Fig. 5: Co-culture with in vitro differentiated lung mesenchyme can induce lung epithelial fate in non-lung epithelial progenitors and can maintain primary epithelial cells.
From: Directed differentiation of mouse pluripotent stem cells into functional lung-specific mesenchyme
a Co-cultures of Nkx2-1mCherry+ cells with primary E12.5 lung mesenchyme (pLM). Created with BioRender.com. b Immunofluorescence showing expression of PDGFRA, E-CADHERIN and NKX2-1 in co-cultures of Nkx2-1mCherry+ epithelial cells and pLM in distal medium. Scale bars = 100 μm. c Yield of Nkx2-1Cherry+ cells after co-culture with iLM or pLM in distal medium N = 7. d Schematic of co-cultures of iLM with Nkx2-1mCherry- day 13 cells. Created with BioRender.com. e Representative images of co-cultures of iLM with Nkx2-1mCherry- cells. Cells were sorted for EPCAM +/mCherry− on day 13 of epithelial differentiation and cultured either alone, with iLM, or with non-lung mesenchyme (MEFs) in distal medium for 7 days. Scale bars = 200 μm. f Percentage and yield of Nkx2-1mCherry+ cells for Nkx2-1mCherry- cells cultured alone, with iLM or with MEFs in distal medium for 7 days. N = 3. g Schematic of co-cultures of primary Nkx2-1GFP + E12.5 lung epithelial cells (pE) with iLM. Created with BioRender.com. h Immunofluorescence images showing expression of PDGFRA and NKX2-1 in co-cultures of pE and iLM in distal medium. Scale bars = 100 μm. i Yield of Nkx2-1GFP+ cells after 1 week of distal culture alone or with iLM. N = 4. j RT-qPCR data showing fold change expression over day 0 iPSCs of Sox9, Etv5, Sftpc, and Ager in pE cultured alone or with iLM in distal medium. N = 4. k Schematic of co-cultures of iLM with SFTPCTomatoTr lineage traced adult primary alveolar epithelial type 2 (AT2) cells. Created with BioRender.com. l Immunofluorescence images showing expression of GFP, Tomato and pro-SFTPC in primary SFTPCTomatoTr lineage traced AT2 cells cultured either with iLM or with primary PdgfraGFP+ lung mesenchyme. Scale bars = 30 μm. Bars show mean ± sd. P values were determined by paired, two-tailed (c and f) or one-tailed (i and j) Student’s t test. Significant (p < 0.05) p values are highlighted in bold font.
