Generating 3D Spheres and 2D Air-Liquid Interface Cultures of Human Induced Pluripotent Stem Cell-Derived Type 2 Alveolar Epithelial Cells

Abstract

In the lung, the alveolar epithelium is a physical barrier from environmental stimuli and plays an essential role in homeostasis and disease. Type 2 alveolar epithelial cells (AT2s) are the facultative progenitors of the distal lung epithelium. Dysfunction and injury of AT2s can result from and contribute to various lung diseases. Improved understanding of AT2 biology is, thus, critical for understanding lung biology and disease; however, primary human AT2s are generally difficult to isolate and limited in supply. To overcome these limitations, human induced pluripotent stem cell (iPSC)derived type 2 alveolar epithelial cells (iAT2s) can be generated through a directed differentiation protocol that recapitulates in vivo lung development. iAT2s grow in feeder-free conditions, share a transcriptomic program with human adult primary AT2s, and execute key functions of AT2s such as production, packaging, and secretion of surfactant. This protocol details the methods for maintaining self-renewing iAT2s through serial passaging in three-dimensional (3D) culture or adapting iAT2s to airliquid interface (ALI) culture. A single-cell suspension of iAT2s is generated before plating in 3D solubilized basement membrane matrix (hereafter referred to as "matrix"), where they self-assemble into monolayered epithelial spheres. iAT2s in 3D culture can be serially dissociated into single-cell suspensions to be passaged or plated in 2D ALI culture. In ALI culture, iAT2s form a polarized monolayer with the apical surface exposed to air, making this platform readily amenable to environmental exposures. Hence, this protocol generates an inexhaustible supply of iAT2s, producing upwards of 1 x 10 30 cells per input cell over 15 passages while maintaining the AT2 program indicated by SFTPC tdTomato expression. The resulting cells represent

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“…iAT2s were then maintained in CK+DCI, feeding every two days, and in serially passaged every two weeks, as described. 29 Following single cell dissociation, iAT2s were maintained in CK+DCI+Y-27632 for 3 days, then switched to CK+DCI media. Cell counts were taken from triplicate wells at each passage to calculate cell yield.…”

“…In some experiments, iAT2s were plated on Transwells to establish air-liquid interface (ALI) cultures, as described. 26 , 29 In brief, iAT2s were dissociated to single cells and 200,000 cells were then plated on growth-factor reduced Matrigel coated Costar 6.5 mm Clear Transwells (Millipore-Sigma) in CK+DCI+Y. After two days, the apical surface was aspirated (“air-lift”).…”

CRISPR interference interrogation of COPD GWAS genes reveals the functional significance of desmoplakin in iPSC-derived alveolar epithelial cells

“…To maintain pure cultures, cells were sorted when necessary to further enrich for NKX2-1 + cells expressing the SFTPC-tdTomato reporter. iAT2s were maintained in culture by refeeding every other day with CK + DCI medium and were serially passaged every 2 weeks at a density of 400 cells/μl, according to our previously published protocol ( 22 , 61 , 62 ). To knock down genes of interest in CRISPRi-targeted PSC lines, cells were maintained in either CBRa or CK + DCI medium containing 2 μM dox (Sigma-Aldrich, D9891).…”

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“…To this regard, efforts have been made to develop culture protocols that enable the expansion of induced (i)AT2 cells. These cells cultured in three-dimensional (3D) Matrigel to generate alveolospheres proliferate while retaining their ability to produce SPC [ 39 ].…”