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Supervisors: Professor Paolo De Coppi, Dr Frederica Michielin
Investigating the role of mechanical compression in alveolar specification during fetal lung development
Background:
Congenital malformations such as Congenital Diaphragmatic Hernia cause a physical impairment to fetal lung growth and development by generating exceptional mechanical compression. In most cases, this results in severe lung hypoplasia and neonatal death or in lung disease that lasts into adulthood. The differentiation of epithelial progenitor stem cells into Alveolar Type-1 (AT1) cells, flat cells responsible for gas exchange and highly sensitive to mechanical forces, and Alveolar Type-2 (AT2) cells, secretory cells that reduce surface tension in the alveolus, is essential to guarantee gas exchange function at birth1.
How mechanical compression alters the specification of lung progenitor stem cells to alveolar cells and what implication this alteration has on fetal lung development and lung hypoplasia is still unclear.
The mechanical signals that the cells receive from their surrounding environment are emerging as key determinants of their behaviour and developmental fate in the lung2. However, major challenges to recapitulating the key extracellular cues regulating lung development still exist. Our lab has recently developed a novel ex vivo model of lung development based on an innovative 3D-printing technology that prevents the spontaneous growth of lung tissue, therefore allowing to dissect the role of mechanical compression in the early stages of lung development (Michielin et al., in preparation).
Aims/Objectives:
The overall goal of this project is to investigate the effects of mechanical compression on the alveolar differentiation of lung epithelial stem cells. The specific objectives will be:
1. To characterize the cell identity of compressed fetal lung tissue;
2. To develop a representative 3D in vitro model of alveolar differentiation;
3. To tune the stiffness of the extracellular environment in the developed model;
4. To correlate stem cell trajectories and fate at single-cell resolution with different mechanical inputs.
Methods:
For Objective 1, an already available model based on the ex vivo culture of mouse fetal lungs coupled with the 3D-printing3 of mechanical con will be used. Cell identity will be investigated by means of bulk and single-cell transcriptomic analysis, immunostaining and FACS analysis.
To achieve objectives 2 and 3, 3D organoids cultures will be established either starting from mouse fetal lung progenitor cells and from human induced pluripotent stem cells (hiPSCs) cultures will be established and induced to differentiate into the alveolar fate4,5. For the human model, NKX2.1-GFP/SFTPC-tdTomato hiPSC line will be used to have a direct readout AT2 specification5 during hiPSC differentiation. Then, different chemically modified gels (PEG-HCC) available in the lab will be used to specifically investigate alveolar differentiation in conditions that mimic pathological tissue compliance. Specifically, increasing stiffnesses will be achieved by modulating the concentration of PEG-HCC.
Finally, to achieve Objective 4 single-cell RNA sequencing analysis will be used to generate for the first time and ultimately identify the stiffness-regulated transcriptional signatures of stem cell progenitor organoids and their differentiated counterparts. Results will be validated by using either the compressed lung model as well as biopsies of aborted human fetuses affected by CDH available at GOSH Pathology.
References:
1. Morrisey, E. E. & Hogan, B. L. M. Preparing for the First Breath: Genetic and Cellular Mechanisms in Lung Development. Dev. Cell 18, 8–23 (2010).
2. Li, J. et al. The Strength of Mechanical Forces Determines the Differentiation of Alveolar Epithelial Cells. Dev. Cell 44, 297-312.e5 (2018).
3. Urciuolo, A. et al. Intravital three-dimensional bioprinting. Nat. Biomed. Eng. 1–15 (2020) doi:10.1038/s41551-020-0568-z.
4. Gkatzis, K., Panza, P., Peruzzo, S. & Stainier, D. Y. Differentiation of mouse fetal lung alveolar progenitors in serum-free organotypic cultures. eLife 10, e65811 (2021).
5. Jacob, A. et al. Derivation of self-renewing lung alveolar epithelial type II cells from human pluripotent stem cells. Nat. Protoc. 14, 3303–3332 (2019).