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Rapid and Efficient Differentiation of Pluripotent Stem Cells into Intermediate That Forms Expressing Proximal Tubular Markers

† † ‡ † Albert Q. Lam,* Benjamin S. Freedman,* Ryuji Morizane,* Paul H. Lerou, § † † M. Todd Valerius,* and Joseph V. Bonventre*

*Renal Division, Department of Medicine, and §Department of Newborn Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts; †Harvard Stem Institute, Cambridge, Massachusetts; and ‡Internal Medicine, Keio University School of Medicine, Tokyo, Japan

ABSTRACT Human pluripotent stem cells (hPSCs) can generate a diversity of cell types, but few methods have been developed to derive cells of the kidney lineage. Here, we report a highly efficient system for differentiating human embryonic stem cells and induced pluripotent stem cells (referred to collectively as hPSCs) into cells expressing markers of the intermediate mesoderm (IM) that subsequently form -like structures. Treatment of hPSCs with the glycogen synthase kinase-3b inhibitor CHIR99021 induced +MIXL1+ mesendoderm differentiation with nearly 100% efficiency. In the absence of addi- tional exogenous factors, CHIR99021-induced mesendodermal cells preferentially differentiated into cells expressing markers of with minimal IM differentiation. However, the sequential treatment of hPSCs with CHIR99021 followed by fibroblast -2 and retinoic acid generated PAX2+LHX1+ cells with 70%–80% efficiency after 3 days of differentiation. Upon growth factor withdrawal, these PAX2+LHX1+ cells gave rise to apically ciliated tubular structures that coexpressed the proximal tubule markers Lotus tetragonolobus lectin, N-cadherin, and kidney-specific and partially inte- grated into embryonic kidney explant cultures. With the addition of FGF9 and activin, PAX2+LHX1+ cells specifically differentiated into cells expressing SIX2, SALL1, and WT1, markers of cap progenitor cells. Our findings demonstrate the effective role of fibroblast growth factor signaling in in- ducing IM differentiation in hPSCs and establish the most rapid and efficient system whereby hPSCs can be differentiated into cells with features characteristic of kidney lineage cells.

J Am Soc Nephrol 25: 1211–1225, 2014. doi: 10.1681/ASN.2013080831

CKD is a significant global public health problem1 whole kidney from a patient’s own tissue, offers and is the leading risk factor for cardiovascular dis- the potential for new therapeutic strategies to treat ease. Despite advances in the quality of dialysis therapy, patients with CKD experience significant morbidity and mortality and reduced quality of life. Received August 5, 2013. Accepted November 9, 2013. For selected patients, kidney transplantation is an alternative renal replacement therapy to dialysis; A.Q.L., B.S.F., and R.M. contributed equally to this work. however, this option is limited by the shortage of Published online ahead of print. Publication date available at compatible organs and requires the use of lifelong www.jasn.org. immunosuppressive medication to prevent graft Correspondence: Dr.AlbertQ.Lam,HarvardInstitutesof rejection. For these reasons, research in regenera- Medicine, Room 550, 4 Blackfan Circle, Boston, MA 02115. tive medicine, with the ultimate aim of generating Email: [email protected] functional replacement kidney tissue or even a Copyright © 2014 by the American Society of Nephrology

J Am Soc Nephrol 25: 1211–1225, 2014 ISSN : 1046-6673/2506-1211 1211 BASIC RESEARCH www.jasn.org

CKD and ESRD. Human pluripotent stem cells (hPSCs) have hPSCs into multipotent cells expressing mesendodermal the potential to revolutionize our ability to generate functional markers in a manner that recapitulates mesendoderm forma- cells and tissues for purposes of regenerative medicine and tion in the developing . We demonstrate that hPSCs disease modeling. Both human embryonic stem cells (hESCs) treated with CHIR preferentially differentiate into cells ex- and human induced pluripotent stem cells (hiPSCs), collec- pressing lateral plate mesoderm markers. With the precisely tively referred to as hPSCs in this manuscript, possess the timed addition of specific growth factors, this default fate can ability to self-renew and to differentiate into cells of all three be diverted into definitive or other types of meso- germ layers of the embryo,2,3 making them ideal starting sub- derm. Importantly, we show that in cells treated with CHIR, strates for generating cells of the kidney lineage. the combination of FGF2 and retinoic acid (RA) efficiently gen- While other organs, such as the heart, liver, pancreas, and erates cells coexpressing PAX2 and LHX1 (markers of IM) within central , have benefited from more established 3daysofdifferentiation.Thisisthemostefficient method to differentiation protocols for deriving their functional cell types generate PAX2+LHX1+ IM cells and the first demonstration of fromhPSCs,considerably fewermethods have beendeveloped to the role of FGF signaling as a potent inducer of IM-specific effect kidney differentiation. This may be partly explained by the PAX2 expression in differentiating hPSCs. These PAX2+LHX1+ complex architecture of the kidney and its functional units, cells spontaneously form tubular-like structures that express , which are composed of highly specialized epithelial apical cilia and markers of proximal tubular epithelial cells and cell types, such as glomerular , proximal tubular integrateintomouseembryonickidneyexplantcultures. epithelial cells, cells of the thick and thin limbs of the loop of PAX2+LHX1+ cells can also be specifically differentiated Henle, , and collecting duct cells. No into cells expressing SIX2, SALL1, and WT1, markers of the single protocol is likely to generate the multitude of these cell multipotent nephron progenitor cells of the cap mesenchyme types, but a system to differentiate hPSCs into the nephron (CM), further demonstrating their capacity to give rise to IM progenitor cell populations, namely the intermediate mesoderm derivatives. When placed under the kidney capsule, cells ex- (IM) and the metanephric mesenchyme, may offer a common press aquaporin-1. point from which more specific kidney lineages can be derived. Although several studies have attempted to differentiate mouse ESCs into kidney cells,4–15 only a few studies have re- RESULTS ported protocols in hESCs and hiPSCs.16–19 These previous reports have produced cells that share characteristics expected CHIR Efficiently Induces hPSCs to Differentiate into of human kidney progenitor or epithelial cells, although the Cells Expressing Markers Characteristic of identities of these differentiated cells have yet to be conclu- Mesendoderm sively verified. In addition, the efficiencies of these protocols Todevelop a platform to target generation of IM, it was vital to for generating cells of the renal lineage are low, necessitating first identify which conditions most efficiently differentiate the use of cell sorting to enrich populations of cells using hPSCs into mesendoderm (Figure 1A). During , markers that are not entirely specific to the kidney. For exam- cells of the ingress through the to ple, OSR1, used as a marker by Mae and colleagues to label form the endodermal and mesodermal germ layers of the cells of the intermediate mesoderm,17 is also expressed in lat- developing embryo,22 and this process depends on the se- eral plate mesoderm,20 which gives rise during embryonic creted molecules of the Wnt and nodal/activin signaling development to the adult heart, hematopoietic system, and pathways.23,24 To determine the minimal signals required vasculature. Narayanan and colleagues isolated populations to induce mesendoderm expression in hPSCs, we dif- of AQP1+ proximal tubular-like cells,18 but this marker is ex- ferentiated cells under serum-free, feeder-free conditions in pressed not only in the kidney but also broadly in the gastro- the absence of other chemical additives except for the precise intestinal system, lungs, and blood cells.21 In both instances, signals tested. We first tested the efficacy of WNT3A and the sorted cells were heterogeneous and included a small per- activin A to induce expression of the primitive streak marker centage of cells that exhibited properties and behaviors of cells BRACHYURY (BRACHY). WNT3A alone, at a concentra- of the kidney lineage. While these and earlier studies have tion of 500 ng/ml, induced BRACHY expression in 26.7% suggested a role for Wnt, activin, bone morphogenetic protein 61.9% of cells after 24 hours of treatment (Figure 1, B and (BMP), and retinoic acid signaling in the induction of cells of C). The addition of activin A to WNT3A increased the per- the kidney lineage, inductive effects of other signaling path- centage of BRACHY+ cells to 70.3%65.5% of the total pop- ways, such as fibroblast growth factor (FGF) signaling, on ulation. However, when cells were treated with 5 mMCHIR,a kidney differentiation from hPSCs have not been reported. potent GSK-3b inhibitor/Wnt pathway agonist, 98.7% Here we report a simple, efficient, and highly reproducible 61.3% of cells expressed BRACHY at 24 hours (Figure 1, system to induceIMdifferentiation in hESCs and hiPSCsunder B and C). profiling of CHIR-induced cells chemically defined, monolayer culture conditions. Chemical revealed that expression of primitive streak (BRACHY, induction with the potent small molecule inhibitor of GSK-3b, MIXL1, EOMES, FOXA2, and GSC) was rapidly upregulated CHIR99021 (CHIR), robustly and rapidly differentiates within 24 hours of treatment, peaked between 36–48 hours,

1212 Journal of the American Society of Nephrology J Am Soc Nephrol 25: 1211–1225, 2014 www.jasn.org BASIC RESEARCH

Figure 1. CHIR99021 efficiently induces human pluripotent stem cells to differentiate into mesendoderm-like cells. (A) Diagram of differentiation of human PSCs into mesendoderm using CHIR. (B) hPSCs treated with DMSO (vehicle), Wnt3a 500 ng/ml, Wnt3a 500 ng/ ml+activin 50 ng/ml, or CHIR 5 mM were immunostained for BRACHYURY after 24 hours of differentiation. (C) Quantification of cells with positive immunofluorescence staining for BRACHYURY after treatment with vehicle, Wnt3a at increasing doses, Wnt3a at in- creasing doses with activin, or CHIR for 24 hours. Data represent mean6SEM (n=3). (D) Time course of gene expression in hPSCs treated with CHIR. Quantitative RT-PCR of genes expressed in mesendoderm and in pluripotent stem cells. Data represent mean6SEM (n.3). (E) Immunofluorescence staining for BRACHYURY and MIXL1 in CHIR-treated hPSCs over the first 48 hours of differentiation. (F) Immunofluorescence staining for BRACHYURY and MIXL1 after 24 hours of differentiation in hESC and hiPSC lines treated with CHIR. (G) Phase contrast images of CHIR-treated human PSCs over 36 hours showing epithelial to mesenchymal transition. (H) Immunoflu- orescence staining for E-cadherin and N-cadherin in CHIR-treated human PSCs. Scale bars, 100 mm. BRACHY, BRACHYURY; CHIR, CHIR99021; ME, mesendoderm; PSC, pluripotent . BJ, iPS cells derived from human foreskin fibroblasts; DAPI, 49,6- diamidino-2-phenylindole; HDF, iPS cells derived from adult human dermal fibroblasts. and decreased by 72 hours, a pattern consistent with the expression, and undergo an epithelial–mesenchymal transi- transient expression of these genes during gastrulation. In tion.25,26 Consistent with these observations, CHIR induc- parallel, the induction of mesendoderm genes was accompa- tion resulted in the upregulation of SNAI1 as early as 24 hours nied by the loss of pluripotency, as reflected by a reduction of after treatment (Figure 1D). This was associated with epi- OCT4 and NANOG mRNA (Figure 1D). Protein levels paral- thelial–mesenchymal transition with waves of cells migrat- leled this gene expression pattern, as immunostaining for ing away from the periphery of differentiating colonies BRACHYandMIXL1 over the first 48 hours of differentiation between 36 and 48 hours of differentiation (Figure 1G) revealed coexpression of these markers in CHIR-induced and with a switch in cadherin expression from E- to N-cadherin cells as early as 12 hours with coexpression peaking at 48 (Figure 1H). Together, these findings demonstrated the hours (Figure 1, E and F). As cells migrate through the prim- potency of CHIR to induce differentiation of hPSCs into itive streak during development, they express SNAI1,down- mesendoderm-like cells via a program that mimics normal regulate E-cadherin expression, upregulate N-cadherin development in vivo.

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Timed Addition of Exogenous Factors Modulates Cell and duration of signaling factors with regards to cell fate deter- Fate of CHIR-Induced hPSCs mination in our differentiation system. To determine whether treatment with CHIR alone was sufficient to induce differentiation toward IM, we next tested the intrinsic FGF2 Induces PAX2 Expression in CHIR-Induced Cells multipotency of CHIR-induced cells by withdrawing CHIR from Having demonstrated that we could manipulate the fate of the culture media after 24 or 48 hours of induction and allowing CHIR-treated cells with the time-sensitive addition of specific the cells to differentiate for another 3–4days(Figure2A).CHIR- inducingfactors, we then screened candidate growth factorsfor induced cells spontaneously differentiated into heterogeneous the ability to induce the expression of the IM marker PAX2. populations of cells expressing transcriptional markers of defin- PAX2 is an early marker of IM, and, unlike the markers OSR1 or itive endoderm (SOX17) and mesodermal subtypes (FOXF1, LHX1, which are also expressed in the adjacent lateral plate KDR, TBX6) without significant upregulation of the IM marker mesoderm, PAX2 expression is restricted in mesoderm to the PAX2 or neuroectodermal markers (SOX1, PAX6)(Figure2B). IM.20 hPSCs were induced with CHIR for 24 hours, at which Immunocytochemistry of these cell cultures revealed a depen- time CHIR was withdrawn and cells were treated with increasing dence of cell fate on the duration of CHIR treatment. The hPSCs doses of activin A, BMP-2, BMP-4, BMP7, FGF2, or RA. On day 4 pulsed with CHIR for 24 hours generated approximately 40% of differentiation, modest increases in PAX2 expression, com- FOXF1+ cells, a marker that is characteristic of lateral plate me- pared with a vehicle control, were seen in cells treated with low soderm (Figure 2, C and D). A significant proportion of cells doses of BMP-2, BMP7, and RA, and no PAX2 expression was (30%–40%), however, expressed the pluripotency marker seen in cells treated with activin or BMP-4 (Figure 2I). We ob- OCT4 (Figure 2, C and D), suggesting that the withdrawal of served, however, that approximately 50%–60% of cells treated CHIR after 24 hours may have resulted in the reversion of in- withFGF2atadoseof100ng/mlwerepositiveforPAX2by completely differentiated cells back to the pluripotent state. Pro- immunostaining, suggesting that FGF2 could be a potential in- longing the duration of CHIR treatment to 48 hours eliminated ducer of IM. The ability of FGF2 to induce PAX2 expression the appearance of OCT4+ cells, with .80% of cells staining pos- depended on cells being pretreated with CHIR. The addition of itive for FOXF1. The protein expression of the paraxial meso- FGF2 to hPSCs not initially induced with CHIR resulted in the derm marker TBX6 and the IM marker PAX2 was seen in ,10% absenceofPAX2expressiononday4ofdifferentiation(Figure2J). and 1% of cells, respectively, signifying that CHIR treatment was insufficient to efficiently induce these mesodermal subtypes (Fig- FGF2 and RA Induce PAX2+LHX1+ Intermediate ure 2C). Similarly, ,1% of cells expressed SOX17 or PAX6, sug- Mesoderm Cells gesting that endodermal or ectodermal differentiation was not Retinoic acid signaling has previously been demonstrated to play significantly induced by CHIR treatment. Because proper differ- an important role in the early stages of .32,33 entiation of lateral plate mesoderm during embryonic develop- Because we observed a small inductive effect on PAX2 expression ment depends on higher BMP-4 signaling gradients, we evaluated with RA, we then tested low-dose RA in combination with FGF2 the expression of BMP-4 transcripts in CHIR-induced cells by to determine whether it could have a synergistic effect on PAX2 quantitative RT-PCR. The hPSCs treated with CHIR for 24 or 48 expression. Although we did not see a significant increase in hours significantly upregulated BMP-4 on day 4 compared with PAX2 expression, the addition of 1 mM RA to FGF2 resulted DMSO-treated controls, demonstrating that induction with in a marked increase in LHX1 expression, with most cells coex- CHIR stimulated endogenous expression of BMP-4 (Figure 2E). pressing both PAX2 and LHX1 (Figure 3A). Because PAX2 and To determine whether we could divert CHIR-induced cells LHX1 are both expressed in the developing IM, we considered from the default lateral plate mesoderm fate, we applied high these cells as a putative IM cell population. doses of activin Ato hPSCs after 24 hours of CHIRtreatment and We then sought to optimize the efficiency of generating IM successfully generatedcellsexpressingSOX17+FOXA2+,markers cells. We first tested the effects of different durations of CHIR of definitive endoderm, with .95% efficiency after 2–3daysof pretreatmentandassayedPAX2expressionfromdays2through7 subsequent differentiation (Figure 2F, Supplemental Figure 1, of differentiation. Induction of hPSCs with CHIR for 36 hours A–C). These cells could be differentiated further into a variety followed by FGF2 and RA resulted in PAX2 expression in .70% of endodermal cells expressing the pancreatic markers of cells as early as day 3 of differentiation (Figure 3B). Longer and somatostatin27,28 (Figure 2G), the mature hepatocyte pretreatment with CHIR for 48 hours resulted in less PAX2 marker albumin,29 and markers of anterior foregut endoderm expression at all time points compared with CHIR treatment (precursors to lung and thyroid tissue)30 or hindgut endoderm for 24 or 36 hours. Regardless of the duration of CHIR pretreat- (precursors to intestine)31 (Supplemental Figure 1, D–F). How- ment, the proportion of cells expressing PAX2 significantly de- ever, when activin A was added after 48 hours of CHIR treat- creased after day 4 of differentiation, with ,10% of cells at day 7 ment, the number of SOX17+ cells markedly decreased after 4 retaining PAX2 expression. Because previous studies have days of differentiation (Supplemental Figure 1B), indicating a identified a role for BMP7 in inducing IM cells,5,14,16,17 we distinct window of time during which CHIR-induced cells could next determined whether the addition of BMP7 to FGF2 and be differentiated into different mesendodermal lineages. Collec- RA could enhance IM cell differentiation. In contrast to these tively, these results highlighted the critical importance of timing other reports, we found that the addition of BMP7 significantly

1214 Journal of the American Society of Nephrology J Am Soc Nephrol 25: 1211–1225, 2014 www.jasn.org BASIC RESEARCH

Figure 2. Timed addition of exogenous factors modulates cell fate of CHIR-induced hPSCs. (A) Diagram depicting time course of differentiation. (B) Time course of gene expression in human PSCs treated with CHIR for 24 hours, CHIR for 48 hours, or DMSO (vehicle). Data shown represent mean6SEM (n=4). Representative immunostaining (C) and quantification (D) of markers of pluripotency, me- soderm, definitive endoderm, and in human PSCs treated with DMSO (vehicle), CHIR for 24 hours, or CHIR for 48 hours, day 4 of differentiation. Data shown in graph represent mean6SEM (n.5). (E) Expression of BMP-4 by quantitative RT-PCR in hPSCs treated with CHIR for 24 hours, CHIR for 48 hours, or DMSO. Data shown represent mean6SEM (n=3). (F) Representative immunostaining for SOX17 and FOXA2 in hESCs and hiPSCs treated with CHIR for 24 hours followed by activin A 100 ng/ml for 3 days. Numbers represent the mean percentage6SEM of SOX17+ cells from at least two independent experiments. (G) Cells at the definitive endoderm stage were differentiated using a three-step protocol into hormone-expressing pancreatic endocrine cells producing insulin, proinsulin C-peptide, and somatostatin. Number represents the mean percentage6SEM of insulin+C-peptide+ cells from at least two independent experiments. (H) Diagram of directed differentiation of hPSCs into intermediate mesoderm. (I) Quantification of cells with positive immunostaining for PAX2. Data shown in the graph represent mean6SEM (n=2). (J) Immunostaining for PAX2 in hPSCs treated with or without CHIR for 24 hours followed by FGF2 100 ng/ml for 3 days, day 4. Scale bars, 100 mm. DAPI, 49,6-diamidino-2- phenylindole.

J Am Soc Nephrol 25: 1211–1225, 2014 Intermediate Mesoderm from hPSCs 1215 BASIC RESEARCH www.jasn.org

Figure 3. FGF2 and RA induce PAX2+LHX1+ intermediate mesoderm cells. (A) Immunostaining for PAX2 and LHX1 in hPSCs cells treated with CHIR for 24 hours followed by FGF2 or FGF2+RA for 3 days, day 4 of differentiation. (B) Quantification of PAX2+ cells on days 2–7 of differentiation in hPSCs treated with CHIR for 24, 36, or 48 hours followed by FGF2+RA. Data shown represent mean6SEM (n.4). (C) Immunostaining for PAX2 and LHX1 and (D) quantification of PAX2+ cells in three hESC lines and two hiPSC lines treated with CHIR for 36 hours followed by FGF2+RA (ChFR), day 3. Data shown represent mean6SEM (n=3). (E) Quantification of PAX2+ or LHX1+

1216 Journal of the American Society of Nephrology J Am Soc Nephrol 25: 1211–1225, 2014 www.jasn.org BASIC RESEARCH decreased the percentage of cells expressing PAX2 but had a continued under these conditions, and as early as day 7, tubular minimal effect on the expression of LHX1 (Figure 3E). epithelial structures formed in parallel with the downregulation To determine the reproducibility of our protocol in different of PAX2 expression (Figure 4, A–D). Immunostaining for mark- hPSC lines, we tested the combination of CHIR induction for 36 ers of more differentiated kidney cell types revealed that the cells hours followed by the addition of FGF2 and RA in three hESC comprising these tubular structures expressed the following: Lo- lines andtwo hiPSC lines. Similar patternsofcostaining for PAX2 tus tetragonolobus lectin (LTL), which localizes to the apical sur- and LHX1 were observed in all cell lines with nearly identical face of kidney proximal tubules; N-cadherin, which is the pre- efficiencies of differentiation (70%–80%) in four of the five cell dominant cadherin expressed on proximal tubular cells37,38;and lines and a slightly reduced differentiation efficiency in one kidney-specific protein (KSP), a cadherin that is known to be hiPSC line (Figure 3, C and D). To confirm these findings, we expressed on all kidney tubular epithelial cells and marks mouse used flow cytometry to quantify PAX2 and LHX1 expression in –derived kidney tubular cells15,39,40 (Figure hESCs and hiPSCs treated with this protocol. Interestingly, even 4, E and F). The formation of laminin-bounded tubular struc- higher proportions of differentiated cells were positive for PAX2 tures coexpressing LTL, KSP, and N-cadherin was reproducible or LHX1 by flow cytometry, and 80%–85% of cells were double in structures derived from both hESC and hiPSC lines (Figure 4, positive for PAX2 and LHX1 (Figure 3F). E and F). Furthermore, we observed the presence of primary cilia Wethen performed geneexpressionprofiling ofthese putative expressing the ciliary protein polycystin-2 on the apical surface IM cells using quantitative RT-PCR. Consistent with our protein of many of the tubular structures (Figure 4G), another feature expression data, we observed a marked upregulation of IM genes, of polarized kidney tubules that confirms the polarity of the including PAX2, LHX1, OSR1,andPAX8,onday3ofdifferen- epithelial cells.41,42 We then evaluated the expression of more tiation, followed by a reduction in gene expression at day 5 (Fig- differentiated kidney markers in cells treated with our IM- ure 3G), suggesting that IM differentiation in hPSCs may be a inducing protocol from days 0 to 9 and observed a significant, transient state which can be rapidly induced but lasts only 2–3 time-dependent upregulation in the expression of SIX2,a days. The expression of lateral plate and marker for multipotent nephron progenitor cells of the markers KDR and MEOX1, respectively, was not significantly metanephric mesenchyme, and markers of mature kidney upregulated by our IM induction protocol, and, as expected epithelial cells, such as NEPHRIN (), SYNAPTOPO- with differentiation, we noted downregulation of the pluripo- DIN (podocyte), AQP1 (proximal tubule), MEGALIN (prox- tency marker OCT4. Expression of WT1, a marker expressed imal tubule), UMOD (), and AQP2 (collecting first in the IM and then in the urogenital ridge, which is derived duct) (Figure 4H). from IM,34 was expressed at a low level on day 3 and was strongly To further confirm the identity of these cells as embryonic upregulated on day 5 of differentiation (Figure 3, G and H). kidneycells, we subjected them to kidneyexplant reaggregation Because PAX2 and LHX1 are also expressed in the developing assays.43 Cells expressing IM markers were dissociated on day ear, eye, and during embryogenesis,35,36 3(PAX2+LHX1+) or 9 (LTL+KSP+) of differentiation and were we evaluated our IM cells for the expression of markers (EYA2, recombined with dissociated cells from wild-type E12.5 SOX3, OTX2, FOXI3, SIX4) that are coexpressed with PAX2 and/ mouse embryonic kidneys. Human cells from day 3 were in- or LHX1 at relevant stages of neuroectodermal development. We corporated into mouse metanephric tissues, distributing in found that their expression was downregulated or unchanged the interstitium; however, no tubular integration was ob- compared with undifferentiated hPSCs (Figure 3I). Wetherefore served. Human cells from day 9 were found not only in the concluded that our PAX2+LHX1+ cell population was most mouse metanephric interstitium but also within organized likely to be representative of IM. laminin-bounded structures that contained mouse cells (Fig- ure 4I). These structures were similar in morphology to other hPSC-Derived PAX2+LHX1+ Cells Form Tubule laminin-bounded structures containing only mouse meta- Structures That Express Proximal Tubular Markers nephric cells in the coculture reaggregate. However, tubular- To determine whether hPSC-derived PAX2+LHX1+ IM cells like structures lined by human cells could not be visualized, have the capacity to give rise to more differentiated cell and suggesting that their integration into the mouse metanephric tissue derivatives of IM, we withdrew FGF2 and RA from the tissue was incomplete. When LTL+KSP+ tubular cells were culture media on day 3 of differentiation and cultured them in implanted beneath the kidney capsule of immunodeficient serum-free media, containing no additional growth factors or mice, they generated human growths expressing AQP1 (Sup- chemicals, for another week. Cell growth and proliferation plemental Figure 2, A–C).

cells in hPSCs treated with CHIR for 36 hours followed by FGF2+RA or FGF2+RA+BMP7, day 3. Data shown represent mean6SEM (n=3). (F) Quantification of PAX2+ and LHX1+ cells by flow cytometry. (G) Quantitative RT-PCR of intermediate mesoderm genes in hPSCs treated with ChFR. Data shown represent mean6SEM (n=3). (H) Immunostaining for LHX1 and WT1 in hPSCs treated with ChFR. (I) Quantitative RT-PCR of non-IM genes in hPSCs treated with ChFR. Data shown represent mean6SEM (n=2). Scale bar, 100 mm. DAPI, 49,6-diamidino-2- phenylindole.

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Figure 4. PAX2+LHX1+ intermediate mesoderm cells form polar tubular structures expressing polycystin-2+ cilia and kidney proximal tubular markers. (A) Immunostaining time course from days 3 to 9 for PAX2, KSP, and LTL in hPSCs treated with FGF2+RA (ChFR) for 3 days, then cultured in media without additional growth factors for an additional 6 days. Scale bar, 50 mm. (B) Immunostaining for KSP and LTL in tubular structures formed by PAX2+LHX1+ IM cells, day 9. Inset shows tubular structure at higher magnification. Scale bar,

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FGF9 and Activin A Induce Expression of CM Markers of CHIR treatment, we observed distinct changes in cell mor- in PAX2+LHX1+ Cells phology and the formation of tubular-like structures (Figure 5, During embryonic kidney development, the CM comprises a F and G). Immunocytochemistry of these structures on day 8 of population of multipotent nephron progenitor stem cells that differentiation revealed a downregulation of SIX2 expression and express the SIX2 and give rise to nearly all the increased expression of the proximal tubule marker LTL in epithelial cells of the nephron, with the exception of the collecting CHIR-induced cells compared with cells not treated with CHIR duct cells.44 Although SIX2 mRNA levels increased during sto- (Figure 5H), suggesting that treatment with CHIR had induced chastic differentiation into tubular structures (Figure 4H), SIX2 changes similar to that seen with induction of CM and the initi- protein was not clearly detectable by immunofluorescence, sug- ation of tubulogenesis in vivo. Furthermore, hPSC-derived SIX2+ gesting that its stable expression may require additional factors.44 cells explanted into dissociated-reaggregated mouse embryonic In addition to forming chimeric laminin-bounded structures in kidneys formed organizing clusters of cells that expressed LTL recombination explant culture (Figure 4I), stochastically differ- (Figure 5I). We therefore concluded that the ability of hPSC- entiated human PAX2+LHX1+ cells incorporated into clusters of derived PAX2+LHX1+ cells to be differentiated into cells express- Six2+ mouse metanephric cells but did not express SIX2 protein ing multiple markers of kidney CM and that could form tubule- by immunofluorescence (Figure 5A). To identify conditions that like structures in response to Wnt signaling was consistent with promote and sustain a SIX2+ cell population in vitro,wescreened the behavior and function of nephrogenic IM cells. growth factors added on day 3 of differentiation for the ability to induce SIX2 expression detectable by immunofluorescence (Fig- ure 5B). From our initial screen, we observed small populations DISCUSSION of SIX2+ cells when PAX2+LHX1+ cells were treated with FGF9 100 ng/ml or activin A 10 ng/ml for 5 additional days, whereas no We report a rapid, efficient, and highly reproducible system to SIX2+ cells were seen with treatment with a vehicle control (Fig- induce intermediate mesoderm cells from hESCs and hiPSCs ure 5C). The combination of FGF9 and activin A, together with under precise,chemicallydefined,monolayerculture conditions. decreasing the initial cell plating density, markedly improved the Robust generation of a BRACHYURY+MIXL1+ cell population efficiency of SIX2 induction and demonstrated that SIX2 expres- with the use of CHIR confirmed the potency of GSK-3b inhib- sion could be seen as early as day 6 of differentiation (Figure 5D). itors to generate mesendoderm-like cells47–49 and established the To confirm that this SIX2 expression was consistent with differ- proper platform for us to screen compounds which could ef- entiation toward CM, we evaluated the expression of SALL1 and fectively promote IM differentiation. By investigating the differ- WT1, two other important markers of CM.34,45 Nearly all SIX2+ entiation kinetics of CHIR-treated hPSCs, we established that cells coexpressed SALL1 as seen by immunocytochemistry, and a increasing exposure to CHIR resulted in differentiation toward a subset of SIX2+ cells also coexpressed WT1 (Figure 5E). lateral plate mesoderm fate, but this default pathway could be During embryonic kidney development, Wnt signals from altered by the precisely-timed addition of fate-altering growth the induce the CM to condense into a pretubular factors. This affirms the findings of prior reports demonstrating aggregate, which subsequently develops into a renal vesicle, that mesodermal and endodermal cell fates are determined by a comma-shaped body, S-shaped body, and ultimately a neph- delicate time- and dose-dependent balance of Wnt, activin, ron.20 In vitro, FACS isolated mouse Six2+ cells transiently BMP, and FGF signaling.24,25,47,50–62 Our findings, however, induced by Wnt signaling (using the GSK-3b inhibitor BIO) contrast with those of a recent study by Tan and colleagues, undergo epithelialization and begin expressing markers of who showed that prolonged exposure to GSK-3b inhibition, nephron development.46 To test the competence of hPSC- specifically with CHIR, promoted an endodermal rather than derived SIX2+ cells to respond to canonical Wnt signaling, mesodermal fate.49 With our differentiation conditions, defini- we treated cells on day 6 of differentiation with 5 mMCHIR tive endoderm differentiation could be achieved only with the for 24 hours, followed by withdrawal of CHIR. Within 24 hours synergistic effect of CHIR and high-dose activin.

50 mm. (C) Brightfield stereomicroscope imaging of tubular structures, day 9. Scale bar, 200 mm. (D) Quantification of tubular structures formed from PAX2+LHX1+ IM cells, day 9. Data shown represent mean6SEM (n=4 for one hESC and n=4 for one hiPSC line). (E) Im- munostaining for KSP, LTL, and laminin in tubular structures derived from two hESC lines and one iPSC line, day 9. Scale bar, 50 mm. (F) Immunostaining for LTL and N-cadherin in tubular structures derived from one hESC and one iPSC line, day 9. Scale bar, 50 mm. (G) Immunostaining for acetylated a-tubulin and polycystin-2 in tubular structures, day 9. Inset shows higher magnification of cilia localized to the apical surface. Scale bar, 50 mm. (H) Quantitative RT-PCR of genes associated with kidney development and mature kidney epithelial cells. Data shown represent mean6SEM (n=2). (I) Whole-mount immunohistochemistry for anti–human nuclear antigen (HNA) and laminin in chimeric kidney explant cultures. Dissociated hPSC-derived IM cells on day 3 (n=10) and day 9 (n=3) of differentiation were mixed with dissociated E12.5 mouse embryonic kidneys in single cell suspension, reaggregated by centrifugation, and cultured for 3 days in kidney explant culture. Arrowhead, laminin-bounded structures containing human and mouse cells. Scale bar, 50 mm. AQP1, aquaporin-1; AQP2, aquaporin-2; DAPI, 49,6-diamidino-2-phenylindole; HNA, human nuclear antigen; UMOD, uromodulin.

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Figure 5. FGF-9 and activin differentiate PAX2+LHX1+ cells into cells expressing markers of CM. (A) Whole-mount immunohisto- chemistry for anti–human nuclear antigen (HNA) and SIX2 in chimeric kidney explant cultures. Dissociated hESC-derived IM cells on day 9(n=3) of differentiation were mixed with dissociated E12.5 mouse embryonic kidneys in single cell suspension, reaggregated by centrifugation, and cultured for 5 days in kidney explant culture. Arrowhead, HNA+ cells present within clusters of mouse Six2+ cells. Scale bar, 50 mm. (B) Diagram showing the stepwise differentiation of hESCs into metanephric CM. (C) Immunostaining for SIX2 in hESCs treated with FGF2+RA (ChFR) for 3 days then either 100 ng/ml FGF-9, 10 ng/ml activin, or vehicle for 3 days, day 6. Scale bar, 100 mm. (D) Immunostaining for SIX2 in hESCs plated at different densities and treated with ChFR for 3 days then 100 ng/ml FGF-9+10 ng/ml activin for 3 days, day 6. Scale bar, 100 mm. (E) Immunostaining for SIX2, SALL1, and WT1 in hESCs treated with ChFR for 3 days then 100 ng/ml FGF-9+10 ng/ml activin for 3 days, day 6. Dashed line encompasses the population of cells that stain positive for WT1.

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Although the differentiation of hPSCs into cells of the cardiac, to form polarized, ciliated tubular structures that express mark- hepatic, pancreatic, and neuronal lineages has been widely ers of kidney proximal tubular cells and partially integrate into reported, relatively few previous studies have attempted to derive mouse metanephric cultures. As demonstrated by Hendry et al., cells of the kidney lineage from hPSCs.16–19 An alternative to this property is a strong indication that the cells may be nephron directed differentiation was recently demonstrated by means of progenitors because other types of cells do not typically integrate direct of immortalized human kidney cells into into this compartment.63 These polarized tubular structures nephron progenitor–like cells; however, the efficiency of integra- could reproducibly form in monolayer culture, in contrast to pre- tion into kidney explant cultures was reportedly low.63 Mae and vious reports in which tubular structures derived from differen- colleagues demonstrated induction of an intermediate meso- tiated hPSCs cells could form only with three-dimensional culture derm population using a stepwise combination of CHIR, activin, in vitro or after incorporation into mouse metanephric kidneys and BMP7 signaling in engineered OSR1-GFP hiPSC cell lines, ex vivo.17,18 Furthermore, using the combination of FGF9 and achieving efficiencies of .90% of OSR1-GFP+ cells after 11–18 activin, we could specifically direct the differentiation of PAX2+ days of differentiation.17 Because OSR1 is expressed in both the LHX1+ cells into cells coexpressing multiple markers of neph- lateral plate and the intermediate mesoderm during early ron progenitor cells in the CM, particularly SIX2, SALL1, and mesoderm specification,20 this expression pattern does not dis- WT1. tinguish intermediate from lateral plate mesoderm, and the pro- Toour knowledge, this is the first report of the generation of portion of OSR1+ cells that coexpressed other important IM SIX2+ cells from hPSCs, and our method of using FGF9 to markers, such as PAX2 or WT1, was comparatively low. We induce SIX2 expression is consistent with the important role selected PAX2 and LHX1 as more specific markers of IM for of FGF9 in maintaining the nephron progenitor population the purpose of defining IM inducing culture conditions. It is during embryonic kidney development.65 When these SIX2+ important to note that the expression of either PAX2 or LHX1 cells were transplanted ex vivo into mouse metanephric cul- is not limited to the developing kidney during embryogenesis tures, they organized into structures that expressed LTL and and can be seen at other stages of development in the eye, ear, laminin. In parallel, activation of canonical Wnt signaling in and central nervous system35,36; however, coexpression of PAX2 the SIX2+ cell population using CHIR resulted in the rapid and LHX1 within the same domain has been described only in formation of tubule-like structures in vitro in which cells the developing kidney and dorsal spinal cord.20,64 Importantly, downregulated SIX2 and expressed LTL. Although this result we identified for the first time that FGF2 is a potent factor in suggests that hPSC-derived SIX2+ cells can be induced to con- inducing PAX2 expression in CHIR-induced mesendodermal dense and epithelialize in a manner similar to that seen with cells, and when combined with RA, we were able to robustly CM in vivo, further studies are needed to determine the precise generate a PAX2+LHX1+ IM cell population as confirmed by conditions for activating a program of kidney tubulogenesis. both immunocytochemistry and flow cytometry. In conclusion, we demonstrate that sequential treatment Our protocol was capable of achieving efficient IM differen- with CHIR99021 and FGF2 and RA induces efficient differ- tiation within 3 days, which is considerably quicker than existing entiation of hPSCs into PAX2+LHX1+ cells characteristic of protocols while maintaining a high level of efficiency, and was intermediate mesoderm and that these cells are capable of highly reproducible in multiple hESC and hiPSC lines without spontaneously forming polar ciliated tubular structures that the need for flow sorting. Interestingly, with our culture express markers of kidney proximal tubular epithelial cells. conditions we observed that the addition of BMP7, which has Stochastically differentiated PAX2+LHX1+ cells also express been used asa component of other kidney-lineage differentiation markers of multiple differentiated, mature kidney cell types. protocols,5,16,17 did not have a synergistic effect in inducing IM The addition of FGF-9 and activin more specifically differen- differentiation. Although the precise conditions for specifically tiates PAX2+LHX1+ cells into cells expressing SIX2, SALL1, generating other IM derivatives, including the adrenal cortices and WT1, markers of the nephron progenitor stem cell pool in and , have yet to be defined, we demonstrated that in- the CM, further demonstrating that PAX2+LHX1+ cells have ducing PAX2+LHX1+ cells is sufficient for these cells to auton- the potential to give rise to IM derivatives. The establishment omously express WT1, a later marker of IM differentiation, and of this system will facilitate and improve the directed

Scale bar, 100 mm. (F) Brightfield microscopy of hESCs treated with ChFR for 3 days, 100 ng/ml FGF-9+10 ng/ml activin for 3 days, then either 5 mM CHIR (+CHIR) or FGF-9+activin (2CHIR) for 24 hours. Scale bar, 100 mm. (G) Higher magnification of tubular epithelial-like structures seen in SIX2+ cells treated on day 6 with 5 mM CHIR for 24 hours, day 7. Scale bar, 100 mm. (H) Immunostaining (day 8) for SIX2 and LTL in hESCs treated with ChFR for 3 days, 100 ng/ml FGF-9+10 ng/ml activin for 3 days, then either 5 mM CHIR (+CHIR) or FGF9 +activin (2CHIR) for 24 hours. Scale bar, 100 mm. (I) Whole-mount immunohistochemistry for anti–human nuclear antigen (HNA), laminin, and LTL in chimeric kidney explant cultures. Dissociated hESC-derived SIX2+ cells on day 6 (n=10) of differentiation were mixed with dissociated E12.5 mouse embryonic kidneys in single cell suspension, reaggregated by centrifugation, and cultured for 3 days in kidney explant culture. Dashed line encompasses an organizing cluster of HNA+ cells, which express laminin and LTL. Scale bar, 50 mm. DAPI, 49,6-diamidino-2-phenylindole; ME, mesendoderm; MM, metanephric cap mesenchyme; PSC, pluripotent stem cell.

J Am Soc Nephrol 25: 1211–1225, 2014 Intermediate Mesoderm from hPSCs 1221 BASIC RESEARCH www.jasn.org differentiation of hPSCs into cells of the kidney lineage for the +2 mM retinoic acid (Sigma-Aldrich)+0.25 mM 3-keto-N-(aminoethyl- purposes of bioengineering kidney tissue and iPS cell disease aminocaproyl-dihydrocinnamoyl)-cyclopamine (EMD Millipore) modeling.41 +100 ng/ml recombinant human (R&D Systems) for 4 days, then high-glucose DMEM+1% B27+100 ng/ml Noggin+300 nM indo- lactam V (Stemgent)+1 mM ALK5 inhibitor II (Axxora) for 4 days. For CONCISE METHODS anterior foregut endoderm differentiation, cells at the definitive endo- derm stage were treated with DMEM/F12+13 L-glu+13 B27+200 ng/ m hES and hiPS Cell Culture ml Noggin+10 M SB431542 (Stemgent) for 3 days. For hindgut en- Human foreskin (American Type Culture Collection) and human dermal doderm differentiation, cells at the definitive endoderm stage were trea- fibroblast-a (Invitrogen) fibroblasts were reprogrammed into iPS cells ted with A-RPMI+13 L-glu+13 P/S+2% FBS+500 ng/ml FGF4 (R&D by two rounds of overnight transduction with murine stem cell virus– Systems)+5mM CHIR for 4 days. For intermediate mesoderm differen- internal ribosome entry site–green fluorescent protein retroviruses for tiation, cells were treated with A-RPMI+13 L-glu+13 P/S+5 mMCHIR OCT4, , ,andc- (Addgene) produced in 293FT cells for 24, 36, or 48 hours, then A-RPMI+13 L-glu+13 P/S+100 ng/ml (Invitrogen). H1, H9, and CHB8-H2B-GFP hESCs (passages 30–50) FGF2+1 mM retinoic acid for 2–3 days. For CM differentiation, cells and human foreskin and human dermal fibroblast iPSCs (passages were treated with A-RPMI+13 L-glu+13 P/S+5 mM CHIR for 36 12–40) were routinely cultured on irradiated mouse embryonic fibro- hours, then A-RPMI+13 L-glu+13 P/S+100 ng/ml FGF2+1 mMreti- blasts (GlobalStem) in DMEM/F12 (Invitrogen) supplemented with noic acid for 36–42 hours, then A-RPMI+13 L-glu+13 P/S+100 ng/ml 20% KnockOut Serum Replacement (Invitrogen), 1 mM nonessential FGF-9 (R&D Systems)+10 ng/ml activin A for 3 days. amino acids (Invitrogen), 2 mM GlutaMAX (Invitrogen), 0.55 mM 2-mercaptoethanol (Invitrogen), penicillin/streptomycin (Invitrogen), Immunofluorescence and 10 ng/ml recombinant human bFGF/FGF2 (Invitrogen). Cultures For immunofluorescence studies, cultures were washed once with PBS were passaged using collagenase type IV (STEMCELLTechnologies) at a (Invitrogen) and fixed in 4% paraformaldehyde for 15 minutes at room 1:3 split ratio every 5–7 days. For feeder-free culture, hESCs grown on temperature (RT). Fixed cells were then washed three times in PBS and mouse embryonic feeder fibroblasts were initially passaged using colla- incubated in blocking buffer (0.3% Triton X-100 [Fisher Scientific] and genase type IVonto plates coated with Geltrex hESC-qualified reduced 5%normaldonkeyserum[EMDMillipore]inPBS)for1houratRT.The growth factor basement membrane matrix (Invitrogen) according to cells were then incubated with primary antibody overnight at 4°C or for 2 manufacturer’s instructions and cultured in mTeSR1 medium (STEM- hours at RT in antibody dilution buffer (0.3% Triton X-100 and 1% BSA CELL Technologies) supplemented with penicillin/streptomycin or [Roche] in PBS). Cells were then washed three times in PBS and incubated ReproFF2 medium (ReproCELL) supplemented with FGF2. with Alexa Fluor 488–,555–,or647–conjugated secondary antibodies (1:500) (Molecular Probes) in antibody dilution buffer for 1 hour at RT. Differentiation For immunostaining with biotinylated LTL (Vector Labs), a streptavidin/ Forall differentiation experiments,hESCs or hiPSCsgrown onGeltrex biotin blocking (Vector Labs) and Alexa Fluor 488- or 647-conjugated were washed once with PBS and dissociated into single cells with streptavidin (Molecular Probes) were used according to manufacturer’s Accutase (STEMCELL Technologies). Cells were then plated at a instructions. Nuclei were counterstained with DAPI (Sigma-Aldrich). A density of 43104 cells/cm2 onto Geltrex-coated plates in mTeSR1 list of primary antibodies can be found in Supplemental Table 1. Immu- medium supplemented with the ROCK inhibitor Y27632 10 mM nofluorescence was visualized using an inverted fluorescence microscope (Stemgent). Cells were then fed daily with mTeSR1 without Y27632 (Nikon Eclipse Ti, Tokyo, Japan). Quantification was performed by for 2–3 days until they reached 50% confluency. To induce mesen- counting a minimum of five random fields at 310 magnification. doderm differentiation, the media were changed to Advanced RPMI (A-RPMI; Invitrogen) supplemented with 13 L-GlutaMAX (L-glu) Quantitative RT-PCR and 13 penicillin/streptomycin (P/S) and CHIR (Stemgent), human Total RNA was harvested and isolated from cells using the RNeasy Mini Wnt3a (R&D systems), and/or human activin A (R&D Systems) at Kit (Qiagen). One microgram of RNAwas used for reverse transcription the doses described. To induce definitive endoderm differentiation, with the M-MLV Reverse Transcription System (Promega), or 500 ng of cells were treated with A-RPMI+13 L-glu+13 P/S+5 mMCHIRfor RNA was used for High Capacity cDNA Reverse Transcription Kits 24 hours, then A-RPMI+13 L-glu+13P/S+100 ng/ml activin A for (Applied Biosystems). RT-PCR reactions were run in duplicate using 2–3 days. For hepatic differentiation, cells at the definitive endoderm cDNA (diluted 1:10), 300 or 400 nM forward and reverse primers, and stage were treated with A-RPMI+13 L-glu+13 P/S+13 B27 supple- iQ SYBR Green Supermix (Bio-Rad) or iTAQ SYBR Green Supermix ment (Invitrogen)+20 ng/ml BMP-4 (R&D systems)+10 ng/ml FGF2 (Bio-Rad). Quantitative RT-PCR was performed using the iQ5 Multi- (Invitrogen) for 5 days, then A-RPMI+13 L-glu+13 P/S+13 B27 color Real-Time PCR Detection System (Bio-Rad). All samples were run +10 ng/ml for 5 days, then hepatocyte cul- with two technical replicates. b-Actin was used as the housekeeping ture medium (Lonza) supplemented with 20 ng/ml Oncostatin M gene. Primer sequences are listed in Supplemental Table 2. and 10 ng/ml hepatocyte growth factor (Peprotech) for 5 days. For pancreatic differentiation, cells at the definitive endoderm stage were Flow Cytometry treated with DMEM/F12+2% FBS (Hyclone)+50 ng/ml FGF-7 (R&D Cells were dissociated using Accutase for 15 minutes, and cell clumps systems) for 2 days, then high-glucose DMEM (Mediatech)+1% B27 were removed with a 40-mm cell strainer (BD Biosciences). Cells were

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fixed with 2% paraformaldehyde for 15 minutes on ice and then with a primary antibody solution of diluted antibodies in PBT with permeabilized with 0.1% Triton for 15 minutes on ice. Cells were 1% donkey serum and samples incubated overnight at 4°C. Antibody then blocked with PBS+5% donkey serum for 15 minutes on ice and dilutions used were mouse anti–human nuclear antigen, 1:250 incubated with primary antibodies (PAX2 1:1000, LHX1 1:100) for 30 (Millipore), and rabbit anti-laminin, 1:500 (Sigma-Aldrich). Explants minutes. After washing three times with 1% BSA in PBS, cells were were then washed with PBT three times for 1 hour each with rocking at incubated with secondary antibodies (Alexa Fluor 488–conjugated don- room temperature. Secondary antibody solution (PBT+1% donkey se- key anti-rabbit 1:5000, Cy5-conjugated donkey anti-mouse 1:2500 rum) with 1:250 dilutions of Alexa Fluor 488–conjugated donkey anti- [Jackson ImmunoResearch]) for 20 minutes on ice. Cells were then mouse and Alexa Fluor 568–conjugated donkey anti-rabbit antibodies washed three times with 1% BSA in PBS. Flow cytometry was performed (Invitrogen) were added and incubated for 1–2 hours at room temper- using MACSQuant (Miltenyi Biotec), and data analysis was performed ature. Samples were then washed with PBT three times for 30 minutes using FlowJo software. Optimal dilution ratios of antibodies were de- each at room temperature, followed by a 10-minute incubation with termined using a negative control human proximal tubular cell line DAPI, and three additional 5-minute washes with PBS. Explant samples (HKC-8) that does not express PAX2 or LHX1. HKC-8 was kindly pro- were then mounted with Vectashield (Vector Labs) and examined vided by Dr. Lorraine Racusen (Johns Hopkins Hospital). using a Nikon C1 confocal microscope.

Chimeric Kidney Explant Cultures Supcapsular Implantation Chimeric kidney explants were createdusingpublishedreaggregation Two wells of a confluent 24-well plate containing LTL+KSP+ tubular techniques.43,66 Briefly, embryonic kidneys at stage E12.5 (day of structures were scored by needle, scraped off in clumps in 100 mlof plug=E0.5) were isolated from timed pregnant females (Charles River). DMEM/F12, and injected through an insulin syringe beneath the Complete urogenital systems were placed in DMEM (Corning Cellgro) kidney capsule of adult NOD-SCID mice. Growths were harvested without serum and further dissected to isolate single E12.5 kidneys. Four 3 weeks later, photodocumented, fixed in 4% paraformaldehyde, and to six kidneys were incubated in TrypLE Express (Invitrogen) at 37°C for paraffin-embedded or cryopreserved for serial sectioning. 4 minutes. The enzyme was then quenched by adding kidney culture media (DMEM + 13 P/S + 10% FBS or renal epithelial cell basal media [Lonza] with 0.5% FBS) and incubating at 37°C for 10 minutes for re- ACKNOWLEDGMENTS covery. Digested kidney rudiments were then transferred to a microcen- trifuge tube with additional kidney culture medium and dissociated by The authors thank Dr. Andrew Elefanty for the rat anti-MIXL1 an- repeated trituration. The cell suspension was then passed through a 100- tibody and Drs. Hiroshi Itoh and Toshiaki Monkawa for the mouse mm-pore-size cell strainer before visualizing to confirm single cell sus- anti-KSP antibody. pension and counting. Differentiated human pluripotent stem cells from This study was supported by National Institutes of Health grants day 3, day 6, or day 9 were dissociated with TrypLE, visualized, and RC1 DK0864406 (J.V.B., A.Q.L.), R01 DK39773 (J.V.B.), F32 DK084692 counted. Reaggregation was done by mixing 130,000 dissociated mouse (A.Q.L.), F32 DK092036 and NIH LRP (B.S.F.), and R00HD061981 kidney cells with 13,000 differentiated human cells in a microcentrifuge (P.L.); American Heart Association grant 11FTF7320023 (A.Q.L.); the tube and centrifuging the chimeric mixture into a pellet at 700g.The Harvard Stem Cell Institute (A.Q.L., M.T.V., J.V.B.) and the Uehara resulting chimeric pellet was then placed onto a Nucleopore Track-Etch Memorial Foundation (R.M.); and a Grant-in-Aid for JSPS Postdoctoral Membrane filter disk (pore size=1 mm) (Whatman),67 and the filter was Fellowship for Research Abroad (R.M.). floated on 1 ml kidney culture medium (or renal epithelial cell basal media)+10 mMY2763268 in a 24-well tissue culture plate (two explants per 13-mm circular filter) and incubated for 24 hours. After the initial DISCLOSURES incubation, the media with Y27632 were replaced with kidney culture None. medium (or renal epithelial cell basal media) only and cultured for an additional 2 days. No qualitative difference in morphology or growth rate was observed between the two media recipes. REFERENCES

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