal disease.9Shankland S.J. The podocyte's response to injury: role in proteinuria and glomerulosclerosis.Kidney Int. 2006; 69: 2131-2147Abstract Full Text Full Text PDF PubMed Scopus (659) Google Scholar Notably, there are still no adequate in vitro cellular models to mimic glomerular function that faithfully recapitulates the inherent in vivo properties of podocytes and the cellular cross-talk taking place in the glomerulus.10Dimke H. Maezawa Y. Quaggin S.E. Crosstalk in glomerular injury and repair.Curr Opin Nephrol Hypertens. 2015; 24: 231-238PubMed Google Scholar, 11Lennon R. Hosawi S. Glomerular cell crosstalk.Curr Opin Nephrol Hypertens. 2016; 25: 187-193Crossref PubMed Scopus (28) Google Scholar Isolation and culture of glomeruli result in outgrowth of a fraction of podocytes that have re-entered the cell cycle. These cells regain a limited proliferation potential and can be transiently maintained in culture; this subculture does, however, induce de-differentiation as reflected by loss of foot processes and differentiation-specific markers such as nephrin (NPHS1) and synaptopodin (SYNPO).12Mundel P. Heid H.W. Mundel T.M. et al.Synaptopodin: an actin-associated protein in telencephalic dendrites and renal podocytes.J Cell Biol. 1997; 139: 193-204Crossref PubMed Scopus (488) Google Scholar, 13Mundel P. Reiser J. Kriz W. Induction of differentiation in cultured rat and human podocytes.J Am Soc Nephrol. 1997; 8: 697-705Crossref PubMed Google Scholar, 14Mundel P. Reiser J. Zuniga Mejia Borja A. et al.Rearrangements of the cytoskeleton and cell contacts induce process formation during differentiation of conditionally immortalized mouse podocyte cell lines.Exp Cell Res. 1997; 236: 248-258Crossref PubMed Scopus (765) Google Scholar, 15Yaoita E. Yamamoto T. Takashima N. et al.Visceral epithelial cells in rat glomerular cell culture.Eur J Cell Biol. 1995; 67: 136-144PubMed Google Scholar, 16van der Woude F.J. Michael A.F. Muller E. Lymphohaemopoietic antigens of cultured human glomerular epithelial cells.Br J Exp Pathol. 1989; 70: 73-82PubMed Google Scholar SIX1 and SIX2 are developmental markers of nephron commitment, and it has been shown that an auto-cross regulatory loop drives continued SIX1 and SIX2 expression during active nephrogenesis.17O'Brien L.L. Guo Q. Lee Y. et al.Differential regulation of mouse and human nephron progenitors by the Six family of transcriptional regulators.Development. 2016; 143: 595-608Crossref PubMed Scopus (82) Google Scholar However, because SIX2 has been suggested to be the predominant SIX factor in human nephron progenitors,17O'Brien L.L. Guo Q. Lee Y. et al.Differential regulation of mouse and human nephron progenitors by the Six family of transcriptional regulators.Development. 2016; 143: 595-608Crossref PubMed Scopus (82) Google Scholar this factor was chosen together with NPHS1 when developing a kidney-specific reporter assay. Combining CRISPR/Cas9 technology with a 3D differentiation protocol, we have established a system on which kidney differentiation, glomerular maturation, and podocyte health can be monitored in living cells using fluorescently tagged kidney lineage markers. This article builds on the published kidney organoid work performed in the Little and Bonventre laboratories2Freedman B.S. Brooks C.R. Lam A.Q. et al.Modelling kidney disease with CRISPR-mutant kidney organoids derived from human pluripotent epiblast spheroids.Nat Commun. 2015; 6: 8715Crossref PubMed Scopus (444) Google Scholar, 3Morizane R. Lam A.Q. Freedman B.S. et al.Nephron organoids derived from human pluripotent stem cells model kidney development and injury.Nat Biotechnol. 2015; 33: 1193-1200Crossref PubMed Scopus (509) Google Scholar, 5Takasato M. Er P.X. Chiu H.S. et al.Kidney organoids from human iPS cells contain multiple lineages and model human nephrogenesis.Nature. 2015; 526: 564-568Crossref PubMed Scopus (879) Google Scholar and extends it to build a higher-throughput system, in which long-term assessment of glomerular maturation and health can be achieved using a 3D live imaging environment. Because de-differentiation of nephrin-expressing podocytes is a hallmark of chronic kidney disease, we can use our system to monitor podocyte health through increase or decrease of nephrin-reporter expression in living cells. This innovative technology is currently being applied in safety and toxicology studies as well as for assessment of targeted delivery approaches for the kidney using new molecular entities. CRISPR/Cas9 nickases with truncated dual guides in combination with plasmid homology donors were used to generate 3 kidney-specific reporter cell lines, 2 single reporters (SIX2-GFP and NPHS1-GFP), and 1 dual SIX2-GFP/NPHS1-mKate. Correctly knocked-in clones were identified using junction polymerase chain reaction (PCR) (see Figure 1 for primer locations). Multiplex digital droplet PCR was used to determine the number of knocked-in sequences (data not shown). Heterozygous knock-in clones were selected to ascertain retained wild-type protein function. Three clones from each line were quality controlled, cryopreserved, and used throughout the optimization procedures to avoid any differentiation bias caused by unknown CRISPR/Cas9 off-target effects. Retained pluripotency was verified by fluorescence-activated cell sorting (FACS) analyses of pluripotency marker expression (Figure 2a) and genomic stability was assessed by g-banding (Figure 2b). Using the combined actions of GSK3β inhibition and FGF9 stimulation we observed an increasing and concomitant expression of green fluorescent protein (GFP; via FACS) and SIX2 (via quantitative PCR [qPCR]) throughout differentiation. SIX2 gene expression appeared around day 7 and increased 9-fold to day 18 (via qPCR), mirroring the GFP expression pattern measured by FACS, starting at 7% (day 7) and reaching 48% at day 18 (Figure 3a). GFP-positive populations were sorted out at various time points, and a high enrichment of SIX2 and NPHS1 transcripts, respectively, was verified (Figure 3b). Based on the 2 landmark papers on kidney organoids,3Morizane R. Lam A.Q. Freedman B.S. et al.Nephron organoids derived from human pluripotent stem cells model kidney development and injury.Nat Biotechnol. 2015; 33: 1193-1200Crossref PubMed Scopus (509) Google Scholar, 5Takasato M. Er P.X. Chiu H.S. et al.Kidney organoids from human iPS cells contain multiple lineages and model human nephrogenesis.Nature. 2015; 526: 564-568Crossref PubMed Scopus (879) Google Scholar we have combined and further developed the protocols for maturation of nephron progenitors into kidney organoids using a standardized assay process in a format suitable for drug discovery. To transfer the protocols we extended the monolayer phase to 10 days and assessed different cell densities (10–500 k), in the end resulting in a robust, higher-throughput protocol generating smaller 10k organoids (>7000 organoids per AggreWell plate [Stemcell Technologies, Vancouver, Canada]) that after transfer to ultralow-attachment plates grow and mature in suspension culture. This methodological expansion in combination with the reporters results in organoids that are highly valuable and useful for drug discovery. The reporters facilitate quality control of each experiment, ensuring that the organoids delivered for downstream applications are fully matured (Figure 3c and d). A comparison of marker expression in different organoid sizes can be found in Supplementary Figure S1. The 3D culture differentiation protocol induced an impressive maturation, as seen by gene expression analysis. Several markers specific for early kidney differentiation (Figure 4a), podocytes (Figure 4b), proximal tubules (Figure 4c), and endothelial cells and extracellular matrix molecules (Figure 4d) have been analyzed over time to define the maturation stage of the organoid. Some additional markers of nephron commitment and maturation can be found in Supplementary Figure S2. Notably, expression of nephron progenitor markers SIX1 and SIX2 appeared to increase even after the organoids were formed and matured. Whole-mount immunostaining of 10k organoids at day 25 of differentiation revealed numerous nephron-like structures with features of podocytes (WT1+) and surrounding proximal tubular networks that stained positive for Lotus tetragonolobus lectin (LTL), a marker specific for mature proximal convoluted tubules and E-cadherin (ECAD), a marker for distal tubules. Infiltrating blood vessel–like structures stained positive for the endothelial marker CD31 (Figure 5a–d and Supplementary Movies S1 and S2). To confirm the structural organization of the organoids, paraffin-embedded sections were stained with periodic acid–Schiff as well as with antibodies against NPHS1, WT1, and CD31 at day 25 (Figure 6). Visual examination of the staining revealed that both NPHS1 and WT1-positive cells were organized in glomerulus-like structures along with CD31-positive vessel-like structures. To further elucidate ultrastructural features characteristic of mature renal epithelia, transmission and scanning electron microscopy were performed around day 35 of 2 different organoid experiments. Figure 7a shows representative images of the 2 major cellular clusters found during transmission electron microscopy analyses, revealing tubular structures with polarized cells, and epithelial tight junctions and desmosomes similar to kidney tubules. These analyses also showed structures resembling foot processes protruding from podocyte-like cells, surrounded by a layer of cells reminiscent of Bowman’s capsule. Notably, structures showing surface areas resembling podocyte secondary foot processes with slit diaphragms were also observed using scanning electron microscopy (Figure 7b). To further validate mature kidney characteristics of cells within the organoid, we used single-cell RNA sequencing technology, which enables profiling of gene expression patterns at the individual cell level, thus allowing characterization of cell types within complex multi-cellular tissues. We dissociated the kidney organoid to achieve single-cell suspension, sorted individual cells onto 384-well plates, and used Smart-Seq2 protocol to sequence the cellular transcriptomes. Single-cell consensus clustering (SC3), a tool for unsupervised clustering, was used to classify different cell types within the organoid,18Kiselev V.Y. Kirschner K. Schaub M.T. et al.SC3: consensus clustering of single-cell RNA-seq data.Nat Methods. 2017; 14: 483-486Crossref PubMed Scopus (689) Google Scholar dividing the cells into 3 clusters. Interestingly, using the differentially expressed test by SC3 we observed that cluster 2 was highly enriched in podocyte marker genes, whereas tubular marker genes such as SPP1 were differentially expressed in cluster 3 (Supplementary Figure S3). A second differentially expressed test was used to analyze the differentially expressed genes of clusters 2 and 3,19Kharchenko P.V. Silberstein L. Scadden D.T. Bayesian approach to single-cell differential expression analysis.Nat Methods. 2014; 11: 740-742Crossref PubMed Scopus (689) Google Scholar confirming that podocyte and tubular markers were among the top highly differentially expressed genes in clusters 2 and 3, respectively (Supplementary Table S1). To illustrate and confirm the SC3 classification, we used t-distributed stochastic neighbor embedding (t-SNE),20van der Maaten L. Hinton G. Visualizing data using t-SNE.J Mach Learning Res. 2008; 9: 2579-2605Google Scholar highlighting the podocyte marker NPHS1, the tubular marker SPP1, and the progenitor markers SIX1 and SIX2 (Figure 8a–d). Of note, the progenitor markers SIX1 and SIX2 were not expressed in the cell clusters 2 and 3, suggesting their complete differentiation. Cluster 1 likely represents a mix of immature cells at different stages of differentiation because they expressed SIX1 while lacking renal cell-specific markers. Furthermore, we assessed similarity of expression patterns between organoid cells and the adult human kidney using previously reported micro-array data (GEO GSE37460 and GSE37455) from micro-dissected living donor kidney biopsies.21Berthier C.C. Bethunaickan R. Gonzalez-Rivera T. et al.Cross-species transcriptional network analysis defines shared inflammatory responses in murine and human lupus nephritis.J Immunol. 2012; 189: 988-1001Crossref PubMed Scopus (130) Google Scholar As shown in Figure 9, the podocyte group (cluster 2) and tubular group (cluster 3) were clustered closely with glomerular and tubulointerstitial renal compartments, respectively, based on a set of known markers.22Ju W. Greene C.S. Eichinger F. et al.Defining cell-type specificity at the transcriptional level in human disease.Genome Res. 2013; 23: 1862-1873Crossref PubMed Scopus (155) Google Scholar Expression of kidney-specific transcription factors was also studied to further validate the maturity of the clusters 2 and 3 (Supplementary Figure S4). Similar results were obtained in single-cell RNA sequencing of 100k organoids (Supplementary Figure S5). Gene expression similarity between the organoid cells and the kidney speaks of physiological relevance and strengthens the translatability of our in vitro model to the human setting. During the past decade 2 major advances in science have truly set the stage for the use of stem cells in drug discovery: the discovery of cellular reprogramming in 200623Takahashi K. Tanabe K. Ohnuki M. et al.Induction of pluripotent stem cells from adult human fibroblasts by defined factors.Cell. 2007; 131: 861-872Abstract Full Text Full Text PDF PubMed Scopus (14964) Google Scholar, 24Takahashi K. Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.Cell. 2006; 126: 663-676Abstract Full Text Full Text PDF PubMed Scopus (18864) Google Scholar and the landmark papers showing the utility of the CRISPR–Cas9 systems in human cells.25Cho S.W. Kim S. Kim J.M. et al.Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease.Nat Biotechnol. 2013; 31: 230-232Crossref PubMed Scopus (1375) Google Scholar, 26Cong L. Ran F.A. Cox D. et al.Multiplex genome engineering using CRISPR/Cas systems.Science. 2013; 339: 819-823Crossref PubMed Scopus (9981) Google Scholar, 27Mali P. Yang L. Esvelt K.M. et al.RNA-guided human genome engineering via Cas9.Science. 2013; 339: 823-826Crossref PubMed Scopus (6424) Google Scholar The combination of these 2 techniques has given the scientific community access to previously inaccessible human somatic cells and has facilitated the creation of novel tools with impact in both basic science and drug discovery. In an effort to generate tools for both drug discovery and drug safety within the area of kidney disease, we aimed to develop an in vitro model for glomerular health using hiPSCs. To date, some recent progress has been made toward this end,1Ciampi O. Iacone R. Longaretti L. et al.Generation of functional podocytes from human induced pluripotent stem cells.Stem Cell Res. 2016; 17: 130-139Crossref PubMed Scopus (52) Google Scholar, 2Freedman B.S. Brooks C.R. Lam A.Q. et al.Modelling kidney disease with CRISPR-mutant kidney organoids derived from human pluripotent epiblast spheroids.Nat Commun. 2015; 6: 8715Crossref PubMed Scopus (444) Google Scholar, 3Morizane R. Lam A.Q. Freedman B.S. et al.Nephron organoids derived from human pluripotent stem cells model kidney development and injury.Nat Biotechnol. 2015; 33: 1193-1200Crossref PubMed Scopus (509) Google Scholar, 4Sharmin S. Taguchi A. Kaku Y. et al.Human induced pluripotent stem cell-derived podocytes mature into vascularized glomeruli upon experimental transplantation.J Am Soc Nephrol. 2016; 27: 1778-1791Crossref PubMed Scopus (141) Google Scholar, 5Takasato M. Er P.X. Chiu H.S. et al.Kidney organoids from human iPS cells contain multiple lineages and model human nephrogenesis.Nature. 2015; 526: 564-568Crossref PubMed Scopus (879) Google Scholar, 28Musah S. Mammoto A. Ferrante T.C. et al.Mature induced-pluripotent-stem-cell-derived human podocytes reconstitute kidney glomerular-capillary-wall function on a chip.Nat Biomed Eng. 2017; 1: 0069Crossref PubMed Scopus (285) Google Scholar but there are still no established functional tools mimicking the human situation in the screening format required for drug discovery. By combining CRISPR/Cas9 technology with a 3D differentiation protocol, we have for the first time established a system in which kidney differentiation, glomerular maturation, and podocyte health can be monitored in living cells using fluorescently tagged kidney lineage markers. The pre-differentiation stage is simple and can easily be scaled up in cell factories yielding millions of progenitor cells, and the organoids are formed in AggreWell plates (easily generating thousands of organoids per batch) that subsequently are transferred to a suspension culture format that is experimentally accessible, scalable, and potentially high-throughput using automation. All major components of the developing nephron, such as endothelial cells, podocyte-like cells, tubular cells (both proximal and distal), and nephron progenitors, are present within each individual organoid, in physiologically relevant 3D structures. In this context, after a total of 25 days of differentiation, hiPSC-derived podocytes share characteristics similar to native podocytes, where formation of structures resembling foot processes and slit diaphragms appeared when visualized by electron microscopy. Using antibody staining on whole mount organoids as well as paraffin-embedded sections, we could confirm the identity of these structures through positive nephrin staining in the glomerular structures, LTL and ECAD staining in the tubular areas, and CD31-positive capillary structures. We detected the formation of CD31-positive capillaries surrounding and entering the WT1- and NPHS1-positive glomerular structures, an observation in agreement with results reported previously.5Takasato M. Er P.X. Chiu H.S. et al.Kidney organoids from human iPS cells contain multiple lineages and model human nephrogenesis.Nature. 2015; 526: 564-568Crossref PubMed Scopus (879) Google Scholar Interestingly, using single-cell RNA sequencing we can visualize 3 separate cell populations in the kidney organoids: 1 glomerular population, 1 tubular population, and 1 progenitor population. The 2 mature populations cluster very well with glomerular and tubular cell fractions when compared with micro-array data from micro-dissected adult kidneys (Figure 9), suggesting that these organoid populations indeed are translatable to adult kidney structures. The progenitor population, characterized by expression of SIX1 and SIX2, could possibly be explained by a technical difficulty in getting stem cells to go through the differentiation process in a fully synchronized manner. Taking into account the auto-regulatory loop of sustained transcriptional activation of SIX1 and SIX2,17O'Brien L.L. Guo Q. Lee Y. et al.Differential regulation of mouse and human nephron progenitors by the Six family of transcriptional regulators.Development. 2016; 143: 595-608Crossref PubMed Scopus (82) Google Scholar we can hypothesize that the cells that continually express these progenitor markers may be part of a progenitor niche that may supply the organoid with more mature cells over time. Summarizing all results, we can conclude that our organoids contain at least 4 different mature cell types (podocytes, proximal tubules, distal tubules, and endothelial cells). The fact that we can only detect 2 mature cell types using single-cell RNA sequencing could have several root causes: (i) fewer specific markers exist for endothelial cells, mesangial cells, and stromal cells, making these cells more difficult to discriminate in comparison with other kidney cell types, (ii) technical difficulties in getting cells into single-cell suspension for FACS sorting, and (iii) a challenge in numbers, seeing that the endothelial cells in particular are a smaller percentage of the total number of organoid cells. While the organoids formed by our differentiation protocol contain nephrin-expressing cells forming foot processes and even early slit diaphragms in close proximity to cells expressing tubular and endothelial markers, we have no evidence that these glomerular-like structures have all the components needed to mimic glomerular filtration. However, the close proximity of the glomerular cell compartments will allow for cross-talk between the cells. Cellular cross-talk is vital for maintenance of the integrity and permselectivity of the glomerular barrier and is generally very challenging to mimic in vitro.10Dimke H. Maezawa Y. Quaggin S.E. Crosstalk in glomerular injury and repair.Curr Opin Nephrol Hypertens. 2015; 24: 231-238PubMed Google Scholar, 11Lennon R. Hosawi S. Glomerular cell crosstalk.Curr Opin Nephrol Hypertens. 2016; 25: 187-193Crossref PubMed Scopus (28) Google Scholar This organoid model is useful as an advanced 3D screening in vitro model where various stressors, stimuli, and toxins can be used to affect relevant cells types, their cross-talk, and their properties, and where compounds could be identified to reverse the injured phenotype. Furthermore, development of human nephron-like structures in vitro fills a major gap in assessing effectiveness of treatments for chronic kidney disease and also in the possibility of clinical translation. The fact that the organoids constitute a compact and dense 3D structure reproduces conditions closer to the in vivo situation, which potentially renders this model more translatable than a 2-dimensional cellular model. We have developed a 3D human kidney model that provides us with a high-throughput tool to study cellular cross-talk, cellular uptake of pharmaceutical modalities, and drug toxicity. By implementing cutting-edge molecular and cellular biological tools and techniques, we are attempting to build the next generation of kidney disease models to facilitate the discovery and development of the kidney therapeutics of tomorrow." @default.
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• W2887161891 title "A CRISP(e)R view on kidney organoids allows generation of an induced pluripotent stem cell–derived kidney model for drug discovery" @default.
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