Based on previous evidence [17,20] and our preliminary results in combination with renal-specific TFs, it selected five TFs that potentially participate in inducing the NPC phenotype (OSR1, SIX1, SIX2, and PAX2) or promote reprogramming as an EMT regulator (SLUG) for screening NPC-inducting TFs. To convert to an NPC phenotype, we retrovirally transduced human UCs at early passages 2–5 with these factors and then cultured these cells in a chemically defined medium containing FGF2, FGF9, activin A, and retinoic acid for 9–15 days, which was based on the study that reported the renal lineage differentiation of human ESCs and iPSCs [21]. At day 15 post-induction, few putative iNPC-like colonies were observed, but more importantly little or no expression of endogenous SIX2 (Figure S1a), widely regarded as one of the critical makers for nephron progenitors, was detected while expanding the picked colonies. Afterward, Yamanaka factors were examined for generating NPCs because their transient overexpression has been well-established to change cell fates between developmentally distant cell types [22]. It attempted to generate three putative NPC lines from UCs overexpressing OSKM. mRNA level expression of typical nephron progenitor makers such as SIX2, CITED1, EYA1, WT1, and GDNF was detected in these cells (Figure S1b). Although expression of the pluripotency markers OCT4 and NANOG were lower in OSKM-transduced UCs than in iPSCs [23], their expression exhibited considerable levels in acquiring a pluripotent status. Based on these results, we investigated whether OSKM in combination with SLUG (referred to as OSKM-SLUG), enabling to regulate EMT, has a synergistic effect on the generation of iNPCs from UCs and allows these cells to maintain their phenotype. Surprisingly, expression of SIX2 and CITED1, which are cap mesenchyme (CM) markers, was elevated in OSKM-SLUG-transduced UCs, while expression of pluripotency markers (NANOG and OCT4) was reduced (Figure S1c). These findings indicate that the generated iNPCs have different pluripotent behaviors from iPSCs, but are similar to ESC-NPCs. ESC-NPCs served as a positive control (Figure S2a,b), previously well-established [24]. Although the combination of OSKM and SLUG exhibited the highest similarity to ESC-NPCs in the phenotype, these cells were not expandable under the present culture condition and gradually lost their progenitor properties (data not shown).

Figure 1a outlines the process by which functional NPCs were successfully generated from human UCs. To maintain and expand undifferentiated NPCs, OSKM-SLUG-transduced UCs were immediately exposed to the nephron progenitor niche, which contained FGF9, Heparin, BMP7, LDN-193189, CHIR99021, and Y-27632. This signaling environment was established by modifying nephron progenitor expansion medium (mNPEM) as previously described [4], in which SIX2+/CITED1+ CM progenitors derived from embryonic kidneys or human ESCs can be propagated by manipulating the BMP, FGF, and WNT signaling pathways while preserving the potential for renal differentiation. At 12 days post-transfection, colonies were observed and expanded as single cells on Matrigel-coated plates by manual picking (Passage 1). Upon further passaging, putative NPCs with a small, spindle-shaped mesenchymal morphology similar to that of ESC-NPCs were established (Figure 1a and Figure S2a). During the conversion process, expression of the NPC markers WT1, SIX2, CITED1, and NCAM1 increased in these cells, comparable to ESC-NPCs (Figure 1b) [25]. Moreover, mRNA expression of pluripotency (NANOG) and NPC (SIX2, CITED1, GDNF, and NCAM1) markers were analyzed in four established lines of iNPCs (Figure 1c). Specification of posterior intermediate mesoderm into NPCs of the metanephric mesenchyme (MM) is characterized by co-expression of WT1, SALL1, PAX2, and GDNF [26,27]. The reciprocal interaction between the MM and ureteric bud tips contributes to the formation of dense clusters called the CM in which SIX2 and CITED1 expression is activated [28,29]. Accordingly, up-regulation of endogenous SIX2 and CITED1 is considered to be a strong indicator of successful conversion into NPCs [30]. Meanwhile, NCAM1+ cells, found in human fetal kidneys, retain their nephrogenic potential during in vitro culture and elicit beneficial effects on the progression of kidney disease [31]. NCAM1 is downregulated during differentiation into kidney epithelial cells and re-activated in a specific subset of cells that undergo dedifferentiation to behave as highly stem/progenitor cells (e.g., in the regenerative response following kidney damage) [32]. Furthermore, GDNF signaling through the Ret receptor is required for ureteric bud growth and branching morphogenesis during kidney development [33].

In an attempt to determine the minimally required set of TFs for the generation of iNPCs from human UCs, individual factors were removed from the pool of OSKM-SLUG. Conversion of human UCs into iNPCs involved EMT-like morphologic changes and mRNA expression of SIX2 and CITED1 (Figure 1d,e and Figure S1d). mRNA expression of SIX2 was markedly reduced by removal of each of the five TFs and was highest in cells co-overexpressing all five TFs, rather than in ESC-NPCs. These findings were consistent with the iNPC colony-forming efficiencies of female- and male-derived UCs (referred to as F- and M-UCs, respectively) (Figure 1f).

The iNPCs established from F-UCs (Figure 2a,b) expressed the CM markers SIX2 and CITED1, consistent with the immunostaining and western blot results obtained with ESC-NPCs. ESC-NPCs served as a positive control. The exogenous expression of each of the reprogramming genes OSKM and EMT regulator SLUG was evaluated by RT-PCR, which demonstrated that all five genes (OSKM and SLUG) were expressed in iNPCs at passage 8 (Figure S3a). By contrast, except for endogenous SLUG, there was only minimal or no expression of the endogenous OSKM genes in the iNPCs (P8) compared with human iPSCs-positive control (Figure S3b). We next injected these cells into immunodeficient mice and monitored tumorigenesis over 12 weeks (Figure S3c); no tumor formation was observed in comparison with the U87MG cell- and iPSC-injected sites, implying the absence of tumorigenic potential of iNPCs.

In vitro expansion of iNPCs is an important prerequisite for potential medical applications, including cell therapy, renal disease modeling, drug screening, and reconstitution of the functional kidney in vitro. Related studies proposed that an in vitro nephron progenitor niche can expand NPCs derived from embryonic kidneys or human ESCs by modulating the FGF, BMP, and WNT pathways [4,5]. Based on previous evidence, it optimized serum-free conditions for the propagation of iNPCs generated from UCs. To investigate the effect of the coating material on cell growth, it compared the proliferation of iNPCs on non-, gelatin- and Matrigel-coated plates (Figure 2c and Figure S4a), which are widely used for stem cell culture, in modified NPEM was evaluated by removing each one from mNPEM (Figure 2d and Figure S4b). Removal of each additive reduced cell proliferation, while FGF9 was a key supplement for in vitro expansion of these cells. The established iNPCs were propagated in mNPEM on Matrigel-coated plates for 30 passages, with repeated freeze-thaw cycles, and continued to robustly express the CM markers SIX2 and CITED1; SIX2+ population purity was 90.93% and 84.85% at passages 8 and 30, respectively (Figure 2e,f). iNPCs generated from F- and M-UCs retained a normal karyotype for at least 20 (Figure 2g) and 10 passages (Figure S4c), respectively, demonstrating their stable expandability in vitro.

Next, we compared the global gene expression patterns of iNPCs with those of parental UCs and ESC-NPCs by RNA sequencing (RNA-seq). Hierarchical clustering of the whole transcriptome (26,256 genes) showed the genome-wide conversion of F- and M-UCs into iNPCs and demonstrated a high degree of similarity between ESC-NPCs and iNPCs (Figure 3a). In particular, the scatter plot of the first two principal components revealed a close relationship between iNPCs (3 independent F- and M-iNPC lines) and ESC-NPCs (3 independent BG01-ESC- and H9-ESC-NPC lines), while clearly demonstrating the successful separation of iNPCs and UCs (Figure 3b). While UCs exhibited no or low expression in a gene set related to nephron development (SIX2, CITED1, EYA1, WT1, OSR1, PODXL, DLL1, BMP7, DCHS1, GPC3, NOTCH3, and PDGFRA), these genes were highly upregulated in both iNPCs and ESC-NPCs (Figure 3c), which is supported by the qPCR analysis in SIX2, CITED1, EYA1, WT1 and OSR1 (Figure S5). These comparisons indicate that UCs were converted into iNPCs with renal progenitor characteristics comparable to ESC-NPCs. Furthermore, similar to ESC-NPCs, the gene ontology (GO) categories significantly enriched in iNPCs were gene sets related to kidney development and nephrogenesis (Figure 3d). These results suggest that the generated iNPCs were almost entirely converted to NPC fate during the conversion process and thus possessed lineage specificity at the global RNA level.

To verify that the established iNPCs could differentiate into the main components of the nephron, human female UC-derived iNPCs were transferred to fibronectin-coated plates in mNPEM and 1 day later exposed to previously reported media with specific compositions for inducing differentiation into glomerular podocytes or renal tubular cells (Figure 4a and Figure 5a) [34,35]. F-UCs and ESC-NPCs served as negative and positive controls, respectively. After 1 week of differentiation, it observed a homogeneous population of multinucleated cells with a large cell body and an arborized morphology (Figure S6a), which is consistent with a previous study showing podocytes generated from human iPSCs [36]. qRT-PCR analysis indicated that expression of podocyte-specific markers, such as podocalyxin, synaptopodin, and nephrin, was significantly increased in the podocytes differentiated from iNPCs and ESC-NPCs when compared to UCs cultured under the same condition (Figure 4b). Meanwhile, the expression of SIX2 in iNPCs and ESC-NPCs was decreased during podocyte differentiation (Figure S6b). These results are in agreement with the previous finding that SIX2+ NPCs gradually give rise to nephron epithelia in which cells exhibit downregulation of SIX2 and express renal subtype-specific markers [21,37]. Moreover, immunostaining analysis demonstrated that iNPC- differentiated podocytes expressed the podocyte-specific protein SYNAPTOPODIN, PODOCYLAXIN, and NEPHRIN, which was similar to ESC-differentiated podocytes (Figure 4c and Figure S6c). Functional activity of the differentiated podocytes was determined by endocytic uptake of FITC-labeled albumin at 4 °C (inhibits albumin endocytosis) or 37 °C (permits endocytosis) [38,39]. Podocytes play a key role in the glomerular filtration barrier that impedes the passage of large proteins and macromolecules such as albumin from the blood to the urinary ultrafiltrate. As shown in Figure 4d and Figure S6d, albumin-containing vesicles were observed within the podocytes placed at 37 °C, but very low at 4 °C. There was a significant increase in the amount of albumin taken up by iNPC- and ESC-NPC-differentiated podocytes compared to the cells derived from UCs. Next, differentiation of iNPC lines into renal tubular cells was observed after 3 weeks of induction, with mesenchymal-to-epithelial morphological changes. These cells expressed the proximal tubular cell-specific markers CD13, AQP1, and LTL and epithelial maker E-CADHERIN, as determined by qRT-PCR and immunostaining (Figure 5b,c). Moreover, the differentiated cells showed the functional activity of the proximal tubule via uptake of fluorescently labeled dextran as previously reported [40]. While a substantial amount of dextran was accumulated in iNPC- and ESC-differentiated tubular cells, very little dextran was found in the cells induced from UCs (Figure 5d and Figure S6e). Thus, these results demonstrate the differentiation potential of iNPCs in directed differentiation towards functional podocytes and renal tubular cells. Human ESC and iPSC-derived NPCs undergo the mesenchymal-to-epithelial transition (MET) and form glomeruli and renal tubules when exposed to FGF9 and a low dose CHIR (Figure 6a) [37]. Similarly, the generated iNPCs and ESC-NPCs were aggregated and underwent nephrogenesis (Figure 6b,c and Figure S7). These clonally derived aggregates expressed segmental markers of the nephron, including glomerular podocytes (PODOCYLAXIN) and renal tubules (E-CADHERIN), and had lumens (Figure 6c). These observations are consistent with earlier findings concerning nephron structures induced from ESCs and iPSCs [5,21,41]. In the human kidney, E-CADHERIN is abundant in the distal tubule, while PODOCYLAXIN is more dominant in the glomeruli (E-CADHERIN+/PODOCYLAXIN) [37,42]. Interestingly, the immunostaining images reveal the expression of these two markers, indicating the co-existence of immediate precursors of distal tubule epithelial cells and podocytes. These results imply the potential for generating a kidney organoid originated from the immature cell clusters consisting of multiple cell types, including podocytes it  tubular cells. To further support the nephrogenic potential of UC-derived iNPCs, we attempted to perform a chimeric aggregate assay by mixed culture with E12.5 mouse embryonic kidney cells at the liquid-air interface for 7 days, as previously described [43,44,45]. The chimeric aggregates were dynamically changed to the morphology of tubular branches and renal vesicles, similar to the complex tubular epithelial networks (Figure 6d). The converted cells (HuNu+) were found to integrate into E-cadherin+ nephron segments (Figure 6e). Nevertheless, the chimeric aggregates were maintained for up to 7 days and after then, they were disintegrated, which is similar to what was observed for chimeric aggregate analysis with iPSC-derived kidney progenitors [18,45].