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. T he distal alveolar epithelium contains type I and II cells. Type II cells comprise 75% of distal lung epithelium, 7% of the surface area, and produce surfactant. Type II cells have many well-characterized markers, including lamellar bodies, surfactant proteins A, B, C, and D (SP-A, B, C, and D) (1), and the aspartic protease pepsinogen C (PGC) (2).Type I cells, although less numerous, cover 93% of adult lung surface area and provide the gas exchange surface (1). Other type I functions, such as ion transport and fluid homeostasis, are less known. Type I cells form a tight monolayer with low permeability (3). However, the utility of previously described type I biochemical markers (4) has been limited by variable reproducibility and expression between species.Challenges in isolation and culture have limited progress in type I cell biology. Despite isolated reports (5,6), there are no widespread primary culture models. Instead, rat type II cells transdifferentiated toward a type I cell on tissue culture plastic (7) have served as a proxy for type I cells (8,9). There are limited data with regards to transdifferentiation in adult human alveolar epithelium (10), with no previous in vivo descriptions in human fetal lung. Rodent model transdifferentiation and the observation that type II cells serve as progenitors of type I cells after alveolar injury in mature lung (11) have led to the assumption that type I cells are derived from type II cells. However, the possibility that both are derived from a common precursor and retain some degree of plasticity has not been examined in the human fetal lung.Because of its potential plasticity, fetal alveolar epithelium provides unique opportunities to study differentiation/ transdifferentiation pathway(s) in the developing lung. We previously demonstrated that type II cells from human fetal lung can maintain a differentiated phenotype with dexamethasone, cAMP, and isobutylmethylxanthine (DCI) (12) and that these conditions induce differentiation of type II cells from naive human fetal lung epithelium (13). Here, we demonstrate that DCI withdrawal from cultured human fetal type II cells results in transdifferentiation toward type I-like cells. Transdifferentiation is associated with diminution of type II morphology, decreased expression of type II markers, and induction of type I cell markers. Importantly, transdifferentiated cells behave like type I cells, with decreased permeability and increased transepithelial resistance (T...

Immortalization of Human Alveolar Epithelial Cells to Investigate Nanoparticle Uptake

“…SSCM of live cells at high magnification also showed that only immortal, AT1-like cells internalized 50-nm particles, which initially deposited at the cell margins and then relocated to central regions that became enriched with microvilli before particle internalization, suggesting an active process involving microvilli. This is interesting, since fetal AT2-derived AT1 cells also exhibited microvilli (37). Our study suggests that AT1 cells may exhibit microvilli when activated.…”

“…An alternative is to use primary AT2 cells that have lost their phenotype in vitro to resemble AT1 cells (28,36). Recently, AT1-like cells have been obtained from transdifferentiating human fetal primary AT2 cells (37). However, the authors did not present evidence that these cells could be passaged as can the immortal cells herein.…”

Transdifferentiation: a cell and molecular reprogramming process

“…These models are: (1) very restricted transdifferentiation or a cell-type conversion between cell types belonging to the same class, such as conversion of Rh5 into Rh6 photoreceptors in D. melanogaster eye development (Sprecher and Desplan 2008), and also conversion of human fetal alveolar type II cells into alveolar type I-like cells in lung epithelium (Foster et al 2007). …”

“…Also, during development and postnatal tissue repair, type II pneumocytes of alveolar epithelium can transdifferentiate into type I pneumocytes, which participate in gaseous exchange and secreting surfactants, respectively (Flecknoe et al 2000). In addition, it has been demonstrated that human fetal type II cells can switch into type I-like cells after addition of hormones in vitro (Foster et al 2007). The conversion of sprouting venous endothelial cells into arteries and capillary endothelial cells is another newly identified example of transdifferentiation in mammals (RedHorse et al 2010).…”

“…Nowadays, transdifferentiation is defined as reprogramming of one somatic cell, such as fibroblast, into an altogether different type of cell, such as muscle or neuron, without an experimental step of reversion to a pluripotent cell (Graf and Enver, 2009). Several studies have demonstrated that transdifferentiation occurs in various types of fully differentiated cells (Ali et al 1999;Shen et al 2000;Kotton et al 2001;Horb et al 2003;Foster et al 2007;Sprecher and Desplan 2008;Szabo et al 2010). Moreover, some studies have shown that switch in the developmental fate of a stem or progenitor cell to another cell type can also take place.…”