Time series single-cell transcriptomic analysis of AEC2 directed differentiation

We perform time-series global transcriptomic profiling of a directed differentiation protocol for generating alveolar epithelial type II cells (AEC2s) from pluripotent stem cells (PSCs). We analyzed 3 different timepoints in the RUES2 differentiation: 1) Day 0 undifferentiated cells, 2) Day 15 lung progenitors highly enriched in NKX2-1+ cells by CD47hi/CD26lo sorting, and 3) the outgrowth of these purified progenitors in 3D culture sorted again on Day 35 based on SFTPCtdTomato+ and SFTPCtdTomato- gating. For comparison to primary cells, we simultaneously sequenced RNA from purified primary fetal (21 week gestation) distal alveolar epithelial progenitors and purified adult human (HTII-280 sorted) AEC2s. In order to evaluate the effect of cell culture on primary fetal alveolar cells, parallel aliquots of the fetal cells were also exposed to 4 days of culture in DCI media. We find that AEC2 maturation involves downregulation of Wnt signaling activity, and that the highest differentially expressed transcripts in iPSC-derived AEC2s encode genes associated with lamellar body and surfactant biogenesis. Overall design: Transcriptional trajectories of individual cell states over time by scRNA-seq performed at the time intervals selected by our TPS algorithm. For each time point we profiled ~4000 cells, following PSC-derived cells sorted on NKX2-1GFP at the lung primordial progenitor stage of differentiation (day 15; Figure 3B) through day 31 of alveolar directed differentiation without further sorting. As a negative control we included a 7th sample, the day 15 non lung population, isolated based on GFP exclusion (NKX2-1GFP negative sorted). Flow cytometry monitoring of NKX2-1 and SFTPC locus activity at each of the 6 time points of alveolar differentiation (Figure 3B) indicated the expected emergence of SFTPCtdTomato expression over time in some cells, peaking on day 29 of differentiation, but a loss of NKX2-1GFP expression in other cells over time, predicting potential loss of lung cell fate in a subset of the population, consistent with our prior reports (McCauley et al., 2018)