Development of a novel ex vivo model of Congenital Diaphragmatic Hernia

Background:
Lung hypoplasia is a significant reduction in size of the otherwise normally developed lungs, which causes high morbidity and mortality among babies at birth. The most common cause is CDH, a congenital malformation characterized by an incidence of 0.8-5 every 10000 births (Chandrasekharan et al., 2017, Matern. Health Neonatol. Perinatol.) in which a defect in the diaphragm causes the protrusion of the abdominal contents into the thoracic cavity, affecting the normal development of the lungs. Both environmental and genetic factors are thought to contribute to the aetiology of CDH. However, the mechanisms by which decreased intrathoracic volume causes the premature arrest of lung development seen in CDH patients are still unclear (Kardon et al., 2017, Dis. Model. Mech.).

Increasing evidence confirms that lung branching morphogenesis is strongly affected by the surrounding environment, including extracellular matrix remodelling and stiffening, which regulates force transmission and epithelium polarity to ensure correct tissue development  (Nelson et al., 2012, Annu. Rev. Biomed. Eng.). Thus, improving the understanding of how mechanosensory mechanisms are involved in lung development could guide strategies to promote compensatory lung growth in CDH patients.

Aims/Objectives:
The overall goal of this project is the development of a novel ex vivo model of lung hypoplasia.
1. To develop a novel model of congenital diaphragmatic hernia, effectively mimicking the spatial restriction of normal lobe expansion.
2. To investigate the signalling pathways impaired by the mechanical compression.
3. To test in the ex vivo model a potential rescue with stem cells-derived extracellular vesicles, EVs (in collaboration with the University of Toronto).

Methods:
An innovative 3D micro-printing technique, will be used. The building of a stiff wall around the left lobe of explanted lungs will prevent the natural expansion of the lobe while growing.

Morphological and biochemical analysis (including high-throughput RNA-seq analysis) will be performed in order to compare lung left lobes in the control and CDH model. In order to validate potential target of impaired lung development, mouse and human lung organoids in vitro cultures (Nikolic et al., 2017, eLife), eventually engineered with the technology described before, will be included.

Collaboration with University of Toronto:
A promising experimental therapy has been described by our collaborators at the University of Toronto, which relies on the use of stem cell derived extracellular vesicles (EVs), small nanoparticles that carry bioactive cargo and recently recognized as the key mediators of the paracrine signalling of stem cells. Dr. Zani’s group has built an expertise in isolating and characterizing EVs derived from amniotic fluid stem cells (AFSC) (Antounians et al., 2019, Sci. Rep.), and they have shown that these specific stem cells can rescue proliferation, and reduce apoptosis in an in vitro lung injury model.

The student will spend a period of 6-12 months between the 2nd and 3rd year of studentship at the University of Toronto to test the ability of AFSC-EVs in promoting lung growth and maturation on the explants will be evaluated through the optimization the number of EVs required to reach a biological difference. Molecular biology techniques such as quantitative PCR and Western blot analysis will be used, alongside histology to assess changes in air spaces.

References:
1.   Antounians, L. et al. The Regenerative Potential of Amniotic Fluid Stem Cell Extracellular Vesicles: Lessons Learned by Comparing Different Isolation Techniques. Sci. Rep. 9, (2019).
2.   Tzanetakis, A. et al. Endoplasmic reticulum stress response is activated in pulmonary hypoplasia secondary to congenital diaphragmatic hernia, but is decreased by administration of amniotic fluid stem cells. Pediatr. Surg. Int. 35, 63–69 (2019).
3.   Nichane, M. et al. Isolation and 3D expansion of multipotent Sox9+ mouse lung progenitors. Nat. Methods 14, 1205–1212 (2017).
4.   Nikolić, M. Z. et al. Human embryonic lung epithelial tips are multipotent progenitors that can be expanded in vitro as long-term self-renewing organoids. eLife 6, e26575 (2017).
5.   Antounians L, Catania VD, Montalva L, Liu BD, Hou H, Chan C, Matei AC, Tzanetakis A, Li B, Figueira RL, da Costa KM, Wong AP, Mitchell R, David AL, Patel K, De Coppi P, Sbragia L, Wilson MD, Rossant J, Zani A. Fetal lung underdevelopment is rescued by administration of amniotic fluid stem cell extracellular vesicles in rodents. Sci Transl Med. 2021 Apr 21;13(590):eaax5941