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Supervisors:
Paolo De Coppi and Federica Michielin
Project Description:
Background:
Alveoli formation in the developing lung is essential for breathing. Impaired alveolar development is a major cause of neonatal death or serious lung disease that continue to adulthood. Each alveolus consists of an epithelium layer that includes flattened AT1 cells, responsible for the gas exchange, and cuboidal AT2 cells, which produce surfactants. In addition, endothelial cells (ECs) help forming the air-blood barrier which enables breathing at birth.
Although the mechanisms that regulate the commitment of epithelial AT1 and AT2 cells during alveolar development have been well-explored, less is known about the contribution of alveolar ECs. This is mostly due to the limited access to fetal tissues but also to the absence of reliable human models able to recapitulate the endothelial-epithelial crosstalk in vitro[1].
Characterizing the role of ECs in the alveolar niche may not only help to understand the mechanisms of lung biology, but also lead to the development of efficient therapeutic strategies for various lung diseases.
Aims and timeline:
The overall goal of this study is to establish a novel in-vitro model of human alveolar development to elucidate the epithelial-endothelial interactions in the developing human lung. Specific aims of the projects are:
- To optimize a protocol of alveolar differentiation of human induced pluripotent stem cells (hiPSCs)-derived alveolar progenitor organoids (alveolospheres) to obtain AT1 and AT2 cells in the same culture, including the optimization of ECM stiffness. (Year 1)
- To develop a guided vascular network of endothelial progenitor cells by leveraging the self-assembly potential to form patent vessels and integrate the differentiating organoids within the vascular bed and assess the maturation of the AT1 and AT2 cells. (Year 2)
- To identify mechanisms of epithelial-endothelial crosstalk that contribute to alveologenesis. (Year 3)
Methods:
HiPSCs will be used to model alveolar differentiation by leveraging an already established protocol that relies on a double NKX2.1-GFP/SFTPC-TdTomato reporter to select for lung progenitor cells-forming alveoloshperes[2]. This differentiation protocol will be further optimized to obtain alveolar organoids containing both AT1 or AT2 cells from alveolospheres with higher degree of maturation, close to the perinatal lung. The maturation of AT1 cells will be evaluated by analysing the degree of cell flattening and the functional activity of sodium channels ENAC, whereas AT2 maturation will be evaluated by quantification of secreted surfactant proteins through ELISA assay.
Human umbilical vein ECs (HUVECs) transduced with ETV2 (R-VECs)[3], a vascular transcription factor that enhance the angiogenic potential of ECs in culture will be used. An EGFP reporter allows to identify culture conditions that can support the co-culture while enabling vasculogenesis. In addition, the stiffness of the 3D extracellular environment which has will be tuned by using photo-crosslinkable gels that we pioneered[4].
Results of alveolar differentiation, RVECs culture and the co-culture will be assessed through live imaging, immunofluorescence, flow-cytometry, and qPCR. Single-cell multiome analysis will be performed through 10x single-cell multiome technology to simultaneously analyse gene expression and chromatin accessibility in both epithelial and endothelial cell populations and reconstruct mechanisms of paracrine interactions.
References:
[1] Mammoto, A. & Mammoto, T. Front. Bioeng. Biotechnol. 7, (2019).
[2] Jacob, A. et al. Nat. Protoc. 14, 3303–3332 (2019).
[3] Palikuqi, B. et al. Nature 585, 426–432 (2020).
[4] Urciuolo, A. et al. Nat. Commun. 14, 3128 (2023).
Contact Information:
Federica Michielin