Fig. 1: In vitro differentiation of iPSCs toward the lung mesenchyme lineage through a mesodermal progenitor.
From: Directed differentiation of mouse pluripotent stem cells into functional lung-specific mesenchyme
a Schematic of the Tbx4 lung enhancer reporter/tracer (LER) line, and image of an E10 embryo (dox from E6.5 to E10). Scale bar = 0.5 mm. Created with BioRender.com. b Directed differentiation of iPSCs into lung mesenchyme through a mesodermal progenitor. cSFDM = complete serum-free differentiation medium, LIF = Leukemia inhibitory factor. Created with BioRender.com. c Expression relative to day 0 iPSCs of Foxf1 (lateral plate mesoderm), Pax2 (intermediate mesoderm) and Tbx6 (paraxial mesoderm) in KDR± cells on day 5. N = 3. d Image and flow cytometry plot showing expression of GFP (green) in the Tbx4-LER iPSC line on day 13. Red = Tomato in non-recombined cells. Scale bar = 100 μm. e GFP percentage on day 13 using all 4 medium factors (RA, PMA, BMP4, WNT3A), or one or two factors removed at a time. Dox from day 5 on. N = 9. f Day 13 GFP percentage in RA&PMA medium, with either the XAV or recombinant mouse WNT3A. Dox from day 5 on. N = 3. g GFP percentage on day 13 in RA&PMA medium, in RA or PMA only medium, or upon addition of Cyclopamine. Dox from day 5 on. N = 4 for “RA&PMA” and “RA”, N = 3 for “PMA” and “RA&Cyclo”. h GFP percentage over time from day 5 to 13 of differentiation. Dox from day 5 on. N = 4 for day 8-12, N = 3 for day 13. i GFP percentage on day 13 in RA&PMA medium after sorting for KDR± on day 5 of differentiation. N = 3. Created with BioRender.com. j Representative image showing reporter activation on day 13 of lung epithelial differentiation and GFP percentage in the mesenchymal versus epithelial (i.e., co-development, cLM) differentiation protocol. Dox from day 5 on in the mesenchymal differentiation protocol, and from day 6 (anterior foregut endoderm stage) on in the epithelial (co-development) protocol. Scale bar = 100 μm. N = 4 for “Mesenchymal”, N = 5 for “Co-development”. All bars show mean ± sd. p values were determined by unpaired, two-tailed Student’s t test. Significant (p < 0.05) p values are highlighted in bold font.
