Promoting Maturation of Human Pluripotent Stem Cell-Derived Renal Microtissue by Incorporation of Endothelial and Mesenchymal Cells.

Directed differentiation of human pluripotent stem cells (hPSCs) uses a growing number of small molecules and growth factors required for in vitro generation of renal lineage cells. Although current protocols are relatively inefficient or expensive. The first objective of the present work was to establish a new differentiation protocol for generating renal precursors. We sought to determine if inducer of definitive endoderm 1 (IDE1), a cost-effective small molecule, can be used to replace activin A. Gene expression data showed significantly increased expressions of nephrogenic markers in cells differentiated with 20 nM IDE1 compared with cells differentiated with activin A. Thus, renal lineage cells could be generated by this alternative approach. Afterward, we determined whether coculture of endothelial and mesenchymal cells could increase the maturation of three-dimensional (3D) renal structures. For this purpose, we employed a 3D coculture system in which hPSC-derived kidney precursors were cocultured with endothelial cells (ECs) and mesenchymal stem cells (MSCs), hereafter named RMEM (renal microtissue derived from coculture of renal precursors with endothelial and mesenchymal stem cells). hPSC-derived kidney precursors were cultured either alone [renal microtissue (RM)] or in coculture with human umbilical vein endothelial cells and human bone marrow-derived mesenchymal stem cells at an approximate ratio of 10:7:2, respectively. Immunofluorescent staining showed expressions of kidney-specific markers synaptopodin, LTL, and E-cadherin, as well as CD31 ECs that were distributed throughout the RMEMs. Quantitative real-time polymerase chain reaction analysis confirmed a significant increase in gene expressions of the renal-specific markers in RMEMs compared with RMs. These findings demonstrated that renal precursors cocultured with endothelial and MSCs showed greater maturity compared with RMs. Moreover, ex ovo transplantation induced further maturation in the RMEM constructs. Our novel approach enabled the generation of RMEM that could potentially be used in high-throughput drug screening and nephrotoxicology studies.