Abstract
In order to circumvent the limited access and donor variability of human primary alveolar cells, directed differentiation of human pluripotent stem cells (hiPSCs) into alveolar-like cells, provides a promising tool for respiratory disease modeling and drug discovery assays. In this work, a unique, miniaturized 96-Transwell microplate system is described where hiPSC-derived alveolar-like cells were cultured at an air–liquid interface (ALI). To this end, hiPSCs were differentiated into lung epithelial progenitor cells (LPCs) and subsequently matured into a functional alveolar type 2 (AT2)-like epithelium with monolayer-like morphology. AT2-like cells cultured at the physiological ALI conditions displayed characteristics of AT2 cells with classical alveolar surfactant protein expressions and lamellar-body like structures. The integrity of the epithelial barriers between the AT2-like cells was confirmed by applying a custom-made device for 96-parallelized transepithelial electric resistance (TEER) measurements. In order to generate an IPF disease-like phenotype in vitro, the functional AT2-like cells were stimulated with cytokines and growth factors present in the alveolar tissue of IPF patients. The cytokines stimulated the secretion of pro-fibrotic biomarker proteins both on the mRNA (messenger ribonucleic acid) and protein level. Thus, the hiPSC-derived and cellular model system enables the recapitulation of certain IPF hallmarks, while paving the route towards a miniaturized medium throughput approach of pharmaceutical drug discovery.
Highlights
In order to circumvent the limited access and donor variability of human primary alveolar cells, directed differentiation of human pluripotent stem cells into alveolar-like cells, provides a promising tool for respiratory disease modeling and drug discovery assays
Within this work the generation of functional human pluripotent stem cells (hiPSCs)-derived alveolar type 2 (AT2)-like cells was successfully achieved through the directed in vitro differentiation of hiPSCs involving the recapitulation of the complex in vivo development of the lung
lung epithelial progenitor cells (LPCs) maturation toward AT2-like cells was carried out on 96-Transwell inserts at air–liquid interface (ALI) to resemble the physiological environment of the mature human alveolar epithelium
Summary
In order to circumvent the limited access and donor variability of human primary alveolar cells, directed differentiation of human pluripotent stem cells (hiPSCs) into alveolar-like cells, provides a promising tool for respiratory disease modeling and drug discovery assays. A unique, miniaturized 96-Transwell microplate system is described where hiPSC-derived alveolar-like cells were cultured at an air–liquid interface (ALI) To this end, hiPSCs were differentiated into lung epithelial progenitor cells (LPCs) and subsequently matured into a functional alveolar type 2 (AT2)like epithelium with monolayer-like morphology. Idiopathic pulmonary fibrosis (IPF) is the most common and pernicious type among ILDs with unknown etiology It is characterized through progressive fibrosis within the lung e pithelium[1,2,3], involving alveolar epithelial cells and macrophages, monocytes and local fibroblasts[4]. To better understand the various mechanisms driving the disease, it is important to have a predictive in vitro system that can resemble the human physiological situation Immortalized cell lines, such as lung adenocarcinoma (A549) cells, have been widely used instead of primary c ells[20,21]. The access to non-diseased human primary alveolar cells is highly limited and cost intensive, rendering them less suitable for the application in large-scale cultures and medium throughput screenings[27]
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