Navin Gupta, Emre Dilmen, and Ryuji Morizane. 10/9/2020. “3D kidney organoids for bench-to-bedside translation.” Journal of Molecular Medicine.Abstract
The kidneys are essential organs that filter the blood, removing urinary waste while maintaining fluid and electrolyte homeostasis. Current conventional research models such as static cell cultures and animal models are insufficient to grasp the complex human in vivo situation or lack translational value. To accelerate kidney research, novel research tools are required. Recent developments have allowed the directed differentiation of induced pluripotent stem cells to generate kidney organoids. Kidney organoids resemble the human kidney in vitro and can be applied in regenerative medicine and as developmental, toxicity, and disease models. Although current studies have shown great promise, challenges remain including the immaturity, limited reproducibility, and lack of perfusable vascular and collecting duct systems. This review gives an overview of our current understanding of nephrogenesis that enabled the generation of kidney organoids. Next, the potential applications of kidney organoids are discussed followed by future perspectives. This review proposes that advancement in kidney organoid research will be facilitated through our increasing knowledge on nephrogenesis and combining promising techniques such as organ-on-a-chip models.
Sandrine Ettou, Youngsook L. Jung, Tomoya Miyoshi, Dhawal Jain, Ken Hiratsuka, Valerie Schumacher, Mary E. Taglienti, Ryuji Morizane, Peter J. Park, and Jordan A. Kreidberg. 7/24/2020. “Epigenetic transcriptional reprogramming by WT1 mediates a repair response during podocyte injury.” Science Advances, 6, 30, Pp. eabb5460. Publisher's VersionAbstract
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.
Seiji Kishi, Craig R Brooks, Kensei Taguchi, Takaharu Ichimura, Yutaro Mori, Akinwande Akinfolarin, Navin Gupta, Pierre Galichon, Bertha C Elias, Tomohisa Suzuki, Qian Wang, Leslie Gewin, Ryuji Morizane, and Joseph V Bonventre. 11/1/2019. “Proximal tubule ATR regulates DNA repair to prevent maladaptive renal injury responses.” The Journal of Clinical Investigation, 11, 129, Pp. 4797-4816. Publisher's VersionAbstract
Maladaptive proximal tubule (PT) repair has been implicated in kidney fibrosis through induction of cell-cycle arrest at G2/M. We explored the relative importance of the PT DNA damage response (DDR) in kidney fibrosis by genetically inactivating ataxia telangiectasia and Rad3-related (ATR), which is a sensor and upstream initiator of the DDR. In human chronic kidney disease, ATR expression inversely correlates with DNA damage. ATR was upregulated in approximately 70% of Lotus tetragonolobus lectin-positive (LTL+) PT cells in cisplatin-exposed human kidney organoids. Inhibition of ATR resulted in greater PT cell injury in organoids and cultured PT cells. PT-specific Atr-knockout (ATRRPTC-/-) mice exhibited greater kidney function impairment, DNA damage, and fibrosis than did WT mice in response to kidney injury induced by either cisplatin, bilateral ischemia-reperfusion, or unilateral ureteral obstruction. ATRRPTC-/- mice had more cells in the G2/M phase after injury than did WT mice after similar treatments. In conclusion, PT ATR activation is a key component of the DDR, which confers a protective effect mitigating the maladaptive repair and consequent fibrosis that follow kidney injury.
Takuya Matsumoto, Olivier J.M. Schäffers, Wenqing Yin, and Ryuji Morizane. 7/30/2019. “Renal Regeneration: Stem Cell-Based Therapies to Battle Kidney Disease.” EMJ Nephrol., 7, 1, Pp. 54-64. Publisher's VersionAbstract

While the worldwide prevalence of kidney disease is increasing rapidly, the current therapeutic repertoire for these patients is often limited to dialysis and organ transplantation. However, advances in developmental and stem cell biology have highlighted the potential of stem cells for the development of novel renal regeneration therapies. While there are currently no approved stem cell-based treatments for kidney disease, various types of stem cells have been shown to facilitate regeneration of kidney tissue in preclinical models of both acute and chronic kidney injury. This review summarises the current status of stem cell-based therapies to battle kidney disease. In addition, future directions for the clinical translation of stem cell research towards development of novel renal regeneration therapies are discussed.

Miyoshi T, Hiratsuka K, Garcia Saiz E, and Morizane R. 3/6/2019. “Kidney Organoids in Translational Medicine: Disease Modeling and Regenerative Medicine.” Developmental Dynamics. Publisher's VersionAbstract
The kidney is one of the most complex organs composed of multiple cell types, functioning to maintain homeostasis via the filtering of metabolic wastes, balancing of blood electrolytes, and adjustment of blood pressure. Recent advances in 3D culture technologies in vitro enabled the generation of 'organoids' which mimic the structure and function of in vivo organs. Organoid technology has allowed for new insights into human organ development and human pathophysiology, with great potential for translational research. Increasing evidence shows that kidney organoids are a useful platform for disease modeling of genetic kidney diseases when derived from genetic patient iPSCs and/or CRISPR-mutated stem cells. Though single cell RNA-seq studies highlight the technical difficulties underlying kidney organoid generation reproducibility and variation in differentiation protocols, kidney organoids still hold great potential to understand kidney pathophysiology as applied to kidney injury and fibrosis. In this review, we summarize various studies of kidney organoids, disease modeling, genome-editing, and bioengineering, and additionally discuss the potential of and current challenges to kidney organoid research.
Morizane R. 2/18/2019. “Modelling diabetic vasculopathy with human vessel organoids.” Nature Reviews Nephrology. Publisher's VersionAbstract
A new study reports that human blood vessel organoids can be generated through the directed differentiation of human pluripotent stem cells. Use of these blood vessel organoids to model diabetic vasculopathy led to the identification of a new potential therapeutic target, suggesting that this system could have translational value for studies of diabetes complications.
Kimberly A. Homan, Navin Gupta, Katharina T. Kroll, David B. Kolesky, Mark Skylar-Scott, Tomoya Miyoshi, Donald Mau, M. Todd Valerius, Thomas Ferrante, Joseph V. Bonventre, Jennifer A. Lewis, and Ryuji Morizane. 2/11/2019. “Flow-enhanced vascularization and maturation of kidney organoids in vitro .” Nature Methods. Publisher's VersionAbstract

Kidney organoids derived from human pluripotent stem cells have glomerular- and tubular-like compartments that are largely avascular and immature in static culture. Here we report an in vitro method for culturing kidney organoids under flow on millifluidic chips, which expands their endogenous pool of endothelial progenitor cells and generates vascular networks with perfusable lumens surrounded by mural cells. We found that vascularized kidney organoids cultured under flow had more mature podocyte and tubular compartments with enhanced cellular polarity and adult gene expression compared with that in static controls. Glomerular vascular development progressed through intermediate stages akin to those involved in the embryonic mammalian kidney’s formation of capillary loops abutting foot processes. The association of vessels with these compartments was reduced after disruption of the endogenous VEGF gradient. The ability to induce substantial vascularization and morphological maturation of kidney organoids in vitro under flow opens new avenues for studies of kidney development, disease, and regeneration.

Morizane R. 1/31/2019. “Revealing potential cardiac manifestation of ADPKD using iPS cell-derived cardiomyocytes.” EBioMedicine. Publisher's Version
Hiratsuka K, Monkawa T, Akiyama T, Nakatake Y, Oda M, Goparaju SK, Kimura H, Chikazawa-Nohtomi N, Sato S, Ishiguro K, Yamaguchi S, Suzuki S, Morizane R, Ko SBH, Itoh H, and Ko MSH. 1/29/2019. “Induction of human pluripotent stem cells into kidney tissues by synthetic mRNAs encoding transcription factors.” Scientific Reports, 9, 1, Pp. 913.Abstract
The derivation of kidney tissues from human pluripotent stem cells (hPSCs) and its application for replacement therapy in end-stage renal disease have been widely discussed. Here we report that consecutive transfections of two sets of synthetic mRNAs encoding transcription factors can induce rapid and efficient differentiation of hPSCs into kidney tissues, termed induced nephron-like organoids (iNephLOs). The first set - FIGLA, PITX2, ASCL1 and TFAP2C, differentiated hPSCs into SIX2+SALL1+ nephron progenitor cells with 92% efficiency within 2 days. Subsequently, the second set - HNF1A, GATA3, GATA1 and EMX2, differentiated these cells into PAX8+LHX1+ pretubular aggregates in another 2 days. Further culture in both 2-dimensional and 3-dimensional conditions produced iNephLOs containing cells characterized as podocytes, proximal tubules, and distal tubules in an additional 10 days. Global gene expression profiles showed similarities between iNephLOs and the human adult kidney, suggesting possible uses of iNephLOs as in vitro models for kidneys.
Hill SJ, Decker B, Roberts EA, Horowitz NS, Muto MG, Worley MJ Jr, Feltmate CM, Nucci MR, Swisher EM, Nguyen H, Yang C, Morizane R, Kochupurakkal BS, Do KT, Konstantinopoulos PA, Liu JF, Bonventre JV, Matulonis UA, Shapiro GI, Berkowitz RS, Crum CP, and D'Andrea AD. 11/2018. “Prediction of DNA Repair Inhibitor Response in Short-Term Patient-Derived Ovarian Cancer Organoids.” Cancer Discov., 8, 11, Pp. 1404-1421.Abstract
Based on genomic analysis, 50% of high-grade serous ovarian cancers (HGSC) are predicted to have DNA repair defects. Whether this substantial subset of HGSCs actually have functional repair defects remains unknown. Here, we devise a platform for functional profiling of DNA repair in short-term patient-derived HGSC organoids. We tested 33 organoid cultures derived from 22 patients with HGSC for defects in homologous recombination (HR) and replication fork protection. Regardless of DNA repair gene mutational status, a functional defect in HR in the organoids correlated with PARP inhibitor sensitivity. A functional defect in replication fork protection correlated with carboplatin and CHK1 and ATR inhibitor sensitivity. Our results indicate that a combination of genomic analysis and functional testing of organoids allows for the identification of targetable DNA damage repair defects. Larger numbers of patient-derived organoids must be analyzed to determine whether these assays can reproducibly predict patient response in the clinic.Significance: Patient-derived ovarian tumor organoids grow rapidly and match the tumors from which they are derived, both genetically and functionally. These organoids can be used for DNA repair profiling and therapeutic sensitivity testing and provide a rapid means of assessing targetable defects in the parent tumor, offering more suitable treatment options.
Dario R Lemos, Michael McMurdo, Gamze Karaca, Julia Wilflingseder, Irina A Leaf, Navin Gupta, Tomoya Miyoshi, Koichiro Susa, Bryce G Johnson, Kirolous Soliman, Guanghai Wang, Ryuji Morizane, Joseph V Bonventre, and Jeremy S Duffield. 5/8/2018. “Interleukin-1β Activates a MYC-Dependent Metabolic Switch in Kidney Stromal Cells Necessary for Progressive Tubulointerstitial Fibrosis.” Journal of the American Society of Nephrology.Abstract

Background Kidney injury is characterized by persisting inflammation and fibrosis, yet mechanisms by which inflammatory signals drive fibrogenesis remain poorly defined.

Methods RNA sequencing of fibrotic kidneys from patients with CKD identified a metabolic gene signature comprising loss of mitochondrial and oxidative phosphorylation gene expression with a concomitant increase in regulators and enzymes of glycolysis under the control of PGC1α and MYC transcription factors, respectively. We modeled this metabolic switch in vivo, in experimental murine models of kidney injury, and in vitro in human kidney stromal cells (SCs) and human kidney organoids.

Results In mice, MYC and the target genes thereof became activated in resident SCs early after kidney injury, suggesting that acute innate immune signals regulate this transcriptional switch. In vitro, stimulation of purified human kidney SCs and human kidney organoids with IL-1β recapitulated the molecular events observed in vivo, inducing functional metabolic derangement characterized by increased MYC-dependent glycolysis, the latter proving necessary to drive proliferation and matrix production. MYC interacted directly with sequestosome 1/p62, which is involved in proteasomal degradation, and modulation of p62 expression caused inverse effects on MYC expression. IL-1β stimulated autophagy flux, causing degradation of p62 and accumulation of MYC. Inhibition of the IL-1R signal transducer kinase IRAK4 in vivo or inhibition of MYC in vivo as well as in human kidney organoids in vitro abrogated fibrosis and reduced tubular injury.

Conclusions Our findings define a connection between IL-1β and metabolic switch in fibrosis initiation and progression and highlight IL-1β and MYC as potential therapeutic targets in tubulointerstitial diseases.

Navin Gupta, Koichiro Susa, Yoko Yoda, Joseph V. Bonventre, M. Todd Valerius, and Ryuji Morizane. 2018. “CRISPR/Cas9‐based Targeted Genome Editing for the Development of Monogenic Diseases Models with Human Pluripotent Stem Cells.” Current Protocols in Stem Cell Biology.Abstract
Human pluripotent stem cells (hPSCs) represent a formidable tool for disease modeling, drug discovery, and regenerative medicine using human cells and tissues in vitro. Evolving techniques of targeted genome editing, specifically the CRISPR/Cas9 system, allow for the generation of cell lines bearing gene‐specific knock‐outs, knock‐in reporters, and precise mutations. However, there are increasing concerns related to the transfection efficiency, cell viability, and maintenance of pluripotency provided by genome‐editing techniques. The procedure presented here employs transient antibiotic selection that overcomes reduced transfection efficiency, avoids cytotoxic flow sorting for increased viability, and generates multiple genome‐edited pluripotent hPSC lines expanded from a single parent cell. Avoidance of xenogeneic contamination from feeder cells and reduced operator workload, owing to single‐cell passaging rather than clump passaging, are additional benefits. The outlined methods may enable researchers with limited means and technical experience to create human stem cell lines containing desired gene‐specific mutations. © 2018 by John Wiley & Sons, Inc.
Ryuji Morizane and Joseph Bonventre. 2018. “Organoids for modeling kidney disease.” In Organs and Organoids, Pp. 227-245. Elsevier Inc.Abstract
There is an urgent need for human-based models of kidney disease. Recent advances in genome editing using CRISPR/Cas9 systems provide the tools to generate specific mutations at desired sites. This enables the mutation of target genes in normal hPSCs: kidney organoids differentiated from these cells can then be used to model kidney disease caused by these genetic modifications, and the parental hPSC line is an ideal control, as all other genes will be the same. This approach complements disease modeling studies using human induced pluripotent stem cells derived from patients with kidney disease. Using a protocol based on normal development, we generate nephron progenitor cells (NPCs) with up to 80%–90% purity within 8–9 days of differentiation, without additional subpopulation selection. The hPSC-derived NPCs possess the developmental potential of their in vivo counterparts, forming renal vesicles that self-pattern into nephron structures with characteristics of podocytes, proximal tubules, loops of Henle, and distal tubules with vascular and interstitial stromal cells. These are very suitable for studying specific disease-associated phenotypes. With these two novel technologies (organoids and CRISPR/Cas9), we can study kidney diseases with human kidney tissues in vitro.
Ryuji Morizane, Tomoya Miyoshi, and Joseph V. Bonventre. 9/4/2017. “Concise Reviews: Kidney Generation with Human Pluripotent Stem Cells.” Stem Cells. Publisher's VersionAbstract
Chronic kidney disease (CKD) is a world-wide healthcare problem, resulting in increased cardiovascular mortality and often leading to end-stage kidney disease (ESKD) where patients require kidney replacement therapies such as hemodialysis or kidney transplantation. Loss of functional nephrons contributes to the progression of CKD, which can be attenuated but not reversed due to inability to generate new nephrons in human adult kidneys. Human pluripotent stem cells (hPSCs), by virtue of their unlimited self-renewal and ability to differentiate into cells of all three embryonic germ layers, are attractive sources for kidney regenerative therapies. Recent advances in stem cell biology have identified key signals necessary to maintain stemness of human nephron progenitor cells (NPCs) in vitro, and led to establishment of protocols to generate NPCs and nephron epithelial cells from human fetal kidneys and hPSCs. Effective production of large amounts of human NPCs and kidney organoids will facilitate elucidation of developmental and pathobiological pathways, kidney disease modeling and drug screening as well as kidney regenerative therapies. We summarize the recent studies to induce NPCs and kidney cells from hPSCs, studies of NPC expansion from mouse and human embryonic kidneys, and discuss possible approaches in vivo to regenerate kidneys with cell therapies and the development of bioengineered kidneys.
Navin Gupta, Koichiro Susa, and Ryuji Morizane. 8/17/2017. “Regenerative Medicine, Disease Modelling, and Drug Discovery in Human Pluripotent Stem Cell-Derived Kidney Tissue.” EMJ Repro Health., 3, 1, Pp. 57-67. Publisher's VersionAbstract
The multitude of research clarifying critical factors in embryonic organ development has been instrumental in human stem cell research. Mammalian organogenesis serves as the archetype for directed differentiation protocols, subdividing the process into a series of distinct intermediate stages that can be chemically induced and monitored for the expression of stage-specific markers. Significant advances over the past few years include established directed differentiation protocols of human embryonic stem cells and human induced pluripotent stem cells (hiPSC) into human kidney organoids in vitro. Human kidney tissue in vitro simulates the in vivo response when subjected to nephrotoxins, providing a novel screening platform during drug discovery to facilitate identification of lead candidates, reduce developmental expenditures, and reduce future rates of drug-induced acute kidney injury. Patient-derived hiPSC, which bear naturally occurring DNA mutations, may allow for modelling of human genetic diseases to enable determination of pathological mechanisms and screening for novel therapeutics. In addition, recent advances in genome editing with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 enable the generation of specific mutations to study genetic disease, with non-mutated lines serving as an ideal isogenic control. The growing population of patients with end-stage kidney disease is a worldwide healthcare problem, with high morbidity and mortality rates, that warrants the discovery of novel forms of renal replacement therapy. Coupling the outlined advances in hiPSC research with innovative bioengineering techniques, such as decellularised kidney and three-dimensional printed scaffolds, may contribute to the development of bioengineered transplantable human kidney tissue as a means of renal replacement therapy.
Ryuji Morizane and Joseph V Bonventre. 2017. “Generation of nephron progenitor cells and kidney organoids from human pluripotent stem cells.” Nat Protoc, 12, 1, Pp. 195-207.Abstract
A variety of protocols have been developed that demonstrate the capability to differentiate human pluripotent stem cells (hPSCs) into kidney structures. Our goal was to develop a high-efficiency protocol to generate nephron progenitor cells (NPCs) and kidney organoids to facilitate applications for tissue engineering, disease modeling and chemical screening. Here, we describe a detailed protocol resulting in high-efficiency production (80-90%) of NPCs from hPSCs within 9 d of differentiation. Kidney organoids were generated from NPCs within 12 d with high reproducibility using 96-well plates suitable for chemical screening. The protocol requires skills for culturing hPSCs and careful attention to morphological changes indicative of differentiation. This kidney organoid system provides a platform for studies of human kidney development, modeling of kidney diseases, nephrotoxicity and kidney regeneration. The system provides a model for in vitro study of kidney intracellular and intercompartmental interactions using differentiated human cells in an appropriate nephron and stromal context.
Ryuji Morizane and Joseph V Bonventre. 2017. “Kidney Organoids: A Translational Journey.” Trends Mol Med, 23, 3, Pp. 246-263.Abstract
Human pluripotent stem cells (hPSCs) are attractive sources for regenerative medicine and disease modeling in vitro. Directed hPSC differentiation approaches have derived from knowledge of cell development in vivo rather than from stochastic cell differentiation. Moreover, there has been great success in the generation of 3D organ-buds termed 'organoids' from hPSCs; these consist of a variety of cell types in vitro that mimic organs in vivo. The organoid bears great potential in the study of human diseases in vitro, especially when combined with CRISPR/Cas9-based genome-editing. We summarize the current literature describing organoid studies with a special focus on kidney organoids, and discuss goals and future opportunities for organoid-based studies.
Shintaro Yamaguchi, Ryuji Morizane, Koichiro Homma, Toshiaki Monkawa, Sayuri Suzuki, Shizuka Fujii, Muneaki Koda, Ken Hiratsuka, Maho Yamashita, Tadashi Yoshida, Shu Wakino, Koichi Hayashi, Junichi Sasaki, Shingo Hori, and Hiroshi Itoh. 2016. “Generation of kidney tubular organoids from human pluripotent stem cells.” Sci Rep, 6, Pp. 38353.Abstract
Recent advances in stem cell research have resulted in methods to generate kidney organoids from human pluripotent stem cells (hPSCs), which contain cells of multiple lineages including nephron epithelial cells. Methods to purify specific types of cells from differentiated hPSCs, however, have not been established well. For bioengineering, cell transplantation, and disease modeling, it would be useful to establish those methods to obtain pure populations of specific types of kidney cells. Here, we report a simple two-step differentiation protocol to generate kidney tubular organoids from hPSCs with direct purification of KSP (kidney specific protein)-positive cells using anti-KSP antibody. We first differentiated hPSCs into mesoderm cells using a glycogen synthase kinase-3β inhibitor for 3 days, then cultured cells in renal epithelial growth medium to induce KSP+ cells. We purified KSP+ cells using flow cytometry with anti-KSP antibody, which exhibited characteristics of all segments of kidney tubular cells and cultured KSP+ cells in 3D Matrigel, which formed tubular organoids in vitro. The formation of tubular organoids by KSP+ cells induced the acquisition of functional kidney tubules. KSP+ cells also allowed for the generation of chimeric kidney cultures in which human cells self-assembled into 3D tubular structures in combination with mouse embryonic kidney cells.
Ryuji Morizane, Shizuka Fujii, Toshiaki Monkawa, Ken Hiratsuka, Shintaro Yamaguchi, Koichiro Homma, and Hiroshi Itoh. 2016. “miR-363 induces transdifferentiation of human kidney tubular cells to mesenchymal phenotype.” Clin Exp Nephrol, 20, 3, Pp. 394-401.Abstract
BACKGROUND: microRNAs (miRNAs) are non-coding small RNAs that regulate embryonic development, cell differentiation and pathological processes via interaction with mRNA. Epithelial-mesenchymal transition (EMT) is pathological process that involves in a variety of diseases such as cancer or fibrosis. METHODS: In this study, we identified miR-363 as a potent inducer of EMT by microarray analysis in human kidney tubular cells, and analyzed the function and mechanisms of miR-363. RESULTS: Overexpression of miR-363 induced mesenchymal phenotypes with loss of epithelial phenotypes in human kidney tubular cells. In addition, in vitro scratch assay demonstrated that miR-363 promotes cell migration of primary culture of human kidney tubular cells. We identified TWIST/canonical WNT pathway as the downstream effecter of miR-363, and inhibition of canonical WNT by small molecule, IWR-1, attenuated EMT induced by miR-363. CONCLUSION: miR-363 induces transdifferentiation of human kidney tubular cells via upregulation of TWIST/canonical WNT pathway.
Ryuji Morizane and Albert Q Lam. 2015. “Directed Differentiation of Pluripotent Stem Cells into Kidney.” Biomark Insights, 10, Suppl 1, Pp. 147-52.Abstract
Pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), represent an ideal substrate for regenerating kidney cells and tissue lost through injury and disease. Recent studies have demonstrated the ability to differentiate PSCs into populations of nephron progenitor cells that can organize into kidney epithelial structures in three-dimensional contexts. While these findings are highly encouraging, further studies need to be performed to improve the efficiency and specificity of kidney differentiation. The identification of specific markers of the differentiation process is critical to the development of protocols that effectively recapitulate nephrogenesis in vitro. In this review, we summarize the current studies describing the differentiation of ESCs and iPSCs into cells of the kidney lineage. We also present an analysis of the markers relevant to the stages of kidney development and differentiation and propose a new roadmap for the directed differentiation of PSCs into nephron progenitor cells of the metanephric mesenchyme.