Expression of stem cell markers in the human fetal kidney

Abstract

In the human fetal kidney (HFK) self-renewing stem cells residing in the metanephric mesenchyme (MM)/blastema are induced to form all cell types of the nephron till 34(th) week of gestation. Definition of useful markers is crucial for the identification of HFK stem cells. Because wilms' tumor, a pediatric renal cancer, initiates from retention of renal stem cells, we hypothesized that surface antigens previously up-regulated in microarrays of both HFK and blastema-enriched stem-like wilms' tumor xenografts (NCAM, ACVRIIB, DLK1/PREF, GPR39, FZD7, FZD2, NTRK2) are likely to be relevant markers. Comprehensive profiling of these putative and of additional stem cell markers (CD34, CD133, c-Kit, CD90, CD105, CD24) in mid-gestation HFK was performed using immunostaining and FACS in conjunction with EpCAM, an epithelial surface marker that is absent from the MM and increases along nephron differentiation and hence can be separated into negative, dim or bright fractions. No marker was specifically localized to the MM. Nevertheless, FZD7 and NTRK2 were preferentially localized to the MM and emerging tubules (<10% of HFK cells) and were mostly present within the EpCAM(neg) and EpCAM(dim) fractions, indicating putative stem/progenitor markers. In contrast, single markers such as CD24 and CD133 as well as double-positive CD24(+)CD133(+) cells comprise >50% of HFK cells and predominantly co-express EpCAM(bright), indicating they are mostly markers of differentiation. Furthermore, localization of NCAM exclusively in the MM and in its nephron progenitor derivatives but also in stroma and the expression pattern of significantly elevated renal stem/progenitor genes Six2, Wt1, Cited1, and Sall1 in NCAM(+)EpCAM(-) and to a lesser extent in NCAM(+)EpCAM(+) fractions confirmed regional identity of cells and assisted us in pinpointing the presence of subpopulations that are putative MM-derived progenitor cells (NCAM(+)EpCAM(+)FZD7(+)), MM stem cells (NCAM(+)EpCAM(-)FZD7(+)) or both (NCAM(+)FZD7(+)). These results and concepts provide a framework for developing cell selection strategies for human renal cell-based therapies.

Figure 1. Immunostaining of putative stem cell markers.

(a–j) Representative immunostaining of SIX2, NCAM1, FZD7, ACVR2B and NTRK2 in paraffin embedded sections of HFK (12, 13 and 18 weeks of human gestation); (a–b) localization of SIX2 to the MM, predominantly to the CM. (c–d) predominant staining of NCAM1 in the MM (including CM) and its derivatives (S- and comma- shaped bodies) and renal stroma, but not mature tubules or UBs. (e–f) FZD7 demonstrates preferential localization to the nephrogenic zone including MM and its derivatives, UBs, and newly forming tubules but not the stroma. (g–h) ACVRIIB immunostaining demonstrates predominant expression in the nephrogenic cortex; MM and its derivatives (S and comma shaped bodies), UBs, parietal epithelium of fetal glomeruli but not in the stroma. (i–j) NTRK2 is detected in the MM (including condensates) and its derivatives, UBs and some differentiated tubules. Figures c, e and g are shown in low magnification (original×4), Figures a, b, d, f and h–j are shown in higher magnifications (original×40; I, original×20).

Figure 2. FACS analysis of single putative stem cell surface markers in HFK cells.

(a) Representative flow-cytometry histograms of surface marker molecules (green) EpCAM, NCAM1, FZD7, NTRK2, CD90, CD34, CD24, CD133, and their respective isotype controls (red) in HFK (19 weeks of gestation). (b) Summarizing bar graph of single marker staining in HFK (15–19 weeks of gestation). Each marker was tested on at least 3 independent samples. Data were calculated as average % of expressing cell±SD. Each marker was tested in 10 HFK.

Figure 3. FACS analysis of putative stem cell markers in EpCAM subpopulations.

(a) Representative zebra graph of EpCAM staining and the subpopulation gating. EpCAM subpopulations were gated according to EpCAM staining intensity (negative, dim or bright) versus FSC. (b) Representative dot plot graphs of NCAM1, PSA-NCAM, FZD7, NTRK2, CD24 and CD133 expression levels in EpCAM subpopulations of HFK. Quadrates were placed according to isotype control confiding the negative staining to the lower left quadrant. Percentage of cells in each subgroup appears on the lower right quadrant. (c) Summarizing bar graphs of NCAM1, PSA- NCAM, NTRK2, FZD7, CD24 and CD133 expression levels in EpCAM subpopulations. Data are average % of cells in each subgroup±SD. Analysis of each marker was performed at least three times.

Figure 4. FACS analysis of putative stem cell markers in NCAM/EpCAM subpopulations.

(a) Representative dot plot graphs of EpCAM staining. Cells were gated in two groups: EpCAM negative (neg) and EpCAM positive (pos) versus FSC. (b) Representative dot plot graphs of PSA- NCAM, FZD7, NTRK2, CD24 and CD133 co-staining with NCAM in EpCAM positive or negative populations of mid-gestation HFK. Quadrates were placed according to the isotype control confiding the negative staining to the lower left quadrant. Percentage of cells for each quadrant appears in the quadrant. (c) Representative dot plot graphs of CD24 and CD133 co-staining in EpCAM subpopulations of HFK. Quadrates were placed according to the isotype control confiding the negative staining to the lower left quadrant. Percentage of cells for each marker combination appears in the quadrant. (d) Summarizing bar graphs of CD24 and CD133 co-staining in EpCAM Subpopulations. Data are average % of cells in each subgroup±SD. Analysis of each marker was performed at least three times.

Figure 5. Immunostaining of putative stem cells markers.

(a–l) Representative immunostaining of FZD2, GPR39, DLK1, CD34, CD90 and CD24 in paraffin embedded sections of HFK (12, 13 or 18 weeks of gestation); (a–b) FZD2 immunostaining demonstrates widespread staining of renal tubules. (c–d) GPR39 immunostaining demonstrates ubiquitous expression in differentiated renal tubular and to a lesser extent in componenets of the nephrogenic cortex. (e–f) Dlk1 immunostaining demonstrates ubiquitous expression in differentiated renal tubular but not in MM and its derivatives renal, UBs or stroma. (g–h) CD34 immunostaining demonstrates exclusive localization to endothelial cells (glomerular and peri-tubular) in all parts of the HFK, including in the nephrogenic cortex. (i–j) CD90 immunostaining demonstrates predominant staining in renal tubular cells but not in MM and its derivatives, UBs or stroma. (k–l) CD24 immunostaining demonstrates widespread expression in mature tubules. Figure (c) is shown in low magnification (original×4), Figures a, e, g, i, k and b, d, f, h, j, l are shown in higher magnifications (original×20 and×40, respectively).

Figure 6. Gene expression analysis in sorted NCAM/EpCAM subpopulations.

Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis of (a) renal stem/progenitor genes (Six2, Cited1, Sall1, Wt1 and Pax2), (b) vimentin and E-cadherin (c) ‘stemness’ genes (β-catenin/CTNNB1, EZH2, BMI1, Nanog and Oct4) and (d) surface marker (FZD7, ACR2B, NTRK2, CD24 and CD133) gene expression in NCAM/EpCAM magnetically separated cells from HFK (15–19 weeks of gestation). Normalization was performed against control HPRT expression and RQ calculated relative to the NCAM- fraction. Data were calculated as average±SD of at least 3 independent samples. ***P<0.001, *P<0.05 versus NCAM-. Sall1 expression in NCAM⁺ EpCAM⁺ cells was near significance (p<0.059).

Figure 7. Gene expression analysis in sorted PSA-NCAM subpopulations.

Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis of (a) renal stem/progenitor genes (Six2, Cited1, Sall1, Wt1 and Pax2), (b) vimentin and E-cadherin genes expression in PSA-NCAM magnetically separated cells from HFK (15–19 weeks of gestation). Normalization was performed against control HPRT expression and RQ calculated relative to the PSA-NCAM⁻ fraction. Data were calculated as average±SD of at least 3 independent samples. ***P<0.001, *P<0.05 versus PSA-NCAM⁻. Sall1 expression in NCAM⁺ EpCAM⁺ cells was near significance (p<0.059).

Figure 8. FACS analysis of single putative stem cell markers in HAK cells.

(a) Summarizing bar graph of single marker staining in HAK cells. Data were calculated as average % of expressing cell±SD. Each marker was tested in 3 HAK. (b) Representative dot plot graphs of CD24 and CD133 co-staining demonstrate a large fraction of CD24⁺CD133⁺ cells in HAK. Quadrates were placed according to the isotype control confiding the negative staining to the lower left quadrant. Percentage of cells for each marker combination appears in the quadrant.

Figure 9. Changes in surface marker expression along renal progenitor differentiation.

A hypothetical model of changes in surface marker expression along differentiation of the nephric-lineage relevant for isolation of cell subpopulations. Abbreviations: MM, metanephric mesenchyme; CM, condensed mesenchyme; LM, loose mesenchyme; UB, ureteric bud.