Directed Differentiation of Human Pluripotent Stem Cells for the Generation of High-Order Kidney Organoids.

Abstract

Our understanding in the inherent properties of human pluripotent stem cells (hPSCs)have made possible the development of differentiation procedures to generate three-dimensional tissue-like cultures, so-called organoids.Here we detail a stepwise methodology to generate kidney organoids from hPSCs. This is achieved through direct differentiation of hPSCs in two-dimensional monolayer culture toward the posterior primitive streak fate, followed by induction of intermediate mesoderm-committed cells, which are further aggregated and cultured in three-dimensionsto generate kidney organoids containing segmented nephron-like structures in a process that lasts 20 days. We alsoprovidea concise description on howto assess renal commitmentduring the time course of kidney organoid generation. This includes the use offlow cytometry and immunocytochemistry analyses for the detection of specific renal differentiation markers.