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Simultaneous isolation of endothelial and alveolar epithelial type I and type II cells during mouse lung development in the absence of a transgenic reporter.

American journal of physiology. Lung cellular and molecular physiology (2020-02-06)
Yves Donati, Sanja Blaskovic, Isabelle Ruchonnet-Métrailler, Josefina Lascano Maillard, Constance Barazzone-Argiroffo
ABSTRACT

Mouse lung developmental maturation and final alveolarization phase begin at birth. During this dynamic process, alveolar cells modify their morphology and anchorage to the extracellular matrix. In particular, alveolar epithelial cell (AEC) type I undergo cytoplasmic flattening and folding to ensure alveoli lining. We developed FACS conditions for simultaneous isolation of alveolar epithelial and endothelial cells in the absence of specific reporters during the early and middle alveolar phase. We evidenced for the first time a pool of extractable epithelial cell populations expressing high levels of podoplanin at postnatal day (pnd)2, and we confirmed by RT-qPCR that these cells are already differentiated but still immature AEC type I. Maturation causes a decrease in isolation yields, reflecting the morphological changes that these cell populations are undergoing. Moreover, we find that major histocompatibility complex II (MHCII), reported as a good marker of AEC type II, is poorly expressed at pnd2 but highly present at pnd8. Combined experiments using LysoTracker and MHCII demonstrate the de novo acquisition of MCHII in AEC type II during lung alveolarization. The lung endothelial populations exhibit FACS signatures from vascular and lymphatic compartments. They can be concomitantly followed throughout alveolar development and were obtained with a noticeable increased yield at the last studied time point (pnd16). Our results provide new insights into early lung alveolar cell isolation feasibility and represent a valuable tool for pure AEC type I preparation as well as further in vitro two- and three-dimensional studies.

MATERIALS
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Product Description

Sigma-Aldrich
胰岛素-转铁蛋白-亚硒酸钠培养基添加剂, γ-irradiated, lyophilized powder, BioXtra, suitable for cell culture
Sigma-Aldrich
抗-Prosurfactant蛋白C(proSP-C)抗体, serum, Chemicon®
Sigma-Aldrich
弹性蛋白酶,猪胰腺,高纯度,结晶, Elastase, Porcine Pancreas, High Purity, Crystallized, CAS 39445-21-1, is a serine protease that hydrolyzes proteins and peptides, especially at bonds adjacent to neutral amino acid residues.