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Additional Supporting Information may be found in the online version of this article.

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STEM_1412_sm_SuppFigure1.tif595KSupplementary Fig. 1 – C-kit expression in adult rat kidney in the thick ascending limb of Henle's loop. (A,B) C-kit+ cells (green) were detected in the cortex and in the medulla, and exhibited co-localization with E-cadherin (red, upper panels) and Na-K-2Cl co-transporter (red, lower panels).
STEM_1412_sm_SuppFigure2.tif261KSupplementary Fig. 2- FACS analyses for c-kit positivity at different passages of c-kit+ cells in comparison to rat bone cells and using anti-mouse CD117-PE conjugated antibody. The vertical or horizontal axes on each histogram indicate the proportion of positive cells. (A) In early passage cells, the left panel reflects the isotype control and the right panel shows the proportion of positive cells (88.6 ± 5.5%). (B) In late passage cells, the left panel reflects the isotype control and the right panel shows the proportion of positive cells (76.2 ±8.6%). (C) Rat bone cells were isolated and comprised two different populations according to the size (first panel). The middle panels correspond to the isotype controls and the lower panels show the proportion of c-kit positive cells on the horizontal axis (average 7.6 ± 6.8%).
STEM_1412_sm_SuppFigure3.tif315KSupplementary Fig. 3 – Characterization of c-kit+/Lin– cells by flow cytometric analyses. (A) Flow cytometric analysis of c-kit+/Lin– cells: The horizontal axis of each histogram indicates the proportion of positive cells for each intra-nuclear marker OCT4, SOX2, KLF4, and C-MYC (orange line) in comparison to unstained cells (red line) and to secondary antibody alone (blue line). All secondary antibodies were Alexa-Fluor 568. Values are expressed as mean ± SD percentage of positive cells found. (B) Flow cytometric analysis of c-kit+/Lin– cells for Nestin, NF-H, Pax2, CD24, CD133, PECAM-1, Acta2, and CD90. The horizontal axis of each histogram indicates the proportion of positive cells for each intracellular or surface protein (orange line) in comparison to unstained cells (red line) and to secondary antibody alone (blue line). All secondary antibodies were Alexa-Fluor 568, except CD90-conjugtaed FITC. Values are expressed as mean ± SD percentage of positive cells found. Abbreviations: Neurofilamentheavy chain (NF-H), paired-box gene 2 (Pax2), platelet-endothelial cell adhesion molecule (PECAM-1), and alpha-actin 2 (Acta2).
STEM_1412_sm_SuppFigure4.tif172KSupplementary Fig. 4 – Characterization of c-kit+/Lin– cells in culture. (A) c-kit+/Lin– late passage cells grow in monolayers. C-kit+/Lin- cells were subcultured for more than a year (> 100 passages) without any evidence of senescence or growth arrest. The image reflects an example of a late passage cell (passage 50). Scale bar represents 100 μm. (B) Telomerase activity. Positive telomerase activity expressed by Log10 (delta Fluorescein/delta Rhodamine) in relative fluorescence units analyzed in passages (P) 11-24, 43-52, 65-66 of c-kit+/Lin– cells in comparison to the positive control (cell extract) and to the neonatal rat kidney (P = NS). (C) Karyotyping by G-banding method: C-kit+/Lin– cells exhibited normal karyotyping ∼5 passages after sorting.
STEM_1412_sm_SuppFigure5.tif641KSupplementary Fig. 5 - C-kit+ early and late passage cells undergo adipogenic, osteogenic, and epithelial differentiation. (A) C-kit+ early (P17-P22) and late (P48-P50) passage cells exhibited lipid droplet accumulation that stained positive for Oil Red O after adipogenic differentiation. Mesenchymal stem cells (MSCs) were used as a positive control. (B) Oil Red O quantification by absorbance (at 510 nm) showing a higher degree of staining in MSCs group in comparison to early passage and late passage cells (**P<0.01 and ***P<0.001, respectively). (C) PPAR-γ and adiponectin calculated as relative fold change in differentiated c-kit+ cells in comparison to MSCs (P=0.11 and **P=0.0024, respectively). (D) C-kit+ early (P17-P22) and late (P46-P50) passage cells exhibited calcium deposition that was positive for Alizarin Red S. MSCs were used as a positive control. (E) Alizarin Red S quantification by absorbance (at 550 nm) exhibiting a higher degree of staining in MSCs in comparison to early and late passage cells (***P<0.0001). (F) Runx2, osteopontin, and alkaline phosphatase (AP) calculated as relative fold change in differentiated c-kit+ cells in comparison to MSCs (*P=0.029, *P=0.012, ***P=0.0007, respectively). (G) C-kit+ late passage cells (P50-P52) formed packed clusters that detached and acquired an embryoid body-like morphology after 3 weeks growing in epithelium medium. Scale bars represent 50 μm. Error bars represent mean ± SEM.
STEM_1412_sm_SuppFigure6.tif716KSupplementary Fig. 6 – In vitro and in vivo endothelial differentiation of control cells. (A) In vitro Matrigel tube formation assay performed with c-kit+ late passage (P50-71) cells (scale bar represents 50 μm). (B) Matrigel assay at 6 hours showing that the number of tubes was not different between c-kit+ early (P15-P20) and late passage (P50-P55) cells, but was lower than MSCs (***P<0.001 and ***P<0.001, respectively). At 24h, both c-kit+ early and late passage cells formed more tubes than MSCs (***P<0.001 and ***P<0.001, respectively), although the number of tubes in c-kit+ late passage cells were higher than c-kit+ early passage cells (**P<0.01). MSCs formed longer tubes in comparison to c-kit+ early and late passage cells at both 6h (***P<0.001 and ***P<0.001, respectively) and 24h (***P<0.001 and ***P<0.001, respectively). Pictures represent three different experiments in triplicate. (C) Human umbilical vein endothelial cells (HUVEC) were used as a positive control of in vivo study of endothelial differentiation. They grew for 1 week in endothelial medium (EGM-2), were embedded in a Matrigel plug (n=2; 2×106 cells), and then injected into NOD-SCID mice. After 2 weeks, the Matrigel plug was removed, and stained with H&E. HUVEC formed network connections inside the Matrigel plug (scale bars represent 50 μm). (D) GFP-labeled HUVEC stained for vWF inside the Matrigel plug (confocal images, scale bars represent 10 μm). (E) GFP-labeled HUVEC stained for Acta2 inside the Matrigel plug (scale bar represents 10 μm). (F) Negative control of endothelial differentiation based on the injection of a Matrigel plug containing only EGM-2 into NOD-SCID mice. There were a few cells infiltrating the Matrigel plug and they did not form network connections (H&E staining scale bar represents 50 μm).
STEM_1412_sm_SuppFigure7.tif299KSupplementary Fig. 7 - Calcium (Ca2+) transient of undifferentiated and differentiated ckit+ cells, response of these cells to different vasoactive agents, and qPCR data after differentiation according to time in endothelial medium (EGM-2). (A) There was no difference on Ca2+ peak after Ang II administration at doses of 10 nM and 100 nM (P=0.19) in differentiated cells. (B) The response to endothelin-1 (ET-1; 100 nM) was more intense after differentiation (P<0.0001). (C) ET-1 response was dose-dependent (P<0.0001). (D) ET-1 response was related to extracellular Ca2+ (1.8 nM) and sodium (137 mnol/L), since their absence in the solution attenuated the response in differentiated cells (P<0.0001). (E) Extracellular Ca2+ and sodium absence attenuated ET-1 response more intensely in undifferentiated cells in comparison to differentiated cells (P<0.0001). (F) Endothelin Ca2+ influx-induced was attenuated by 2-APB (60 μM) (P<0.0001). (G) There was a biphasic attenuation of ET-1 response after antagonists administration; ETB antagonist (BQ-788 1 μM; P<0.0001) attenuated the response earlier whereas ETA antagonist (BQ -123 1 μM; P<0.0001) had a later effect. (H) The response to prostaglandin F2α (10 μM) was higher in differentiate cells (P<0.0001). (I) Prostaglandin F2α (PGF-2α, 0.1 μM) Ca2+ influx-induced was attenuated by 2-APB (60 μM) (P<0.0001). (J) Prostaglandin F2α response was attenuated after SQ 29,548 administration (P<0.0001). (K) The response to bradykinin (100 nM) was higher in undifferentiated cells (green line) in comparison to differentiated cells (red line; P<0.0001). (L) Bradykinin Ca2+ influxinduced was attenuated by 2-APB (60 μM) (P<0.0001). (M) The response to bradykinin was specifically mediated by B2 receptor, since HOE 140 (10 μM) attenuated Ca2+ influx (P<0.0001). (N) That response was dose-dependent, since HOE 140 1 μM had no effect. (O). B1 antagonist Lys-(des-Arg9, Leu8)-bradykinin trifluoracetate salt had no effect on bradykinin administration. (P) Gene expression by real-time PCR analyzed by the fold change (2ΔΔCt) according to time in the endothelium medium for endothelin type Ia receptor (P=0.0001), PGF-2α receptor (P=0.0001), and bradykinin B2 receptor (P=0.066). Data represent mean ± SEM.
STEM_1412_sm_SuppFigure8.tif351KSupplementary Fig. 8 - C-kit expression in different clones (1 cell/well) obtained in 96-well plates. C-kit receptor (red) was detected by immunofluorescence in three different clones (single-cell derived) obtained by serial dilutions in 96-well plates. Cell nuclei are stained blue with DAPI.
STEM_1412_sm_SuppTable1.pdf120KTable S1. Gene expression of c-kit+ /Lin- cells by quantitative PCR (qPCR). Results are expressed according to the mean Ct (threshold cycle).
STEM_1412_sm_SuppInfo.pdf115KSupporting Information

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