BACKGROUND:The prevalence of NAFLD is rapidly increasing. NAFLD can progress to NASH, fibrosis, cirrhosis, and HCC, which will soon become the main causes of liver transplantation. To date, no effective drug for NASH has been approved by the Food and Drug Administration. This is partly due to the lack of reliable human in vitro models. Here, we present a novel human liver spheroid model that can be used to study the mechanisms underlying liver fibrosis formation and degradation. METHODS AND RESULTS:Such spheroids, which contain hepatocytes, stellate cells, KC, and LSECs, spontaneously develop fibrosis that is exacerbated by treatment with free fatty acids. Conditioned medium from activated LSECs caused similar activation of fibrosis in spheroids containing primary human hepatocyte and NPCs, indicating the action of soluble mediators from the LSECs. Spheroids containing LSECs treated with free fatty acids produced tissue inhibitor of metalloproteinases inhibitor 1, a matrix metalloproteinases inhibitor important for fibrosis progression. Tissue inhibitor of metalloproteinases inhibitor 1 knockdown using siRNA led to a reduction in collagen and procollagen accumulation, which could be partially rescued using a potent matrix metalloproteinases inhibitor. Interestingly, tissue inhibitor of metalloproteinases inhibitor 1 was found to be expressed at higher levels, specifically in a subtype of endothelial cells in the pericentral region of human fibrotic livers, than in control livers. CONCLUSION:Potential anti-NASH drugs and compounds were evaluated for their efficacy in reducing collagen accumulation, and we found differences in specificity between spheroids with and without LSECs. This new human NASH model may reveal novel mechanisms for the regulation of liver fibrosis and provide a more appropriate model for screening drugs against NASH.

Non-alcoholic steatohepatitis (NASH) is a major health problem leading to liver fibrosis and hepatocellular carcinoma, among other diseases, and for which there is still no approved drug treatment. Previous studies in animal models and in LX-2 cells have indicated a role for serotonin (5-HT) and 5-HT receptors in stellate cell activation and the development of NASH. In the current study, we investigated the extent to which these findings are applicable to a human NASH in vitro model consisting of human liver spheroids containing hepatocytes and non-parenchymal cells. Treatment of the spheroids with 5-HT or free fatty acids (FFA) induced fibrosis, whereas treatment of the spheroids with the 5-HT receptor antagonists ketanserin, pimavanserin, sarpogrelate, and SB269970 inhibited FFA-induced fibrosis via a reduction in stellate cell activation as determined by the expression of vimentin, TGF-β1 and COL1A1 production. siRNA-based silencing of 5-HT2A receptor expression reduced the anti-fibrotic properties of ketanserin, suggesting a role for 5-HT receptors in general and 5-HT2A receptors in particular in the FFA-mediated increase in fibrosis in the human liver spheroid model. The results suggest a contribution of the 5-HT receptors in the development of FFA-induced human liver fibrosis with implications for further efforts in drug development.

Connective tissue growth factor (CTGF) is involved in the regulation of extracellular matrix (ECM) production. Elevated levels of CTGF can be found in plasma from patients with liver fibrosis and in experimental animal models of liver fibrosis, but the exact role of CTGF in, e.g., diet-induced human liver fibrosis is not entirely known. To address this question, we utilized a 3D human liver co-culture spheroid model composed of hepatocytes and non-parenchymal cells, in which fibrosis is induced by TGF-β1, CTGF or free fatty acids (FFA). Treatment of the spheroids with TGF-β1 or FFA increased COL1A1 deposition as well as the expression of TGF-β1 and CTGF. Recombinant CTGF, as well as angiotensin II, caused increased expression and/or production of CTGF, TGF-β1, COL1A1, LOX, and IL-6. In addition, silencing of CTGF reduced both TGF-β1- and FFA-induced COL1A1 deposition. Furthermore, we found that IL-6 induced CTGF, COL1A1 and TGF-β1 production, suggesting that IL-6 is a mediator in the pathway of CTGF-induced fibrosis. Taken together, our data indicate a specific role for CTGF and CTGF downstream signaling pathways for the development of liver inflammation and fibrosis in the human 3D liver spheroid model.

Human lung function is intricately linked to blood flow and breathing cycles, but it remains unknown how these dynamic cues shape human airway epithelial biology. Here we report a state-of-the-art protocol for studying the effects of dynamic medium and airflow as well as stretch on human primary airway epithelial cell differentiation and maturation, including mucociliary clearance, using an organ-on-chip device. Perfused epithelial cell cultures displayed accelerated maturation and polarization of mucociliary clearance, and changes in specific cell-types when compared to traditional (static) culture methods. Additional application of airflow and stretch to the airway chip resulted in an increase in polarization of mucociliary clearance towards the applied flow, reduced baseline secretion of interleukin-8 and other inflammatory proteins, and reduced gene expression of matrix metalloproteinase (MMP) 9, fibronectin, and other extracellular matrix factors. These results indicate that breathing-like mechanical stimuli are important modulators of airway epithelial cell differentiation and maturation and that their fine-tuned application could generate models of specific epithelial pathologies, including mucociliary (dys)function.

A comparatively straightforward approach to accomplish more physiological realism in organ-on-a-chip (OoC) models is through substrate geometry. There is increasing evidence that the strongly, microscale curved surfaces that epithelial or endothelial cells experience when lining small body lumens, such as the alveoli or blood vessels, impact their behavior. However, the most commonly used cell culture substrates for modeling of these human tissue barriers in OoCs, ion track-etched porous membranes, provide only flat surfaces. Here, we propose a more realistic culture environment for alveolar cells based on biomimetically microcurved track-etched membranes. They recreate the mainly spherical geometry of the cells' native microenvironment. In this feasibility study, the membranes were given the shape of hexagonally arrayed hemispherical microwells by an innovative combination of three-dimensional (3D) microfilm (thermo)forming and ion track technology. Integrated in microfluidic chips, they separated a top from a bottom cell culture chamber. The microcurved membranes were seeded by infusion with primary human alveolar epithelial cells. Despite the pronounced topology, the cells fully lined the alveoli-like microwell structures on the membranes' top side. The confluent curved epithelial cell monolayers could be cultured successfully at the air-liquid interface for 14 days. Similarly, the top and bottom sides of the microcurved membranes were seeded with cells from the Calu-3 lung epithelial cell line and human lung microvascular endothelial cells, respectively. Thereby, the latter lined the interalveolar septum-like interspace between the microwells in a network-type fashion, as in the natural counterpart. The coculture was maintained for 11 days. The presented 3D lung-on-a-chip model might set the stage for other (micro)anatomically inspired membrane-based OoCs in the future.

Development of effective treatment strategies for lung tissue destruction as seen in emphysema would greatly benefit from representative human in vitro models of the alveolar compartment. Studying how cellular cross talk and/or (altered) biomechanical cues affect alveolar epithelial function could provide new insight for tissue repair strategies. Preclinical models of the alveolus ideally combine human primary patient-derived lung cells with advanced cell culture applications such as breathing-related stretch, to reliably represent the alveolar microenvironment. To test the feasibility of such a model, we isolated primary alveolar type 2 cells (AEC2s) from patient-derived lung tissues including those from patients with severe emphysema, using magnetic bead-based selection of cells expressing the AEC2 marker HTII-280. We obtained pure alveolar feeder-free organoid cultures using a minimally modified commercial medium. This was confirmed by known AEC2 markers as well as by detection of lamellar bodies using electron microscopy. Following (organoid-based) expansion, cells were seeded on both cell culture inserts and the Chip-S1 Organ-Chip that has a flexible polydimethylsiloxane (PDMS) membrane enabling the application of dynamic stretch. AEC2s cultured for 7 days on inserts or the chip maintained expression of HTII-280, prosurfactant protein C (SP-C), SP-A and SP-B, and zonula occludens-1 (ZO-1) also in the presence of stretch. AEC2s cultured on the chip showed lower expression levels of epithelial-mesenchymal transition-related vimentin expression compared with static cultures on inserts. The combination of a straightforward culture method of patient-derived AEC2s and their application in microfluidic chip cultures supports successful development of more representative human preclinical models of the (diseased) alveolar compartment.

Human lung function is intricately linked to the mechanics of breathing; however, it remains unknown whether and how these mechanical cues shape human lung cellular biology. While respiration-related strains and fluid flows have been suggested to promote alveolar epithelial cell function, the study of such fundamental mechanisms in the conducting airway epithelium has been hindered by the lack of suitable in vitro airway models. Here, we developed a model of human bronchial airway epithelium using well-differentiated primary cell cultures on a commercial Organs-on-Chips platform that enables the application of breathing-associated airflow and cyclic strain. It furthermore features optional endothelial cell co-culture to allow for crosstalk with the vascular compartment. Using this model, we evaluated the impact of airflow and physiological levels of cyclic strain on airway epithelial cell differentiation and function. Our findings suggest that breathing-associated mechanical stimulation changes epithelial composition, reduces secretion of IL-8, and downregulates gene expression of matrix metalloproteinase 9, fibronectin, and other extracellular matrix (ECM) factors. These results indicate that breathing-associated forces are important modulators of airway epithelial cell biology and that their fine-tuned application could generate models of specific epithelial phenotypes and pathologies.

Due to the continuing high impact of lung diseases on society and the emergence of new respiratory viruses, such as SARS-CoV-2, there is a great need for in vitro lung models that more accurately recapitulate the in vivo situation than current models based on lung epithelial cell cultures on stiff membranes. Therefore, we developed an in vitro airway epithelial–endothelial cell culture model based on Calu-3 human lung epithelial cells and human lung microvascular endothelial cells (LMVECs), cultured on opposite sides of flexible porous poly(trimethylene carbonate) (PTMC) membranes. Calu-3 cells, cultured for two weeks at an air–liquid interface (ALI), showed good expression of the tight junction (TJ) protein Zonula Occludens 1 (ZO-1). LMVECs cultured submerged for three weeks were CD31-positive, but the expression was diffuse and not localized at the cell membrane. Barrier functions of the Calu-3 cell cultures and the co-cultures with LMVECs were good, as determined by electrical resistance measurements and fluorescein isothiocyanate-dextran (FITC-dextran) permeability assays. Importantly, the Calu-3/LMVEC co-cultures showed better cell viability and barrier function than mono-cultures. Moreover, there was no evidence for epithelial- and endothelial-to-mesenchymal transition (EMT and EndoMT, respectively) based on staining for the mesenchymal markers vimentin and α-SMA, respectively. These results indicate the potential of this new airway epithelial–endothelial model for lung research. In addition, since the PTMC membrane is flexible, the model can be expanded by introducing cyclic stretch for enabling mechanical stimulation of the cells. Furthermore, the model can form the basis for biomimetic airway epithelial–endothelial and alveolar–endothelial models with primary lung epithelial cells.

Research on acute and chronic lung diseases would greatly benefit from reproducible availability of alveolar epithelial cells (AEC). Primary alveolar epithelial cells can be derived from human lung tissue but the quality of these cells is highly donor dependent. Here, we demonstrated that culture of EpCAM + cells derived from human induced pluripotent stem cells (hiPSC) at the physiological air-liquid interface (ALI) resulted in type 2 AEC-like cells (iAEC2) with alveolar characteristics. iAEC2 cells expressed native AEC2 markers (surfactant proteins and LPCAT-1) and contained lamellar bodies. ALI-iAEC2 were used to study alveolar repair over a period of 2 weeks following mechanical wounding of the cultures and the responses were compared with those obtained using primary AEC2 (pAEC2) isolated from resected lung tissue. Addition of the Wnt/β-catenin activator CHIR99021 reduced wound closure in the iAEC2 cultures but not pAEC2 cultures. This was accompanied by decreased surfactant protein expression and accumulation of podoplanin-positive cells at the wound edge. These results demonstrated the feasibility of studying alveolar repair using hiPSC-AEC2 cultured at the ALI and indicated that this model can be used in the future to study modulation of alveolar repair by (pharmaceutical) compounds.

Multiple studies have indicated that growing up on a traditional farm is associated with protection against virus-induced wheezing, allergies and asthma, suggesting protective effects on the airway epithelial barrier. Efficient re‐epithelialization of the wound is important to restore tissue homeostasis after injury from e.g. allergic inflammatory responses or from respiratory infections. A wide variety of immune cells contribute to wound repair and tissue homeostasis including resident and infiltrating macrophages. We therefore aimed to investigate the effects of farm dust on epithelial wound repair in a co-culture model of differentiated air-liquid interface (ALI) cultures of primary bronchial epithelial cells (PBEC) and monocyte-derived macrophages (MDMs). Circular wounds were made in ALI-PBEC cultures and these were subsequently cultured in presence or in absence of MDMs that had been differentiated towards pro- or anti-inflammatory macrophages in presence of GM-CSF and M-CSF, respectively. The cultures were stimulated with farm dust extract (FD) and LPS in the basal medium and wound closure was monitored at 0, 5, 21, 29 and 45 h after wounding. We observed that exposure to FD significantly enhanced epithelial wound repair after 21 and 29 h in presence of both M(GM-CSF) and M(M-CSF) macrophages, whereas direct effects of FD treatment on ALI-PBEC mono-cultures were non-significant. The results indicate that FD enhances airway epithelial wound repair through activation of both M(GM-CSF) and M(M-CSF) macrophages. However, mechanisms underlying the effects of FD on epithelial wound repair in airway epithelial-macrophage co-cultures require further investigation.

Polymeric membranes are widely applied in biomedical applications, including in vitro organ models. In such models, they are mostly used as supports on which cells are cultured to create functional tissue units of the desired organ. To this end, the membrane properties, e.g., morphology and porosity, should match the tissue properties. Organ models of dynamic (barrier) tissues, e.g., lung, require flexible, elastic and porous membranes. Thus, membranes based on poly (dimethyl siloxane) (PDMS) are often applied, which are flexible and elastic. However, PDMS has low cell adhesive properties and displays small molecule ad- and absorption. Furthermore, the introduction of porosity in these membranes requires elaborate methods. In this work, we aim to develop porous membranes for organ models based on poly(trimethylene carbonate) (PTMC): a flexible polymer with good cell adhesive properties which has been used for tissue engineering scaffolds, but not in in vitro organ models. For developing these membranes, we applied evaporation-induced phase separation (EIPS), a new method in this field based on solvent evaporation initiating phase separation, followed by membrane photo-crosslinking. We optimised various processing variables for obtaining form-stable PTMC membranes with average pore sizes between 5 to 8 µm and water permeance in the microfiltration range (17,000–41,000 L/m2/h/bar). Importantly, the membranes are flexible and are suitable for implementation in in vitro organ models.

With advances brought on by Organ-on-Chip technology, a new level of complexity was introduced into cell systems that allows research into human cellular cross-talk combined with mechanical cues. This added complexity is especially relevant in the lungs where biomechanics play a prominent role. Here we leveraged the commercial (EmulateTM) Lung-Chip and optimized its use for primary bronchial epithelial cell (PBEC) culturing to investigate the impact of airflow and mild stretch on epithelial biology. For this, PBEC culturing had to be optimized on a flexible PDMS, 7 µm pore-size membrane that allows application of stretch and leukocyte migration. To obtain a fully differentiated airway epithelium, cell media selection, coating strategy, and prevention of PBEC migration to the bottom channel was successfully achieved. Inter-chip variability (baseline IL-8 levels (ELISA)) was low (coefficient of variation <0.11 (n=3 chips)) and was comparable between donors (n=2). Furthermore, cells in the chips showed presence of relevant epithelial cell markers (qPCR), e.g. TP63 (basal cells), pIgR (luminal cells), FOXj1 (ciliated cells), MUC5AC (goblet cells). Pilot studies investigated effects of 1 week automatized mild (5%, 0.25 Hz) cyclic stretch and airflow during differentiation (week 2 at air-liquid interface) on survival, morphology, and cellular composition as assessed by qPCR. Results showed no macroscopic changes in morphology, and similar cell marker gene expression as control chips (n=1). To date, we have optimized human PBEC culturing on a flexible 7 µm pore-size membrane chip that allows studies on the impact of biomechanics on lung biology.

Air-liquid interface (ALI) cultures are frequently used in lung research but require substantial cell numbers that cannot readily be obtained from patients. We explored whether organoid expansion [three-dimensional (3D)] can be used to establish ALI cultures from clinical samples with low epithelial cell numbers. Airway epithelial cells were obtained from tracheal aspirates (TA) from preterm newborns and from bronchoalveolar lavage (BAL) or bronchial tissue (BT) from adults. TA and BAL cells were 3D-expanded, whereas cells from BT were expanded in 3D and 2D. Following expansion, cells were cultured at ALI to induce differentiation. The impact of cell origin and 2D or 3D expansion was assessed with respect to 1) cellular composition, 2) response to cigarette smoke exposure, and 3) effect of Notch inhibition or IL-13 stimulation on cellular differentiation. We established well-differentiated ALI cultures from all samples. Cellular compositions (basal, ciliated, and goblet cells) were comparable. All 3D-expanded cultures showed a similar stress response following cigarette smoke exposure but differed from the 2D-expanded cultures. Higher peak levels of antioxidant genes HMOX1 and NQO1 and a more rapid return to baseline, and a lower unfolded protein response was observed after cigarette smoke exposure in 3D-derived cultures compared to 2D-derived cultures. In addition, TA- and BAL-derived cultures were less sensitive to modulation by DAPT or IL-13 than BT-derived cultures. Organoid-based expansion of clinical samples with low cell numbers, such as TA from preterm newborns is a valid method and tool to establish ALI cultures.

Injury and remodeling of the alveolar compartment are central events in various lung diseases, including emphysema and fibrotic lung disease. The role of biomechanical forces in alveolar biology, especially in development and progression of lung diseases, has been understudied. This is in part due to the difficulties in obtaining and maintaining a stable alveolar phenotype in vitro, as well as the inability of standard cell culture systems to facilitate cyclic stretch to recreate the effect of breathing. To this end we developed a feeder-free organoid-based expansion method for tissue-isolated primary alveolar cells. These cells could be propagated for several months in vitro while retaining alveolar type 2 morphology and characteristics like surfactant production and expression of HTII-280. The organoid-expanded cells could be dissociated and seeded onto the commercial (EmulateTM) Lung-Chip to study the effect of airflow and cyclic stretch on alveolar cell biology. After optimization of the culture protocol, we cultured and maintained the primary alveolar epithelial cells on flexible PDMS membranes with a 7 µm pore-size for 7 days. We observed that alveolar type 2 cell-related markers were more stable on this chip compared to standard transwell cultures. When introducing cyclic stretch (10% at 0.25 Hz) to the cultures for 5 consecutive days, we observed alignment of the cells perpendicular to the stretch and a redistribution of tight junction protein ZO-1. These results demonstrate the feasibility of developing an Alveolus Lung-Chip model that emulates the effects of breathing-induced mechanical force on cell behaviour.

Airway epithelial cells and macrophages participate in inflammatory responses to external noxious stimuli, which can cause epithelial injury. Upon injury, epithelial cells and macrophages act in concert to ensure rapid restoration of epithelial integrity. The nature of the interactions between these cell types during epithelial repair is incompletely understood. We used an in vitro human coculture model of primary bronchial epithelial cells cultured at the air-liquid interface (ALI-PBEC) and polarized primary monocyte-derived macrophages. Using this coculture, we studied the contribution of macrophages to epithelial innate immunity, wound healing capacity, and epithelial exposure to whole cigarette smoke (WCS). Coculture of ALI-PBEC with lipopolysaccharide (LPS)-activated M(GM-CSF) macrophages increased the expression ofDEFB4A,CXCL8, andIL6at 24 h in the ALI-PBEC, whereas LPS-activated M(M-CSF) macrophages only increased epithelialIL6expression. Furthermore, wound repair was accelerated by coculture with both activated M(GM-CSF) and M(M-CSF) macrophages, also following WCS exposure. Coculture of ALI-PBEC and M(GM-CSF) macrophages resulted in increasedCAMPexpression in M(GM-CSF) macrophages, which was absent in M(M-CSF) macrophages.CAMPencodes LL-37, an antimicrobial peptide with immune-modulating and repair-enhancing activities. In conclusion, dynamic crosstalk between ALI-PBEC and macrophages enhances epithelial innate immunity and wound repair, even upon concomitant cigarette smoke exposure.

There is increasing evidence that surface curvature at a near-cell-scale influences cell behaviour. Epithelial or endothelial cells lining small acinar or tubular body lumens, as those of the alveoli or blood vessels, experience such highly curved surfaces. In contrast, the most commonly used culture substrates for in vitro modelling of these human tissue barriers, ion track-etched membranes, offer only flat surfaces. Here, we propose a more realistic culture environment for alveolar cells based on biomimetically curved track-etched membranes, preserving the mainly spherical geometry of the cells’ native microenvironment. The curved membranes were created by a combination of three-dimensional (3D) micro film (thermo)forming and ion track technology. We could successfully demonstrate the formation, the growth and a first characterization of confluent layers of lung epithelial cell lines and primary alveolar epithelial cells on membranes shaped into an array of hemispherical microwells. Besides their application in submerged culture, we could also demonstrate the compatibility of the bioinspired membranes for air-exposed culture. We observed a distinct cellular response to membrane curvature. Cells (or cell layers) on the curved membranes reveal significant differences compared to cells on flat membranes concerning membrane epithelialization, areal cell density of the formed epithelial layers, their cross-sectional morphology, and proliferation and apoptosis rates, and the same tight barrier function as on the flat membranes. The presented 3D membrane technology might pave the way for more predictive barrier in vitro models in future.

Pathologies of the respiratory system such as lung infections, chronic inflammatory lung diseases, and lung cancer are among the leading causes of morbidity and mortality, killing one in six people worldwide. Development of more effective treatments is hindered by the lack of preclinical models of the human lung that can capture the disease complexity, highly heterogeneous disease phenotypes, and pharmacokinetics and pharmacodynamics observed in patients. The merger of two novel technologies, Organs-on-Chips and human stem cell engineering, has the potential to deliver such urgently needed models. Organs-on-Chips, which are microengineered bioinspired tissue systems, recapitulate the mechanochemical environment and physiological functions of human organs while concurrent advances in generating and differentiating human stem cells promise a renewable supply of patient-specific cells for personalized and precision medicine. Here, we discuss the challenges of modeling human lung pathophysiology in vitro, evaluate past and current models including Organs-on-Chips, review the current status of lung tissue modeling using human pluripotent stem cells, explore in depth how stem-cell based Lung-on-Chips may advance disease modeling and drug testing, and summarize practical consideration for the design of Lung-on-Chips for academic and industry applications.

Advances in the culture and generation of various cell types from human induced pluripotent stem cells (hiPSCs) allow the development of more realistic and patient-specific tissue models. A major advantage of using hiPSC-derived cells is the possibility to obtain multiple cell types with the same genetic background from the same hiPSC line. The aim of the present study was to develop alveolar epithelial type 2 (AT2) cells from hiPSCs in order to develop a model of alveolar wound repair. Therefore, hiPSCs were gradually differentiated towards an alveolar and endothelial fate by closely controlling the BMP, FGF, WNT, RA and TGF-ß signalling pathways. First, definitive endoderm was induced over a period of 4 days, followed by the formation of anterior foregut endoderm and subsequently ventralized over a period of 12 days. Following ventralization, alveolar progenitors were generated during 8 days and finally the cells were exposed to dexamethasone and KGF for 4-6 weeks to induce expression of alveolar epithelial type 2 markers (surfactant proteins and LP-CAT 1) as well as lamellar bodies in about 20% of the cells. Using the EpCAM isolated cells we managed to setup a long-term culture of induced ATII cells at the air liquid interface (ALI). Using this setup we were able to visualize and monitor alveolar repair following mechanical wounding of the cultures. In a next phase of our research, we will compare wound repair of primary and hiPSC-derived ATII cells. This study was supported by a grant from the Lung Foundation NL (grant# 6.1.14.010).

The interaction between airway epithelial cells with immune and structural cells in the lung is complex and insufficiently studied due to a lack of an appropriate in vitro model. We designed an in vitro model using well-differentiated human primary bronchial epithelial cells cultured at the air-liquid interface (ALI-PBEC). These cells were co-cultured with human CD14+ monocyte-derived pro-inflammatory (Mφ1) or anti-inflammatory, pro-repair (Mφ2) macrophages, resp. Co-culture of ALI-PBEC with LPS-activated Mφ1 induced mRNA expression of hBD-2, IL-8 and IL-6 (36-, 11- and 18-fold increase resp. compared to ALI-PBEC only) at 24h, whereas this response was lower upon co-culture with LPS-stimulated Mφ2 (15-, 2- and 4-fold increase resp.). Furthermore, epithelial wound repair was accelerated by co-culture with both Mφ1 and Mφ2 (100% wound closure at 48h after wounding vs. 60% wound closure in ALI-PBEC alone), but remarkably the effect of Mφ1 cells was more pronounced at earlier time points than that of Mφ2. We demonstrate that this model is suitable for studying macrophage-epithelial interactions, and show that especially Mφ1 are capable of enhancing innate immune responses and accelerating wound repair in ALI-PBEC.

The introduction of human induced pluripotent stem cells (hiPSCs) for the generation of various cell types allows the development of more realistic and patient-specific tissue models. A major advantage is the possibility to obtain multiple cell types with the same genetic background from the same hiPSC line. The aim of the present study was to develop alveolar epithelial type 2 (AT2) cells from hiPSCs in order to develop a model of alveolar wound repair. Therefore, hiPSCs were gradually differentiated towards an alveolar and endothelial fate by closely controlling the BMP, FGF, WNT, RA and TGF-β signalling pathways. First, definitive endoderm was induced over a period of 4 days, followed by the formation of anterior foregut endoderm and subsequently ventralized over a period of 12 days. Following ventralization, alveolar progenitors were generated during 8 days and finally the cells were exposed to dexamethasone and KGF for 4-6 weeks to induce expression of alveolar epithelial type 2 markers (surfactant proteins and LP-CAT 1) as well as lamellar bodies in about 20% of the cells. Using an EpCAM isolation we managed to setup a long-term culture of induced ATII cells at the air liquid interface (ALI). Using this setup we were able to visualize and monitor alveolar repair over the course of 2 weeks following mechanical wounding of the cultures. In a next phase of our research, we will compare wound repair of primary and hiPSC-derived ATII cells.