Direct Transcriptional Reprogramming of Adult Cells to Embryonic Nephron Progenitors

BASIC RESEARCH www.jasn.org

Caroline E. Hendry, Jessica M. Vanslambrouck, Jessica Ineson, Norseha Suhaimi, Minoru Takasato, Fiona Rae, and Melissa H. Little

Institute for Molecular Bioscience, University of Queensland, St. Lucia, Australia

ABSTRACT Direct reprogramming involves the enforced re-expression of key transcription factors to redefine a cellular state. The nephron progenitor population of the embryonic kidney gives rise to all cells within the nephron other than the collecting duct through a mesenchyme-to-epithelial transition, but this population is exhausted around the time of birth. Here, we sought to identify the conditions under which adult proximal tubule cells could be directly transcriptionally reprogrammed to nephron progenitors. Using a combinatorial screen for lineage-instructive transcription factors, we identified a pool of six genes (SIX1, SIX2, OSR1, EYA1, HOXA11,andSNAI2) that activated a network of genes consistent with a cap mesenchyme/nephron progenitor phenotype in the adult proximal tubule (HK2) cell line. Consistent with these reprogrammed cells being nephron progenitors, we observed differential contribution of the reprogrammed population into the Six2+ nephron progenitor fields of an embryonic kidney explant. Dereplication of the pool suggested that SNAI2 can suppress E-CADHERIN, presumably assisting in the epithelial-to-mesenchymal transition (EMT) required to form nephron progenitors. However, neither TGFb-induced EMT nor SNAI2 overexpression alone was sufficient to create this phenotype, suggesting that additional factors are required. In conclusion, these results suggest that reinitiation of kidney development from a population of adult cells by generating embryonic progenitors may be feasible, opening the way for additional cellular and bioengineering approaches to renal repair and regeneration.

J Am Soc Nephrol 24: 1424–1434, 2013. doi: 10.1681/ASN.2012121143

The direct conversion of one differentiated cell type Within the field of nephrology, there is consider- to another through the forced expression of key able interest in regenerative medicine for the transcription factors has been shown to be feasible treatment of ESRD. However, the well characterized for a number of distinct cell types, including the stem cell population responsible for giving rise to conversion of fibroblasts to neurons1 and cardio- the functional units of the adult kidney, the neph- myocytes.2 These advances and other similar ad- rons, exists only during the embryonic state.10,11 vances provide the potential for cellular therapies These nephron progenitor (NP) cells are a mesen- in tissues, including heart, liver, pancreas, and the chymal population residing within the periphery of nervous system.1–7 Such lineage conversion is the developing kidney in a region termed the cap thought to require the reactivation of a critical endogenous gene regulatory network, with the introduced key genes removing epigenetic barriers Received December 3, 2012. Accepted April 5, 2013. to re-establish the attractor state of the cell type C.E.H. and J.M.V. contributed equally to this work. 8,9 required. Although there have been impressive Published online ahead of print. Publication date available at examples of direct reprogramming to well charac- www.jasn.org. fi terized and phenotypically identi able mature cell Correspondence: Prof. Melissa H. Little, Institute for Molecular types, for some tissues, it is the generation of stem/ Bioscience, University of Queensland, St. Lucia, 4072, Australia. progenitor cells that may be required for the regen- Email: [email protected] eration of complex structures. Copyright © 2013 by the American Society of Nephrology

1424 ISSN : 1046-6673/2409-1424 JAmSocNephrol24: 1424–1434, 2013 www.jasn.org BASIC RESEARCH mesenchyme. These cells, in turn, are derivatives of the Odd- expression profile of the nephron progenitor/cap mesenchyme skipped-related 1 (Osr1)+ /Wilmstumor1 (WT1)+ meta- population defined in the mouse kidney,20,21 11 transcriptional nephric mesenchyme, which gives rise to both the Sine oculis regulators potentially playing a role in specification of the neph- homeobox homolog 2 (Six2)+ nephron progenitors and the ron progenitor phenotype were selected. Other genes included Foxd1+ stromal progenitors of the kidney.12 Specification of have previously been identified to either specify epithelial-to- these separate lineages seems to occur from Osr1+ intermedi- mesenchymal transition (EMT; SNAI1 and SNAI2) and/or con- ate mesoderm before the onset of kidney development, al- fer stemness (Octamer-binding transcription factor 4 [OCT4]/ though Osr1 activity is only required for the formation of High mobility group AT-hook 2 [HMGA2]) (Figure 1A and Sup- nephron progenitors and not the stromal progenitors.12 plemental Table 1). Each gene was cloned separately into a len- Throughout kidney development, WT1 continues to be ex- tiviral construct accompanied with green fluorescent protein pressed in the NPs as well as the developing nephrons.13 How- (GFP) to identify successfully infected cells. To reprogram to a ever, Six2 exclusively marks the NP compartment.14 Lineage nephron progenitor phenotype, we undertook combinatorial tracinghasshownthattheseSix2+ NP cells self-renew screening of these 15 different transcriptional regulators through throughout development to give rise to all of the epithelial lentiviral transduction of HK2 cells with each transcription fac- cells of the nephron other than the collecting ducts.15,16 tor either alone or combined with other factors. Infection was In this study, we have used a combinatorial screening approach performed in a 96-well plate format. Reprogramming can be to identify the genes required to reprogram human adult proximal facilitated through the active relaxation of the chromatin marks tubule cells to a kidney nephron progenitor phenotype. A major specifying lineage.22,23 Hence, epigenetic modifying agents, such challenge to such a project is the successful identification of the as the histone deacetylase inhibitor valproic acid (VPA) or 5- nephron progenitor end point. Unlike a mature well characterized azacytadine, can partially remove chromatin marks in fully dif- target cell type, the nephron progenitor has only been identified ferentiated cells, increasing the efficiency of reprogramming.24–26 and characterized in vivo during development. Hence, a robust For this reason, VPA was included during reprogramming and stringent assay of nephron progenitor potential was required. to assist in chromatin relaxation.25 In the screen, results were We also show that our previously described ex vivo organoid re- compared back with HK2 cells infected with lentiviral cassette combination assay can be used to selectively identify the nephron encoding GFP alone in the absence of VPA (HK2-VPA). progenitor capacity of introduced test cell populations. Using Figure 1A outlines the process of selection for successful this recombination assay together with a multistage screen, in- reprogramming events. This process first involved the assess- cluding changes in cellular morphology and the reinduction of ment of a significant morphologic change consistent with endogenous nephron progenitor gene and protein expression, EMT and the induction of Cbp/P300-interacting transactivator we describe the identification of a pool of six genes, SIX1, SIX2, 1 (CITED1) protein, a specific marker of the NP phenotype that OSR1, Eyes absent homolog 1 (EYA1), Homeobox A11 (HOXA11) is not required for NP specification or survival.27 Only 10 dif- and Snail homolog 2 (SNAI2), able to reprogram a proportion of ferent reprogramming pools induced consistent CITED1 pro- adult kidney epithelial cells to adopt a nephron progenitor-like tein, indicating putative induced NPs (Figure 1B). These 10 phenotype. These results show the feasibility of the potential to pools were then screened for the relative gene expression of reinitiate kidney development from an adult cell population. key components of the NP gene regulatory network. In doing this screening, we focused on a coordinated upregulation of NP markers accompanied by low or decreased epithelial cadherin RESULTS (E-CADHERIN) expression. To assess induction of endogenous gene expression rather than expression of the introduced repro- A Combinatorial Screening Approach for NP gramming transcripts, we used primers located in the untrans- Reprogramming Factors lated region of these genes (Supplemental Table 2), because only The efficiency of direct reprogramming correlates with the the coding sequence was delivered by lentivirus. All 10 pools lineage relationship between the start and end cell type.17 Hence, displayed some level of endogenous NP gene expression activa- adult progenitor cells, such as myeloid progenitor cells or pro-B tion. However, only one pool (pool 8), containing SIX1, SIX2, cells, can be reprogrammed to induced pluripotent stem cells OSR1, HOXA11, EYA1,andSNAI2,showedlowerE-CAD- with 300 times greater efficiency than their more differentiated HERIN coupled with coordinated activation of the NP gene progeny under identical conditions.18 For this reason, we used regulatory network (Figure 1C). Pool 8 also showed the highest the adult proximal tubule cell line human kidney 2 (HK2),19 level of SIX2 and CITED1 expression, genes that exclusively which will have initially been derived from an embryonic neph- mark the NP population in the developing kidney.15,16 Thus, ron progenitor followed by subsequent patterning, segmenta- we focused our additional analyses on pool 8 reprogramming. tion, and differentiation (reviewed in ref. 20). HK2 cells are epithelial in morphology and show clear expression of the tight Pool 8 Induced EMT and Specifically Activated the NP junction protein, zona occludens 1 (ZO-1), as well as cortical Gene Regulatory Network F-actin fibers typical of an epithelial morphology (Supplemental Reprogramming of HK2cells using pool 8was reanalyzed using Figure 1, A–E). Based on the known transcription factor morphology, RT-PCR/quantitative real-time PCR (qRT-PCR),

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and immunofluorescence. Results were compared back with either parental HK2 cells (HK2 parental) or HK2 cells infected with lentiviral cassette encoding GFP alone in the presence (HK2+VPA) or absence (HK22VPA) of VPA. Ten days after HK2 transduction with pool 8 reprogramming factors, the cells lost their cobblestone morphology, and they became elongated and spindle-shaped (Figure 2A). EMT markers Matrix metalloproteinase 9 (MMP9)and MMP2 were upregulated approximately 3- and 30-fold, respectively (Figure 2B). The apparent reinduction of NP gene expression was further analyzed to examine the effect of VPA. The addition of VPA alone seemed to result in an upregulation of Paired box gene 2 (PAX2). However, the coordinate upregulation of other NP genes (SIX2, EYA1, OSR1, and CITED1) was restricted to HK2+Pool 8 (Figure 2C). It has been previously reported that VPA also derepresses histone deacetylase- mediated silencing of E-CADHERIN,leading to sustained E-CADHERIN expression.28,29 This result was seen as an upregulation of E-CADHERIN in HK2+VPA cells, with this expression repressed to the level of HK2- VPA in pool 8-treated cells (Figure 2C). This replicated set of pool 8 reprogrammed cells showed evidence of a more convincing EMTevent than was seen in the initial screen. E-CADHERIN levels were on par with control cells as opposed to the 15-fold upregulation in the initial pass. This technical variability may be caused by the variation in response of the cultures to VPA. Because HK2 cells are an immortalized cell line, we also investigated whether a similar gene expression profile could be induced in primary human renal proximal tubule cells (RPTECs). Despite exhibiting some variation in gene upregulation com- Figure 1. Combinatorial screening for transcription factors is able to reprogram to a pared with HK2+Pool 8 cells, RPTEC+Pool nephron progenitor. (A) Combinatorial screening with 15 different factors was performed in 8 showed highly increased expression of the triplicate in 96-well plates. Criteria of morphologic EMT and detection of Cited1+ cells in NP-specific genes SIX2 and CITED1 two of three wells allowed progression to the qRT-PCR phase, which assessed a broader panel of NP markers. qRT-PCR results provided the basis for input populations for the (approximately 200- and 20-fold, respec- recombination assay, which assessed the capacity of reprogrammed cell populations to tively)aswellasSIX2proteinexpression behave as bona fide kidney progenitors. UB; ureteric bud. (B) Ten pools were identified (Supplemental Figure 2, A and B). Further- based on induction of CITED1 protein as assessed by immunofluorescence. E1, EYA1; H11, more, although PAX2 expression did not HOXA11;O1,OSR1;P2,PAX2; S1, SIX1;S2,SIX2; Sn2, SNAI2. (C) qRT-PCR to determine seem increased in RPTECs+Pool 8 when ex- the degree of reprogramming in HK2 cells infected with viral pools. qRT-PCR screening pressed relative to RPTECs-VPA, the level identified only one factor combination (pool 8; black) that showed consistent activation of of PAX2 expression in unmanipulated a variety of NP markers coupled with downregulation of E-CADHERIN. Gene expression is RPTECs was found to be 410-fold higher normalized to glyceraldehyde-3-phosphate dehydrogenase, and it is expressed relative to than the expression in HK2 cells (Supple- HK2 cells infected with GFP-containing lentivirus and cultured without VPA. mental Figure 2C), suggesting that these

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cells already possess a high endogenous level of PAX2. To validate the qRT-PCR and further investigate the specificity of this reprogramming event, we used RT-PCR (Figure 2D). Activation of NP markers SIX2 and CITED1 was differentially higher in HK2+Pool 8 cells, whereas sal-like 1 (SALL1) expression was detected at a similar level in the presence of VPA alone (HK2+VPA). HK2+Pool 8 cells also showed upregulation of forkhead box D1 (FOXD1), a marker of the renal stroma.30,31 However, this result was detected in both parental HK2 and HK2+VPA (Figure 2D). Because the VPA may randomly relax chromatin marks, we also investigated the expression of markers of the three germ layers, early mesoderm (Goosecoid, GSC), endoderm (FOXA2), and ectoderm (PAX6)as well as evidence for re-expression of a pluripotency marker (NANOG). Although we did detect weak expression of the ectodermal/ neural marker, PAX6,thisresultwasalso detected in control conditions. Finally, we examined whether the detectable levels of NP gene expression were accompanied by the endogenous production of proteins characteristic of the NP state but not in the lentiviral pool. Immunofluores- cence for PAX2 and CITED1 proteins in parental HK2, HK2+VPA, and HK2+Pool 8cellsshowedthespecificinductionof these proteins in GFP+ (lentivirally infected) HK2+Pool 8 cells (Figure 2E and Supple- mental Figures 3 and 4).

The Recombination Assay Is a Stringent Assay for NP Potential Because there are no NP cells within the Figure 2. Identiﬁcation of a pool of genes able to induce EMT and endogenous expression of NP markers. (A) Compared with HK2 cells infected with an empty lentiviral vector without postnatal kidney, there is no standard in vivo the addition of VPA (HK2-VPA), pool 8-reprogrammed HK2 cells (HK2+Pool 8) became assay to test NP potential in an adult mouse elongated and spindle-shaped by day 7 of reprogramming, consistent with an EMT. (B) qRT- model. In addition, because the expression PCR revealed that EMT markers MMP2 and MMP9 were upregulated ;30- and 3-fold, constructs delivered continue to be expressed, respectively, in HK2+Pool 8 cells relative to HK2-VPA control cells, whereas E-CADHERIN successfully reprogrammed cells will face con- levels were unchanged. (C) qRT-PCR showing coordinated upregulation of all NP markers stant pressure to remain in an NP state and in HK2+Pool 8 compared with HK2-VPA control cells, with simultaneous downregulation of hence, must be assessed for this phenotype. E-CADHERIN expression. qRT-PCR results are expressed as the average relative fold We have previously developed an assay in 6 change compared with control from three biologic replicates SEM. (D) RT-PCR analysis which to test NP potential using ex vivo orga- of the unmanipulated HK2 cell line (HK2 parental), HK2+VPA, and HK2+Pool 8 to examine noid cultures (Figure 3A).32,33 In this assay, (1) the endogenous expression level of markers of kidney development (SIX2, CITED1, embryonic day 12.5 (E12.5) kidneys were dis- SALL1,andFOXD1),(2) the marker of three germ layers (mesoderm [goosecoid; GSC], endoderm [FOXA1], and ectoderm [PAX6]), and (3) a pluripotency marker (NANOG). (E) sociated to single-cell suspensions, mixed with Immunoﬂuorescence analysis of the induction of PAX2 and CITED1 protein in the GFP+ test cells to form the recombination pellet, and lentivirally transfected cells within cultures of HK2+Pool 8 cells versus HK2 parental cells. cultured above a feeder layer of wingless-type Merge includes 4’,6-diamidino-2-phenylindole to visualize nuclei. Scale bars, 30 mm. MMTV integration site family member 4 (Wnt4)-expressing National Institute of

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Health 3-day transfer, inoculum 33105 cells (NIH3T3).34 The only), and Calbindin+ (ureteric epithelium only) populations recombined organoid culture recapitulates critical aspects of nor- (Figure 3, B–D). To address the stringency of this assay in de- mal kidney development, such as retention of specificsubcom- termining the NP potential of test populations, GFP+ fractions partments and their spatial arrangement (Supplemental Figure were FACS-sorted from the dissociated E13 kidneys of Sall1 5). Specifically, the Six2+ NP compartment is present in these GFP mice35 and recombined with unlabeled wild-type embry- cultures (Figure 3, C and F and Supplemental Figure 5). For this onic kidney. GFP+ cells were readily detectable within the Wt1+ approach to represent a stringent assay for NP potential, it was and Six2+ NP fields (Figure 3, E and F) but not in the Calbindin+ necessary to show (1) the presence of NP cells in such cultures, (2) ureteric epithelial compartment (Figure 3G). The capacity of the capacity for such endogenous NP cells to participate in neph- non-NP cells to integrate into the endogenous NP field was rogenesis, and (3) the evidence that only populations with an NP also examined to determine the stringency of the assay. Non- phenotype are able to integrate into the endogenous NP field. NP cell populations, including the mesonephric cell line M15,36 The ability to detect the origin of individual cells within a HEK293T, an immortalized cell line derived from human recombination was determined by mixing dissociated wild- embryonic kidney,37 primary isolates of murine bone marrow- type (unlabeled) and GFP-labeled E12.5 kidneys. GFP+ cells were derived mesenchymal stem cells (MSCs), and the human prox- able to contribute to all endogenous populations, including Wt1+ imal tubule cells used in this study (HK2),19 were assessed for (metanephric mesenchyme, NP, and nephron), Six2+ (NP their integration capacity in this assay. Each cell line was first infected with a GFP-containing lentivirus and FACS-sorted to isolate a pure population of GFP-labeled cells. All cell types were detected in the recombined organoids but showed negligible integration into the Six2+, Wt1+,orCalbindin+ compartments (Figure 3, H–K and Supplemental Figure 6A). Hence, the only population of cells able to robustly contribute to the NP field was bona fide NPs.

Pool 8 Reprogrammed HK2 Cells Differentially Integrate into the NP Population Ex Vivo The recombination assay was subse- quently used to assess the relative capacity of HK2+Pool 8 cells to integrate into the NP compartment comparison with parental HK2, HK2+VPA, and HK2 cells treated with recombinant TGFb1 for 3 days to induce a fibrogenic transformation. HK2+Pool 8 cells integrated into both Wt1+ and Six2+ compartments in recombination assays, which was evidenced by the surrounding 2 Six2+/GFP cells (Figure 4A, arrows), and showed nuclear expression of both WT1 and SIX2 proteins. However, the latter can be partially attributed to the inclusion of SIX2 in the reprogramming pool. Note that both antibodies recognize murine and Figure 3. Development of a stringent assay for NP potential. (A) E12.5 mouse kidneys human forms of the protein. In contrast, nei- were dissociated to single cells, mixed with GFP-labeled test cells, and processed to ther HK2+VPA nor HK2+TGFb1 cells – form a recombined metanephric kidney ex vivo.(B D) Single cells from a GFP-labeled showed robust integration into the Six2+ E12.5 kidney can be detected in the NP compartment marked by Wt1 and Six2 and + – compartment, although HK2+VPA cells did also in the calbindin ureteric bud (UB) compartment. (E G) Sall1-GFP NPs contribute hi to developing nephron compartments but not to the UB-derived calbindin+ compart- integrate into Wt1 structures likely to rep- fi ment. Arrows highlight integration sites of the test cell population. (H–K) Non-NP cell resent non-NP elds (Figure 4B). Relative lines M15, MSCs, HEK293Ts, and HK2s cannot contribute to the Six2+ NP compartment. quantitation of integration events revealed Cell lines were identified by anti-GFP antibody. Immunofluorescence for Six2 was used to that HK2+Pool 8 cells were approximately assess incorporation into NP compartments. Scale bars, 120 mminB–G; 360 mminH–K. fourfold more likely to integrate into a Six2+

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(Figure 4A). A fraction of these GFP+ cells were also positive for phosphohistone H3 (Supplemental Figure 7), indicating that pool 8-transduced cells not only integrate into endogenous renal structures but can also proliferate within the recombination assay.

EMT Is Not Sufficient for Reprogramming to an NP Phenotype EMT describes a phenotypic change that occurs both during normal development (type 1 EMT) and also in association with postnatal fibrosis and the initiation of metastasis in cancer (reviewed in ref. 38). This change uniformly involves a change in cellular morphology and the loss of expression of cadherins and cell–cell junction proteins accompanied by the re-expression of fibroblastic proteins, such as fibroblast- specific protein (FSP-1), vimentin, and MMPs.39,40 In culture, renal epithelial cells readily undergo EMT in response to vari- ous treatments, including TGFb1 or cyclosporine A.41–43 Whereas in the kidney, this EMT has been associated with the patho- logic progression to interstitial fibrosis,44 it has more recently been challenged.31 Cer- tainly, reprogramming from renal epithelial cells to cap mesenchyme will require an EMT. Although the murine orthologs of all six of the genes within pool 8 are expressed in the murine nephron progenitor population, many also show expression in preceding (MM) or subsequent (RV) structures (Figure Figure 4. Contribution of reprogrammed cells to the NP compartment in re- 4D). In the mouse, Snai2 is expressed only combination assays. (A) GFP+ HK2+Pool 8 cells (block arrows) contributed to both weakly in the endogenous NP population, the Wt1+ and Six2+ NP compartments but not the Calbindin+ compartment ex vivo. with expression more tightly associated 2 Additional Six2+/GFP cells surround the reprogrammed cells, indicating true in- with the cortical stroma (www.gudmap. tegration into the NP compartment. Scale bars, 30 mm. (B) Although unable to effi- org).45 SNAI2 is a potent inducer of EMT; ciently integrate into the Six2+ compartment, HK2+VPA cells did integrate into Wt1+ hence, it was possible that transduction with structures likely to represent early nephrons. Scale bars, 30 mm. (C) Quantitation of SNAI2 alone in conjunction with VPA treat- integration events revealed that, although HK2 cells treated with VPA alone or to- ment may be sufficient for reprogramming gether with pool 8 integrated into Wt1+ structures, HK2+Pool 8 cells preferentially + to an NP phenotype. Sequential removal of integrated into the endogenous ex vivo Six2 compartment. Both HK2 parental one factor at a time from the pool revealed and HK2+TGFb1-treated cells showed poor integration into either compartment. that SNAI2 was able to repress E-CAD- (n=36SEM). (D) Schematic representation of the expression domain in the murine developing kidney of the six reprogramming factors in pool 8. MM, metanephric HERIN, because removal of SNAI2 from mesenchyme; RV, renal vesicle. the pool led to a 40-fold upregulation of E-CADHERIN. In contrast, removal of any other reprogramming factor did not affect NP compartment compared with all other populations tested E-CADHERIN levels significantly (P,0.001) (Figure 5A). How- (P,0.05) (Figure 4C). As anticipated, although able to integrate ever, the expression of the NP marker CITED1 was unaffected into these regions, pool 8-reprogrammed HK2 cells did not by individual removal of any of the six factors, including SNAI2 integrate into the ureteric bud-derived Calbindin+ compartment (Figure 5A).

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Recently, the process of EMT has been linked to the upregulation of PAX2 protein was observed. In this instance, acquisition of a stem cell-like phenotype,46,47 possibly reflect- HK2+TGFb1 cells were seen within recombinations but did ing the reversal of processes involved in the initial differenti- not contribute to the Wt1+,Six2+,orCalbindin+ compart- ation of such cells. We, therefore, investigated the possibility ments (Figures 4C and 5B and Supplemental Figure 6B). that the simple induction of EMT in HK2 cells would This result argues that EMT alone is not sufficient to endow generate a nephron progenitor phenotype. HK2 cells infected aNPphenotype. with lentivirus containing SNAI2 alone underwent marked EMT, with a distinct transition to more elongated and spindle- shaped FSP-1+ cells (Supplemental Figure 8). However, when DISCUSSION SNAI2-overexpressing cells were tested in the recombination assay, only very rare cells were detected (less than five cells per The reprogramming of adult epithelial kidney cells to a NP entire recombined kidney), none of which integrated into phenotype represents a major advance for both kidney de- either the Wt1+ or Six2+ compartments (Figure 5B). Simi- velopmental biology and the reprogramming field. There are larly, the treatment of parental HK2 cells with recombinant no previous reports of kidney cells reprogrammed to a pro- TGFb1for3daysresultedinafibroblastic transformation genitor cell type, whether by forced expression of lineage- with the induction of FSP-1 protein and the loss of corti- instructive genes or manipulation of the local environment cal F-actin (Supplemental Figure 1, F–J). Of note, no with growth factors, epigenetic modifying agents, or small molecules. We have shown that overexpression of SIX1, SIX2, OSR1, EYA1, HOXA11,andSNAI2 in HK2 cells can re- programthemtoundergoanEMTand activate a gene regulatory network consistent with a cap mesenchyme/NP phenotype. Furthermore, a portion of HK2 cells transduced with this pool of genes showed a differential capacity to contribute to the NP compartment of a developing embryonic kidney ex vivo, showing the feasibility of direct reprogramming to generate nephron progenitors. The specific combination of reprogramming factors identified in this study supports much of what is known in kidney developmental biology. All five kidney developmental genes in the pool (OSR1, SIX2, SIX1, HOXA11,andEYA1) are expressed in the cap mesenchyme and essential for formation of this population in vivo (Figure 4D).48–51 PAX2 is also expressed in the cap mesenchyme,52 and accordingly, PAX2 expression in HK2s was upregulated in response to pool 8. PAX2 may not have been Figure 5. The role of EMT in reprogramming to a NP phenotype. (A) Removal of SNAI2 required in the pool, because its expres- from the reprogramming pool leads to dramatic upregulation of E-CADHERIN,with sion may have been activated by EYA1, no effect on CITED1 levels. (B) Reprogramming using SNAI2 alone or HK2 cells in- HOXA11,and/orSIX1, all of which com- duced to undergo EMT through treatment with TGFb1 failed to generate cell pop- plex with PAX2 to regulate kidney develop- ulations able to integrate into embryonic kidney recombinations. (C) Schematic ment.51,53,54 However, it may be able to diagram illustrating the observed outcomes of the reprogramming screen. (C, i) Ad- substitute for one or more of these factors. b fi dition of either recombinant TGF 1orSNAI2 transduction alone resulted in a bro- Six2 is known to upregulate its own expres- genic EMT, with the resulting cells unable to contribute to the nephron progenitor sion, and recent studies investigating Six2 compartment of the developing explants. (C, ii) Undirected chromatin relaxation mediated by VPA treatment biases the HK2s to the NP/cap mesenchyme fate, but the target genes during murine kidney devel- cells cannot overcome existing epigenetic barriers; therefore, they are only partially opment suggest direct regulation of Osr1 reprogrammed. (C, iii) Lineage-instructive transcriptions factors together with SNAI2 and Hox11 paralog expression by Six2 in reduce the existing epigenetic barriers, which then allows (C, iv) transition from the self-renewing undifferentiated cap mesen- HK2 proximal tubular phenotype to the NP/cap mesenchyme phenotype. chyme,55 further reinforcing the interactions

1430 Journal of the American Society of Nephrology J Am Soc Nephrol 24: 1424–1434, 2013 www.jasn.org BASIC RESEARCH between this particular set of transcription factors in defining the average of 0.5 integration events per section and approxi- nephron progenitor attractor state. This finding suggests the mately 140 slides per recombined kidney, we conservatively possibility for remaining redundancy in this pool. As such, ad- estimate a reprogramming efficiency of ;0.875% in response ditional dereplication may enable the substitution of genes to to pool 8 reprogramming. This result represents a 433 higher reduce the total number of transcription factors required. rate of reprogramming than in the initial study showing the SNAI2 was the only reprogramming factor without a pre- generation of induced pluripotent stem cells (1 in 53104 start- viously shown role in kidney development. Removal of SNAI2 ing cells).60 In that instance, the identification of a successful from the reprogramming pool suggested a role in the suppres- outcome relied on a combination of genetic selection, prede- sion of E-CADHERIN and subsequent destabilization of the fined optimal culture conditions, and a characteristic colony epithelial phenotype. Consistent with this finding, SNAI2 has phenotype for pluripotent cells in culture. None of these fea- been reported to repress E-CADHERIN and is involved in tures was available within the screen described here. Having EMT.56–59 In the context of Waddington’s epigenetic land- identified a key gene regulatory network capable of NP reprog- scape,8 which depicts the dynamics of cellular differentiation ramming,itwillnowbefeasibletorecreatethisapproach during development, SNAI2 may serve to overcome epigenetic using inducible, polycistronic constructs. This result will en- barriers and make a smoother and more efficient transition to sure an increased efficiency of integration of all genes and a a NP phenotypic state through this induction of EMT (Figure capacity to shut this program down in vitro or in vivo to show a 5C). However, it is possible that SNAI2 has more specific roles to subsequent capacity to move from nephron progenitor to play in the NPstate. Presumably, the remaining five genes in pool nephron. Furthermore, reducing toxicity by removing the 8 are critical for ensuring survival and integration in the correct need for multiple lentiviral constructs may also improve reprog- developmental context (Figure 5C). If the starting cell type was ramming of primary human cells, for which our preliminary not epithelial, such as a skin fibroblast, SNAI2 may not have been results suggest are amenable to reprogramming with pool 8 fac- required. Importantly, however, proximal tubule cells treated tors. However, regardless of the parental cell type, it will be nec- with either recombinant TGFb1orSNAI2 alonewerenotable essary to optimize the cell culture conditions for the prolonged to contribute to the NP lineage, suggesting that EMTalone is not maintenance of a nephron progenitor population in culture. sufficient to recapitulate the specific NP phenotype. Major challenges to the reprogramming of adult cells to Althoughwe assume that the chromatinrelaxation provided nephronprogenitorsarethetransientnatureofthisattractorstate by the addition of VPA assisted the initial reprogramming, one and the intricate reliance of this population on niche instructive obstacle faced with its usage is the potential for nonspecific cues for its identity. The NP population is a very transient gene activation, which may result in an undesirable phenotypic embryonic population, existing in vivo for only 8–10 days of outcome. Indeed, treatment with VPA alone induced the development in the mouse and unable to survive in the absence upregulation of FOXD1 and SALL1. The further upregulation of an appropriate niche.61 It is also in a highly permissive state, of FOXD1 in the presence of pool 8 may even suggest that at ready to differentiate into nephron epithelia in response to least a portion of the population has adopted an even more Wnt9b-mediated canonical Wnt signaling,62 Wnt4-mediated primitive metanephric mesenchymal state or that both a noncanonical signaling,63,64 or even ectopic notch signaling.65 nephron progenitor and stromal progenitor population are Hence, the challenge during reprogramming, like during kidney present. Better defining the hetereogeneity of the response development, is maintaining the self-renewal of this population. will be important if this approach is to be harnessed for The growth conditions optimal for maintenance of nephron cellular therapy. Indeed, the induction of a heterogeneous, progenitors/cap mesenchyme in vitro have not been completely confused, or intermediate phenotype in a portion of cells un- defined, although there is evidence that it is mediated by fibro- dergoing reprogramming continues to represent a serious blast growth factor and bone morphogenetic protein signaling.66 challenge in the reprogramming field as a whole and cannot Optimization of these conditions will be critical for increasing be avoided in the absence of an appropriate selection marker. the efficiency of this reprogramming process. Thus, the development of a stringent functional assay was Despitetheseremainingbarriers,thisstudy representsamajor critical in this instance to enable detection of successful re- step forward to reprogramming in the renal field. Ultimately, the programming to specific intermediate progenitor phenotypes. generation of such cells may lead to cellular therapies for adult The recombination assay not only opened the way for a sys- renal disease. The observation in zebrafish that a NP population tematic quantitative analysis of putative kidney stem cell can be transplanted from one animal to another and generate populations but also provided the specific niche requirements neonephrons in an adult fish67 raises the possibility that a to support the NP population generated. selected population of NPs may behave similarly in an adult Although this assay allowed us to provide evidence that mammalian kidney. Indeed, it has recently been shown that reprogramming to a nephron progenitor state is feasible, the embryonic kidney organoids placed into adult rat kidney can approach used in thisstudy hasseveral limitations to overcome. undergo onward development and vascular development.68 The Successful reprogramming is known to be an inefficient optimization of methods for the expansion of NP in culture may process, with only a portion of the treated cells adopting the also allow the renewal generation of renal cells for nephrotoxicity desired end point without active selection.60 Based on an screening. Finally, the generation of nephron progenitors from

J Am Soc Nephrol 24: 1424–1434, 2013 Reprogramming to Nephron Progenitors 1431 BASIC RESEARCH www.jasn.org human adult cells may also assist us in developing approaches to For recombinations, slides were prepared for antibody staining as prolong or reinitiate nephrogenesis based on our understanding previously described.32,33 Antibody staining was performed overnight of the genetic regulation of this population. at 4°C for primary antibodies and for 1 hour for secondary antibodies. A list of antibodies and stains used and the manufacturers’ details are supplied in Supplemental Table 4; 4’,6-diamidino-2-phenylindole was CONCISE METHODS diluted to 1 in 1000 and incubated for 5 minutes before slides were mounted in Vectashield or Prolong Gold (Invitrogen) and photo- Cell Culture and Reprogramming graphed using an Olympus BX-51 upright or Zeiss Meta 510 confocal HEK293T (CRL-11268; ATCC), HK2 (CRL-2190; ATCC), and primary microscope. human RPTE (CC-2553; Lonza Australia) cells were maintained as per the manufacturer’s instructions (HEK293Ts and RPTECs) or as previ- Recombination Assay ously described (HK2s) plus 100 U/ml penicillin, 100 g/ml streptomy- Assays were performed as previously described.32,33 A total of 43105 cin, 2 nmol/L L-glutamine, and 0.1 mMhydrocortisone.69 M15 cells were single cells (5% exogenous cells for M15, HEK293T, and MSC cells maintained as previously described with the addition of L-glutamine.36 and 2% exogenous cells for HK2s) was centrifuged to form a single Primary bone marrow-derived murine MSCs were isolated and main- recombination pellet. The chosen ratio of test cells to embryonic tained as previously described.70 For lentiviral reprogramming, the kidney cells was based on both our own (unpublished) findings coding sequence of candidate genes (Supplemental Table 1) was cloned and the findings of others74 that the addition of higher numbers of into the pLV101G vector upstream of a hrGFP reporter through the test cells can have a disruptive effect on the self-organization of dis- Gateway Cloning System (Invitrogen). Third generation lentivirus was aggregated kidney cells. Quantification of integration events was cal- packaged as described elsewhere using 5 ml/ml Lipofectamine 2000 culated as the number of GFP+ cells associated with either a Wt1+ or 71 + fi fi (Invitrogen) instead of CaCl2. For reprogramming studies, HK2s Six2 cluster (a cluster is de ned as ve or more positive cells) in a were serum-deprived for 24 hours and then infected with lentivirus given section. Association was regarded as being in contact with more overnight before treatment with VPA (2 mM for 7 days; P4543; Sigma than one cell within a given cluster. Either 8 (Wt1) or 16 (Six2) sections Aldrich). These cells were split into one-third pieces 48 and 96 hours were analyzed per biologic replicate, where the sections were spaced after the initial treatment exposure. RPTECs were treated similarly to equally to cover the entire recombination pellet. Means were calculated HK2 cells above but were not serum-deprived. Control HK2s and based on three biologic replicates per cell line and expressed 6 SEM. RPTECs underwent identical treatment, and they were infected with a P values were calculated using a two-tailed homoscedastic t test. GFP-only lentivirus and cultured minus VPA. For EMT studies, HK2s (43103 cells/cm) were serum-deprived for 24 hours and then treated b with TGF 1 (20 ng/ml for 3 days; 240-B; R&D). ACKNOWLEDGMENTS qRT-PCR and RT-PCR We thank Dr. Ryuichi Nishinakamura for the provision of the Sall1- RNA was extracted from cell cultures using the RNeasy Mini Kit eGFP mice, Prof. Kerry Atkinson for the provision of bone marrow- ’ (QIAGEN) as per the manufacturer s instructions. Equal amounts of derived MSCs, Prof. Peter Koopman for access to constitutional GFP+ RNA were used to make cDNA using the SuperScript III First Strand mice, Prof. Tom Gonda for lentiviral vectors, and Prof. Andy Synthesis System (Invitrogen). qRT-PCR reactions were performed McMahon for provision of the Wnt4-expressing NIH3T3 cells. using Platinum SYBR Green Qpcr Supermix UDG (Invitrogen) as per This work was supported by Australian Stem Cell Centre Grant P067 ’ the manufacturer s instruction. All qRT-PCR reactions were per- and National Health and Medical Research Council, Australia Grant formed on the ABI Prism 7000 real-time machine with the manufac- ID631362. Microscopy was performed at the Institute for Molecular ’ turer s software. cDNA levels of target genes were analyzed using Bioscience/Australian Cancer Research Foundation Cancer Biology comparative Ct levels, normalized to glyceraldehyde-3-phosphate de- Imaging Facility, whichwas established with the generous support of the hydrogenase, and expressed as fold change in treated cells relative to Australian Cancer Research Foundation. C.E.H. is a Rosamond Siemon 72 control as previously described. Means were calculated based on at postgraduate scholar. M.H.L. is an National Health and Medical 6 least three biologic replicates per treatment and expressed SEM. Research Council Senior Principal Research Fellow. P values were calculated using a two-tailed homoscedastic t test. Conventional RT-PCR analysis was performed using One Taq ’ DNA polymerase (NEB) as per the manufacturer sinstruction. DISCLOSURES Primer sequences for qRT-PCR are supplied in Supplemental Table 2. None. 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