Higher-Order Kidney Organogenesis from Pluripotent Stem Cells

•Functional and molecular profiling reveals insights into in vivo UB maturation•Induction of UB from PSCs and nephron progenitors requires distinct cues•Induced UB and MM generate kidney organoids with higher-order architecture•Induced UBs are useful tools to dissect roles of genes in human kidney development Organogenesis generates higher-order structures containing functional subunits, connective components, and progenitor niches. Despite recent advances in organoid-based modeling of tissue development, recapitulating these complex configurations from pluripotent stem cells (PSCs) has remained challenging. In this study, we report assembly of kidney organoids that recapitulate embryonic branching morphogenesis. By studying the distinct origins and developmental processes of the ureteric bud, which contains epithelial kidney progenitors that undergo branching morphogenesis and thereby plays a central role in orchestrating organ geometry, and neighboring mesenchymal nephron progenitors, we established a protocol for differential induction of each lineage from mouse and human PSCs. Importantly, reassembled organoids developed the inherent architectures of the embryonic kidney, including the peripheral progenitor niche and internally differentiated nephrons that were interconnected by a ramified ureteric epithelium. This selective induction and reassembly strategy will be a powerful approach to recapitulate organotypic architecture in PSC-derived organoids. Organogenesis generates higher-order structures containing functional subunits, connective components, and progenitor niches. Despite recent advances in organoid-based modeling of tissue development, recapitulating these complex configurations from pluripotent stem cells (PSCs) has remained challenging. In this study, we report assembly of kidney organoids that recapitulate embryonic branching morphogenesis. By studying the distinct origins and developmental processes of the ureteric bud, which contains epithelial kidney progenitors that undergo branching morphogenesis and thereby plays a central role in orchestrating organ geometry, and neighboring mesenchymal nephron progenitors, we established a protocol for differential induction of each lineage from mouse and human PSCs. Importantly, reassembled organoids developed the inherent architectures of the embryonic kidney, including the peripheral progenitor niche and internally differentiated nephrons that were interconnected by a ramified ureteric epithelium. This selective induction and reassembly strategy will be a powerful approach to recapitulate organotypic architecture in PSC-derived organoids. Recent progress in biology has enabled the induction of various types of functional organ subunits from pluripotent stem cells (PSCs). In particular, strategies employing the cellular “self-organization” phenomenon have enabled successful generation of three-dimensional (3D) “organoids” in a dish (Lancaster and Knoblich, 2014Lancaster M.A. Knoblich J.A. Organogenesis in a dish: Modeling development and disease using organoid technologies.Science. 2014; 345: 1247125Crossref PubMed Scopus (1504) Google Scholar, Sasai, 2013Sasai Y. Cytosystems dynamics in self-organization of tissue architecture.Nature. 2013; 493: 318-326Crossref PubMed Scopus (312) Google Scholar). However, most of the currently available organoids lack module-module connections and a progenitor niche, namely, the “higher-order structure” of the embryonic organ essential for development of the systemic organ anatomy and functions. Thus, we focused on innate branching morphogenesis by epithelial tissue, which plays a critical role in orchestrating organ geometries (Ochoa-Espinosa and Affolter, 2012Ochoa-Espinosa A. Affolter M. Branching morphogenesis: From cells to organs and back.Cold Spring Harb. Perspect. Biol. 2012; 4Crossref PubMed Scopus (82) Google Scholar). A rudiment of the kidney, the embryonic metanephros, develops by mutual interaction of the metanephric mesenchyme (MM; including nephron progenitors [NPs] and stromal progenitors [SPs]) and the ureteric bud (UB) (Costantini and Kopan, 2010Costantini F. Kopan R. Patterning a complex organ: Branching morphogenesis and nephron segmentation in kidney development.Dev. Cell. 2010; 18: 698-712Abstract Full Text Full Text PDF PubMed Scopus (504) Google Scholar). The UB undergoes branching morphogenesis to form urine-collecting systems, and the tips of the UB signal to maintain undifferentiated NPs and induce differentiation of a subset of NPs. In this process, a transient Wnt signal from the UB induces mesenchymal-to-epithelial transition (MET) of NPs, and each epithelialized nephron then attaches to the UB tips for connection to the collecting duct. In turn, the undifferentiated NPs produce Gdnf to maintain UB tip proliferation, and the surrounding cortical SPs support ureteric branching by maintaining Ret receptor tyrosine kinase expression in the UB tips. This triad interaction enables concomitant NP maintenance and differentiation, thereby producing millions of nephrons with systemic connections. Hence, the roles of the UB, including dichotomous branch formation, NP maintenance, and NP differentiation, are essential for organ-scale kidney morphogenesis. Recently, several groups have reported induction of the renal lineage from PSCs. We and another group demonstrated selective induction of the NP lineage (Taguchi et al., 2014Taguchi A. Kaku Y. Ohmori T. Sharmin S. Ogawa M. Sasaki H. Nishinakamura R. Redefining the in vivo origin of metanephric nephron progenitors enables generation of complex kidney structures from pluripotent stem cells.Cell Stem Cell. 2014; 14: 53-67Abstract Full Text Full Text PDF PubMed Scopus (554) Google Scholar, Morizane et al., 2015Morizane R. Lam A.Q. Freedman B.S. Kishi S. Valerius M.T. Bonventre J.V. Nephron organoids derived from human pluripotent stem cells model kidney development and injury.Nat. Biotechnol. 2015; 33: 1193-1200Crossref PubMed Scopus (519) Google Scholar). Other groups have shown the derivation of a UB-like population by selective (Xia et al., 2013Xia Y. Nivet E. Sancho-Martinez I. Gallegos T. Suzuki K. Okamura D. Wu M.Z. Dubova I. Esteban C.R. Montserrat N. et al.Directed differentiation of human pluripotent cells to ureteric bud kidney progenitor-like cells.Nat. Cell Biol. 2013; 15: 1507-1515Crossref PubMed Scopus (254) Google Scholar) or simultaneous (Takasato et al., 2015Takasato M. Er P.X. Chiu H.S. Maier B. Baillie G.J. Ferguson C. Parton R.G. Wolvetang E.J. Roost M.S. Chuva de Sousa Lopes S.M. Little M.H. Kidney organoids from human iPS cells contain multiple lineages and model human nephrogenesis.Nature. 2015; 526: 564-568Crossref PubMed Scopus (895) Google Scholar) induction with NP and SP populations. Most protocols that aimed to include the NP lineage resulted in epithelial nephron-like structure formation to a certain extent (Taguchi et al., 2014Taguchi A. Kaku Y. Ohmori T. Sharmin S. Ogawa M. Sasaki H. Nishinakamura R. Redefining the in vivo origin of metanephric nephron progenitors enables generation of complex kidney structures from pluripotent stem cells.Cell Stem Cell. 2014; 14: 53-67Abstract Full Text Full Text PDF PubMed Scopus (554) Google Scholar, Morizane et al., 2015Morizane R. Lam A.Q. Freedman B.S. Kishi S. Valerius M.T. Bonventre J.V. Nephron organoids derived from human pluripotent stem cells model kidney development and injury.Nat. Biotechnol. 2015; 33: 1193-1200Crossref PubMed Scopus (519) Google Scholar, Takasato et al., 2015Takasato M. Er P.X. Chiu H.S. Maier B. Baillie G.J. Ferguson C. Parton R.G. Wolvetang E.J. Roost M.S. Chuva de Sousa Lopes S.M. Little M.H. Kidney organoids from human iPS cells contain multiple lineages and model human nephrogenesis.Nature. 2015; 526: 564-568Crossref PubMed Scopus (895) Google Scholar). However, the induced UB-like cells did not show branching morphogenesis and the NP induction/maintenance capacity was not proved, and therefore the inter-nephron connection by the collecting ducts was lacking (Xia et al., 2013Xia Y. Nivet E. Sancho-Martinez I. Gallegos T. Suzuki K. Okamura D. Wu M.Z. Dubova I. Esteban C.R. Montserrat N. et al.Directed differentiation of human pluripotent cells to ureteric bud kidney progenitor-like cells.Nat. Cell Biol. 2013; 15: 1507-1515Crossref PubMed Scopus (254) Google Scholar, Takasato et al., 2015Takasato M. Er P.X. Chiu H.S. Maier B. Baillie G.J. Ferguson C. Parton R.G. Wolvetang E.J. Roost M.S. Chuva de Sousa Lopes S.M. Little M.H. Kidney organoids from human iPS cells contain multiple lineages and model human nephrogenesis.Nature. 2015; 526: 564-568Crossref PubMed Scopus (895) Google Scholar). These findings suggest that the currently available UB induction protocols are not sufficient to induce a functional UB, which could be partly due to the lack of precise knowledge about the differentiation signals for the early-stage UB lineage (Costantini and Kopan, 2010Costantini F. Kopan R. Patterning a complex organ: Branching morphogenesis and nephron segmentation in kidney development.Dev. Cell. 2010; 18: 698-712Abstract Full Text Full Text PDF PubMed Scopus (504) Google Scholar). Previously, we identified spatiotemporally distinct origins of the UB and the MM (NP+SP) (Taguchi et al., 2014Taguchi A. Kaku Y. Ohmori T. Sharmin S. Ogawa M. Sasaki H. Nishinakamura R. Redefining the in vivo origin of metanephric nephron progenitors enables generation of complex kidney structures from pluripotent stem cells.Cell Stem Cell. 2014; 14: 53-67Abstract Full Text Full Text PDF PubMed Scopus (554) Google Scholar, Taguchi and Nishinakamura, 2015Taguchi A. Nishinakamura R. Nephron reconstitution from pluripotent stem cells.Kidney Int. 2015; 87: 894-900Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). The UB differentiates from the T+ immature mesoderm at embryonic day (E) 7.5. Subsequently, at E8.5, the immature mesoderm becomes the Osr1+/Lhx1+/Pax2+/T– anterior intermediate mesoderm (AIM). The anteriorly located committed UB lineage precursors extend and migrate caudally to form an elongated epithelial tube, the Wolffian duct (WD). The precursor of the NP is maintained in the caudal T+ immature state longer than the UB lineage, at least up to E8.5, and then differentiates into the Osr1+/Wt1+/Hox11+ posterior intermediate mesoderm (PIM) at E9.5 (Figure 1A). These results suggested that the period in the T+ state determines the anteroposterior positioning within the intermediate mesoderm, the precursor domain of the urogenital system. Indeed, we succeeded in selective induction of metanephric NPs from PSCs (Taguchi et al., 2014Taguchi A. Kaku Y. Ohmori T. Sharmin S. Ogawa M. Sasaki H. Nishinakamura R. Redefining the in vivo origin of metanephric nephron progenitors enables generation of complex kidney structures from pluripotent stem cells.Cell Stem Cell. 2014; 14: 53-67Abstract Full Text Full Text PDF PubMed Scopus (554) Google Scholar), by recapitulating the prolonged exposure to Wnt signals essential to induce and maintain the T+ nascent mesoderm (Wilson et al., 2009Wilson V. Olivera-Martinez I. Storey K.G. Stem cells, signals and vertebrate body axis extension.Development. 2009; 136: 1591-1604Crossref PubMed Scopus (204) Google Scholar). Consistent with our findings, a recent report showed preferential induction of an AIM-like population by shortening the exposure period to Wnt signaling (Takasato et al., 2015Takasato M. Er P.X. Chiu H.S. Maier B. Baillie G.J. Ferguson C. Parton R.G. Wolvetang E.J. Roost M.S. Chuva de Sousa Lopes S.M. Little M.H. Kidney organoids from human iPS cells contain multiple lineages and model human nephrogenesis.Nature. 2015; 526: 564-568Crossref PubMed Scopus (895) Google Scholar). However, the signal that distinguishes the AIM and PIM besides the Wnt exposure period has remained unclear. Furthermore, it remains to be elucidated how the AIM undergoes MET to differentiate into the epithelialized WD and finally mature to form the UB that possesses the branching capacity. Thus, we addressed the WD developmental process using in vivo analysis and in vitro directed differentiation systems, to identify the key signals that are sufficient to induce the UB from PSCs. To develop our multistep protocol, we used the reverse induction approach established in our previous NP lineage induction study (Taguchi et al., 2014Taguchi A. Kaku Y. Ohmori T. Sharmin S. Ogawa M. Sasaki H. Nishinakamura R. Redefining the in vivo origin of metanephric nephron progenitors enables generation of complex kidney structures from pluripotent stem cells.Cell Stem Cell. 2014; 14: 53-67Abstract Full Text Full Text PDF PubMed Scopus (554) Google Scholar). In brief, we first focused on the latest maturation stage of UB development (from E9.5 WD to E11.5 UB) and then focused on an earlier stage (from E8.75 WD progenitor to E9.5 WD) using sorted embryonic precursors. Finally, the differentiation factors from the pluripotent state to the E8.75 WD progenitor stage were examined by employing mouse embryonic stem cells (mESCs). To evaluate the functional maturation process of the early-stage WD, we employed the Hoxb7-GFP transgenic mouse line (Srinivas et al., 1999Srinivas S. Goldberg M.R. Watanabe T. D’Agati V. al-Awqati Q. Costantini F. Expression of green fluorescent protein in the ureteric bud of transgenic mice: A new tool for the analysis of ureteric bud morphogenesis.Dev. Genet. 1999; 24: 241-251Crossref PubMed Scopus (201) Google Scholar) and established a kidney reconstruction assay by modifying previously reported methods (Auerbach and Grobstein, 1958Auerbach R. Grobstein C. Inductive interaction of embryonic tissues after dissociation and reaggregation.Exp. Cell Res. 1958; 15: 384-397Crossref PubMed Scopus (67) Google Scholar, Ganeva et al., 2011Ganeva V. Unbekandt M. Davies J.A. An improved kidney dissociation and reaggregation culture system results in nephrons arranged organotypically around a single collecting duct system.Organogenesis. 2011; 7: 83-87Crossref PubMed Scopus (59) Google Scholar, Grobstein, 1953Grobstein C. Inductive epitheliomesenchymal interaction in cultured organ rudiments of the mouse.Science. 1953; 118: 52-55Crossref PubMed Scopus (300) Google Scholar). E11.5 MMs, including NPs and SPs, were reaggregated with a WD or UB from E9.5, E10.5, and E11.5 stage embryos (Figures 1B, 1C, and S1A). The UB or WD from the E11.5 embryo showed robust branch formation, whereas the WD from E10.5 and E9.5 embryos showed less branch formation (Figures 1D and 1E). Intriguingly, the branch numbers did not differ significantly between the WD at the caudal and rostral parts of the E10.5 or E11.5 embryos (Figure 1E). These results suggest that the functional MM, but not the in situ neighboring mesonephric mesenchyme, induces branching morphogenesis to the WD. Furthermore, the branching capacity, which is retained regardless of the anteroposterior position in the WD, is acquired during developmental progression. To identify markers that can monitor these maturation processes, we performed gene expression array analyses for each stage of the UB, WD, and progenitors from E8.75 to E11.5 (Figures 1C, 1F, and S1A). Non-biased clustering and similar entity analyses of representative UB marker genes identified several groups showing different gene expression kinetics (data not shown; Figure S1C). One group containing many key transcription factors with roles in early WD development (Pax2, Lhx1, Emx2, Sim1, and Gata3) was enriched in the UB lineage and already expressed in the E8.75 WD progenitor. These factors were maintained regardless of the developmental stage or anteroposterior position (Figures 1F and S1C). Another group was enriched with ureteric tip marker genes (En2, Wnt11, and Ret) that showed higher expression in the leading tip region of the WD or UB. Meanwhile, the expression of the other set of genes (E-cadherin, Calb1, Wnt9b, and Hnf1b) increased with progression of development and could be useful for monitoring maturation. We established a protocol to generate the UB by a reverse induction approach. The first step was to identify factors that induce maturation of the E9.5 WD into E11.5 UB-like cells. Our microarray analysis data identified accumulated expression of retinoic acid (RA) synthetic enzyme (Raldh3), Wnt co-receptor (Lgr5), and FGF receptor/target genes in the WD throughout early development (Figure S2A). Thus, we sorted WD cells from E9.5 embryos and reaggregated the cells in the presence of combinations of these factors. The combination of RA, Wnt agonist CHIR99021 (CHIR), and Fgf9 synergistically maintained lineage markers (Pax2 and Emx2) and a tip type marker (Ret) and induced mature WD/UB markers (Hnf1b, Wnt9b, and Calb1) (Figure 2A). However, the aggregates did not maintain Wnt11 expression and lacked the morphology of bud formation. Therefore, we employed Gdnf, a well-known inducer of Wnt11 and UB attractant (Majumdar et al., 2003Majumdar A. Vainio S. Kispert A. McMahon J. McMahon A.P. Wnt11 and Ret/Gdnf pathways cooperate in regulating ureteric branching during metanephric kidney development.Development. 2003; 130: 3175-3185Crossref PubMed Scopus (384) Google Scholar, Pichel et al., 1996Pichel J.G. Shen L. Sheng H.Z. Granholm A.C. Drago J. Grinberg A. Lee E.J. Huang S.P. Saarma M. Hoffer B.J. et al.Defects in enteric innervation and kidney development in mice lacking GDNF.Nature. 1996; 382: 73-76Crossref PubMed Scopus (1003) Google Scholar, Sainio et al., 1997Sainio K. Suvanto P. Davies J. Wartiovaara J. Wartiovaara K. Saarma M. Arumäe U. Meng X. Lindahl M. Pachnis V. Sariola H. Glial-cell-line-derived neurotrophic factor is required for bud initiation from ureteric epithelium.Development. 1997; 124: 4077-4087Crossref PubMed Google Scholar). As expected, the optimized condition with Gdnf successfully induced Wnt11 expression and bud-like structure formation (Figure 2A; data not shown). These results support previous genetic loss-of-function studies showing the requirement for each growth factor signal (Zhao et al., 2004Zhao H. Kegg H. Grady S. Truong H.T. Robinson M.L. Baum M. Bates C.M. Role of fibroblast growth factor receptors 1 and 2 in the ureteric bud.Dev. Biol. 2004; 276: 403-415Crossref PubMed Scopus (185) Google Scholar, Marose et al., 2008Marose T.D. Merkel C.E. McMahon A.P. Carroll T.J. Beta-catenin is necessary to keep cells of ureteric bud/Wolffian duct epithelium in a precursor state.Dev. Biol. 2008; 314: 112-126Crossref PubMed Scopus (120) Google Scholar, Mendelsohn et al., 1994Mendelsohn C. Lohnes D. Décimo D. Lufkin T. LeMeur M. Chambon P. Mark M. Function of the retinoic acid receptors (RARs) during development (II). Multiple abnormalities at various stages of organogenesis in RAR double mutants.Development. 1994; 120: 2749-2771Crossref PubMed Google Scholar). We then focused on maturation cues at an earlier stage of the WD precursor from E8.75 to E9.5, given that Hoxb7-GFP+ committed WD precursors were first clearly detectable at E8.75 in the anterior body trunk of the embryo (12 somite stage) (Figure S1A). Similar to the E9.5–11.5 stage induction, sorted Hoxb7-GFP+ precursors maintained Emx2 and Ret expression in the presence of RA, Wnt agonist, and Fgf9. Conversely, the Wnt agonist and Fgf9 at these concentrations (3 μM and 100 ng/mL, respectively) were not effective for the expression of certain maturation marker genes (E-cadherin, Calb1, and Hnf1b) at this step (Figure 2B). Thus, we concluded that the combination of RA and low concentrations of Wnt agonist (1 μM) and Fgf9 (5 ng/mL) is optimal for induction. At this step, we could maintain the expression of Wnt11 by Fgf9 without Gdnf. These results suggest the existence of somewhat different modes of gene regulatory circuits between the early and late developmental stages of the WD maturation process. Finally, we sequentially applied the combined 3-day induction protocol to sorted E8.75 WD progenitors (Figure 2C). During the culture, the progenitors showed similar gene expression kinetics to those in vivo. At day 3 of induction, we observed the formation of a UB-like structure and quantitatively comparable gene expression levels to the embryonic UB in E11.5 embryos (Figures 2D and 2E). Next, we investigated the factors that induce the E8.75 WD progenitor-like population from mESCs. First, to enable isolation and quantitative evaluation of the efficiency of WD progenitor induction, we searched for cell-surface molecules that are specifically expressed in committed WD progenitors at E8.75. The in vivo microarray analysis data identified the combination of Cxcr4 and Kit as efficiently sortable markers (Figures 2F, S2B, and 3A ). Most WD progenitors at E8.75 were highly positive for Cxcr4 and Kit (Figure 2F; 94.6%), and the labeled population formed an isolated cluster in the entire E8.75 embryo proper (Figure S2C). For induction of WD progenitors from mESCs, we first considered the in vivo anteroposterior patterning process within the intermediate mesoderm. Our previous lineage segregation model of the UB and NP indicated that the UB lineage differentiates earlier than the NP lineage from the T+ immature mesoderm state that is maintained by strong Wnt signaling (Taguchi et al., 2014Taguchi A. Kaku Y. Ohmori T. Sharmin S. Ogawa M. Sasaki H. Nishinakamura R. Redefining the in vivo origin of metanephric nephron progenitors enables generation of complex kidney structures from pluripotent stem cells.Cell Stem Cell. 2014; 14: 53-67Abstract Full Text Full Text PDF PubMed Scopus (554) Google Scholar). Thus, we first tentatively shortened the incubation period with a high concentration of Wnt agonist to 1.5 day for the UB in contrast to 2.5 days for NP induction (Figure 3A; step 2). Based on the minimally conserved gene expression profile between the AIM (Osr1+, Lhx1+, and Pax2+) and the PIM (Osr1+, Wt1+, and Hox11+), we hypothesized that there would be partly different signaling for the AIM differentiation step compared with that for the PIM differentiation step (Figure 3A; step 3). We identified RA as a common inducer for both the AIM and PIM (Figures 3B and S4A) (Taguchi et al., 2014Taguchi A. Kaku Y. Ohmori T. Sharmin S. Ogawa M. Sasaki H. Nishinakamura R. Redefining the in vivo origin of metanephric nephron progenitors enables generation of complex kidney structures from pluripotent stem cells.Cell Stem Cell. 2014; 14: 53-67Abstract Full Text Full Text PDF PubMed Scopus (554) Google Scholar). Although the endogenous FGF signal was sufficient for PIM induction, the high concentration of Fgf9 further enhanced AIM marker expression. In contrast to the PIM, addition of Activin A (Activin) was inhibitory for the induction of AIM markers. Intriguingly, suppression of the Smad2/3 pathway by SB431542 enhanced AIM marker induction, suggesting a principal role of Activin/Tgfb signaling for AIM versus PIM fate determination. These results may agree with a previous report showing the involvement of these signals in anteroposterior body patterning (Green et al., 2011Green M.D. Chen A. Nostro M.C. d’Souza S.L. Schaniel C. Lemischka I.R. Gouon-Evans V. Keller G. Snoeck H.W. Generation of anterior foregut endoderm from human embryonic and induced pluripotent stem cells.Nat. Biotechnol. 2011; 29: 267-272Crossref PubMed Scopus (280) Google Scholar, McPherron et al., 1999McPherron A.C. Lawler A.M. Lee S.J. Regulation of anterior/posterior patterning of the axial skeleton by growth/differentiation factor 11.Nat. Genet. 1999; 22: 260-264Crossref PubMed Scopus (368) Google Scholar). Conversely, either addition or inhibition of Bmp4 (Bmp) suppressed AIM induction, indicating the requirement for an optimal level of Bmp signaling for AIM specification. We then explored factors that specify the differentiation of the AIM to Cxcr4+/Kit+ WD progenitors (Figure 3A; step 4). At this step, we found a synergistic effect of RA, Wnt, and Fgf9. In particular, removal of the Wnt agonist dramatically reduced the Cxcr4+/Kit+ population induction, suggesting a crucial role for committed WD progenitor induction (Figure 3C). To further fine-tune and understand the UB lineage differentiation process, we re-examined the optimal timing for efficient differentiation of the nascent mesoderm into the AIM by changing the Wnt incubation period (Figure 3A; step 2). Surprisingly, the permissive time window for AIM induction was quite restricted at around day 4.5 (36 hr of Wnt treatment), and the efficiency was dramatically reduced at day 4 or 5 (Figure 3D). This observation may reflect the very narrow anteroposterior AIM domain (pronephric anlagen) in vivo, which initially appears within the 2-somite width (8th–10th somite level) in the intermediate mesoderm at E8.5 (Bouchard et al., 2002Bouchard M. Souabni A. Mandler M. Neubüser A. Busslinger M. Nephric lineage specification by Pax2 and Pax8.Genes Dev. 2002; 16: 2958-2970Crossref PubMed Scopus (411) Google Scholar, Grote et al., 2006Grote D. Souabni A. Busslinger M. Bouchard M. Pax 2/8-regulated Gata 3 expression is necessary for morphogenesis and guidance of the nephric duct in the developing kidney.Development. 2006; 133: 53-61Crossref PubMed Scopus (252) Google Scholar). Previous reports showing early-stage mesoderm patterning prompted us to further investigate earlier fate specification signals within the epiblast and primitive streak/nascent mesoderm (Attia et al., 2012Attia L. Yelin R. Schultheiss T.M. Analysis of nephric duct specification in the avian embryo.Development. 2012; 139: 4143-4151Crossref PubMed Scopus (20) Google Scholar, James and Schultheiss, 2003James R.G. Schultheiss T.M. Patterning of the avian intermediate mesoderm by lateral plate and axial tissues.Dev. Biol. 2003; 253: 109-124Crossref PubMed Scopus (68) Google Scholar) (Figure 3A; steps 1 and 2). Indeed, we observed concentration-dependent patterning by Activin/Bmp signaling from day 2 to day 3 (step 1) and day 3 to day 4.5 (step 2) of the differentiation. During the mesoderm formation/patterning (step 2), UB induction was maximum at a higher Bmp concentration compared with NP induction (Figures 3E and S3). In the epiblast patterning stage (step 1), the UB had a preference for a higher concentration of Activin compared with the NP (Figures 3F and S3). Combination analysis of these two steps showed a reciprocal pattern in the optimal concentration range for the UB or NP (Figure S3). These results suggest that the cell-fate patterning of the UB and NP starts before and during the formation of the immature mesoderm. By employing these optimizations, we obtained an average 35.6% Cxcr4+/Kit+ population at day 6.25 of mESC differentiation (Figure 3G). We analyzed the gene expression kinetics from immature mESCs to day 6.25-induced committed WD progenitors (iWD; corresponding to E8.75 WD progenitors) (Figures 3A and 3H). Notably, the induced spheroids showed quantitatively comparable gene expression levels to E8.75 WD progenitors and were devoid of PIM and metanephric NP marker expression (Figure 3H; Hoxd11, Wt1, and Six2), indicating successful selective induction of the UB lineage. We applied the WD maturation factors to the mESC-derived iWD. To visualize the branching morphogenesis in the following experiments, we established mESCs from Hoxb7-GFP transgenic mice. Minimal modifications in the initial induction steps successfully induced Hoxb7-GFP+/Cxcr4+/Kit+ iWD at day 6.25 of differentiation (Figure S4A). The sorted GFP+/Cxcr4+/Kit+ population was reaggregated and cultured in the WD maturation condition established with the E8.75 WD (Figures 2C and 4A ). The aggregates formed UB-like structures at day 9.25 of induction and expressed UB markers that were comparable to the E11.5 UB (Figures 4B and 4C). To establish a single exit and confirm the branching capacity, we manually isolated a single bud from a spheroid and reaggregated it with an isolated E11.5 MM (including NPs and SPs) (Figure 4B). In the presence of the MM, the induced UB (iUB) underwent dichotomous branching up to the 6th–7th generations (1 generation/day) (Figure 4D). The final tip number from the single iUB grew to 141 ± 12 (n = 6), which was comparable to that induced by the E11.5 embryo-derived UB (Figure 4E). We also assessed the branching capacity of the iUB in a cell-free branching culture condition established by modifying a previously reported method (Rosines et al., 2007Rosines E. Sampogna R.V. Johkura K. Vaughn D.A. Choi Y. Sakurai H. Shah M.M. Nigam S.K. Staged in vitro reconstitution and implantation of engineered rat kidney tissue.Proc. Natl. Acad. Sci. USA. 2007; 104: 20938-20943Crossref PubMed Scopus (70) Google Scholar). We again observed bifurcation up to the 6th generation from the single iUB (Figure S4B). Whole-mount staining of the reconstituted organoid at day 7 identified the Six2+ NPs maintained on each iUB tip in the periphery of the organoid, which was reminiscent of the nephrogenic zone of embryonic kidneys (Figures 4F and S4C). Conversely, on the inner side of the organoid, we observed differentiated nephrons composed of E-cadherin+ distal tubule segments, LTL+ proximal tubule segments, and Nephrin+ glomerular structures, thereby confirming the nephron induction capacity of the iUB (Figure 4G; Movie S1). Importantly, the distal end of each nephron was connected to the ureteric tips, given that interconnection of nephrons is essential for urine drainage (Figure 4H). The typical UB tip marker Sox9 was expressed in the periphery (Reginensi et al., 2011Reginensi A. Clarkson M. Neirijnck Y. Lu B. Ohyama T. Groves A.K. Sock E. Wegner M. Costantini F. Chaboissier M.C. Schedl A. SOX9 controls epithelial branching by activating RET effector genes during kidney development.Hum. Mol. Genet. 2011; 20: 1143-1153Crossref PubMed Scopus (100) Google Scholar), while cytokeratin 8 (CK8) showed stronge