Marcu, R; Choi, YJ; Xue, J; Fortin, CL; Wang, Y; Nagao, RJ; Xu, J; MacDonald, JW; Bammler, TK; Murry, CE; Muczynski, K; Stevens, KR; Himmelfarb, J; Schwartz, SM; Zheng, Y. iScience. vol. 4, 20–35. June 2018.
The endothelium first forms in the blood islands in the extra-embryonic yolk sac and then throughout the embryo to establish circulatory networks that further acquire organ-specific properties during development to support diverse organ functions. Here, we investigated the properties of endothelial cells (ECs), isolated from four human major organs—the heart, lung, liver, and kidneys—in individual fetal tissues at three months’ gestation, at gene expression, and at cellular function levels. We showed that organ-specific ECs have distinct expression patterns of gene clusters, which support their specific organ development and functions. These ECs displayed distinct barrier properties, angiogenic potential, and metabolic rate and support specific organ functions. Our findings showed the link between human EC heterogeneity and organ development and can be exploited therapeutically to contribute in organ regeneration, disease modeling, as well as guiding differentiation of tissue-specific ECs from human pluripotent stem cells.
Howden, SE; Thomson, JA; Little, MH. Nature Protocols. vol. 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.
Daniel, E; Azizoglu, DB; Ryan, AR; Walji, TA; Chaney, CP; Sutton, GI; Carroll, TJ; Marciano, DK; Cleaver, O. Angiogenesis. April 2018.
The kidney vasculature facilitates the excretion of wastes, the dissemination of hormones, and the regulation of blood chemistry. To carry out these diverse functions, the vasculature is regionalized within the kidney and along the nephron. However, when and how endothelial regionalization occurs remains unknown. Here, we examine the developing kidney vasculature to assess its 3-dimensional structure and transcriptional heterogeneity. First, we observe that endothelial cells (ECs) grow coordinately with the kidney bud as early as E10.5, and begin to show signs of speci cation by E13.5 when the rst arteries can be identi ed. We then focus on how ECs pattern and remodel with respect to the developing nephron and collecting duct epithelia. ECs circumscribe nephron progenitor populations at the distal tips of the ureteric bud (UB) tree and form stereotyped cruciform structures around each tip. Beginning at the renal vesicle (RV) stage, ECs form a continuous plexus around developing nephrons. The endothelial plexus envelops and elaborates with the maturing nephron, becoming preferentially enriched along the early distal tubule. Lastly, we perform transcriptional and immuno uorescent screens to characterize spatiotemporal heterogeneity in the kidney vasculature and identify novel regionally enriched genes. A better understanding of development of the kidney vasculature will help instruct engineering of properly vascularized ex vivo kidneys and evaluate diseased kidneys.
Eng, DG; Kaverina, NV; Schneider, RRS; Freedman, BS; Gross, KW; Miner, JH; Pippin, JW; Shankland, SJ. Kidney International. March 2018.
Understanding of cellular transdifferentiation is limited by the technical inability to track multiple lineages in vivo. To overcome this we developed a new tool to simultaneously fate map two distinct cell types in the kidney, and genetically test whether cells of renin lineage (CoRL) can transdifferentiate to a podocyte fate. Ren1cCreER/ tdTomato/Nphs1-FLPo/FRT-EGFP mice (CoRL-PODO mice) were generated by crossing Ren1c-CreER/tdTomato CoRL reporter mice with Nphs1-FLPo/FRT-EGFP podocyte reporter mice. Following tamoxifen administration in these animals, CoRL were labeled with red fluorescence (tdTomato) and co-localized with renin. Podocytes were labeled green (enhanced green fluorescent protein) and co-localized with nephrin. Following podocyte loss by nephrotoxic antibody and subsequent enalapril-enhanced partial replacement, tdTomato-EGFP-labeled CoRL were detected as yellow- colored cells in a subset of glomerular tufts, without the use of antibodies. Co-localization with podocin indicated that these cells are podocytes, derived from CoRL origin. Thus, our novel study shows that two distinct cell types can be simultaneously labeled in the mouse kidney and provide strong genetic evidence in vivo that lost podocytes can be replaced in part by CoRL.
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.. vol. 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.
Lindström, NO; McMahon, JA; Guo, J; Tran, T; Guo, Q; Rutledge, E; Parvez, RK; Saribekyan, G; Schuler, RE; Liao, C; Kim, AD; Abdelhalim, A; Ruffins, SW; Thornton, ME; Basking, L; Grubbs, B; Kesselman, C; McMahon, AP. J Am Soc Nephrol. February 2018.
Human kidney function is underpinned by approximately 1,000,000 nephrons, although the number varies substantially, and low nephron number is linked to disease. Human kidney development initiates around 4 weeks of gestation and ends around 34-37 weeks of gestation. Over this period, a reiterative inductive process establishes the nephron complement. Studies have provided insightful anatomic descriptions of human kidney development, but the limited histologic views are not readily accessible to a broad audience. In this first paper in a series providing comprehensive insight into human kidney formation, we examined human kidney development in 135 anonymously donated human kidney specimens. We documented kidney development at a macroscopic and cellular level through histologic analysis, RNA in situ hybridization, immunofluorescence studies, and transcriptional profiling, contrasting human development (4-23 weeks) with mouse development at selected stages (embryonic day 15.5 and postnatal day 2). The high-resolution histologic interactive atlas of human kidney organogenesis generated can be viewed at the GUDMAP database (www.gudmap.org) together with three-dimensional reconstructions of key components of the data herein. At the anatomic level, human and mouse kidney development differ in timing, scale, and global features such as lobe formation and progenitor niche organization. The data also highlight differences in molecular and cellular features, including the expression and cellular distribution of anchor gene markers used to identify key cell types in mouse kidney studies. These data will facilitate and inform in vitro efforts to generate human kidney structures and comparative functional analyses across mammalian species.
Menon, R; Otto, EA; Kokoruda, A; Zhou, J; Zhang, Z; Yoon, E; Chen, Y; Troyanscaya, O; Spence, J; Kretzler, M; Cebrian, C. bioRxiv. February 2018.
Malone, AF; Wu, H; Humphreys, BD. Semin Nephrol. January 2018.
The renal biopsy provides critical diagnostic and prognostic information to clinicians including cases of acute kidney injury, chronic kidney disease, and allograft dysfunction. Today, biopsy specimens are read using a combination of light microscopy, electron microscopy, and indirect immunofluorescence, with a limited number of antibodies. These techniques all were perfected decades ago with only incremental changes since then. By contrast, recent advances in single-cell genomics are transforming scientists’ ability to characterize cells. Rather than measure the expression of several genes at a time by immunofluorescence, it now is possible to measure the expression of thousands of genes in thousands of single cells simultaneously. Here, we argue that the development of single-cell RNA sequencing offers an opportunity to describe human kidney disease comprehensively at a cellular level. It is particularly well suited for the analysis of immune cells, which are characterized by multiple subtypes and changing functions depending on their environment. In this review, we summarize the development of single-cell RNA sequencing methodologies. We discuss how these approaches are being applied in other organs, and the potential for this powerful technology to transform our understanding of kidney disease once applied to the renal biopsy.
McClelland, AD; Lichtnekert, J; Eng, DG; Pippin, JW; Gross, KW; Gharib, SA; Shankland, SJ. PLOS ONE. vol. 12(12) December 2017.
Renin producing cells of the juxtaglomerulus, herein called cells of renin lineage (CoRL), have garnered recent interest for their propensity to act as a progenitor source for various kidney cell types including podocytes. Despite recent advances, the process of transdifferentiation of CoRL to podocytes is poorly understood. In this study, we employed a transgenic reporter mouse line which permanently labels CoRL with ZsGreen fluorescent protein, allowing for isolation by fluorescence-activated cell sorting. At 5 days following induction of abrupt podocyte ablation via anti-podocyte sheep IgG, mice were sacrificed and CoRL were isolated by FACS. RNA was subsequently analyzed by microarray. Gene set enrichment analysis (GSEA) was performed and revealed that CoRL display a distinct phenotype following podocyte ablation, primarily consisting of downregulation of metabolic processes and upregulation of immuno-modulatory processes. Additionally, RNA-biology and cell cycle-related processes were also upregulated. Changes in gene expression or activity of a core set of transcription factors including HNF1 and E2F were identified through changes in enrichment of their respective target genes. However, integration of results from transcription factor and canonical pathway analysis indicated that ERR1 and PU-box family members may be the major contributors to the post-podocyte ablation phenotype of CoRL. Finally, top ranking genes were selected from the microarray-based analysis and confirmed by qPCR. Collectively, our results provide valuable insights into the transcriptional regulation of CoRL following abrupt podocyte ablation.
Kim, YK; Refaeli, I; Brooks, CR; Jing, P; Gulieva, RE; Hughes, MR; Cruz, NM; Liu, Y; Churchill, AJ; Wang, Y; Fu, H; Pippin, JW; Lin, LY; Shankland, SJ; Vogl, AW; McNagny, KM; Freedman, BS. Stem Cells. vol. 35(12), 2366–2378. December 2017.
A critical event during kidney organogenesis is the differentiation of podocytes, specialized epithelial cells that filter blood plasma to form urine. Podocytes derived from human pluripotent stem cells (hPSC-podocytes) have recently been generated in nephron-like kidney organoids, but the developmental stage of these cells and their capacity to reveal disease mechanisms remains unclear. Here, we show that hPSC-podocytes phenocopy mammalian podocytes at the capillary loop stage (CLS), recapitulating key features of ultrastructure, gene expression, and mutant phenotype. hPSC-podocytes in vitro progressively establish junction-rich basal membranes (nephrin+ podocin+ ZO-1+ ) and microvillus-rich apical membranes (podocalyxin+ ), similar to CLS podocytes in vivo. Ultrastructural, biophysical, and transcriptomic analysis of podocalyxin-knockout hPSCs and derived podocytes, generated using CRISPR/Cas9, reveals defects in the assembly of microvilli and lateral spaces between developing podocytes, resulting in failed junctional migration. These defects are phenocopied in CLS glomeruli of podocalyxin-deficient mice, which cannot produce urine, thereby demonstrating that podocalyxin has a conserved and essential role in mammalian podocyte maturation. Defining the maturity of hPSC-podocytes and their capacity to reveal and recapitulate pathophysiological mechanisms establishes a powerful framework for studying human kidney disease and regeneration. Stem Cells 2017;35:2366-2378
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.
Wu, H; Humphreys, Benjamin D.. Kidney International. vol. 92(6), 1334–1342. December 2017.
Recent techniques for single-cell RNA sequencing (scRNA-seq) at high throughput are leading to profound new discoveries in biology. The ability to generate vast amounts of transcriptomic data at cellular resolution represents a transformative advance, allowing the identification of novel cell types, states, and dynamics. In this review, we summarize the development of scRNA-seq methodologies and highlight their advantages and drawbacks. We discuss available software tools for analyzing scRNA-Seq data and summarize current computational challenges. Finally, we outline ways in which this powerful technology might be applied to discovery research in kidney development and disease.
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.
Mandrycky, C; Phong, K; Zheng, Y. MRS Communications. vol. 7(3), 332–347. September 2017.
Tissue engineering has been recognized as a translational approach to replace damaged tissue or whole organs. Engineering tissue, however, faces an outstanding knowledge gap in the challenge to fully recapitulate complex organ-specific features. Major components, such as cells, matrix, and architecture, must each be carefully controlled to engineer tissue-specific structure and function that mimics what is found in vivo. Here we review different methods to engineer tissue, and discuss critical challenges in recapitulating the unique features and functional units in four major organs-the kidney, liver, heart, and lung, which are also the top four candidates for organ transplantation in the USA. We highlight advances in tissue engineering approaches to enable the regeneration of complex tissue and organ substitutes, and provide tissue-specific models for drug testing and disease modeling. We discuss the current challenges and future perspectives toward engineering human tissue models.
Kaverina, NV; Eng, DG; Largent, AD; Daehn, I; Chang, A; Gross, KW; Pippin, JW; Hohenstein, P; Shankland, SJ. Stem Cell Reports.. vol. pii: S2213-6711(17), 30377–6. September 2017.
Wilms’ tumor suppressor 1 (WT1) plays an important role in cell proliferation and mesenchymal-epithelial balance in normal development and disease. Here, we show that following podocyte depletion in three experimental models, and in patients with focal segmental glomerulosclerosis (FSGS) and membranous nephropathy, WT1 increased significantly in cells of renin lineage (CoRL). In an animal model of FSGS in RenWt1fl/fl reporter mice with inducible deletion of WT1 in CoRL, CoRL proliferation and migration to the glomerulus was reduced, and glomerular disease was worse compared with wild-type mice. To become podocytes, CoRL undergo mesenchymal-to-epithelial transformation (MET), typified by reduced staining for mesenchymal markers (MYH11, SM22, αSMA) and de novo expression of epithelial markers (E-cadherin and cytokeratin18). Evidence for changes in MET markers was barely detected in RenWt1fl/fl mice. Our results show that following podocyte depletion, WT1 plays essential roles in CoRL proliferation and migration toward an adult podocyte fate.
Uzarski, JS; DiVito, MD; Wertheim, JA; Miller, WM. Biomaterials. (129), 163–175. June 2017.
Precise measurement of cellularity within bioartificial tissues and extracellular matrix (ECM) scaffolds is necessary to augment rigorous characterization of cellular behavior, as accurate benchmarking of tissue function to cell number allows for comparison of data across experiments and between laboratories. Resazurin, a soluble dye that is reduced to highly fluorescent resorufin in proportion to the metabolic activity of a cell population, is a valuable, noninvasive tool to measure cell number. We investigated experimental conditions in which resazurin reduction is a reliable indicator of cellularity within three-dimensional (3D) ECM scaffolds. Using three renal cell populations, we demonstrate that correlation of viable cell numbers with the rate of resorufin generation may deviate from linearity at higher cell densities, lower resazurin working volumes, or longer incubation times that all contribute to depleting the pool of resazurin. In conclusion, while the resazurin reduction assay provides a powerful, noninvasive readout of metrics enumerating cellularity and growth within ECM scaffolds, assay conditions may strongly influence its applicability for accurate quantification of cell number. The approach and methodological recommendations presented herein may be used as a guide for application-specific optimization of this assay to obtain rigorous and accurate measurement of cellular content in bioengineered tissues.
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. vol. 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.
Kramann, Rafael; Wongboonsin, Janewit; Chang-Panesso, Monica; Machado, Flavia G; Humphreys, Benjamin D.. J Am Soc Nephrol. (28), 776–784. 2017.
Peritubular capillary rarefaction is hypothesized to contribute to the increased risk of future CKD after AKI. Here, we directly tested the role of Gli1+ kidney pericytes in the maintenance of peritubular capillary health, and the consequences of pericyte loss during injury. Using bigenic Gli1-CreERt2; R26tdTomato reporter mice, we observed increased distance between Gli1+ pericytes and endothelial cells after AKI (mean6 SEM: 3.360.1 mm before injury versus 12.560.2 mm after injury; P,0.001). Using a genetic ablation model, we asked whether pericyte loss alone is sufficient for capillary destabilization. Ten days after pericyte ablation, we observed endothelial cell damage by electron microscopy. Furthermore, pericyte loss led to significantly reduced capillary number at later time points (mean6SEM capillaries/high-power field: 67.664.7 in control versus 44.164.8 at 56 days; P,0.05) and increased cross-sectional area (mean6 SEM: 21.960.4 mm2 in control versus 24.160.6 mm2 at 10 days; P,0.01 and 24.66 0.6 mm2 at 56 days; P,0.001). Pericyte ablation also led to hypoxic focal and subclinical tubular injury, reflected by transient expression of Kim1 and vimentin in scattered proximal tubule segments. This analysis provides direct evidence that AKI causes pericyte detachment from capillaries, and that pericyte loss is sufficient to trigger transient tubular injury and permanent peritubular capillary rarefaction.
Takasato, M; Little, MH. Dev Biol. vol. 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.
Takasat, M; Er, PX; Chiu, HS; Little, MH. Nat Protoc. vol. 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.
. Transplantation. vol. 100(1), 3–4. January 2016.