Kidney Gene Expression within Mouse Embryonic Stem Cells
Purpose: The major focus of this project is to develop an in vitro assay capable of differentiating mouse ES cells towards a renal lineage with the vision of integration of these cells into a damage/repair model of kidney disease. Mouse embryonic stem (ES) cells can be cultured to form aggregates termed embryoid bodies (EBs) that spontaneously produce cells of the three primary embryonic germ lineages. Controlled differentiation of ES cells towards specific cell types may one day prove useful in the cell-based therapy of many diseases.
Methods: Embryoid bodies were differentiated over a 16-day period in serum-free media and collected for total RNA extraction. This differentiation series was then analyzed for kidney gene expression profiles using real-time RT-PCR analysis. Two unique reporter ES cell lines were generated using modified ‘urogenital specific’ Pax2 gene constructs. Transgenic ES cell lines generated from this approach were analyzed by FACS to determine the spontaneous profile of urogenital Pax2 expression.
Results: Grown under serum free conditions, cells within EBs can be enriched towards mesodermal fates with the addition of BMP4 to culture. Here we show spontaneous expression of a panel of kidney genes during EB differentiation. Pax2-GFP and Pax2-lacZ reporter ES cell lines reveal spontaneous urogenital Pax2 expression occurs in mature EBs with greatest expression after two weeks of differentiation.
Conclusions: Mouse ES cells spontaneously differentiate to express genes important to kidney organogenesis. Pax2 transgenic ES cells can be used to define conditions for enhanced kidney cell generation from ES cells.