In vitro transdifferentiation of human fetal type II cells toward a type I-like cell

Abstract

For alveolar type I cells, phenotype plasticity and physiology other than gas exchange await further clarification due to in vitro study difficulties in isolating and maintaining type I cells in primary culture. Using an established in vitro model of human fetal type II cells, in which the type II phenotype is induced and maintained by adding hormones, we assessed for transdifferentiation in culture toward a type I-like cell with hormone removal for up to 144 h, followed by electron microscopy, permeability studies, and RNA and protein analysis. Hormone withdrawal resulted in diminished type II cell characteristics, including decreased microvilli, lamellar bodies, and type II cell marker RNA and protein. There was a simultaneous increase in type I characteristics, including increased epithelial cell barrier function indicative of a tight monolayer and increased type I cell marker RNA and protein. Our results indicate that hormone removal from cultured human fetal type II cells results in transdifferentiation toward a type I-like cell. This model will be useful for continued in vitro studies of human fetal alveolar epithelial cell differentiation and phenotype plasticity.

Figure 1

Transdifferentiation via hormone withdrawal alters type II cell morphology and surface characteristics. Representative electron micrographs of human fetal lung epithelial cells treated with DCI for4d(4d DCI) (A,C,E), or 4 d DCI followed by 72 h without hormones (4 d DCI/3 d Waymouth’s) (B,D,F). TEM of cell pellets (A,B) and cross-sectional slices of plated cells (C,D)at ×5000. SEM images of plated cells (E,F) at ×1000. (A,E): Microvilli indicated by asterisk. (B) Blunting of microvilli indicated by open arrowhead.(E) Lamellar bodies indicated by bold arrowhead.(A–D) Bars = 10 μm. (E,F) bars = 20 μm.

Figure 2

Quantitative morphology changes with transdifferentiation. TEM samples from three experiments were evaluated at ×5000 after 4 d DCI (0 h) and 96 h after hormone withdrawal from 4 d DCI-treated cells (4 d DCI/4 d Waymouth’s). (A) Percentage of cells exhibiting microvilli (*p < 0.001). (B) Lamellar bodies per cell (*p < 0.05 vs 4 d DCI). (C) Lamellar body diameter (p < 0.001).

Figure 3

Increased barrier function with transdifferentiation. Epithelial barrier function was examined in cells cultured on permeable supports with 4 d DCI, 7 d DCI, or 4 d DCI followed by hormone withdrawal (4 d DCI/3 d Waymouth’s). (A) Carboxyfluorescein filtration (*p < 0.05). (B) Texas Red dextran filtration (*p < 0.05). (C) TER (*p < 0.01). (D) Cells immunostained for ZO-1 at ×20 demonstrate confluent monolayers. Bars = 20 μm.

Figure 4

Type II cell markers decrease with transdifferentiation. The type II cell markers SP-A, SP-C, SP-B, and PGC were assessed by real-time RT-PCR (A) and immunoblotting (B) for up to 144 h after hormone withdrawal from 4 d DCI–treated cells. SPA (lightly shaded columns), SP-C (darkly shaded columns), SP-B (open columns), PGC (hatched columns).

Figure 5

Type I cell markers increase with transdifferentiation. Type I cell markers claudin 7, Cav-1, aquaporin 5, and PAI-1 were assessed by real-time RT-PCR (A) and immunoblotting (B) for up to 144 h after hormone withdrawal from 4 d DCI–treated cells. Claudin 7 (lightly shaded columns), Dark gray: Cav-1 (darkly shaded columns), aquaporin 5 (open columns), PAI-1 (hatched columns)(*p < 0.05).