Single-Cell RNA Sequencing Analysis of Kidney Organoids derived in 2D or 3D from SCTi004-A hiPSC line using the STEMdiff™ Kidney Organoid Kit

Kidney organoids differentiated from human pluripotent stem cells are complex structures that capture glomerular, tubular and mesenchymal cell types of the developing human kidney. The STEMdiff™ Kidney Organoid Kit is a serum-free cell culture medium system for the efficient and reproducible generation of kidney organoids in 2D using a simple, 18 day, two-stage differentiation protocol. We recently also showed compatibility of this kit for the generation of 3D organoids using single cell dissociation and re-aggregation on Day 7. Here we evaluate kidney organoids derived from the commercially available hiPSC line SCTi004-A with the STEMdiff™ Kidney Organoid Kit for their cellular diversity and transcriptomic profiles. Datasets were created for kidney organoids differentiated in the 2D and 3D format. Overall design: hiPSC line SCTi004-A, previously maintained in mTeSR™ Plus maintenance medium, was differentiated to kidney organoids at p35 with the STEMdiff™ Kidney Organoid Kit. Organoids were generated in 2D format as described in the manufacturer PIS or as 3D aggregates for 18 days, with the addition of CloneR™2 at the time of seeding. At the end of differentiation, 3D organoids were dissociated with cold 0.05% trypsin and DNase, with multiple rounds of incubations and triturations until a single cell suspension was achieved. For the 2D format, tubules were enriched by a crude removal of the mesenchymal monolayer by a brief incubation with trypsin and forceful trituration. The remaining tubules were dissociated to single cells with fresh trypsin and DNase with multiple rounds of incubations and triturations. Single cell capture and library prep was performed using the Pipseq V T10 3' Single Cell RNA workflow.