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)
[18][19]. 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
[20]. 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
[3][21]. 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)
[22][23]. 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
[24]. 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)
[21]. 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
[17][3][25]. In the human kidney, E-CADHERIN is abundant in the distal tubule, while PODOCYLAXIN is more dominant in the glomeruli (E-CADHERIN+/PODOCYLAXIN)
[21][26]. 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
[27][28][29]. 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
[30][29].