Figure 1

Mesenchymal Stem Cells Increase Lung Organoid Formation in 3D Culture. (a) Schematic of FACS strategy and 3D organoid co-culture methods. Fresh lung cells were isolated from β-actin GFP mice and FACS strategy represents signature used to enrich for Epcam⁺ Sca-1⁻ cells and Epcam⁺ Sca-1⁺ cells. CD45⁺ hematopoietic and CD31⁺endothelial cells were first excluded. Epcam⁺ epithelial cells were selected and Sca-1⁺ cells were enriched for lung progenitors and Sca-1⁻ cells were enriched for AT2 cells. Isolated cells were placed in co-culture with either mouse lung endothelial cells (MEC) or mouse bone marrow derived mesenchymal stem cells (MSC) in growth factor reduced matrigel on an air-liquid interface 3D co-culture system. Representative images of the different stromal cells are shown in the lower panel. Scale bar: 50μM. (b) Representative images of GFP⁺ organoids formed from 3D co-culture of Sca-1⁺ cells with MEC or MSC after 14 days in co-culture. Scale bar: 100μM (c) Organoid forming efficiency (OFE) of Sca1⁺/MEC co-cultures was 0.875 which was significantly decreased compared to Sca1⁺/MSC (1.5 OFE) (p < 0.02). Quantification of number of GFP⁺ lung organoids formed in co-culture after 14 days in culture showed a significant 1.7x increase in total Sca-1⁺ colony number when co-cultured with MSC versus MEC. No significant difference in organoid forming efficiency is observed when Sca-1⁻ cells are cultured with MEC versus MSC, Sca-1^−/MEC OFE was 1.685 and Sca-1⁻/MSC OFE was 1.76. (d) Organoid size measured on GFP⁺ pictures from the indicated co-cultures show an increased colony size with Sca1⁺/MSC co-cultures compared to Sca1⁺/MEC co-cultures, p < 0.05. Data represented is the mean from 4 independent experiments with 3 wells per experimental state in each experiment. Error bars represent SD.