Generation of an alveolar epithelial type II cell line from induced pluripotent stem cells

Abstract

Differentiation of primary alveolar type II epithelial cells (AEC type II) to AEC type I in culture is a major barrier in the study of the alveolar epithelium in vitro. The establishment of an AEC type II cell line derived from induced pluripotent stem cells (iPSC) represents a novel opportunity to study alveolar epithelial cell biology, for instance in the context of lung injury, fibrosis and repair. In the present study, we generated long lasting AECII from iPSC (iPSC-AEC). Long lasting iPSC-AECII (LL)-iPSC-AECII) displayed morphological characteristics of AECII including growth in a cobble stone monolayer, the presence of lamellar bodies and microvilli as shown by electron microscopy. Also, (LL)-iPSC-AECII expressed AEC type II proteins such as cytokeratin, surfactant protein C and lysotracker DND 26 (a marker for lamellar bodies). Furthermore, the (LL)-iPSC-AECII exhibited functional properties of AECII by an increase of transepithelial electrical resistance (TERR) over time, secretion of inflammatory mediators in biologically relevant quantities (interleukin-6, interleukin-8) and efficient in vitro alveolar epithelial wound repair. Consistent with the AECII phenotype, the cell line showed the ability to uptake and release surfactant protein B, to secrete phospholipids and to differentiate into AEC type I. In summary, we established a long lasting, but finite AEC type II cell line derived from iPSC as a novel cellular model to study alveolar epithelial cell biology in lung health and disease.

In vitro modelling of alveolar repair at the air-liquid interface using alveolar epithelial cells derived from human induced pluripotent stem cells

“…In summary, our study provides support for the use of hiPSC in generating relatively mature populations of AEC2 and both confirms and extends previous studies in this area 18,20,22,23,26,35,39 . In addition, the study shows that iAEC2 can be used to study epithelial repair processes.…”

“…In iAEC2 cultures, and not in pAEC2 cultures, this treatment was accompanied by an increase in cells positive for PDPN, a marker associated with an AEC1 phenotype, with a concomitant decrease in pro-SFTPC positive cells at the wound edge. Before our study, other groups had reported the generation of AEC2 from embryonic- 16,17 and induced pluripotent stem cells [18][19][20][21][22][23][24][25][26] . Our protocol was largely based on these published protocols, with some modifications that improved purity and maturity in our hiPSC cell lines.…”

“…A note of caution is needed, since CHIR99021 activates Wnt signalling through GSK3beta inhibition, and its mode of action is therefore not specific. Tamo et al 26 recently reported that the Wnt-signalling inhibitor IWT increased transdifferentiation of iAEC2 to iAEC1 under submerged conditions, further pointing to a possible role of experimental design and agents used to study AEC transdifferentiation. Another mechanism that has been implicated in transdifferentiation of AEC2 to AEC1 is the autocrine action of TGF-β 43 , which may be antagonized by BMP signalling 44 .…”

“…hiPSC lines can be generated from any healthy individual or patient and thus potentially represent an unlimited source of tissue-specific cells, which can be used as in vitro models for screening effectiveness or toxicity of candidate therapeutic agents. Human AEC cultures have been successfully derived from human embryonic stem cells 16,17 and from hiPSC previously [18][19][20][21][22][23][24][25][26] . These latter studies relied on directed differentiation of hiPSC into the endodermal lineage using Activin A, followed by differentiation of this definitive endoderm into foregut endoderm through inhibition of TGF-β and BMP signalling.…”

What have we learned from basic science studies on idiopathic pulmonary fibrosis?

“…Furthermore, several animal models of IPF, including bleomycin-induced lung fibrosis, fail to fully recapitulate IPF as seen in patients. Despite this, basic science contributes to the field by generating novel in vitro and in vivo models such as induced pluripotent stem cell-generated AECs [129], precision-cut ex vivo lung models [130], lung-on-chip technologies [131] and gene-engineered mice. These systems will be important to appropriately study lung fibrosis and develop effective therapies in the future.…”