Figure 3.

Human fetal kidney cells harbor renal epithelial stem/progenitor characteristics and exhibit basal canonical WNT activity. (A–D) HFK-PC cells express renal stem/progenitor markers. (A) Cellular appearance of cultured fetal kidney cells (upper panel) and clone formation by human fetal kidney cells, demonstrating their high clonogenic capacity. Culturing of 0.3 cells per well results in 5%–10% of single clone formation (lower panel). (B) Gene expression analysis of renal stem/progenitor genes. Quantitative RT-PCR (qat-PCR) analysis of Vimentin, PAX2, and SIX2, three representative genes expressed early during nephrogenesis. Normalization is performed against control GAPDH expression and real-time quantitative is calculated relative to the well differentiated HAK-PC cells. (C) Immunofluoresence staining for the expression of two representative early nephrogenesis transcriptional factors: WT1 and SIX2 (×20 and ×100). All nuclei are stained with DAPI (blue). The green florescence signal in the upper and lower panels corresponds to anti-SIX2 protein and anti-WT1 protein staining respectively. Both clearly show nuclear staining (representative stained nuclei are indicated with white arrows). (D) Flow cytometric analysis for CD34, CD45, and CD56/NCAM1 expression and corresponding isotype controls in HFK-PC cells. Results show negligible levels of CD34 (a well known marker of hematopoietic stem cells and vascular endothelial cells) and CD45 (leukocyte common antigen). Moreover, the HFK-PC contains 44% of CD56/NCAM1-positive cells. NCAM1 has been previously shown to be a stem/progenitor cell marker in the human fetal kidney.³¹ (E–G) HFK-PC cells harbor basal canonical WNT activity. (E) qRT-PCR analysis of WNT4 and frizzled 7 (FZD7), a representative receptor of the WNT signaling ligands. (F) Because WNT/β-catenin and SIX2 pathways have opposing actions (commitment and self-renewal of renal stem/progenitors, respectively), we sought to manipulate this balance so as to provide additional support that our HFK-PC cells contain a significant portion of cells with early renal stem/progenitor characteristics. Consequently, qRT-PCR analysis of SIX2, in HFK-PC after the addition of three different WNT pathway antagonists—dickkopf-related protein 1 (DKK1), secreted frizzled-related protein 1 (sFRP1), and Wnt inhibitory factor (WIF)—shows significant SIX2 increment compared with the control. Normalization is performed against control GAPDH expression and real-time quantitative is calculated relative to the well differentiated HAK-PC cells. Data are presented as the mean ± SEM from three separate experiments. (G) HFK-PC immunofluorescence staining for anti-active β-catenin (green) discloses its cytoplasmic and nuclear presence. All nuclei are stained with DAPI (blue). *P<0.05; **P<0.01 (t test). DAPI, 4',6-diamidino-2-phenylindole; NCAM-1, neural cell adhesion molecule 1.