A CRISP(e)R view on kidney organoids allows generation of an induced pluripotent stem cell–derived kidney model for drug discovery

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 found during transmission electron microscopy tubular structures with cells, and epithelial tight junctions and to kidney tubules. These analyses also structures foot processes from cells, by a of cells of Notably, structures showing areas podocyte foot processes with slit were also observed using scanning electron microscopy (Figure To further mature kidney characteristics of cells within the we used which of gene expression at the cell of cell within the kidney organoid to sorted cells and used protocol to the cellular a for was used to different cell within the et of PubMed Scopus Google Scholar the cells into 3 using the by we observed that 2 was highly in podocyte marker tubular marker such as were in 3 Figure A was used to the of 2 and L. to expression PubMed Scopus Google Scholar that podocyte and tubular markers were the highly in 2 and To and confirm the we used der L. using Res. 2008; Scholar the podocyte marker NPHS1, the tubular marker and the progenitor markers SIX1 and SIX2 (Figure the progenitor markers SIX1 and SIX2 were not in the cell 2 and their differentiation. 1 a of cells at different of differentiation because SIX1 renal markers. we assessed of expression between organoid cells and the adult human kidney using previously and from living kidney R. T. et transcriptional in and human PubMed Scopus Google Scholar shown in Figure the podocyte and tubular were with glomerular and renal respectively, on a of W. et at the transcriptional in human Res. PubMed Scopus Google Scholar of kidney-specific factors was also to further the of the 2 and 3 Figure results were in of organoids Figure expression between the organoid cells and the kidney of physiological and the translatability of our in vitro model to the human the 2 major advances in have the stage for the use of stem cells in drug the discovery of cellular in M. et of pluripotent stem cells from adult human by Full Text Full Text PDF PubMed Scopus Google Scholar, S. Induction of pluripotent stem cells from mouse and adult by 2006; Full Text Full Text PDF PubMed Scopus Google Scholar and the landmark papers showing the of the in human S. et in human cells with the Biotechnol. PubMed Scopus Google Scholar, L. D. et using PubMed Scopus Google Scholar, P. L. et human via PubMed Scopus Google Scholar The combination of these 2 has the access to previously human cells and has the of novel tools with in both basic and drug discovery. In an to generate tools for both drug discovery and drug safety within the of kidney disease, we to an in vitro model for glomerular health using To some recent has been made this 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, S. A. et human podocytes kidney function on a PubMed Scopus Google Scholar but there are still no established functional tools the human in the screening format for drug discovery. CRISPR/Cas9 technology with a 3D differentiation protocol, we have for the 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 stage is and can be up in cell of progenitor cells, and the organoids are formed in AggreWell plates generating of organoids per that are to a suspension culture format that is and potentially using major of the developing nephron, such as endothelial cells, cells, tubular cells proximal and and nephron are within each in physiologically relevant 3D structures. In this after a of 25 days of differentiation, podocytes characteristics to where formation of structures foot processes and slit appeared when visualized by electron microscopy. Using staining on organoids as well as paraffin-embedded we could confirm the of these structures through positive nephrin staining in the glomerular structures, and staining in the tubular and CD31-positive capillary structures. the formation of CD31-positive surrounding and the and glomerular structures, an in with results 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 using we can 3 cell populations in the kidney 1 glomerular 1 tubular and 1 progenitor population. The 2 mature populations well with glomerular and tubular cell when with from adult (Figure that these organoid populations are to adult kidney structures. The progenitor by expression of SIX1 and could be by a in stem cells to through the differentiation process in a fully into the loop of transcriptional of SIX1 and 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 that the cells that express these progenitor markers may be of a progenitor that may the organoid with mature cells over all we can that our organoids contain at different mature cell proximal distal and endothelial The that we can only 2 mature cell using could have specific markers for endothelial cells, mesangial cells, and cells, these cells difficult to in comparison with kidney cell in cells into suspension for and a in that the endothelial cells in are a smaller of the number of organoid cells. the organoids formed by our differentiation protocol contain nephrin-expressing cells forming foot processes and even early slit in to cells tubular and endothelial we have no that these structures have all the needed to mimic glomerular However, the of the glomerular cell for cross-talk between the cells. cross-talk is for of the and of the glomerular barrier and is to mimic in 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 and can be used to relevant cells their and their and where could be identified to the development of human nephron-like structures in vitro fills a major gap in of for chronic kidney disease and also in the of clinical translation. The that the organoids a and 3D to the in vivo which potentially this model a cellular model. have developed a 3D human kidney model that with a to cellular cellular of and drug molecular and cellular tools and we are to build the generation of kidney disease models to facilitate the discovery and development of the kidney of