An induced pluripotent stem cell-based model to study human lung mesenchyme development and disease

The lung mesenchyme plays important roles in lung development and is affected in a variety of respiratory diseases. However, little is known about the biology of normal lung mesenchymal progenitors or how they initiate disease, partly due to the inaccessibility of the tissue, particularly in humans, and partly due to the previous lack of early lung mesenchyme-specific reporters. Increasing evidence suggests specific transcriptional signatures for mesenchymal progenitors from different organs, underlining the importance of studying the lung mesenchymal lineage specifically. Thus, the goal of this project is to generate functional lung mesenchyme from mouse and human induced pluripotent stem cells (iPSCs) and to then use these engineered cells to a) model poorly understood respiratory diseases specifically affecting the lung mesenchyme as a first step towards developing new therapies, and b) study lung epithelial-mesenchymal crosstalk in lung development and disease in order to better understand biology and pathogenesis. Using an iPSC line from a published Tbx4 lung enhancer reporter (Tbx4-LER) mouse, which specifically labels lung mesenchymal progenitors by GFP expression, I have established a protocol for the in vitro differentiation of mouse iPSCs towards the lung mesenchymal lineage. GFP+ lung mesenchymal progenitors express many known lung mesenchymal marker genes at levels similar to primary lung mesenchyme from embryonic day (E) 12.5 embryos, are competent to differentiate towards more mature mesenchymal lineages such as the smooth muscle and the adipogenic/lipofibroblast lineage, form 3-dimensional organoids composed of juxtaposed derivatives of epithelium and mesenchyme when co-cultured with engineered lung epithelial progenitors, and can induce lung epithelial fate when co-cultured with engineered non-lung epithelial progenitors, suggesting that they can function as lung mesenchyme. For this proposed project, which represents a follow-up of my Early Postdoc.Mobility project, I aim to focus on a) the in vitro differentiation of human iPSCs towards the lung mesenchyme lineage using a human TBX4 lung enhancer reporter iPSC line, b) the establishment of engineered human lung epithelial-mesenchymal co-cultures, and c) the use of our engineered human lung mesenchyme and epithelial-mesenchymal co-cultures to model pulmonary fibrosis (PF) and pulmonary arterial hypertension (PAH). Based on my preliminary results, I hypothesize that Retinoic Acid (RA), Hedgehog (Hh), and Wnt signaling are required for human lung mesenchyme specification and I will test this hypothesis by performing a media screen using activators and inhibitors of these 3 pathways. As a readout, I will quantify TBX reporter activation, and perform RT-qPCR to detect expression of lung mesenchymal markers. I will also evaluate the capacity of engineered human lung mesenchyme to function as such, by testing its competence to differentiate towards more mature lineages, and its ability to signal to developing epithelium and induce lung epithelial fate in epithelial-mesenchymal co-cultures. Furthermore, I hypothesize that engineered human lung epithelial-mesenchymal co-cultures can be used as a model for PF, and I will test whether these co-cultures can recapitulate a fibrotic phenotype by comparing mesenchymal cell proliferation and expression of fibrotic markers in co-cultures with engineered lung alveolar epithelial type II (iAT2) cells derived from a PF patient and co-cultures with gene-corrected iAT2s. Finally, I aim to generate an iPSC-derived model for pulmonary arterial hypertension (PAH), comparing gene expression profiles and differentiation efficiencies of engineered lung mesenchyme generated from a patient-derived iPSC line carrying a mutation in the BMPR2 gene and its gene corrected version. The lung mesenchymal differentiation protocols and epithelial-mesenchymal co-culture systems generated in this project will foster significant advancement in our understanding of poorly characterized respiratory diseases specifically affecting the lung mesenchyme or involving epithelial-mesenchymal crosstalk, and will serve as a platform for further studies of lung mesenchymal development and disease, to perform drug screens and for personalized medicine.