Figure 5. Global Transcriptomic Profiling of PSC-Derived Lung Progenitors and Their Differentiated iAEC2 Progeny.

(A) Time points in alveolosphere differentiation from which samples were taken for RNA-seq.

(B) Principal-component analysis (PCA) of gene expression variance across all samples based on 30,000 transcripts.

(C) Top: qRT-PCR of each sample, with mean fold change (2^−ΔΔCt) ± SD, n = 3 biological replicates (RUES2 samples), cells isolated for RNA separately (Wk 21), cells cultured for 4 days separately (Wk 21 DCI), and cells from separate lungs (Adult AEC2). *p % 0.05, **p % 0.01 by unpaired, two-tailed Student’s t test. Bottom: Log2 expression of SFTPC across all samples, with the dotted line representing “noise” because these levels of expression are not consistently detected by qRT-PCR.

(D) Heatmap of row-normalized expression of selected lineage markers across PSC-derived and primary samples.

(E) Heatmap of the top 10 genes upregulated in day 35 Tom+ cells versus day 15 progenitors (ranked by fold change, FDR % 0.05). Known AEC2 genes are shown in bold.

(F) Heatmap of the top 50 genes differentially expressed in day 35 Tom+ cells versus day 15 cells (ranked by FDR, FDR % 0.01). Known AEC2 genes are shown in bold.

(G) Heatmap of supervised hierarchical clustering based on the top 300 genes differentially expressed in day 35 Tom+ versus day 15 cells (ranked by absolute fold change, FDR % 0.01).

(H) Left: experimental design. Center: western blot for IκB-alpha, phospho-Stat3 (Tyr705), and pan-actin. Right: qRT-PCR of each sample, with mean fold change (2^−ΔΔCt) ± SD, n = 3 biological replicates separated on day 35; *p % 0.05, **p % 0.01 by unpaired, two-tailed Student’s t test.

See also Figure S5.