Generating Kidney Organoids and Mature Renal Cell Types Via Directed iPSC Differentiation
Key Personnel
Melissa H. Little (PI)
Murdoch Childrens Research Institute
- Andrew Elefanty
Murdoch Childrens Research Institute - Pei Er
Murdoch Childrens Research Institute - Sara Howden
Murdoch Childrens Research Institute - Alicia Oshlack
Murdoch Childrens Research Institute - Belinda Phipson
Murdoch Childrens Research Institute - Ed Stanley
Murdoch Childrens Research Institute - Jess Vanslambrouck
Murdoch Childrens Research Institute
Project Description
We have previously shown that human kidney tissue can be generated from human pluripotent stem cells. Our project focuses on characterizing and optimizing this approach to improve tubular maturation and cellular function and generating reporter lines for the isolation of specific cell types. In the long term, human kidney tissue generated in this way may be used for drug screening, tissue regeneration or cell therapy.
Publications
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Development of the Human Fetal Kidney from Mid to Late Gestation in Male and Female Infants
Ryan, D; Sutherland, MR; Flores, TJ; Kent, AL; Dahlstrom, JE; Puelles, VG; Bertram, JF; McMahon, AP; Little, MH; Moore, L; Black, MJ. EBioMedicine . 27:275–283. January 2018.
BACKGROUND: During normal human kidney development, nephrogenesis (the formation of nephrons) is complete by term birth, with the majority of nephrons formed late in gestation. The aim of this study was to morphologically examine nephrogenesis in fetal human kidneys from 20 to 41weeks of gestation. METHODS: Kidney samples were obtained at autopsy from 71 infants that died acutely in utero or within 24h after birth. Using image analysis, nephrogenic zone width, the number of glomerular generations, renal corpuscle cross-sectional area and the cellular composition of glomeruli were examined. Kidneys from female and male infants were analysed separately. FINDINGS: The number of glomerular generations formed within the fetal kidneys was directly proportional to gestational age, body weight and kidney weight, with variability between individuals in the ultimate number of generations (8 to 12) and in the timing of the cessation of nephrogenesis (still ongoing at 37weeks gestation in one infant). There was a slight but significant (r2=0.30, P=0.001) increase in renal corpuscle cross-sectional area from mid gestation to term in females, but this was not evident in males. The proportions of podocytes, endothelial and non-epithelial cells within mature glomeruli were stable throughout gestation. INTERPRETATION: These findings highlight spatial and temporal variability in nephrogenesis in the developing human kidney, whereas the relative cellular composition of glomeruli does not appear to be influenced by gestational age.
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An illustrated anatomical ontology of the developing mouse lower urogenital tract
Georgas, KM; Armstrong, J; Keast, JR; Larkins, CE; McHugh, KM; Southard-Smith, EM; Cohn, MJ; Batourina, E; Dan, H; Schneider, K; Buehler, DP; Wiese, CB; Brennan, J; Davies, JA; Harding, SD; Baldock, RA; Little, MH; Vezina, CM; Mendelsohn, C. Development . 142(10):1893–908. May 2015.
Malformation of the urogenital tract represents a considerable paediatric burden, with many defects affecting the lower urinary tract (LUT), genital tubercle and associated structures. Understanding the molecular basis of such defects frequently draws on murine models. However, human anatomical terms do not always superimpose on the mouse, and the lack of accurate and standardised nomenclature is hampering the utility of such animal models. We previously developed an anatomical ontology for the murine urogenital system. Here, we present a comprehensive update of this ontology pertaining to mouse LUT, genital tubercle and associated reproductive structures (E10.5 to adult). Ontology changes were based on recently published insights into the cellular and gross anatomy of these structures, and on new analyses of epithelial cell types present in the pelvic urethra and regions of the bladder. Ontology changes include new structures, tissue layers and cell types within the LUT, external genitalia and lower reproductive structures. Representative illustrations, detailed text descriptions and molecular markers that selectively label muscle, nerves/ganglia and epithelia of the lower urogenital system are also presented. The revised ontology will be an important tool for researchers studying urogenital development/malformation in mouse models and will improve our capacity to appropriately interpret these with respect to the human situation.
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Defining kidney biology to understand renal disease
Little, MH; Brown, D.; Humphreys, BD; McMahon, AP; Miner, JH; Sands, JM; Weisz, OA; Mullins, C; Hoshizaki, D; Kidney Research National Dialogue, (KRND). Clin J Am Soc Nephrol . 9(4):809–11. April 2014.
The Kidney Research National Dialogue represents a novel effort by the National Institute of Diabetes and Digestive and Kidney Diseases to solicit and prioritize research objectives from the renal research and clinical communities. The present commentary highlights selected scientific opportunities specific to the study of renal development, physiology, and cell biology. Describing such fundamental kidney biology serves as a necessary foundation for translational and clinical studies that will advance disease care and prevention. It is intended that these objectives foster and focus scientific efforts in these areas in the coming decade and beyond.
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Wiese, CB; Ireland, S; Fleming, NL; Yu, J; Valerius, MT; Georgas, K; Chiu, HS; Brennan, J; Armstrong, J; Little, MH; McMahon, AP; Southard-Smith, EM. Front Neurosci . 6:130. September 2012.
Relative positions of neurons within mature murine pelvic ganglia based on expression of neurotransmitters have been described. However the spatial organization of developing innervation in the murine urogenital tract (UGT) and the gene networks that regulate specification and maturation of neurons within the pelvic ganglia of the lower urinary tract (LUT) are unknown. We used whole-mount immunohistochemistry and histochemical stains to localize neural elements in 15.5 days post coitus (dpc) fetal mice. To identify potential regulatory factors expressed in pelvic ganglia, we surveyed expression patterns for known or probable transcription factors (TF) annotated in the mouse genome by screening a whole-mount in situ hybridization library of fetal UGTs. Of the 155 genes detected in pelvic ganglia, 88 encode TFs based on the presence of predicted DNA-binding domains. Neural crest (NC)-derived progenitors within the LUT were labeled by Sox10, a well-known regulator of NC development. Genes identified were categorized based on patterns of restricted expression in pelvic ganglia, pelvic ganglia and urethral epithelium, or pelvic ganglia and urethral mesenchyme. Gene expression patterns and the distribution of Sox10+, Phox2b+, Hu+, and PGP9.5+ cells within developing ganglia suggest previously unrecognized regional segregation of Sox10+ progenitors and differentiating neurons in early development of pelvic ganglia. Reverse transcription-PCR of pelvic ganglia RNA from fetal and post-natal stages demonstrated that multiple TFs maintain post-natal expression, although Pax3 is extinguished before weaning. Our analysis identifies multiple potential regulatory genes including TFs that may participate in segregation of discrete lineages within pelvic ganglia. The genes identified here are attractive candidate disease genes that may now be further investigated for their roles in malformation syndromes or in LUT dysfunction.
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Identification of molecular compartments and genetic circuitry in the developing mammalian kidney
Yu, J; Valerius, MT; Duah, M; Staser, K; Hansard, JK; Guo, JJ; McMahon, J; Vaughan, J; Faria, D; Georgas, K; Rumballe, B; Ren, Q; Krautzberger, AM; Junker, JP; Thiagarajan, RD; Machanick, P; Gray, PA; van Oudenaarden, A; Rowitch, DH; Stiles, CD; Ma, Q; Grimmond, SM; Bailey, TL; Little, MH; McMahon, AP. Development . 139(10):1863–73. May 2012.
Lengthy developmental programs generate cell diversity within an organotypic framework, enabling the later physiological actions of each organ system. Cell identity, cell diversity and cell function are determined by cell type-specific transcriptional programs; consequently, transcriptional regulatory factors are useful markers of emerging cellular complexity, and their expression patterns provide insights into the regulatory mechanisms at play. We performed a comprehensive genome-scale in situ expression screen of 921 transcriptional regulators in the developing mammalian urogenital system. Focusing on the kidney, analysis of regional-specific expression patterns identified novel markers and cell types associated with development and patterning of the urinary system. Furthermore, promoter analysis of synexpressed genes predicts transcriptional control mechanisms that regulate cell differentiation. The annotated informational resource (www.gudmap.org) will facilitate functional analysis of the mammalian kidney and provides useful information for the generation of novel genetic tools to manipulate emerging cell populations.
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Thiagarajan, RD; Georgas, KM; Rumballe, BA; Lesieur, E; Chiu, HS; Taylor, D; Tang, DT; Grimmond, SM; Little, MH. PLoS ONE . 6(2). February 2012.
The development of the mammalian kidney is well conserved from mouse to man. Despite considerable temporal and spatial data on gene expression in mammalian kidney development, primarily in rodent species, there is a paucity of genes whose expression is absolutely specific to a given anatomical compartment and/or developmental stage, defined here as ’anchor’ genes. We previously generated an atlas of gene expression in the developing mouse kidney using microarray analysis of anatomical compartments collected via laser capture microdissection. Here, this data is further analysed to identify anchor genes via stringent bioinformatic filtering followed by high resolution section in situ hybridisation performed on 200 transcripts selected as specific to one of 11 anatomical compartments within the midgestation mouse kidney. A total of 37 anchor genes were identified across 6 compartments with the early proximal tubule being the compartment richest in anchor genes. Analysis of minimal and evolutionarily conserved promoter regions of this set of 25 anchor genes identified enrichment of transcription factor binding sites for Hnf4a and Hnf1b, RbpJ (Notch signalling), PPARγ:RxRA and COUP-TF family transcription factors. This was reinforced by GO analyses which also identified these anchor genes as targets in processes including epithelial proliferation and proximal tubular function. As well as defining anchor genes, this large scale validation of gene expression identified a further 92 compartment-enriched genes able to subcompartmentalise key processes during murine renal organogenesis spatially or ontologically. This included a cohort of 13 ureteric epithelial genes revealing previously unappreciated compartmentalisation of the collecting duct system and a series of early tubule genes suggesting that segmentation into proximal tubule, loop of Henle and distal tubule does not occur until the onset of glomerular vascularisation. Overall, this study serves to illuminate previously ill-defined stages of patterning and will enable further refinement of the lineage relationships within mammalian kidney development.
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Identification of novel markers of mouse fetal ovary development
Chen, H; Palmer, JS; Thiagarajan, RD; Dinger, ME; Lesieur, E; Chiu, H; Schulz, A; Spiller, C; Grimmond, SM; Little, MH; Koopman, P; Wilhelm, D. PLoS ONE . 7(7). 2012.
In contrast to the developing testis, molecular pathways driving fetal ovarian development have been difficult to characterise. To date no single master regulator of ovarian development has been identified that would be considered the female equivalent of Sry. Using a genomic approach we identified a number of novel protein-coding as well as non-coding genes that were detectable at higher levels in the ovary compared to testis during early mouse gonad development. We were able to cluster these ovarian genes into different temporal expression categories. Of note, Lrrc34 and AK015184 were detected in XX but not XY germ cells before the onset of sex-specific germ cell differentiation marked by entry into meiosis in an ovary and mitotic arrest in a testis. We also defined distinct spatial expression domains of somatic cell genes in the developing ovary. Our data expands the set of markers of early mouse ovary differentiation and identifies a classification of early ovarian genes, thus providing additional avenues with which to dissect this process.
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Rumballe, BA; Chiu, HS; Georgas, KM; Little, MH. Methods Mol Biol . 886:223–39. 2012.
Studies into the molecular basis of morphogenesis frequently begin with investigations into gene expression across time and cell type in that organ. One of the most anatomically informative approaches to such studies is the use of in situ hybridization, either of intact or histologically sectioned tissues. Here, we describe the optimization of this approach for use in the temporal and spatial analysis of gene expression in the urogenital system, from embryonic development to the postnatal period. The methods described are applicable for high throughput analysis of large gene sets. As such, ISH has become a powerful technique for gene expression profiling and is valuable for the validation of profiling analyses performed using other approaches such as microarrays.
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Access and use of the GUDMAP database of genitourinary development
Davies, JA; Little, MH; Aronow, B; Armstrong, J; Brennan, J; Lloyd-MacGilp, S; Armit, C; Harding, S; Piu, X; Roochun, Y; Haggarty, B; Houghton, D; Davidson, D; Baldock, R. Methods Mol Biol . 886:185–201. 2012.
The Genitourinary Development Molecular Atlas Project (GUDMAP) aims to document gene expression across time and space in the developing urogenital system of the mouse, and to provide access to a variety of relevant practical and educational resources. Data come from microarray gene expression profiling (from laser-dissected and FACS-sorted samples) and in situ hybridization at both low (whole-mount) and high (section) resolutions. Data are annotated to a published, high-resolution anatomical ontology and can be accessed using a variety of search interfaces. Here, we explain how to run typical queries on the database, by gene or anatomical location, how to view data, how to perform complex queries, and how to submit data.
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Thiagarajan, Rathi D.; Georgas, Kylie M.; Rumballe, Bree A.; Lesieur, Emmanuelle; Chiu, Han Sheng; Taylor, Darrin; Tang, Dave T. P.; Grimmond, Sean M.; Little, Melissa H.. PLoS One . 6(2):e17286. February 2011.
The development of the mammalian kidney is well conserved from mouse to man. Despite considerable temporal and spatial data on gene expression in mammalian kidney development, primarily in rodent species, there is a paucity of genes whose expression is absolutely specific to a given anatomical compartment and/or developmental stage, defined here as ’anchor’ genes. We previously generated an atlas of gene expression in the developing mouse kidney using microarray analysis of anatomical compartments collected via laser capture microdissection. Here, this data is further analysed to identify anchor genes via stringent bioinformatic filtering followed by high resolution section in situ hybridisation performed on 200 transcripts selected as specific to one of 11 anatomical compartments within the midgestation mouse kidney. A total of 37 anchor genes were identified across 6 compartments with the early proximal tubule being the compartment richest in anchor genes. Analysis of minimal and evolutionarily conserved promoter regions of this set of 25 anchor genes identified enrichment of transcription factor binding sites for Hnf4a and Hnf1b, RbpJ (Notch signalling), PPARgamma:RxRA and COUP-TF family transcription factors. This was reinforced by GO analyses which also identified these anchor genes as targets in processes including epithelial proliferation and proximal tubular function. As well as defining anchor genes, this large scale validation of gene expression identified a further 92 compartment-enriched genes able to subcompartmentalise key processes during murine renal organogenesis spatially or ontologically. This included a cohort of 13 ureteric epithelial genes revealing previously unappreciated compartmentalisation of the collecting duct system and a series of early tubule genes suggesting that segmentation into proximal tubule, loop of Henle and distal tubule does not occur until the onset of glomerular vascularisation. Overall, this study serves to illuminate previously ill-defined stages of patterning and will enable further refinement of the lineage relationships within mammalian kidney development.
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Nephron formation adopts a novel spatial topology at cessation of nephrogenesis
Rumballe, BA; Georgas, KM; Combes, AN; Ju, AL; Gilbert, T; Little, MH. Dev Biol . 360(1):110–22. December 2011.
Nephron number in the mammalian kidney is known to vary dramatically, with postnatal renal function directly influenced by nephron complement. What determines final nephron number is poorly understood but nephron formation in the mouse kidney ceases within the first few days after birth, presumably due to the loss of all remaining nephron progenitors via epithelial differentiation. What initiates this event is not known. Indeed, whether nephron formation occurs in the same way at this time as during embryonic development has also not been examined. In this study, we investigate the key cellular compartments involved in nephron formation; the ureteric tip, cap mesenchyme and early nephrons; from postnatal day (P) 0 to 6 in the mouse. High resolution analyses of gene and protein expression indicate that loss of nephron progenitors precedes loss of ureteric tip identity, but show spatial shifts in the expression of cap mesenchyme genes during this time. In addition, cap mesenchymal volume and rate of proliferation decline prior to birth. Section-based 3D modeling and Optical Projection Tomography revealed a burst of ectopic nephron induction, with the formation of multiple (up to 5) nephrons per ureteric tip evident from P2. While the distal-proximal patterning of these nephrons occurred normally, their spatial relationship with the ureteric compartment was altered. We propose that this phase of nephron formation represents an acceleration of differentiation within the cap mesenchyme due to a displacement of signals within the nephrogenic niche.
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Defining and redefining the nephron progenitor population
Hendry, C; Rumballe, B; Moritz, K; Little, MH. Pediatr Nephrol . 26(9):1395–406. September 2011.
It has long been appreciated that the mammalian kidney arises via reciprocal interactions between an epithelial ureteric epithelium and the surrounding metanephric mesenchyme. More recently, lineage tracing has confirmed that the portion of the metanephric mesenchyme closest to the advancing ureteric tips, the cap mesenchyme, represents the progenitor population for the nephron epithelia. This Six2(+)Cited1(+) population undergoes self-renewal throughout nephrogenesis while retaining the potential to epithelialize. In contrast, the Foxd1(+) portion of the metanephric mesenchyme shows no epithelial potential, developing instead into the interstitial, perivascular, and possibly endothelial elements of the kidney. The cap mesenchyme rests within a nephrogenic niche, surrounded by the stroma and the ureteric tip. While the role of Wnt signaling in nephron induction is known, there remains a lack of clarity over the intrinsic and extrinsic regulation of cap mesenchyme specification, self-renewal, and nephron potential. It is also not known what regulates cessation of nephrogenesis, but there is no nephron generation in response to injury during the postnatal period. In this review, we will examine what is and is not known about this nephron progenitor population and discuss how an increased understanding of the regulation of this population may better explain the observed variation in final nephron number and potentially facilitate the reinitiation or prolongation of nephron formation.
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Thiagarajan, RD; Cloonan, N; Gardiner, BB; Mercer, TR; Kolle, G; Nourbakhsh, E; Wani, S; Tang, D; Krishnan, K; Georgas, KM; Rumballe, BA; Chiu, HS; Steen, JA; Mattick, JS; Little, MH; Grimmond, SM. BMC Genomics . 12:441. September 2011.
BACKGROUND: The developing mouse kidney is currently the best-characterized model of organogenesis at a transcriptional level. Detailed spatial maps have been generated for gene expression profiling combined with systematic in situ screening. These studies, however, fall short of capturing the transcriptional complexity arising from each locus due to the limited scope of microarray-based technology, which is largely based on "gene-centric" models. RESULTS: To address this, the polyadenylated RNA and microRNA transcriptomes of the 15.5 dpc mouse kidney were profiled using strand-specific RNA-sequencing (RNA-Seq) to a depth sufficient to complement spatial maps from pre-existing microarray datasets. The transcriptional complexity of RNAs arising from mouse RefSeq loci was catalogued; including 3568 alternatively spliced transcripts and 532 uncharacterized alternate 3’ UTRs. Antisense expressions for 60% of RefSeq genes was also detected including uncharacterized non-coding transcripts overlapping kidney progenitor markers, Six2 and Sall1, and were validated by section in situ hybridization. Analysis of genes known to be involved in kidney development, particularly during mesenchymal-to-epithelial transition, showed an enrichment of non-coding antisense transcripts extended along protein-coding RNAs. CONCLUSION: The resulting resource further refines the transcriptomic cartography of kidney organogenesis by integrating deep RNA sequencing data with locus-based information from previously published expression atlases. The added resolution of RNA-Seq has provided the basis for a transition from classical gene-centric models of kidney development towards more accurate and detailed "transcript-centric" representations, which highlights the extent of transcriptional complexity of genes that direct complex development events.
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Defining the molecular character of the developing and adult kidney podocyte
Brunskill, EW; Georgas, K; Rumballe, B; Little, MH; Potter, SS. PLoS ONE . 6(9). 2011.
BACKGROUND: The podocyte is a remarkable cell type, which encases the capillaries of the kidney glomerulus. Although mesodermal in origin it sends out axonal like projections that wrap around the capillaries. These extend yet finer projections, the foot processes, which interdigitate, leaving between them the slit diaphragms, through which the glomerular filtrate must pass. The podocytes are a subject of keen interest because of their key roles in kidney development and disease. METHODOLOGY/PRINCIPAL FINDINGS: In this report we identified and characterized a novel transgenic mouse line, MafB-GFP, which specifically marked the kidney podocytes from a very early stage of development. These mice were then used to facilitate the fluorescent activated cell sorting based purification of podocytes from embryos at E13.5 and E15.5, as well as adults. Microarrays were then used to globally define the gene expression states of podocytes at these different developmental stages. A remarkable picture emerged, identifying the multiple sets of genes that establish the neuronal, muscle, and phagocytic properties of podocytes. The complete combinatorial code of transcription factors that create the podocyte was characterized, and the global lists of growth factors and receptors they express were defined. CONCLUSIONS/SIGNIFICANCE: The complete molecular character of the in vivo podocyte is established for the first time. The active molecular functions and biological processes further define their unique combination of features. The results provide a resource atlas of gene expression patterns of developing and adult podocytes that will help to guide further research of these incredible cells.
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The GUDMAP database–an online resource for genitourinary research
Harding, SD; Armit, C; Armstrong, J; Brennan, J; Cheng, Y; Haggarty, B; Houghton, D; Lloyd-MacGilp, S; Pi, X; Roochun, Y; Sharghi, M; Tindal, C; McMahon, AP; Gottesman, B; Little, MH; Georgas, K; Aronow, B; Potter, SS; Brunskill, EW; Southard-Smith, EM; Mendelsohn, C; Baldock, RA; Davies, JA; Davidson, D. Development . 138(13):2845–53. July 2011.
The GenitoUrinary Development Molecular Anatomy Project (GUDMAP) is an international consortium working to generate gene expression data and transgenic mice. GUDMAP includes data from large-scale in situ hybridisation screens (wholemount and section) and microarray gene expression data of microdissected, laser-captured and FACS-sorted components of the developing mouse genitourinary (GU) system. These expression data are annotated using a high-resolution anatomy ontology specific to the developing murine GU system. GUDMAP data are freely accessible at www.gudmap.org via easy-to-use interfaces. This curated, high-resolution dataset serves as a powerful resource for biologists, clinicians and bioinformaticians interested in the developing urogenital system. This paper gives examples of how the data have been used to address problems in developmental biology and provides a primer for those wishing to use the database in their own research.
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Expression of metanephric nephron-patterning genes in differentiating mesonephric tubules
Georgas, KM; Chiu, HS; Lesieur, E; Rumballe, BA; Little, MH. Dev Dyn . 240(6):1600–12. June 2011.
The metanephros is the functional organ in adult amniotes while the mesonephros degenerates. However, parallel tubulogenetic events are thought to exist between mesonephros and metanephros. Mesonephric tubules are retained in males and differentiate into efferent ducts of the male reproductive tract. By examining the murine mesonephric expression of markers of distinct stages and regions of metanephric nephrons during tubule formation and patterning, we provide further evidence to support this common morphogenetic mechanism. Renal vesicle, early proximal and distal tubule, loop of Henle, and renal corpuscle genes were expressed by mesonephric tubules. Vip, Slc6a20b, and Slc18a1 were male-specific. In contrast, mining of the GUDMAP database identified candidate late mesonephros-specific genes, 10 of which were restricted to the male. Among the male-specific genes are candidates for regulating ion/fluid balance within the efferent ducts, thereby regulating sperm maturation and genes marking tubule-associated neurons potentially critical for normal male reproductive tract function.
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Chiu, HS; Szucsik, JC; Georgas, KM; Jones, JL; Rumballe, BA; Tang, D; Grimmond, SM; Lewis, AG; Aronow, B; Lessard, JL; Little, MH. Developmental Biology . 344(2):1071–87. August 2010.
Here we describe the first detailed catalog of gene expression in the developing lower urinary tract (LUT), including epithelial and mesenchymal portions of the developing bladder, urogenital sinus, urethra, and genital tubercle (GT) at E13 and E14. Top compartment-specific genes implicated by the microarray data were validated using whole-mount in situ hybridization (ISH) over the entire LUT. To demonstrate the potential of this resource to implicate developmentally critical features, we focused on gene expression patterns and pathways in the sexually indeterminate, androgen-independent GT. GT expression patterns reinforced the proposed similarities between development of GT, limb, and craniofacial prominences. Comparison of spatial expression patterns predicted a network of Wnt7a-associated GT-enriched epithelial genes, including Gjb2, Dsc3, Krt5, and Sostdc1. Known from other contexts, these genes are associated with normal epidermal differentiation, with disruptions in Dsc3 and Gjb2 showing palmo-plantar keratoderma in the limb. We propose that this gene network contributes to normal foreskin, scrotum, and labial development. As several of these genes are known to be regulated by, or contain cis elements responsive to retinoic acid, estrogen, or androgen, this implicates this pathway in the later androgen-dependent development of the GT.
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Molecular anatomy of the kidney: what have we learned from gene expression and functional genomics?
Rumballe, B; Georgas, K; Wilkinson, L; Little, MH. Pediatr Nephrol . 25(6):1005–16. June 2010.
The discipline of paediatric nephrology encompasses the congenital nephritic syndromes, renal dysplasias, neonatal renal tumours, early onset cystic disease, tubulopathies and vesicoureteric reflux, all of which arise due to defects in normal kidney development. Indeed, congenital anomalies of the kidney and urinary tract (CAKUT) represent 20-30% of prenatal anomalies, occurring in 1 in 500 births. Developmental biologists have studied the anatomical and morphogenetic processes involved in kidney development for the last five decades. However, with the advent of transgenic mice, the sequencing of the genome, improvements in mutation detection and the advent of functional genomics, our understanding of the molecular basis of kidney development has grown significantly. Here we discuss how the advent of new genetic and genomics approaches has added to our understanding of kidney development and paediatric renal disease, as well as identifying areas in which we are still lacking knowledge.
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Georgas, K; Rumballe, B; Valerius, MT; Chiu, HS; Thiagarajan, RD; Lesieur, E; Aronow, B; Brunskill, EW; Combes, AN; Tang, D; Taylor, D; Grimmond, SM; Potter, SS; McMahon, AP; Little, MH. Dev Biol . 332(2):273–86. August 2009.
While nephron formation is known to be initiated by a mesenchyme-to-epithelial transition of the cap mesenchyme to form a renal vesicle (RV), the subsequent patterning of the nephron and fusion with the ureteric component of the kidney to form a patent contiguous uriniferous tubule has not been fully characterized. Using dual section in situ hybridization (SISH)/immunohistochemistry (IHC) we have revealed distinct distal/proximal patterning of Notch, BMP and Wnt pathway components within the RV stage nephron. Quantitation of mitoses and Cyclin D1 expression indicated that cell proliferation was higher in the distal RV, reflecting the differential developmental programs of the proximal and distal populations. A small number of RV genes were also expressed in the early connecting segment of the nephron. Dual ISH/IHC combined with serial section immunofluorescence and 3D reconstruction revealed that fusion occurs between the late RV and adjacent ureteric tip via a process that involves loss of the intervening ureteric epithelial basement membrane and insertion of cells expressing RV markers into the ureteric tip. Using Six2-eGFPCre x R26R-lacZ mice, we demonstrate that these cells are derived from the cap mesenchyme and not the ureteric epithelium. Hence, both nephron patterning and patency are evident at the late renal vesicle stage.
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Combes, AN; Lesieur, E; Harley, VR; Sinclair, AH; Little, MH; Wilhelm, D; Koopman, P. Dev Dyn . 238(5):1033–41. May 2009.
Testis cords are specialized tubes essential for generation and export of sperm, yet the mechanisms directing their formation, and the regulation of their position, size, shape, and number remain unclear. Here, we use a novel fluorescence-based three-dimensional modeling approach to show that cords initially form as a network of irregular cell clusters that are subsequently remodeled to form regular parallel loops, joined by a flattened plexus at the mesonephric side. Variation in cord number and structure demonstrates that cord specification is not stereotypic, although cord alignment and diameter becomes relatively consistent, implicating compensatory growth mechanisms. Branched, fused, and internalized cords were commonly observed. We conclude that the tubule-like structure of testis cords arise through a novel form of morphogenesis consisting of coalescence, partitioning, and remodeling. The methods we describe are applicable to investigating defects in testis cord development in mouse models, and more broadly, studying morphogenesis of other tissues.
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Atlas of gene expression in the developing kidney at microanatomic resolution
Brunskill, EW; Aronow, B; Georgas, K; Rumballe, B; Valerius, MT; Aronow, B; Kaimal, V; Jegga, AG; Yu, J; Grimmond, SM; McMahon, AP; Patterson, LT; Little, MH; Potter, SS. Dev Cell . 15(5):781–91. November 2008.
Kidney development is based on differential cell-type-specific expression of a vast number of genes. While multiple critical genes and pathways have been elucidated, a genome-wide analysis of gene expression within individual cellular and anatomic structures is lacking. Accomplishing this could provide significant new insights into fundamental developmental mechanisms such as mesenchymal-epithelial transition, inductive signaling, branching morphogenesis, and segmentation. We describe here a comprehensive gene expression atlas of the developing mouse kidney based on the isolation of each major compartment by either laser capture microdissection or fluorescence-activated cell sorting, followed by microarray profiling. The resulting data agree with known expression patterns and additional in situ hybridizations. This kidney atlas allows a comprehensive analysis of the progression of gene expression states during nephrogenesis, as well as discovery of potential growth factor-receptor interactions. In addition, the results provide deeper insight into the genetic regulatory mechanisms of kidney development.
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Georgas, K; Rumballe, B; Wilkinson, L; Chiu, HS; Lesieur, E; Gilbert, T; Little, MH. Histochem Cell Biol . 130(5):927–42. November 2008.
The kidney is the most complex organ within the urogenital system. The adult mouse kidney contains in excess of 8,000 mature nephrons, each of which can be subdivided into a renal corpuscle and 14 distinct tubular segments. The histological complexity of this organ can make the clarification of the site of gene expression by in situ hybridisation difficult. We have defined a panel of seven antibodies capable of identifying the six stages of early nephron development, the tubular nephron segments and the components of the renal corpuscle within the embryonic and adult mouse kidney. We have analysed in detail the protein expression of Wt1, Calb1 Aqp1, Aqp2 and Umod using these antibodies. We have then coupled immunohistochemistry with RNA in situ hybridisation in order to precisely identify the expression pattern of different genes, including Wnt4, Umod and Spp1. This technique will be invaluable for examining at high resolution, the structure of both the developing and mature nephron where standard in situ hybridisation and histological techniques are insufficient. The use of this technique will enhance the expression analyses of genes which may be involved in nephron formation and the function of the mature nephron in the mouse.
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Rumballe, B; Georgas, K; Little, MH. CSH Protoc . 2008. July 2008.
Section in situ hybridization (SISH) is a high-resolution tool used to analyze gene expression patterns. This protocol utilizes the Tecan Freedom EVO150 platform to perform high-throughput SISH on paraffin sections to detect mRNA with a digoxigenin (DIG)-labeled probe. The slide is mounted and imaged before performing immunohistochemistry (IHC) on the same section. The dual reaction enables a marker of protein expression to be localized on the same section as the mRNA and facilitates more accurate annotation of the gene expression.
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GUDMAP: the genitourinary developmental molecular anatomy project
McMahon, AP; Aronow, B; Davidson, DR; Davies, JA; Gaido, KW; Grimmond, SM; Lessard, JL; Little, MH; Potter, SS; Wilder, EL; Zhang, P; GUDMAP, Project. J Am Soc Nephrol . 19(4):667–71. April 2008.
In late 2004, an International Consortium of research groups were charged with the task of producing a high-quality molecular anatomy of the developing mammalian urogenital tract (UGT). Given the importance of these organ systems for human health and reproduction, the need for a systematic molecular and cellular description of their developmental programs was deemed a high priority. The information obtained through this initiative is anticipated to enable the highest level of basic and clinical research grounded on a 21st-century view of the developing anatomy. There are three components to the Genitourinary Developmental Molecular Anatomy Project GUDMAP; all of these are intended to provide resources that support research on the kidney and UGT. The first provides ontology of the cell types during UGT development and the molecular hallmarks of those cells as discerned by a variety of procedures, including in situ hybridization, transcriptional profiling, and immunostaining. The second generates novel mouse strains. In these strains, cell types of particular interest within an organ are labeled through the introduction of a specific marker into the context of a gene that exhibits appropriate cell type or structure-specific expression. In addition, the targeting construct enables genetic manipulation within the cell of interest in many of the strains. Finally, the information is annotated, collated, and promptly released at regular intervals, before publication, through a database that is accessed through a Web portal. Presented here is a brief overview of the Genitourinary Developmental Molecular Anatomy Project effort.
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A high-resolution anatomical ontology of the developing murine genitourinary tract
Little, MH; Brennan, J; Georgas, K; Davies, JA; Davidson, DR; Baldock, RA; Beverdam, A; Bertram, JF; Capel, B; Chiu, HS; Clements, D; Cullen-McEwen, L; Fleming, J; Gilbert, T; Herzlinger, D; Houghton, D; Kaufman, MH; Kleymenova, E; Koopman, PA; Lewis, AG; McMahon, AP; Mendelsohn, C; Mitchell, EK; Rumballe, BA; Sweeney, DE; Valerius, MT; Yamada, G; Yang, Y; Yu, J. Gene Expr Patterns . 7(6):680–99. June 2007.
Cataloguing gene expression during development of the genitourinary tract will increase our understanding not only of this process but also of congenital defects and disease affecting this organ system. We have developed a high-resolution ontology with which to describe the subcompartments of the developing murine genitourinary tract. This ontology incorporates what can be defined histologically and begins to encompass other structures and cell types already identified at the molecular level. The ontology is being used to annotate in situ hybridisation data generated as part of the Genitourinary Development Molecular Anatomy Project (GUDMAP), a publicly available data resource on gene and protein expression during genitourinary development. The GUDMAP ontology encompasses Theiler stage (TS) 17-27 of development as well as the sexually mature adult. It has been written as a partonomic, text-based, hierarchical ontology that, for the embryological stages, has been developed as a high-resolution expansion of the existing Edinburgh Mouse Atlas Project (EMAP) ontology. It also includes group terms for well-characterised structural and/or functional units comprising several sub-structures, such as the nephron and juxtaglomerular complex. Each term has been assigned a unique identification number. Synonyms have been used to improve the success of query searching and maintain wherever possible existing EMAP terms relating to this organ system. We describe here the principles and structure of the ontology and provide representative diagrammatic, histological, and whole mount and section RNA in situ hybridisation images to clarify the terms used within the ontology. Visual examples of how terms appear in different specimen types are also provided.
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Regrow or Repair: An Update on Potential Regenerative Therapies for the Kidney
Little, Melissa H.; Humphreys, Benjamin D. Journal of the American Society of Nephrology: JASN . 33(1):15–32. January 2022.
Fifteen years ago, this journal published a review outlining future options for regenerating the kidney. At that time, stem cell populations were being identified in multiple tissues, the concept of stem cell recruitment to a site of injury was of great interest, and the possibility of postnatal renal stem cells was growing in momentum. Since that time, we have seen the advent of human induced pluripotent stem cells, substantial advances in our capacity to both sequence and edit the genome, global and spatial transcriptional analysis down to the single-cell level, and a pandemic that has challenged our delivery of health care to all. This article will look back over this period of time to see how our view of kidney development, disease, repair, and regeneration has changed and envision a future for kidney regeneration and repair over the next 15 years.
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Djenoune, Lydia; Tomar, Ritu; Dorison, Aude; Ghobrial, Irene; Schenk, Heiko; Hegermann, Jan; Beverly-Staggs, Lynne; Hidalgo-Gonzalez, Alejandro; Little, Melissa H.; Drummond, Iain A.. Journal of the American Society of Nephrology . 32(7):1697–1712. July 2021.
Podocytes are critical to maintaining the kidney glomerular filtration barrier. Mutations in genes associated with development of nephrotic syndrome lead to elevated cytoplasmic calcium in podocytes and cause disruption of filtration barrier function. Whether calcium signaling plays a role in the initial formation of the filtration barrier is not known. Using live calcium imaging in two models, larval zebrafish and human kidney organoids, the authors demonstrate that podocyte calcium signaling is active during podocyte differentiation, is podocyte-cell autonomous, occurs independently of neighboring cell types, and is required for foot process and slit diaphragm formation. Their findings also show that developmental calcium signaling occurs by a different mechanism than disease-associated calcium perturbations, and represents a critical regulatory signal for podocyte morphogenesis and filtration barrier formation.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.
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Returning to kidney development to deliver synthetic kidneys
Little, Melissa H. Developmental Biology . 2020.
There is no doubt that the development of transplantable synthetic kidneys could improve the outcome for the many millions of people worldwide suffering from chronic kidney disease. Substantial progress has been made in the last 6 years in the generation of kidney tissue from stem cells. However, the limited scale, incomplete cellular complexity and functional immaturity of such structures suggests we are some way from this goal. While developmental biology has successfully guided advances to date, these human kidney models are limited in their capacity for ongoing nephrogenesis and lack corticomedullary definition, a unified vasculature and a coordinated exit path for urinary filtrate. This review will reassess our developmental understanding of how the mammalian embryo manages to create kidneys, how this has informed our progress to date and how both engineering and developmental biology can continue to guide us towards a synthetic kidney.
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Howden, Sara E.; Wilson, Sean B.; Groenewegen, Ella; Starks, Lakshi; Forbes, Thomas A.; Tan, Ker Sin; Vanslambrouck, Jessica M.; Holloway, Emily M.; Chen, Yi-Hsien; Jain, Sanjay; Spence, Jason R.; Little, Melissa H. Cell Stem Cell . 2020.
Summary During development, distinct progenitors contribute to the nephrons versus the ureteric epithelium of the kidney. Indeed, previous human pluripotent stem-cell-derived models of kidney tissue either contain nephrons or pattern specifically to the ureteric epithelium. By re-analyzing the transcriptional distinction between distal nephron and ureteric epithelium in human fetal kidney, we show here that, while existing nephron-containing kidney organoids contain distal nephron epithelium and no ureteric epithelium, this distal nephron segment alone displays significant in vitro plasticity and can adopt a ureteric epithelial tip identity when isolated and cultured in defined conditions. “Induced” ureteric epithelium cultures can be cryopreserved, serially passaged without loss of identity, and transitioned toward a collecting duct fate. Cultures harboring loss-of-function mutations in PKHD1 also recapitulate the cystic phenotype associated with autosomal recessive polycystic kidney disease.
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Cellular extrusion bioprinting improves kidney organoid reproducibility and conformation
Lawlor, Kynan T.; Vanslambrouck, Jessica M.; Higgins, J. William; Chambon, Alison; Bishard, Kristina; Arndt, Derek; Er, Pei Xuan; Wilson, Sean B.; Howden, Sara E.; Tan, Ker Sin; Li, Fanyi; Hale, Lorna J.; Shepherd, Benjamin; Pentoney, Stephen; Presnell, Sharon C.; Chen, Alice E.; Little, Melissa H. Nature Materials . November 2020.
Directed differentiation of human pluripotent stem cells to kidney organoids brings the prospect of drug screening, disease modelling and the generation of tissue for renal replacement. Currently, these applications are hampered by organoid variability, nephron immaturity, low throughput and limited scale. Here, we apply extrusion-based three-dimensional cellular bioprinting to deliver rapid and high-throughput generation of kidney organoids with highly reproducible cell number and viability. We demonstrate that manual organoid generation can be replaced by 6- or 96-well organoid bioprinting and evaluate the relative toxicity of aminoglycosides as a proof of concept for drug testing. In addition, three-dimensional bioprinting enables precise manipulation of biophysical properties, including organoid size, cell number and conformation, with modification of organoid conformation substantially increasing nephron yield per starting cell number. This facilitates the manufacture of uniformly patterned kidney tissue sheets with functional proximal tubular segments. Hence, automated extrusion-based bioprinting for kidney organoid production delivers improvements in throughput, quality control, scale and structure, facilitating in vitro and in vivo applications of stem cell-derived human kidney tissue.
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Kidney organoids: accurate models or fortunate accidents.
Little, Melissa H.; Combes, Alexander N.. Genes & development . 33(19-20):1319–1345. October 2019.
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.
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Vanslambrouck, Jessica M.; Wilson, Sean B.; Tan, Ker Sin; Soo, Joanne Y.-C.; Scurr, Michelle; Spijker, H. Siebe; Starks, Lakshi T.; Neilson, Amber; Cui, Xiaoxia; Jain, Sanjay; Little, Melissa Helen; Howden, Sara E.. Journal of the American Society of Nephrology . 30(10):1811–1823. 2019.
Kidney organoids generated from human induced pluripotent stem cells (iPSCs) show great potential for modeling kidney diseases and studying disease pathogenesis. However, the relative accuracy with which kidney organoids model normal morphogenesis, as well as the maturity and identity of the renal cell types they comprise, remain to be fully investigated. The authors describe the generation and validation of ten fluorescent CRISPR/Cas9 gene-edited iPSC reporter lines specifically designed for the visualization, isolation, and characterization of cell types and states within kidney organoids, and demonstrate the use of these lines for cellular isolation, time-lapse imaging, protocol optimization, and lineage-tracing applications. These tools offer promise for better understanding this model system and its congruence with human kidney morphogenesis.Background The generation of reporter lines for cell identity, lineage, and physiologic state has provided a powerful tool in advancing the dissection of mouse kidney morphogenesis at a molecular level. Although use of this approach is not an option for studying human development in vivo, its application in human induced pluripotent stem cells (iPSCs) is now feasible.Methods We used CRISPR/Cas9 gene editing to generate ten fluorescence reporter iPSC lines designed to identify nephron progenitors, podocytes, proximal and distal nephron, and ureteric epithelium. Directed differentiation to kidney organoids was performed according to published protocols. Using immunofluorescence and live confocal microscopy, flow cytometry, and cell sorting techniques, we investigated organoid patterning and reporter expression characteristics.Results Each iPSC reporter line formed well patterned kidney organoids. All reporter lines showed congruence of endogenous gene and protein expression, enabling isolation and characterization of kidney cell types of interest. We also demonstrated successful application of reporter lines for time-lapse imaging and mouse transplantation experiments.Conclusions We generated, validated, and applied a suite of fluorescence iPSC reporter lines for the study of morphogenesis within human kidney organoids. This fluorescent iPSC reporter toolbox enables the visualization and isolation of key populations in forming kidney organoids, facilitating a range of applications, including cellular isolation, time-lapse imaging, protocol optimization, and lineage-tracing approaches. These tools offer promise for enhancing our understanding of this model system and its correspondence with human kidney morphogenesis.
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Kumar, Santhosh V.; Er, Pei X.; Lawlor, Kynan T.; Motazedian, Ali; Scurr, Michelle; Ghobrial, Irene; Combes, Alexander N.; Zappia, Luke; Oshlack, Alicia; Stanley, Edouard G.; Little, Melissa H. Development . 146(5):dev172361. March 2019.
Kidney organoids have potential uses in disease modelling, drug screening and regenerative medicine. However, novel cost-effective techniques are needed to enable scaled-up production of kidney cell types in vitro. We describe here a modified suspension culture method for the generation of kidney micro-organoids from human pluripotent stem cells. Optimisation of differentiation conditions allowed the formation of micro-organoids, each containing six to ten nephrons that were surrounded by endothelial and stromal populations. Single cell transcriptional profiling confirmed the presence and transcriptional equivalence of all anticipated renal cell types consistent with a previous organoid culture method. This suspension culture micro-organoid methodology resulted in a three- to fourfold increase in final cell yield compared with static culture, thereby representing an economical approach to the production of kidney cells for various biological applications.
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Howden, Sara E; Vanslambrouck, Jessica M; Wilson, Sean B; Tan, Ker Sin; Little, Melissa H. EMBO Rep . March 2019.
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.EMBO Reports (2019) e47483
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Evaluation of variability in human kidney organoids
Phipson, Belinda; Er, Pei X.; Combes, Alexander N.; Forbes, Thomas A.; Howden, Sara E.; Zappia, Luke; Yen, Hsan-Jan; Lawlor, Kynan T.; Hale, Lorna J.; Sun, Jane; Wolvetang, Ernst; Takasato, Minoru; Oshlack, Alicia; Little, Melissa H. Nature Methods . 16(1):79–87. January 2019.
The utility of human pluripotent stem cell–derived kidney organoids relies implicitly on the robustness and transferability of the protocol. Here we analyze the sources of transcriptional variation in a specific kidney organoid protocol. Although individual organoids within a differentiation batch showed strong transcriptional correlation, we noted significant variation between experimental batches, particularly in genes associated with temporal maturation. Single-cell profiling revealed shifts in nephron patterning and proportions of component cells. Distinct induced pluripotent stem cell clones showed congruent transcriptional programs, with interexperimental and interclonal variation also strongly associated with nephron patterning. Epithelial cells isolated from organoids aligned with total organoids at the same day of differentiation, again implicating relative maturation as a confounder. This understanding of experimental variation facilitated an optimized analysis of organoid-based disease modeling, thereby increasing the utility of kidney organoids for personalized medicine and functional genomics.
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Single-cell analysis reveals congruence between kidney organoids and human fetal kidney
Combes, Alexander N.; Zappia, Luke; Er, Pei Xuan; Oshlack, Alicia; Little, Melissa H. Genome Medicine . 11(1):3. January 2019.
Human kidney organoids hold promise for studying development, disease modelling and drug screening. However, the utility of stem cell-derived kidney tissues will depend on how faithfully these replicate normal fetal development at the level of cellular identity and complexity.
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3D organoid-derived human glomeruli for personalised podocyte disease modelling and drug screening
Hale, Lorna J.; Howden, Sara E.; Phipson, Belinda; Lonsdale, Andrew; Er, Pei X.; Ghobrial, Irene; Hosawi, Salman; Wilson, Sean; Lawlor, Kynan T.; Khan, Shahnaz; Oshlack, Alicia; Quinlan, Catherine; Lennon, Rachel; Little, Melissa H. Nature Communications . 9(1):5167. December 2018.
The podocytes within the glomeruli of the kidney maintain the filtration barrier by forming interdigitating foot processes with intervening slit diaphragms, disruption in which results in proteinuria. Studies into human podocytopathies to date have employed primary or immortalised podocyte cell lines cultured in 2D. Here we compare 3D human glomeruli sieved from induced pluripotent stem cell-derived kidney organoids with conditionally immortalised human podocyte cell lines, revealing improved podocyte-specific gene expression, maintenance in vitro of polarised protein localisation and an improved glomerular basement membrane matrisome compared to 2D cultures. Organoid-derived glomeruli retain marker expression in culture for 96 h, proving amenable to toxicity screening. In addition, 3D organoid glomeruli from a congenital nephrotic syndrome patient with compound heterozygous NPHS1 mutations reveal reduced protein levels of both NEPHRIN and PODOCIN. Hence, human iPSC-derived organoid glomeruli represent an accessible approach to the in vitro modelling of human podocytopathies and screening for podocyte toxicity.
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Simultaneous reprogramming and gene editing of human fibroblasts
Howden, SE; Thomson, JA; Little, MH. Nature Protocols . 13(5):875–898. April 2018.
The utility of human induced pluripotent stem cells (iPSCs) is enhanced by an ability to precisely modify a chosen locus with minimal impact on the remaining genome. However, the derivation of gene-edited iPSCs typically involves multiple steps requiring lengthy culture periods and several clonal events. Here, we describe a one-step protocol for reliable generation of clonally derived gene-edited iPSC lines from human fibroblasts in the absence of drug selection or FACS enrichment. Using enhanced episomal-based reprogramming and CRISPR/Cas9 systems, gene-edited and passage-matched unmodified iPSC lines are obtained following a single electroporation of human fibroblasts. To minimize unwanted mutations within the target locus, we use a Cas9 variant that is associated with decreased nonhomologous end-joining (NHEJ) activity. This protocol outlines in detail how this streamlined approach can be used for both monoallelic and biallelic introduction of specific base changes or transgene cassettes in a manner that is efficient, rapid (∼6–8 weeks), and cost-effective.
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van den Berg, CW; Ritsma, L; Avramut, MC; Wiersma, LE; van den Berg, BM; Leuning, DG; Lievers, E; Koning, M; Vanslambrouck, JM; Koster, AJ; Howden, SE; Takasato, M; Little, MH; Rabelink, TJ. Stem Cell Reports. . 10(3):751–765. March 2018.
Human pluripotent stem cell (hPSC)-derived kidney organoids may facilitate disease modeling and the generation of tissue for renal replacement. Long-term application, however, will require transferability between hPSC lines and significant improvements in organ maturation. A key question is whether time or a patent vasculature is required for ongoing morphogenesis. Here, we show that hPSC-derived kidney organoids, derived in fully defined medium conditions and in the absence of any exogenous vascular endothelial growth factor, develop host-derived vascularization. In vivo imaging of organoids under the kidney capsule confirms functional glomerular perfusion as well as connection to pre-existing vascular networks in the organoids. Wide-field electron microscopy demonstrates that transplantation results in formation of a glomerular basement membrane, fenestrated endothelial cells, and podocyte foot processes. Furthermore, compared with non-transplanted organoids, polarization and segmental specialization of tubular epithelium are observed. These data demonstrate that functional vascularization is required for progressive morphogenesis of human kidney organoids.
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Combes, AN; Phipson, B; Zappia, L; Lawlor, KE; Er, PX; Oshlack, A; Little, MA. bioRxiv . December 2017.
Recent advances in our capacity to differentiate human pluripotent stem cells to human kidney tissue are moving the field closer to novel approaches for renal replacement. Such protocols have relied upon our current understanding of the molecular basis of mammalian kidney morphogenesis. To date this has depended upon population based-profiling of non-homogenous cellular compartments. In order to improve our resolution of individual cell transcriptional profiles during kidney morphogenesis, we have performed 10x Chromium single cell RNA-seq on over 6000 cells from the E18.5 developing mouse kidney, as well as more than 7000 cells from human iPSC-derived kidney organoids. We identified 16 clusters of cells representing all major cell lineages in the E18.5 mouse kidney. The differentially expressed genes from individual murine clusters were then used to guide the classification of 16 cell clusters within human kidney organoids, revealing the presence of distinguishable stromal, endothelial, nephron, podocyte and nephron progenitor populations. Despite the congruence between developing mouse and human organoid, our analysis suggested limited nephron maturation and the presence of off target populations in human kidney organoids, including unidentified stromal populations and evidence of neural clusters. This may reflect unique human kidney populations, mixed cultures or aberrant differentiation in vitro. Analysis of clusters within the mouse data revealed novel insights into progenitor maintenance and cellular maturation in the major renal lineages and will serve as a roadmap to refine directed differentiation approaches in human iPSC-derived kidney organoids.
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Phipson, B; Er, PX; Hale, L; Yen, DH; Lawlor, KE; Takasato, M; Sun, J; Wolvetang, E; Oshlack, A; Little, MH. bioRxiv . December 2017.
We have previously reported a protocol for the directed differentiation of human induced pluripotent stem cells to kidney organoids comprised of nephrons, proximal and distal epithelium, vasculature and surrounding interstitial elements. The utility of this protocol for applications such as disease modelling will rely implicitly on the developmental accuracy of the model, technical robustness of the protocol and transferability between iPSC lines. Here we report extensive transcriptional analyses of the sources of variation across the timecourse of differentiation from pluripotency to complete kidney organoid, focussing on repeated differentiations to day 18 organoid. Individual organoids generated within the same differentiation experiment show Spearmans correlation coefficients of \textgreater0.99. The greatest source of variation was seen between experimental batch, with the enrichment for genes that also varied temporally between day 10 and day 25 organoids implicating nephron maturation as contributing to transcriptional variance between individual differentiation experiments. A morphological analysis revealed a transition from renal vesicle to capillary loop stage nephrons across the same time period. Distinct iPSC clones were also shown to display congruent transcriptional programs with inter-experimental and inter-clonal variation most strongly associated with nephron patterning. Even epithelial cells isolated from organoids showed transcriptional alignment with total organoids of the same day of differentiation. This data provides a framework for managing experimental variation, thereby increasing the utility of this approach for personalised medicine and functional genomics.
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Oxburgh, L; Carroll, TJ; Cleaver, O; Gossett, DR; Hoshizaki, DK; Hubbell, JA; Humphreys, BD; Jain, S; Jensen, J; Kaplan, DL; Kesselman, C; Ketchum, CJ; Little, MH; McMahon, AP; Shankland, SJ; Spence, JR; Valerius, MT; Wertheim, JA; Wessely, O; Zheng, Y; Drummond, IA. J Am Soc Nephrol . 28(5):1370–1378. May 2017.
(Re)Building a Kidney is a National Institute of Diabetes and Digestive and Kidney Diseases-led consortium to optimize approaches for the isolation, expansion, and differentiation of appropriate kidney cell types and the integration of these cells into complex structures that replicate human kidney function. The ultimate goals of the consortium are two-fold: to develop and implement strategies for in vitro engineering of replacement kidney tissue, and to devise strategies to stimulate regeneration of nephrons in situ to restore failing kidney function. Projects within the consortium will answer fundamental questions regarding human gene expression in the developing kidney, essential signaling crosstalk between distinct cell types of the developing kidney, how to derive the many cell types of the kidney through directed differentiation of human pluripotent stem cells, which bioengineering or scaffolding strategies have the most potential for kidney tissue formation, and basic parameters of the regenerative response to injury. As these projects progress, the consortium will incorporate systematic investigations in physiologic function of in vitro and in vivo differentiated kidney tissue, strategies for engraftment in experimental animals, and development of therapeutic approaches to activate innate reparative responses.
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Takasato, M; Little, MH. Dev Biol . 420(2):210–220. December 2016.
Directed differentiation of human pluripotent stem cells (hPSCs) can provide us any required tissue/cell types by recapitulating the development in vitro. The kidney is one of the most challenging organs to generate from hPSCs as the kidney progenitors are composed of at least 4 different cell types, including nephron, collecting duct, endothelial and interstitium progenitors, that are developmentally distinguished populations. Although the actual developmental process of the kidney during human embryogenesis has not been clarified yet, studies using model animals accumulated knowledge about the origins of kidney progenitors. The implications of these findings for the directed differentiation of hPSCs into the kidney include the mechanism of the intermediate mesoderm specification and its patterning along with anteroposterior axis. Using this knowledge, we previously reported successful generation of hPSCs-derived kidney organoids that contained all renal components and modelled human kidney development in vitro. In this review, we explain the developmental basis of the strategy behind this differentiation protocol and compare strategies of studies that also recently reported the induction of kidney cells from hPSCs. We also discuss the characterization of such kidney organoids and limitations and future applications of this technology.
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Generation of kidney organoids from human pluripotent stem cells.
Takasat, M; Er, PX; Chiu, HS; Little, MH. Nat Protoc . 11(9):1681–92. September 2016.
The human kidney develops from four progenitor populations-nephron progenitors, ureteric epithelial progenitors, renal interstitial progenitors and endothelial progenitors-resulting in the formation of maximally 2 million nephrons. Until recently, the reported methods differentiated human pluripotent stem cells (hPSCs) into either nephron progenitor or ureteric epithelial progenitor cells, consequently forming only nephrons or collecting ducts, respectively. Here we detail a protocol that simultaneously induces all four progenitors to generate kidney organoids within which segmented nephrons are connected to collecting ducts and surrounded by renal interstitial cells and an endothelial network. As evidence of functional maturity, proximal tubules within organoids display megalin-mediated and cubilin-mediated endocytosis, and they respond to a nephrotoxicant to undergo apoptosis. This protocol consists of 7 d of monolayer culture for intermediate mesoderm induction, followed by 18 d of 3D culture to facilitate self-organizing renogenic events leading to organoid formation. Personnel experienced in culturing hPSCs are required to conduct this protocol.
