Sanjay Jain (PI)
Washington University in St. Louis
We propose to generate modified human pluripotent stem cell (hPSC) reporter lines with a genetically encoded activity sensor that tags key cell types important in development and function of the urinary collecting system. We will leverage an existing WTC11 hiPSC line that harbors GCaMP6 as an activity sensor thereby enhancing the functional utility of these lines by superimposing physiology onto development during or after a desired cell type or kidney organoid is formed. Multi-labeled reporter hiPSC cell lines of the collecting system in this proposal will enable RBK members to incorporate this essential component of the kidneys in efforts to optimize nephron formation, patterning and organization in scaffolds with the goal of making a kidney that maintains homeostasis and successfully expels urine.
Hoshi, Masato; Batourina, Ekatherina; Mendelsohn, Cathy; Jain, Sanjay. Development . 139(13):2405–2415. July 2012.
Mutations in the receptor tyrosine kinase RET are associated with congenital anomalies of kidneys or urinary tract (CAKUT). RET tyrosine Y1015 is the docking site for PLCgamma, a major regulator of RET signaling. Abrogating signaling via Y1015 causes CAKUT that are markedly different than renal agenesis in Ret-null or RetY1062F mutant mice. We performed analysis of Y1015F mutant upper and lower urinary tracts in mice to delineate its molecular and developmental roles during early urinary tract formation. We found that the degeneration of the common nephric ducts (CND), the caudal-most Wolffian duct (WD) segment, depends on Y1015 signals. The CNDs in Y1015F mutants persist owing to increased proliferation and reduced apoptosis, and showed abundance of phospho-ERK-positive cells. In the upper urinary tract, the Y1015 signals are required for proper patterning of the mesonephros and metanephros. Timely regression of mesonephric mesenchyme and proper demarcation of mesonephric and metanephric mesenchyme from the WD depends on RetY1015 signaling. We show that the mechanism of de novo ectopic budding is via increased ERK activity due to abnormal mesenchymal GDNF expression. Although reduction in GDNF dosage improved CAKUT it did not affect delayed mesenchyme regression. Experiments using whole-mount immunofluorescence confocal microscopy and explants cultures of early embryos with ERK-specific inhibitors suggest an imbalance between increased proliferation, decreased apoptosis and increased ERK activity as a mechanism for WD defects in RetY1015F mice. Our work demonstrates novel inhibitory roles of RetY1015 and provides a possible mechanistic explanation for some of the confounding broad range phenotypes in individuals with CAKUT.
Golden, Judith P.; Hoshi, Masato; Nassar, Mohammed A.; Enomoto, Hideki; Wood, John N.; Milbrandt, Jeffrey; Gereau, Robert W. 4th; Johnson, Eugene M. Jr; Jain, Sanjay. J Neurosci . 30(11):3983–3994. March 2010.
Small unmyelinated sensory neurons classified as nociceptors are divided into two subpopulations based on phenotypic differences, including expression of neurotrophic factor receptors. Approximately half of unmyelinated nociceptors express the NGF receptor TrkA, and half express the GDNF family ligand (GFL) receptor Ret. The function of NGF/TrkA signaling in the TrkA population of nociceptors has been extensively studied, and NGF/TrkA signaling is a well established mediator of pain. The GFLs are analgesic in models of neuropathic pain emphasizing the importance of understanding the physiological function of GFL/Ret signaling in nociceptors. However, perinatal lethality of Ret-null mice has precluded the study of the physiological role of GFL/Ret signaling in the survival, maintenance, and function of nociceptors in viable mice. We deleted Ret exclusively in nociceptors by crossing nociceptor-specific Na(v)1.8 Cre and Ret conditional mice to produce Ret-Na(v)1.8 conditional knock-out (CKO) mice. Loss of Ret exclusively in nociceptors results in a reduction in nociceptor number and size, indicating that Ret signaling is important for the survival and trophic support of these cells. Ret-Na(v)1.8 CKO mice exhibit reduced epidermal innervation but normal central projections. In addition, Ret-Na(v)1.8 CKO mice have increased sensitivity to cold and increased formalin-induced pain, demonstrating that Ret signaling modulates the function of nociceptors in vivo. Enhanced inflammation-induced pain may be mediated by decreased prostatic acid phosphatase (PAP), as PAP levels are markedly reduced in Ret-Na(v)1.8 CKO mice. The results of this study identify the physiological role of endogenous Ret signaling in the survival and function of nociceptors.
Jain, Sanjay. Organogenesis . 5(4):177–190. October 2009.
Signaling pathways that are activated upon interaction of glial cell-line derived neurotrophic factor (Gdnf), its coreceptor Gfra1, and receptor tyrosine kinase Ret are critical for kidney development and ureter maturation. Outside the kidney, this pathway is implicated in a number of congenital diseases including Hirschsprung disease (intestinal aganglionosis, HSCR) and hereditary cancer syndromes (MEN 2). Total lack of Gdnf, Gfra1 or Ret in mice results in perinatal lethality due to bilateral renal agenesis or aplasia. In humans, RET mutations have been identified in a spectrum of congenital malformations involving the RET axis including isolated HSCR, isolated congenital anomalies of kidney or urinary tract (CAKUT), or CAKUT and HSCR together. The molecular basis for these pleiotropic effects of RET has just begun to be unraveled. In an effort to delineate the pathogenetic mechanisms that underlie these congenital malformations, we and others have characterized Ret’s role in early kidney and urinary system development. Here we present a brief overview of the "many faces" of Ret dysfunction in kidney with particular emphasis on Ret’s signaling specificity and intergenic interactions that confer normal urinary system development.
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.
Ettou, Sandrine; Jung, Youngsook L.; Miyoshi, Tomoya; Jain, Dhawal; Hiratsuka, Ken; Schumacher, Valerie; Taglienti, Mary E.; Morizane, Ryuji; Park, Peter J.; Kreidberg, Jordan A.. Science advances . 6(30):eabb5460. July 2020.
In the context of human disease, the mechanisms whereby transcription factors reprogram gene expression in reparative responses to injury are not well understood. We have studied the mechanisms of transcriptional reprogramming in disease using murine kidney podocytes as a model for tissue injury. Podocytes are a crucial component of glomeruli, the filtration units of each nephron. Podocyte injury is the initial event in many processes that lead to end-stage kidney disease. Wilms tumor-1 (WT1) is a master regulator of gene expression in podocytes, binding nearly all genes known to be crucial for maintenance of the glomerular filtration barrier. Using murine models and human kidney organoids, we investigated WT1-mediated transcriptional reprogramming during the course of podocyte injury. Reprogramming the transcriptome involved highly dynamic changes in the binding of WT1 to target genes during a reparative injury response, affecting chromatin state and expression levels of target genes.
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.
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.