Fig. 3: Co-culture of induced lung mesenchyme with Nkx2-1^mCherry+ lung epithelial progenitors leads to the formation of three-dimensional organoids and increases yield of Nkx2-1^mCherry+ cells in distal and proximal epithelial differentiation conditions.

a Schematic of lung epithelial-mesenchymal co-culture experiments. Nkx2-1^mCherry ESCs and Tbx4-LER iPSCs were differentiated separately using the lung epithelial or lung mesenchymal differentiation protocol. Day 11 Tbx4-LER^GFP + (=iLM) and Day13 Nkx2-1^mCherry + (Ch + ) cells were then mixed at a 20:1 ratio and cultured for 7 days in either distal or proximal differentiation medium. Created with BioRender.com. b Representative images of day 20 co-cultures. Day 13 Nkx2.1^mCherry + (Ch + ) cells were cultured in distal or proximal medium, either alone, with iLM cells or with a non-lung mesenchyme control (Mouse embryonic fibroblasts, MEFs). Scale bars = 200 μm. c Zoomed in versions of distal and proximal co-culture images shown in panel b. Scale bars = 200 μm. d Immunofluorescence images showing expression of NKX2-1 and GFP in Nkx2-1^mCherry+ cells cultured in distal or proximal conditions alone or co-cultured with iLM for 1 week. Scale bars = 100 μm. e Percentage of Nkx2-1^mCherry+ cells and yield (i.e., number of Nkx2-1^mCherry+ cells per day 13 Nkx2-1^mCherry+ input cell) for Nkx2-1^Cherry+ cells cultured alone, with iLM or with MEFs in distal or proximal medium. N = 8. Bars show mean ± sd. P values were determined by paired, two-tailed Student’s t test. Significant (p < 0.05) p values are highlighted in bold font.