Subfractionation of differentiating human embryonic stem cell populations allows the isolation of a mesodermal population enriched for intermediate mesoderm and putative renal progenitors

Human embryonic stem (ES) cells are pluripotent and are believed to be able to generate all cell types in the body. As such, they have potential applications in regenerative therapy for kidney disease. However, before this can be achieved, a protocol to differentiate human ES cells to mesodermal renal progenitor lineages is required. Reduction of serum concentration and feeder layer density reduction cultures were used to differentiate human ES cells for 14 days. Differentiated ES cells were then fractionated by flow cytometry based on expression of the markers CD24, podocaylxin and GCTM2 to isolate putative renal cells. These cells upregulated the expression of the renal transcription factors PAX2, LHX1 and WT1 when compared to unfractionated human ES cells. Immunohistochemical assays confirmed that a subset of cells within this fraction co-expressed nuclear WT1 and PAX2 proteins. Transcriptome profiling also showed that the most differentially upregulated genes in this fraction differentially associated with kidney development in comparison to any other lineage. When compared to a transcriptome profile database of urogenital development (GUDMAP), the top 200 differentially upregulated genes in this fraction strongly clustered into a group of genes associated with the metanephric mesenchyme at E11.5 and the corticonephrogenic interstitium at E15.5 of murine kidney development. Hence, this approach confirms an ability to direct human ES cells towards a renal progenitor state.