Single-Cell RNA-Seq Analysis of long-term transplanted single-cell-derived mouse DASC in host lung

Distal airway stem cell (DASC) expressing basal cell restrictive transcription factor p63 and keratin-5 (Krt5) has a strong ability of regeneration after lung injury. Such cells are originated from primitive progenitors in distal airways and could expand/migrate to inflamed damaged lung parenchymal region to form ‘KRT5 pods’ once activated by various types of tissue injury. We isolated the mouse DASC and transplanted the GFP-labelled mDASC into the bleomycin-injured mouse lung by intratracheal instillation. Mice were sacrificed at 30 and 90 days post transplantation and their lung tissue were harvest to detect GFP signal by fluorescence stereomicroscope, the region of which was dissect for single cell-RNA-seq (scRNA-seq). We utilized scRNA-seq to trace the fate of transplanted mDASCs at 30 days and 90 days post transplantation, which revealed cell types differentiated from the transplanted DASCs. Bleomycin was intratracheally administrated to isoflurane-anesthetized mice at a concentration of 3U/kg 7 days prior transplantation (n=8). DASCs suspended in PBS (106 cells per mouse) were transplanted via trachea. At 30dpt and 90dpt, mice were sacrificed and their lung tissue were harvest to detect GFP signal by fluorescence stereomicroscope (MVX10, Olympus, Japan), the region of which was dissect for further work (30dpt= 3, 90dpt= 4). Firstly, the GFP+ tissues of lung were collected and immersed in cold F12 medium with 5% FBS, followed by being minced into small pieces and digested with dissociation buffer on shaker for 1~1.5h. Dissociated cells were filtered through 100 μm cell strainer, and Red Blood Cell Lysis Buffer was used to remove erythrocyte. Cell pellets were resuspended in DMEM containing with 1% FBS following washing twice, and then passed through 30 μm strainers. Sorting and subsequent quantification were performed on BD FACS Aria ll cytometers.