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The establishment of an alveolar epithelial cell (AEC) line derived from induced pluripotent stem cells (iPSC) represents a novel opportunity in lung disease modelling and tissue engineering. In the present study, we succeeded in generating type II AECs from iPSCs. To ensure homogeneous and long term propagation of these cells, iPSC-derived type II AECs, were immortalized by human telomerase reverse transcriptase andpolycomb complex protein BMI-1 lentiviral vectors. The generated type II AEC line displayed morphological characteristics of type II AECs including growing as a cobble stone monolayer. Thus, electron microscopy analysis of this cell line confirmed the presence of lamellar body and microvilli. Also this cell line expressed type II AEC proteins such as cytokeratin, surfactant protein C and lysotracker DND 26 (marker for lamellar body). Furthermore, the type II AEC line exhibited functional properties of type II AECs including an increase of their transepithelial electrical resistance over time and an improved wound closure in presence of serum in the invitro wound healing assay. Upon TNF-α stimulation, these cells released IL-6 an IL-8 cytokines. Consistent with type II AEC phenotype, the cell line showed the ability to uptake and release of surfactant protein B and to differentiate towards type I AECs. Altogether, this study established immortalized type II AECs derived from iPSCs as a novel cellular model for lung disease modelling and tissue engineering.

Background: Macrophages are involved in the pathogenesis and progression of pulmonary fibrosis (PF). Imbalance between M1 and M2 macrophages could play role in progression of PF. The secretome from human induced pluripotent stem cells (iPS) reduce bleomycin induced fibrosis in the rat lung. We investigate the effect of iPS conditioned media (iPS-cm) on macrophages. Methods: Human monocytes were polarized towards M1 and M2 phenotype, and treated with iPS-cm; migration and secretory properties were tested in vitro . Bleomycin injured rats were treated on d7 either with iPSC-cm or CCD1-cm; FACS analysis was performed on d14. Results: iPS-cm increased migration of macrophages in vitro . Levels of proinflammatory and profibrotic mediators were reduced after in vitro treatment with iPSC-cm. In vivo , percentage of total macrophages increased in bleomycin injured lungs compared to control. Moreover, M1 and M2 populations were both increased in bleomycin group. Interestingly, iPS-cm treatment reduced total percentage of macrophage; moreover, the original macrophage phenotype was partially restored. These effects couldn9t be detected using CCD1-cm. Conclusions: iPS-cm modulates migration and alters the macrophage phenotype by reducing pro inflammatory and pro fibrotic mediators in vitro and alters macrophage percentage and phenotype in vivo in a lung injury and fibrosis model. Targeting the macrophage phenotypical switch could be a possible therapy for PF.

Endoplasmic Reticulum stress (ER stress) plays an important role in initiation of the inflammatory response in the lung. In the present study we investigate the effects of Alpha1-Antitrypsin (AAT) on the ER stress response, inflammation and lung epithelial wound healing.We hypothesize that AAT restores ER homeostasis and reduces inflammation in bronchial and alveolar epithelial cells. Bronchial epithelial cells B2B and alveolar epithelial-like cells A549 were treated with Tunicamycin (TN) to induce ER stress, followed by treatment AAT. ER stress markers: Grp78, XBP1 and CHOP where measured by RT PCR. ELISA was performed to measure the inflammatory markers and wound healing was measured over time. Gene expression of ER stress markers was significantly reduced after AAT treatment compared to placebo: Grp78 (49.43±16.61 vs 105.87±11.76), XBP1 (34.00±6.22 vs 107.2±24.12), CHOP (58.93±15.54 vs 297.2±39.58) in A549 cells and Grp78 (42.77±9.95 vs 789.20±9.28), XBP1 (37.33±5.114 vs 120.5±14.58), CHOP (48.93±5.53 vs 307.2±31.35) in B2B cells. Levels of IL-6 in cells treated with AAT decreased significantly in A549 (193.8±16.01 vs 1321±120.2), in B2B (193.8±16.01 vs 1388±167.3). In accordance, IL-8 decreased significantly after AAT treatment for A549 (36.17±8.78 vs 93.90±1.044) and for B2B (36.17±8.78 vs 122.3±28.55). AAT increased epithelial wound healing in ER stressed B2B and A549 cells (25%+/-SD vs 18%+/-SD). AAT treatment decreases ER stress and inflammation and improves wound repair in both alveolar epithelial and bronchial epithelial cells.