SPECIAL ARTICLE www.jasn.org

(Re)Building a Kidney

†‡ †‡ Leif Oxburgh,* Thomas J. Carroll, Ondine Cleaver, Daniel R. Gossett,§ Deborah K. Hoshizaki,§ | †† ‡‡ Jeffrey A. Hubbell, Benjamin D. Humphreys,¶ Sanjay Jain,¶ Jan Jensen,** David L. Kaplan, || ††† Carl Kesselman,§§ Christian J. Ketchum,§ Melissa H. Little,¶¶*** Andrew P. McMahon, ‡‡‡ ||| †††† Stuart J. Shankland, Jason R. Spence,§§§ M. Todd Valerius,¶¶¶ Jason A. Wertheim,**** |||| Oliver Wessely,§§§§ Ying Zheng, and Iain A. Drummond¶¶¶¶

*Center for Molecular Medicine, Maine Medical Center Research Institute, Scarborough, Maine; †Department of Molecular Biology and ‡Center for Regenerative Science and Medicine, University of Texas Southwestern Medical Center, Dallas, Texas; §Division of Kidney, Urologic, & Hematologic Diseases, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland; |Institute for Molecular Engineering, University of Chicago, Chicago, Illinois; ¶Division of Nephrology, Washington University School of Medicine, St. Louis, Missouri; **Department of Stem Cell Biology and Regenerative Medicine and §§§§Department of Cellular and Molecular Medicine, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio; ††Trailhead Biosystems, Inc, Cleveland, Ohio; ‡‡Department of Biomedical Engineering, Tufts University, Medford, Massachusetts; §§Epstein Department of Industrial and Systems Engineering and Information Sciences Institute, Viterbi School of Engineering and ||Department of Preventive Medicine, Keck School of Medicine, University of Southern California, Los Angeles, California; ¶¶Cell Biology Theme, Murdoch Childrens Research Institute, Parkville, Melbourne, Australia; ***Department of Pediatrics, Faculty of Medicine, Dentistry and Health Sciences, University of Melbourne, Parkville, Melbourne, Australia; †††Department of Stem Cell Biology and Regenerative Medicine, Keck School of Medicine of the University of Southern California, Los Angeles, California; ‡‡‡Division of Nephrology and ||||Department of Bioengineering, Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle; §§§Department of Internal Medicine, Division of Gastroenterology and |||Department of Cell and Developmental Biology and Center for Organogenesis, University of Michigan Medical School, Ann Arbor, Michigan; ¶¶¶Department of Medicine, Brigham and Women’s Hospital, Boston, Massachusetts; ****Department of Surgery, Jesse Brown VA Medical Center, and ††††Comprehensive Transplant Center, Department of Surgery, Northwestern University Feinberg School of Medicine, Chicago, Illinois; and ¶¶¶¶Nephrology Division, Massachusetts General Hospital, Charlestown, Massachusetts

ABSTRACT (Re)Building a Kidney is a National Institute of Diabetes and Digestive and Kidney Diseases- for kidney disease is a global health care led consortium to optimize approaches for the isolation, expansion, and differentiation of priority.1 Kidney disease and injury affect appropriate kidney cell types and the integration of these cells into complex structures that diverse renal structures. Some, such as the replicate human kidney function. The ultimate goals of the consortium are two-fold: to proximal tubule, are regenerative and develop and implement strategies for in vitro engineering of replacement kidney tissue, show highly proliferative responses to in- and to devise strategies to stimulate regeneration of nephrons in situ to restore failing jury,2,3 whereas others, such as glomerular kidney function. Projects within the consortium will answer fundamental questions regard- podocytes, do not.4 Recent advances in ing human gene expression in the developing kidney, essential signaling crosstalk between our understanding of tissue regeneration, distinct cell types of the developing kidney, how to derive the many cell types of the kidney stem cell biology, and bioengineering through directed differentiation of human pluripotent stem cells, which bioengineering or make it feasible to develop new strategies scaffolding strategies have the most potential for kidney tissue formation, and basic pa- for restoring kidney function. rameters of the regenerative response to injury. As these projects progress, the consor- tium will incorporate systematic investigations in physiologic function of in vitro and in vivo differentiated kidney tissue, strategies for engraftment in experimental animals, and de- Published online ahead of print. Publication date velopment of therapeutic approaches to activate innate reparative responses. available at www.jasn.org.

Correspondence: Dr. Leif Oxburgh, Maine Medical J Am Soc Nephrol 28: 1370–1378, 2017. doi: https://doi.org/10.1681/ASN.2016101077 Center Research Institute, 81 Research Drive, Scarborough, ME 04074, or Dr. Iain Drummond, Nephrology Division, Massachusetts General Hos- pital, Charlestown, MA 02129. E-mail: oxburl@ By current clinical definitions, approxi- Epidemiologic studies suggest a similar mmc.org or [email protected] mately 10% of the adult population of the incidence worldwide, and as populations Copyright © 2017 by the American Society of United States suffers from CKD.1 age it is clear that finding new therapies Nephrology

1370 ISSN : 1046-6673/2805-1370 JAmSocNephrol28: 1370–1378, 2017 www.jasn.org SPECIAL ARTICLE

THE (RE)BUILDING A KIDNEY CONSORTIUM

The (Re)Building a Kidney (RBK) con- sortium (www.rebuildingakidney.org) was formed by the National Institute of Diabetes and Digestive and Kidney Dis- ease (NIDDK) in 2015 to coordinate and integrate research on strategies to restore kidney function on the basis of our un- derstanding of kidney development, and the response of the adult organ to injury. The consortium focuses on two funda- mentally different but complementary strategies that have the potential to transform our approach to treating kid- ney disease and solving the kidney short- age crisis. The first is to engineer and engraft new kidney tissue. The second Figure 1. The RBK Consortium is taking multiple approaches to achieve its goals of engineering is to identify endogenous repair and re- and engrafting new kidney tissue as well as enhancing kidney repair and regeneration to restore . generation processes in the injured kid- kidney function. (1)iPSC/ESC NPCs: propagating human progenitor cells in their un- ney that can be exploited therapeutically. differentiated state and using them to generate kidney cells and organoids, populate tissue scaffolds, and recellularize kidney matrix are key goals of the RBK consortium. Starting with iPSCs As depicted in Figure 1, the RBK aims to or established ESC lines, NPCs will be generated using differentiation protocols for nephrogenic develop the tools, resources, and knowl- mesoderm. Propagation and self-renewal of these cells and mouse cap mesenchyme NPCs will edge to (1) characterize fetal and adult be optimized using transgenic reporters of the progenitor cell type, quantitative RT-PCR of human kidney cell types to determine qualified human marker genes, and single-cell mRNA phenotyping as end points to detect which cellular phenotypes should be tar- expansion of the progenitor cell type. (2) Kidney organoids/kidney cell types: differentiation of gets for directed differentiation of hu- self-organizing organoids or directed differentiation of individual kidney cell types will also be man induced pluripotent stem cells achieved by optimizing medium conditions and assaying outcomes using cell-type specific (iPSCs), (2) produce and characterize fluorescent protein transgenic reporter iPSC/ESC lines, quantitative RT-PCR of qualified human human kidney progenitor cells from marker genes, and single-cell mRNA phenotyping. Fluorescent protein reporter human iPSC pluripotent cells while optimizing sys- lines will also allow live imaging of ex vivo kidney differentiation as well as reisolation and tran- fi tems for their propagation, (3) generate scriptional pro ling of organoid-derived kidney cells, including nephron and stromal progenitors, podocytes, proximal tubules, distal tubules, and endothelium. Rigorously defined human kidney complex and well organized kidney tis- cell transcriptional signatures as well as cell injury markers derived from single-cell RNA se- sue through controlled differentiation of quencing and MARIS will be essential for organoid and cell type quality control and to establish natural and iPSC-derived kidney pro- baseline phenotypes for further functional characterization, disease modeling, and potential genitor cells, (4) use biologic scaffolds therapeutic use. (3) Scaffold tissue assembly: multiple approaches, including decellularized to promote tissue assembly and growth, kidneys, silk, and synthetic polymers, and three-dimensional printed arrays of nephron tubules (5) connect filtering nephrons to and blood vessels, will be tested to generate structures into which differentiated kidney cells or tubule networks to act as conduits for organoids can be seeded, instructed to differentiate, and interconnect nephron tubules to form urine, (6) interrogate the potential of functioning kidney tissue with the potential to replace failed kidney function. (4)Celltherapy/ cell therapy to repair kidney damage, repair and regeneration factors: defined growth factor activities optimized for progenitor cell self- and (7) exploit endogenous repair renewal and organoid differentiation as well as for directed differentiation of renal cell types will mechanisms in the adult human kidney be integrated into new approaches to promote endogenous repair of nephrons. Adult kidney podocyte and interstitial progenitor cells will be profiled and characterized to develop new ways to enhance regeneration. to promote endogenous repair. Contributions of nonepithelial cells, the endothelium, inter- stitium, and pericytes/vascular smooth muscle to kidney cell differentiation will also be assayed and modeled using fluorescent reporter lines and coculture with nephron cells in two- CLUES FROM KIDNEY dimensional and three-dimensional cultures. ESC, embryonic stem cell lines. DEVELOPMENT: GENERATING COMPLEX KIDNEY STRUCTURES FROM HUMAN IPSCS lines to form human kidney organoids.5–8 collecting ducts, renal interstitium, and These organoids have been shown to endothelium (Figure 2).8 As with proto- Within the consortium, protocols on the contain all of the anticipated compo- cols for the directed differentiation of basis of recapitulating renal development nents of the fetal human kidney, includ- iPSC to other human tissues (reviewed have been adopted to coach human iPSC ing patterned and segmented nephrons, in Clevers9), morphology and gene

J Am Soc Nephrol 28: 1370–1378, 2017 (Re)Building a Kidney 1371 SPECIAL ARTICLE www.jasn.org expression analysis suggests that kidney New and powerful technologies for the exists for self-assembly of glomerular organoids represent first trimester hu- capture of single cells are being used capillaries within some organoid glo- man tissue.8 The presence of derivatives such as methods for analyzing RNA fol- meruli, the majority remain avascular.8 of all major progenitors (cap mesen- lowing intracellular sorting (MARIS), in Endothelial reporter iPSC lines are being chyme, ureteric bud, vascular progeni- which fixed cells are FACS-isolated for generated to facilitate the isolation and tors, and stroma) suggests that these RNA sequencing on the basis of expres- characterization of this endothelium for organoids could represent a promising sion of intracellular antigens.11 comparison with the profiles of endoge- source of human kidney tissue, although Achieving high-throughput efficiency nous embryonic mouse kidney endothe- nephrons within the organoids are not in optimizing kidney organoid forma- lium and human embryonic kidney fully differentiated and lack functioning tion will require reliable and rapid means tissue.14 vasculature. to detect the differentiation of different Key concerns in developing a directed The ability to evaluate similarities be- renal cell types. Currently there is a pau- differentiation protocol are robustness tween individual cell types present city of human iPSC lines expressing re- and reproducibility; i.e.,itisessential within iPSC-derived kidney organoids porters of cellular differentiation suitable to determine a differentiation strategy and the corresponding cells in human for the development of directed differen- that can be transferred from one stem kidney is a high priority for the consor- tiation protocols for kidney. Capitalizing cell line to another with reproducible tium. Although the transcriptional char- on knowledge gained from mouse and outcomes in terms of organoid gene acterization of metanephric mouse human kidney cell-type specificgeneex- expression profiles and cellular com- kidney cells is well established, and the pression, fluorescently tagged human plexity. With these concerns in mind, core pathways controlling nephrogenesis iPSC reporter lines12,13 are being gener- substantial focus is placed on sources of may be orthologously conserved, differ- ated using CRISPR/Cas9 gene editing variation and the transferability of the ences in gene expression presumably approaches. These will allow both live approach between iPSC lines. Within underlie important differences in imaging of ex vivo kidney differentiation the consortium, a Quality-by-Design renal architecture and function between and the isolation and transcriptional strategy is used to establish strict param- mouse and human kidneys.10 Thus, a key profiling of organoid-derived progeni- eters for each component in the directed objective of the RBK is to generate a tors of the nephron, collecting duct, differentiation process.15,16 This strategy comprehensivegeneexpressiondata- and stromal lineages, as well as differen- allows for .12 dimensions within a de- base of the human kidney against which tiated podocytes, proximal tubules, and sign space in which morphogens and tissue derived from iPSCs can be com- distal tubules. It is interesting to note growth factors are tested simultaneously pared. A rigorous microanatomic map that kidney organoids generated from and interactions between individual sig- will be established through RNA se- human iPSC spontaneously form endo- naling pathways can be determined. The quencing at the single cell level and ex- thelial cell networks with accompanying end point of the experiment is expres- haustive in situ hybridization analysis. perivascular cells.8 Although evidence sion analysis of .50 markers of renal

Figure2. iPSC-derivedkidneyorganoids containallcomponentsof thefetal humankidney,includingpatternedandsegmentednephrons, collecting ducts, renal interstitium, and endothelium. Comparison of mature and developing cell types between normal human fetal kidney (left) and iPSC-derived kidney organoids (right). Structures: CD, collecting duct; Endo, endothelium; Glom, glomerulus; PC, perivascular cell; PT, proximal tubule; SSB, s-shaped body nephron; STR, stroma. Markers: CALB1, collecting duct in early kidney and mature distal tubule in nephrons; CD31, endothelial cells; CUBN, proximal tubule; DAPI, nuclei; ECAD, distal epithelial cells; GATA3, collecting duct; KRT8, collecting duct in early kidney and mature distal tubule in nephrons; LTL, proximal tubule; MAFB, podocytes and glomeruli; MEIS1, interstitial cells; NPHS1, podocytes; PAX2, collecting duct and nephron; PDGFRA, pericytes; SOX17, endothelial cells. Images of human fetal kidney supplied by McMahon laboratory (Tracy Tran and Nils Lindström). Images of human iPSC-derived kidney organoid supplied by Little laboratory (Han Chiu, Minoru Takasato, Pei Er, Jessica Vanslambrouck, and Sara Howden).

1372 Journal of the American Society of Nephrology J Am Soc Nephrol 28: 1370–1378, 2017 www.jasn.org SPECIAL ARTICLE cell types, enabling evaluation of subtle required is to isolate them from iPSC- function.29 Adult decellularized organs differences between differentiation derivedorganoidsanddeviseculture provide the complex extracellular matrix states. Robust differentiation protocols methods to expand them. An alternate environment required for attachment developed using this approach will approach is to differentiate each of the and function of differentiated kidney be particularly important in disease individual cell types in isolation for use cell types.27,30 Engineering the matrix modeling applications where uniform in building synthetic tissue. Protocols to present growth factors in a spatially organoid composition is essential for re- have been reported to differentiate ure- organized fashion provides another ap- producibility of high-throughput drug teric bud cells,21 NPCs,6,22 podo- proach to drive organoid differentiation screening. cytes,23 and tubular epithelial cells.24 on a larger scale.28 The ultimate scaffold- Understanding how human renal To date, no attempts have been report- ing solution may incorporate aspects of progenitor cells may be propagated in ed to recombine these distinct cell types each of these bioengineering ap- their undifferentiated state to numbers to form kidney tissue, and this proaches. Further, it is important that sufficient for trials of scaffolding and re- presents a fascinating future challenge the systems are also considered in the cellularization of organs is an important for the consortium. context of future needs in vivo to maxi- goal of the consortium. On the basis of in mize translation potential for the work vitro mouse work that identified a cock- in the consortium. tail of factors that mimic the in vivo renal PUTTING THE PIECES TOGETHER: Development of renal blood vessels is progenitor cell niche,17 efforts are cur- SCAFFOLDING APPROACHES FOR essential for the generation of functional rently focused on methods to culture BUILDING HUMAN KIDNEY nephrons, whether within bioengineered and provide a source of phenotypically TISSUE tissue or organoids.31–33 Recent results normal human nephron progenitor cells reveal that there is a surprising hetero- (NPCs) sufficient to generate synthetic Another priority within the RBK is ex- geneity in endothelial cell gene expres- kidney tissue scaled to the human. ploring different approaches to generate sion within the developing kidney. How Both monolayer and aggregate culture synthetic kidney tissue that could then be spatial and temporal differences in en- technologies have shown promise in transplanted into a host (Figure 3). In dothelial cell phenotype might affect propagating NPCs, and procedures one approach, purified silk is being nephron progenitor self-renewal or dif- have been reported for both propaga- used to generate structures into which ferentiation is an important and under- tion of mouse and human cells.18,19 kidney cells derived from directed differ- studied area. Furthermore, regeneration Comparisons of these culture methods entiation can be seeded and instructed to of the nephron will require both peri- have revealed that they differ in their differentiate into mature kidney struc- cytes and vascular smooth muscle cells capacities to propagate cells from dif- tures.25 A second approach, also on the for stabilizing capillaries and larger ferent developmental stages, and that basis of the use of polymers, involves vessels, respectively. The consortium propagation conditions also may skew three-dimensional printing arrays of is developing approaches for proper the differentiation potential of cells, nephron tubules that can be layered to vascularization of organoids seeded particularly the glomerular podocyte. establish tissue.26 A third approach, is in silk or other three-dimensional The NPC resides within a niche in the recellularization of decellularized scaffolds by (1) generating primary vivo, and studies are being carried out kidneys,27 for which transplant donor and conditionally immortalized Gli1- to understand the extracellular envi- kidneys that do not fulfill quality control positive mesenchymal stem cells (e.g., ronment that confines highly motile criteria constitute a plausible source. A interstitial pericytes and perivascular NPCs within the cap mesenchyme, fourth complementary approach aims to fibroblasts), (2) developing methods yet allows differentiation to all nephro- engineer extracellular matrix to maxi- for pericyte and vascular smooth mus- nalcelltypes.20 mize retention and presentation of rele- cle cell differentiation, (3) determining Althoughtheself-organizationofthe vant growth factors.28 transcriptional profiles and molecular iPSC-derived organoid provides an at- Each of these approaches has distinct signatures of endothelial cell classes, tractive solution to generating tissue advantages. Scaffolds made from silk are and (4) identifying endothelial cell sig- containing all relevant cell types in vi- extremely robust, and can easily be ster- nals and crosstalk with interstitial and tro,itmaybedifficult to generate suffi- ilized by autoclaving, modified with epithelial cells that affect kidney differ- cient tissue volumes using this method. growth factors, and manipulated for en- entiation or proliferation within syn- A complementary strategy for building graftment.25 Also, silk is in regular sur- thetic kidney tissue. new kidney tissue is to recombine spe- gical use, suggesting minimal regulatory The patterning of the kidney stroma cific cell types of the developing kidney hurdles for clinical application. Printing and interstitium is likely to be an essential in appropriate proportions and in ap- of nephrons has the advantage that event in the functional development of propriate locations within biologic structures can be easily organized in the nephrons. The renal interstitium scaffolds. One possible strategy to gen- the stereotypic pattern seen in vivo, arises along the cortical–medullary erate the distinct cell types that are which is important for physiologic axis from two distinct progenitor

J Am Soc Nephrol 28: 1370–1378, 2017 (Re)Building a Kidney 1373 SPECIAL ARTICLE www.jasn.org

Figure 3. Multiple different bioengineering approaches to generating tissue scaffolds offer unique advantages for rebuilding kidney tissue. Polymer-based (A and B), decellularized tissue (C and D), and organ-on-a-chip (E and F) approaches are shown. (A) Scanning electron micrograph of 6% silk scaffold before cell seeding; a network of channels is formed into which NPCs can be seeded. Photo: Jeannine Coburn. (B) E-cadherin (epithelial cells) and DAPI (nuclei) staining reveals a network of tubules with lumens within the silk approximately 2 weeks after seeding NPCs. Photo: Jessica Davis-Knowlton. (C) Scanning electron micrograph of decellularized kidney tissue showing the extracellular matrix framework for tubules and glomeruli. Photo: Joseph Uzarski. (D) Immunostaining of a decellularized kidney that has been recellularized with MDCK cells 7 days after cell seeding. E-cadherin (green) and luminal cilia (acetylated tubulin, red) demonstrate epithelialization. Photo: Joseph Uzarski. (E) Schematic and top view picture of a vascular network-on-a-chip device that allows flow through the network via inlet (i) and outlet (o). Photo: Zheng laboratory. (F) An example view of a three dimensional microvessel network formed by mouse kidney endothelial cells. Red: CD31, blue: DAPI. The inset shows fluorescence immunostaining of a device in which podocytes (green) were cocultured with the vascular endothelial network (red). Photo: Zheng laboratory. EHT, extra high tension voltage setting; WD, working distance. populations marked by Foxd1 and recapitulated in efforts to engineer or re- S-shaped bodies fuse lumens with the Tbx18;theTbx18-positive population generate kidney tissue.36 With the goal of branched collecting system to generate gives rise to the interstitium surround- understanding interstitial cell differenti- functioning nephrons.37,38 The signals ing the ureter, the renal pelvis, and some ation, fluorescent reporter mouse strains guiding epithelial cells to invade an ex- of the papilla, whereas Foxd1-positive are being generated with the purpose of isting tubule, rearrange cell junctions, progenitors give rise to the remaining isolating interstitial cell types, and tech- and form a new patent lumen are not interstitium, including the pericytes, niques are being developed for coculture known. The regenerating zebrafish kid- peritubular fibroblasts, and mesangial with nephron and endothelial cells both ney provides a quantitative model to test cells.34,35 Loss of the Foxd1-positive cell in two-dimensional culture and three- signaling pathways required for tubule population affects the development of dimensional silk scaffolds. Transcrip- invasion and nephron fusion. After in- kidney tubules and vasculature nonau- tional profiles derived from interstitial jury, the adult fish kidney regenerates tonomously,20 and it seems likely that cell subpopulations should yield impor- by a stem cell–mediated process, creat- different interstitial zones could estab- tant insights into interstitial cell pattern- ing new nephrons that fuse to existing lish distinct molecular niches, affecting ing and signaling roles. branched collecting ducts.39 The consor- the final differentiation and function of To take full advantage of newly gen- tium is taking genetic and pharmaco- kidney epithelia and endothelia. Also, erated iPSC-derived replacement tissue logic approaches to manipulating cell much of the endocrine function of to restore kidney function, filtering signaling during this process and trans- the kidney, including the production nephrons must connect to a collecting lating new insights to how nephrons can of renin and erythropoietin, is per- duct network to pass fluid. In kidney de- be directed to “plumb into” existing col- formed by interstitial cells that must be velopment, new nephrons derived from lecting duct networks.

1374 Journal of the American Society of Nephrology J Am Soc Nephrol 28: 1370–1378, 2017 www.jasn.org SPECIAL ARTICLE

ENHANCING ENDOGENOUS REPAIR

Understanding mechanisms of endoge- nous repair remains an important goal for regenerative nephrology. The consor- tium aims to understand cell-specificin- jury responses and the potential of adult progenitors to repair damaged kidney through regenerative processes. The proximal tubule epithelium has a robust capacity for repair after injury through the proliferative potential of surviving proximal tubule cells.2,3 Agoalofthe consortium is to determine whether dedifferentiation confers unique func- tional properties or tubulogenic poten- tial on proximal tubule cells and to define the transcriptional profiles of these cells during the full course of repair. Cell types of particular interest include injured and dedifferentiated proximal tubule cells (defined by expres- sion of Havcr1),40 interstitial pericytes and perivascular fibroblasts (defined by expression of Gli1),41 and cells of the re- nin lineage.42,43 These studies will also help inform efforts to generate high- quality kidney organoids by providing transcriptional snapshots of injured ver- sus healthy proximal tubule cells. Adult podocytes are injured in many forms of glomerular diseases44 and show very little, if any, ability to self-replicate.4 However, there are two podocyte cell progenitor populations under study in the consortium: the glomerular parietal epithelial cells4,45,46 and cells of the renin fi lineage.42,43 Inducible reporter mice Figure4. Integrationofvasculature,overcomingkidney brosis,andconnectingbioengineered kidney tubules to the collecting system remain important challenges in advancing renal re- have enabled lineage tracing of both po- placement therapy. The kidney vasculature represents a variety of unexplored endothelial cell docyte progenitor types under normal subtypes that may affect nephron differentiation through reciprocal signaling events. (A) In the and experimental disease states. Isolation developing mouse kidney, vascular progenitor endothelial cells positive for Pecam (PE)/CD31 of each cell type is being performed to de- (green) are intimately associated with the nephrogenic cortical fetal kidney domain populated by rive their transcriptional profiles with the Six2-positive nephrogenic progenitor cells (red). E-cadherin (white) marks polarized epithelium of goal of defining regulatory mechanisms the branching ureter tips. Photo: Edward Daniel. (B) Kidney injury and fibrosis represents a major controlling podocyte progenitor differen- barrier to regeneration. Kidney injury recruits myofibroblasts, leading to fibrosis and CKD. Kim-1 tiation in health and disease. The cells immunostaining (green) identifies injured and dedifferentiated human proximal tubule whereas will also be used to develop microfluidic PDGFRb immunostaining reveals activated adjacent interstitial kidney fibroblasts (red). Photo: “organs-on-chips,” to provide efficient, Monica Chang-Panesso and Farid Kadyrov. (C) Nephron interconnection is an essential processin fi fi reproducible, and cost-effective multicellu- generating a ltering kidney. New insights from the regenerating zebra sh kidney, where many new nephrons are added to the existing collecting system, can help define signaling pathways lar cell culture models in native matrices.47 that stimulate cell rearrangements that lead to interconnected tubule lumens. The lhx1a:GFP A goal of the consortium is to produce transgene (green) marks new nephron cell projections, or invadopodia (arrowheads), that pen- bioengineered three-dimensional micro- etrate the collecting system as a prelude to interconnection. Photo: Caramai N. Kamei. CD, 47,48 physiologic flow-directed systems Collecting duct; nN, new nephron; blue fluorescence, DAPI nuclear stain. to recapitulate the glomerular filtration

J Am Soc Nephrol 28: 1370–1378, 2017 (Re)Building a Kidney 1375 SPECIAL ARTICLE www.jasn.org unit and provide a platform to examine genome sequencing data of the parent The content is solely the responsibility of replacement of depleted adult podocytes iPSC lines will be available on the web- the authors and does not necessarily repre- by local stem/progenitor cells: the glo- site as an aid to design efficient genome sent the official views of either funding merular parietal epithelial cells and cells editing strategies. organization. of the renin lineage.

RBK CONSORTIUM TRAJECTORY: DISCLOSURES CONSORTIUM ORGANIZATION FUTURE CHALLENGES The following authors declare that they have no financial conflict of interest: B.D.H., C.J.K., C.K., The current composition of the consor- This review provides a snapshot of the con- D.K.H., D.L.K., D.R.G., I.A.D., J.A.H. J.A.W., J.R.S., tium is described on the RBK website sortium in its present form. As the individual L.O., M.T.V., O.C., O.W., S.J., S.J.S., T.J.C., and Y.Z. (www.rebuildingakidney.org). The con- projects within the consortium progress, the A.P.M. receives royalty income from Harvard Uni- sortium will continue to add new mem- focus of the program will evolve. Advances versity (Cambridge, MA) for licensing agreements around the Hedgehog pathway, and receives re- bers and ideas through a Partnership made during the initial phase will answer search support from Biogen (Cambridge, MA) on Project Program administrated by the fundamental questions regarding human kidney disease mechanisms. J.J. owns stock in the RBK coordinating center. Requests for gene expression in the developing kidney, following companies for activities as a founder applications in research areas that com- essential signaling crosstalk between distinct and inventor: Alive Technologies Group, LLC plement ongoing consortium studies cell types of the developing kidney, how to (Cleveland, OH) and Trailhead Biosystems will be announced annually. The coordi- derive the many cell types of the human (Cleveland,OH).M.H.L.hasreceivedresearch nating center is a central data and re- kidney through directed differentiation, contract income from and consulted for Organovo source repository for the consortium which scaffolding strategies have the Inc (San Diego, CA). and the broader research community. most potential for kidney tissue forma- On the basis of current consortium proj- tion in vitro, and basic parameters of the REFERENCES ects, the following types of data will be regenerative response to injury. In the provided. Gene expression: RNA se- next phase we anticipate focusing on 1. Stevens PE, Levin A; Kidney Disease: Im- quencing data will be uploaded to the physiologic function of in vitro and in proving Global Outcomes Chronic Kidney National Center for Biotechnology In- vivo differentiated kidney tissue, systematic Disease Guideline Development Work formation Gene Expression Omnibus investigation of strategies for integration of Group Members: Evaluation and manage- and links will be provided on the RBK urinary and vascular conduits between ment of chronic kidney disease: Synopsis of the kidney disease: Improving global out- website. In addition, the following will graft and host in experimental animal comes 2012 clinical practice guideline. Ann be archived and made publically avail- systems, and development of therapeutic Intern Med 158: 825–830, 2013 able on the RBK website. Protocols: de- approaches to promote innate reparative 2. Humphreys BD, Czerniak S, DiRocco DP, tailed protocols covering new methods responses (Figure 4). Hasnain W, Cheema R, Bonventre JV: Repair developed in the RBK consortium. Ex- of injured proximal tubule does not involve amples include iPSC differentiation specialized progenitors. Proc Natl Acad Sci – procedures, methods to generate bioma- USA 108: 9226 9231, 2011 3. Kusaba T, Lalli M, Kramann R, Kobayashi A, terials for use as scaffolds for kidney tis- ACKNOWLEDGMENTS Humphreys BD: Differentiated kidney epi- sue, validation of quantitative PCR for thelial cells repair injured proximal tubule. marker genes to identify key cell types We would like to thank Allie Ware for de- Proc Natl Acad Sci USA 111: 1527–1532, and stages of differentiation. Reagents: signing Figure 1. 2014 databases for growth factor and small Research reported in this publication 4. Wanner N, Hartleben B, Herbach N, Goedel M, Stickel N, Zeiser R, Walz G, Moeller MJ, is supported by the National Institutes molecule activities and concentrations Grahammer F, Huber TB: Unraveling the role as well as a list of validated antibodies of Health/National Institute of Diabetes of podocyte turnover in glomerular aging will be maintained. Images: annotated and Digestive and Kidney Diseases under and injury. JAmSocNephrol25: 707–716, micrographs, three-dimensional repre- award numbers U01DK094526 (A.P.M.), 2014 sentations of tissue structures, in situ 1UH2DK107374 (B.D.H.), R24DK106743 5. Freedman BS, Brooks CR, Lam AQ, Fu H, hybridization data, and chemical struc- (O.C., D.L.K., L.O., and T.J.C.), U01DK107350 Morizane R, Agrawal V, Saad AF, Li MK, Hughes MR, Werff RV, Peters DT, Lu J, Baccei tures. Reporter iPSC lines: reporter lines (C.K., S.J.S., M.T.V., and S.J.), UH2DK10732 A, Siedlecki AM, Valerius MT, Musunuru K, are being generated from consortium- (I.A.D.), UH2DK107357 (J.J. and O.W.), McNagny KM, Steinman TI, Zhou J, Lerou selected parent iPSC lines to enable the UH2DK107344 (M.H.L.), DP3DK108215 PH, Bonventre JV: Modelling kidney disease tagging of specific cell types for isolation (J.A.H.), U01DK107350 (S.J.), and with CRISPR-mutant kidney organoids derived and characterization. Once validated for UH2DK107343 (S.J.S. and Y.Z.). Research from human pluripotent epiblast spheroids. Nat Commun 6: 8715, 2015 pluripotency, genetic integrity, and ap- is also supported by the US Department 6. Morizane R, Lam AQ, Freedman BS, Kishi S, propriatereportergeneexpression, of Veterans Affairs under award number Valerius MT, Bonventre JV: Nephron organoids these will be listed on the website. Whole I01BX002660 (J.A.W.). derived from human pluripotent stem cells

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