Figure 1.
KSC-derived proximal tubule cells display normal absorptive function.
(A) Immunofluorescence staining shows that some KSC-derived cells expressed the proximal tubule marker megalin (green). Nuclei are stained with DAPI (blue). (B) Immunostaining shows that FBSA (red) was uptaken by megalin+ KSCs (green); nuclei are stained with DAPI (blue). Asterisks indicate cells co-stained for megalin and FBSA. (C) In vitro functionality assay demonstrates that in the presence of either receptor-associated protein (RAP), or an excess of unlabeled BSA, the uptake of FBSA (red) was almost completely blocked. Nuclei are stained with Hoechst 33342 (blue). Scale bars are 50 µm (A), 25 µm (B) and 100 µm (C).
Figure 2.
E11.5 re-aggregated kidneys form organotypic renal structures.
(A, B) Immunostaining for Wt1 (red) and Pax2 (green) (A), or Wt1 (green) and laminin-111 (red) (B) shows the presence of condensed MM (pink arrowheads) surrounding the UB (white arrowheads) after 3 days of culture, thus demonstrating that the induction of the mesenchyme had taken place. (C) Immunostaining for megalin (green) and Pax2 (blue) at day 3 of rudiment culture reveals the presence of proximal tubules (open arrowheads). (D) Immunostaining for synaptopodin (green) demonstrates the presence of nascent podocytes within developing glomerular structures (open arrowheads) at 5 days of culture. Scale bars are 75 µm (A), 150 µm (B, C) and 100 µm (D).
Figure 3.
KSCs generate proximal tubule cells and podocytes within the developing nephrons.
(A, B) Chimeric organoids were stained for megalin (green). Arrowheads indicate QD-labeled (white), megalin+ KSCs within a nascent proximal tubule. Note that megalin is present on the apical surface of the nascent proximal tubule (dotted lines in B), thus recapitulating its normal expression pattern observed in vivo. (C, D) Chimeric organoids were stained for synaptopodin (green) and laminin-111 (red). Arrowheads point to QD-labeled (white), synaptopodin+ KSCs within a nascent glomerulus. Scale bars are 20 µm (A, B) and 8 µm (C, D).
Figure 4.
Population growth of cultured MM.
Growth curve of MM cells cultured in the presence of Bmp7 and Fgf2 for the following times: days 1–4 (P1, black), days 5–8 (P2, blue), days 9–12 (P3, red). The MM cell population expanded by >12-fold during the P1 and P2 culture periods, but ceased expanding during P3. Data are expressed as mean ± SD; n = 6 for P1 and P2; n = 5 for P3.
Figure 5.
Expression of kidney progenitor markers in culture MM.
(A) MM cells were cultured for the indicated time periods, and immunostained for Wt1 (green) and Pax2 (red). Wt1 was expressed in MM cells throughout the period studied, but there were noticeably fewer Wt1+ cells by day 8. Pax2 was only detected for the initial 24 hours of culture. Nuclei were stained with DAPI (blue). Scale bars for all images, 100 µm. (B) RT-PCR shows the expression profile of key MM markers during the 12-day culture period. NTC, no template control.
Figure 6.
The extent of KSC integration into developing nephrons within chimeric rudiments is similar to that of MM cells.
(A) Chimeras generated with QD+ (white)-labelled (i) freshly isolated MM, (ii) MM that had been cultured for 4 days, or (iii) KSCs, were cultured for 3 days and immunostained for Wt1 (green) and laminin-111 (red), or for Pax2 (green). Arrowheads point to QD+ cells integrated within the forming nephrons. Scale bars are 30 µm, 15 µm and 15 µm (upper row), and 36 µm, 31 µm and 10 µm (lower row). (B) The percentage of KSCs that integrated into developing nephrons was significantly lower than that of freshly isolated MM cells, but not statistically different to that of MM cells cultured for 4 days (Student’s t-test; P>0.05). Results are expressed as mean ± SE. n = 3 in each group; for each organoid, 7 random developing nephrons were selected for analysis.