by Boston University School of Medicine
In vitro differentiation of iPSCs toward the lung mesenchyme lineage through a mesodermal progenitor. 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. Credit: Nature Communications (2023). DOI: 10.1038/s41467-023-39099-9
Lung mesenchymal cells, which are critical components of the lung’s unique structure, also play important roles in disease and recovery from injury, yet knowledge is limited about their biology or how they initiate diseases like pulmonary fibrosis. While experimental models have helped to identify some regulators of lung mesenchyme behavior, the understanding of how the lung mesenchyme is identified during human development is unknown.
To better understand these processes, researchers from Boston University Chobanian & Avedisian School of Medicine have developed an in-vitro induced pluripotent stem cell (iPSC)-based model system for the derivation and study of early lung-specific mesenchyme with potential benefit for comprehending basic mechanisms regulating tissue-specific mesenchymal fate decisions and future applications for regenerative medicine.
“Our study has implications for the study of lung diseases, such as pulmonary fibrosis and interstitial lung diseases that arise from dysfunction of the part of the lung known as mesenchyme. These diseases currently have very limited treatment options and we hope our model system will provide new tools to understand what goes wrong in these diseases and to screen for better drugs,” said corresponding author Darrell Kotton, MD, the David C. Seldin Professor of Medicine and director of the BU/Boston Medical Center Center for Regenerative Medicine (CReM).
The researchers used an experimental model with an iPSC line carrying a lung mesenchyme-specific fluorescent reporter, meaning that cells that become lung mesenchymal were marked by green fluorescence. Using this model, they tested several growth factors and small molecules to stimulate pathways with known roles in lung development.
They found that stimulating the retinoic acid and hedgehog signaling pathways, both known to play essential roles in embryonic development, resulted in the maximum percentage of green fluorescent cells indicative of the potential presence of lung mesenchyme. They then isolated those cells and compared their gene expression profile to primary cells from embryonic lungs of the experimental model to determine how similar these cells are to primary lung mesenchymal cells.
Finally, they used their recombinant organoid system to test whether these cells can actually function as lung mesenchyme.
“An important role of the developing lung mesenchyme in the experimental model is their ability to interact with and signal to the neighboring epithelium. We found that our engineered cells can recapitulate some of those signaling interactions, suggesting that they have functional capacity,” explained first author Andrea Alber, Ph.D., a postdoctoral fellow in Kotton’s laboratory.
According to the researchers, the part of the study where the engineered lung mesenchymal cells are combined with lung epithelial cells in culture dishes (so called “recombinants”) is particularly exciting as this resulted in organoids, living cells assembled together in a 3D culture gel that helps scientists understand how cells are organized and communicate. “We are now working to apply these types of new organoid models to better understand pulmonary fibrosis,” added Alber.
These findings appear in the journal Nature Communications.
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