End-stage renal disease (ESRD) is a leading public health issue associated with increased morbidity and mortality. Currently, renal transplantation remains the curative treatment yet with limited organ availability. The organ structural complexity is a major hurdle in restoring kidney function and overcoming this hurdle is in critical need to advance renal replacement medicine. Recent advances of 3D/2D pluripotent stem cell-based approaches and the development of kidney organoids have opened up the possibility of novel renal replacement systems. However, it remains to be answered whether the stem cells generated in 3D or 2D have the equivalent nephrogenic potential.

We generated a Rosa26-E2-Crimson/T-GFP mouse embryonic stem cell line and differentiated the cells under 3D and 2D culture conditions, respectively. E2-Crimson-expressing T-GFP+ mesodermal cells were isolated and analysed for the expression profile of key nephrogenic genes. Their renal differentiation potential was also investigated by incorporating them within an ex vivo 3D self-assembly embryonic kidney organoid model.

Representative nephrogenic gene expression patterns were confirmed. In the assembled organoid model, abundant nephron features including proximal tubules and glomeruli with mature podocytes were observed. It was also revealed that the majority of T-GFP+ mesodermal cells derived from 3D culture condition displayed a vascular progenitor-like property and eventually differentiated into endothelial cells in the organoids. In contrast, the T-GFP+ mesodermal cells arising from 2D culture system appeared to develop into renal stroma.

This study showed distinct nephrogenic potential between T+ mesodermal cells arising in 3D and 2D differentiation culture. By integrating the differentiation strategies, our kidney organoid model can provide novel insights into the construction of hierarchical renal structure towards a synthetic kidney for renal replacement therapy to improve patient outcomes.