Generation of human iPS cell-derived mesenchymal cells capable of forming alveolar organoids [scRNA-Seq]

We have developed a method to generate human induced pluripotent stem cell (iPSC)-derived mesenchymal cells (iMES) that were able to induce AT1 and AT2 epithelial cells within their organoids (iMES-AO). Single-cell transcriptome analysis comparing iMES-AO with our previously reported alveolar organoids using human fetal lung fibroblasts delineated not only differences in composition of epithelial lineages but also distinctive mesenchymal lineages. Overall design: Lung progenitors differentiated from SFTPC-GFP reporter iPSC were co-cultured with human fetal lung fibroblasts (HFLF) or iMES differentiated from its parental iPSC (201B7) to form alveolar organoids in matrigel (P0). Then, SFTPC-GFP positive cells were recovered by fluorescence-activated cell sorting (FACS) and passaged for generating alveolar organoids with newly differentiated iMES or HFLF (P1). The passage was repeated again to form alveolar organoids (P2). Single-cell RNA sequencing (scRNA-seq) of alveolar organoids at P0 and P2 was performed. Single-cell RNA libraries were prepared by using a 10x genomics Chromium device according to the manufacturer's protocol (Single Cell 3' Reagent Kits v3.1 or Single Cell 3' Reagent Kits v3.1(Dual Index)). Alveolar organoids with iMES (iMES-AO) at P0, Alveolar organoids with HFLF at P0 and P2 were prepared using Dual Index Kit TT Set A. iMES-AO at P2 was prepared using Single Index Kit T Set A. The libraries were sequenced using NovaSeq 6000 (Illumina).