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Figure S1. Gene expression coordinately regulated with NCAM1 during mouse kidney development. HeatMap of gene expression in different renal compartments and time points during mouse kidney development and adulthood, that their expression was coordinately regulated with NCAM1 expression. Genomic data was retrieved from the GUDMAP database (Harding et al, 2011) and analysed using Pearson correlation assay across the normalized gene expression profiles.
Figure S2. NCAM1 expression module. A scheme of the NCAM1 module network. Genomic data was retrieved from GUDMAP database (Harding et al, 2011) and analysed using Toppcluster (Kaimal et al, 2010). Genes in the NCAM1 expression module that are known to play a role in neurogenesis at some level are highlighted.
Figure S3. Characterization of hFK cells (A) FACS analysis of surface marker expression in hFK cell cultures. Each marker was tested on at least three independent samples. Data was calculated as the average percentage of expressing cells ± SDEV. (B and C) Immunofluorescence staining of hFK cells cultured with SFM for CD24 (B, red) and EpCAM (C, green). Nuclei were stained with Dapi (blue). Images were obtained using Olympus DP72 camera attached to Olympus BX51 fluorescence microscope and processed via cellSens standard software. (D and E) Isolation of NCAM1+ cells from hFK cells. Representative FACS sorting analysis of NCAM1 expression in hFK cells cultured in SFM at passage #1. Data are presented in histogram graphs, mean fluorescence intensities (MFI) and percentage of NCAM1 expressing cells. Shown are NCAM1 (right panel) and isotype control (left panel) staining (D). (E) Validation of sorting purity of NCAM1− (left panel) and NCAM1+ (right panel) sub-populations. (F) Characterization of NCAM1 cells sorted from SCM cultures. qRT-PCR analysis of gene expression in NCAM1 cell fractions grown in SCM. Normalization was performed against control GAPDH expression and RQ calculated relative to NCAM1− cell fraction. Data were analysed using SDS 3.2 software and presented as average RQ ± SDEV of three replicates. ***p < 0.001, *p < 0.05 versus NCAM1.
Figure S4. Calibration of immunoflorescence staining of SIX2. Double IF staining of SIX2 (green) and NCAM1 (red) in hFK NCAM1+ (B) and NCAM1− (D) sorted cells. Cells from primary cultures of Wilms tumour (A) or adult kidney (D) were used as positive or negative controls (respectively) of the SIX2 staining. Nuclei were stained with Dapi (blue). Images were obtained using Zeiss SLM 510 microscope and processed in ImageJ/Fiji software. Scale: 50 µm.
Figure S5. In vitro characterization of NCAM+
cells. (A) Representative graph of self-renewal in NCAM1+ cells sorted from hFK that was cultured in SCM. Data are presented for each passage as relative number of clones developed from the total number of cells plated. (B) Percentage of surviving NCAM1+ cells after sorting and culturing with or without BMP7 for 7 days. Each treatment was tested on three independent samples. Number of cells was calculated as percentage of the control group and presented as average of three replicates ± SDEV. (C) Percentage of surviving NCAM1+ cells after sorting and culturing with or without BIO. Number of cells was calculated as percentage of the control group. (D) Proliferation analysis of hFK cells treated with different dosages of IMGN901 (0–1.675 μM) as demonstrated by MTS proliferation assay. OD levels are presented as percentage of the control sample (0 μM). (E) FACS analysis of the NCAM1 population in hFK treated with different dosages of IMGN901 (0–55 nM). Results are presented as percentage of the control sample (0 μM). FIB, fibronectin coating.
Figure S6. Renal differentiation of hFK cells on chick CAM. (A) hFK cells grafted on the chick CAM. Graft images were obtained using Scion Corporation colour digital camera attached to Olympus SZX12. (B) Summarizing table of the amount of hFK cells grafted on the CAM (grafted cells, replicates, visible grafts and grafts with tubule-like formations). (C–E) H&E staining of paraffin embedded sections of 0.35 × 106 (C), 1.25 × 106 (D) and 2.5 × 106 (E) grafted cells. Images were obtained using Scion colour digital cameras attached to Olympus BX51TF microscope. (F–K) IF staining of Ki67 (F–H, red) and pan-CK (I–K, green) in paraffin embedded sections of 2.5 × 106 cell grafts. Hoechst 33342 (Blue) was used for nuclear staining. Images were obtain using Olympus AX70 motorized microscope and spectral unmixing using multispectral imaging system (NuanceFX camera and software).
Figure S7. Chronic renal disease model of 5/6-Nephrectomy (5/6 Nx) in NOD/SCID mice. (A) Schema of in vivo experiments: 5/6 Nx in NOD/SCID mice was performed in two steps (Nx1-unilateral nephrectomy and Nx2-2/3 nephrectomy). One week after the second step, hNPCs were injected directly into the remnant kidney (transplantation #1–3) in three cycles, with 3 weeks intervals between each injection. Blood and urine were collected 2 weeks after each cell injection (sample collection #1–3). Starting on the 10th week mice were fed by high protein food followed by another blood and urine sample collection (#4). Fourteen-week after the second step of the Nx the mice were sacrificed and the kidneys were removed for IHC or gene expression analysis. (B) hNPCs were labelled with CM-Dil Cell Tracker Mice injected with saline served as controls. Sections of the injected kidneys were then analysed by confocal microscopy. While saline injected kidney show no detectable signal, hNPCs are seen in the injected kidneys as soon as 24 h after the injection and are still detectable at 2 weeks. (C) RT-PCR for human-specific β2-microglobulin (hB2m) was performed to validate the presence of human cells in the injected mouse kidney. Representative real-time PCR amplification plot demonstrates amplification in the hNPC-injected mouse kidneys, but not in untreated (naïve) or saline-injected kidneys. Mouse β-actin (mβ-actin) served as endogenous control. (D) IHC with human specific antibody for pan-cytokeratin/MNF116 is shown in positive control hFK tissue (left panel), and in two negative control NOD/SCID kidney tissues: healthy/naive mouse kidney (MK-naïve, middle panel) and in 5/6 Nx kidney injected with saline (MK-5/6 saline, right panel). (E) IHC with human specific antibody for EMA in MK-5/6 saline (right panel) and in positive control human adult kidney (AK) tissue (right panel). (F) IHC with human antibody for ENPEP is shown in both MK-5/6 saline (left panel) and AK (right panel) tissues. Bar represents 200 μm.
Table S1. Expression analysis of NCAM1+ cells with oligonucleotide microarrays.
Table S2. Differential expressed genes in NCAM1+ cells.
Table S3. Primary antibodies used in FACS assays.
Table S4. Secondary antibodies used in the FACS assays.
Table S5. Antibodies used in IHC or IF staining..