Figure 4. In vitro-derived airway organoid cells recapitulate molecular features of native fetal airway cells.

A) Feature plots showing expression of bud tip progenitor markers SFTPC and SOX9 and basal cell marker TP63 from scRNA-seq of in vitro cells at day 0 (bud tip progenitor maintenance), day 3 (after 3 days of DSA) and day 21 (3 days DSA, 18 days DSI). Additional feature plots of the Day 0 scRNA-seq data are shown in Figure 1C. B) tSNE projections from day 0, day 3 and day 21 in vitro derived cells showingthe sum of z-transformed expression levels of 331 fetal basal cell cluster markers, as identified from human fetal basal cells in Fig. 3, in day 0, day 3 and day 21 in vitro samples. C-E) Heatmap showing Pearson’s correlation coefficients (PCCs) of transcriptome between each of 2,000 randomly selected cell from each time point (day 0 [C], day 3 [D] and day 21 [E]) and the 12 fetal epithelial sub-clusters. PCCs were calculated using log-normalized expression levels of genes that are top 50 markers in any of the fetal epithelial sub-clusters and detected in in vitro cells. Colors represent range of PCCs for all cells in each time point. F) 21 day organoids were subjected to scRNA-seq and visualized by tSNE. Colors represent the fetal cell type identity as described in Fig 3 with the highest PCC to each individual 21 day cell. Top right corner: feature plot showing expression pattern of EPCAM across day 21 cells. C-F) MCP=multiciliated precursor, MC=multiciliated, NE=neuroendocrine, SP=secretory progenitor, BP=bud tip progenitor, BA=bud tip adjacent, BC=basal cell, SMG=submucosal gland, SMB=submucosal basal, GS=goblet-like secretory, IC=intermediate ciliated, CS=club-like secretory.