Figure 1. LuMECs support BASC self-renewal and differentiation in vitro and in vivo.

(A) Schematic of FACS strategy to enrich for AT2 cells and BASCs from β-actin-GFP mice and 3D co-culture with LuMECs. CD45+ hematopoietic and CD31+ endothelial cells were excluded. EpCAM+ epithelial cells were selected. From these selections, Sca1-positive cells were BASCs and Sca1-negative cells were AT2 cells. (B) Representative images of GFP fluorescent colonies from 3D co-culture of AT2 cells (left) or BASCs (right) with LuMECs after 14 days. Arrowhead, bronchiolar colony; arrow, alveolar colony; asterisk, bronchioalveolar colony. Scale bar, 500um. (C) Self-renewal of AT2 cells and BASCs in 3D LuMEC co-cultures. Primary colonies (1°) were dissociated a nd GFP+ cells were replated for secondary (2°) and subsequent (3°, 4°, 5°, 6°) colony formati on. Colony forming efficiency: number of colonies formed/number of cells plated per well as a percentage. Data presented are the mean of three independent experiments with triplicate wells. Error bars indicate standard deviation. (D) Representative GFP images of alveolar colonies from AT2 cells (top, left) and bronchiolar, alveolar, and bronchioalveolar colonies from BASCs (top, right). H&E (middle) and IF (bottom) for CCSP (red), SPC (green), and DAPI (blue) show BASC differentiation into club (Clara) cells and AT2 cells. Scale bar, 100um. (E) Quantification of each colony type from AT2 cell (n=687) or BASC co-cultures (n=842). 25.4% of colonies were bronchiolar, 53.5% were alveolar, and 21.1% were mixed. The mean percentage of total colonies per well represented by each type of colony is shown. n=number of colonies scored. Data presented are the mean of seven independent experiments with triplicate wells. Error bars indicate standard deviation.

(F) Subcutaneous co-transplantation of AT2 cells or BASCs mixed with LuMEC/Matrigel. H&E (top) and IF (bottom) for CCSP (red), SPC (green), and DAPI (blue) show that only BASCs co-injected with LuMECs formed epithelial structures with cells positive for CCSP, SPC, or both; BASCs, n=7/8 mice injected formed epithelial structures; AT2 cells, n=9/9 mice injected did not yield epithelial structures. Images are representative of three independent experiments. Scale bar, 100um. (G) Schematic of clonal serial passage analysis. 1° colonies were plated for 2° or 3° colony formation (I) and half of the cells were used for qPCR (H and J). Data shown are from 20 individual colonies per type analyzed over four independent experiments. (H) Representative qPCR analysis validating expression of SPC (white bars) and CCSP (black bars) in cells from two different individual colonies. B1, B2: primary bronchiolar colony; A1, A2: primary alveolar colony; BA1, BA2: primary bronchioalveolar colony. Normalized to Gapdh. Data presented are the mean of triplicate wells. Error bars indicate standard deviation (**, p<0.001). (I) Representative GFP images of 2° colo nies from passage of each colony type. Arrowhead, bronchiolar colony; arrow, alveolar colony; asterisk, bronchioalveolar colony. Scale bar, 500um (top); H&E (middle) and IF (bottom) analysis for CCSP (red), SPC (green), and DAPI (blue) in tissue sections from subcutaneous transplantation of cells from BASC-derived bronchiolar (left)(n=3/8 mice formed epithelial structures), alveolar (middle)(n=15/15 mice did not yield epithelial structures), or bronchioalveolar colonies (right)(n=9/9 mice generated epithelial structures). Scale, 100um. (J) Representative qPCR analysis in tertiary colonies as in H. 3°B1, 3°B2: tertiary bronchiolar colony; 3°A1, 3°A2: tertiary alveolar colony; 3°BA1, 3°BA2: tertiary bronchioalveolar colony. N ormalized to Gapdh. Data presented are the mean of triplicate wells. Error bars indicate standard deviation (**, p<0.001). See also Figure S1.