Isolation and Characterisation of Renal Precursor Cells Derived from Human Embryonic Stem Cells. Homo sapiens

HES4 hESC were cultured in serum media and maintained on a layer of mouse embryonic fibroblast feeder cells at a density of 6 x 104 cells/cm2. For differentiation: hESC were differentiated for a total of 14 days. Differentiation was induced by passaging 4 human ES cell pieces onto 12 well plates seeded with 0.67 x 10E4 cells/cm2. Cells were maintained in media containing 20% FCS for 2 days before media containing 5% FCS was used. Reduced serum media was changed every second day for the remaining 12 days ESC cells were taken at time zero and RNA was isolated (hESC_undiff). Cells differentiated by the above procedure at day 14 were isolated and RNA extracted (Diff). The remaining cells were FACs sorted with antibodies to Podocalyxin, CD24 and GCTM-2. Two populations were isolated, 1: Positive for podocalyxin and CD24 and low level of expression of GCTM-2 (POD+CD24+GCTM2Low) and 2: Positive for podocalyxin and CD24 and negative for GCTM-2 (POD+CD24+GCTM2Neg). The experiment was repeated in triplicate. The final aim was to determine the gene expression enrichment in cells with the marker profile (Podocalyxin+, CD24+, GCTM-2-neg) which is predicted to be enriched for kidney precursors. Keywords: cell type comparison Overall design: Three consecutive passages of HES4 cells were grown in standard conditions, RNA derived or differentiated for 14 days subject to FACs sorting, collection and RNA isolated. Microarray was performed on a total of 12 samples, (3 replicates of 4 populations).