International Journal of Molecular Sciences Communication Lung Extracellular Matrix Hydrogels Enhance Preservation of Type II Phenotype in Primary Alveolar Epithelial Cells Esther Marhuenda 1,2,† , Alvaro Villarino 1,† , Maria Leonor Narciso 1,3 , Marta Camprubí-Rimblas 2,4 , Ramon Farré 1,2,5 , Núria Gavara 1,2,3 , Antonio Artigas 2,4 , Isaac Almendros 1,2,5 and Jorge Otero 1,2,3, * 1 Unitat de Biofísica i Bioenginyeria, Facultat de Medicina i Ciències de la Salut, Universitat de Barcelona, 08036 Barcelona, Spain; marhuenda.esther@gmail.com (E.M.); alvaro.villarino.romero@gmail.com (A.V.); mnarciso@ibecbarcelona.eu (M.L.N.); rfarre@ub.edu (R.F.); ngavara@ub.edu (N.G.); ialmendros@ub.edu (I.A.) 2 CIBER de Enfermedades Respiratorias, 28029 Madrid, Spain; mcamprubi@tauli.cat (M.C.-R.); aartigas@tauli.cat (A.A.) 3 The Institute for Bioengineering of Catalonia (IBEC), The Barcelona Institute of Science and Technology (BIST), 08028 Barcelona, Spain 4 Corporació Sanitària Universitària Parc Tauli, I3PT, Universitat Autònoma de Barcelona, 08193 Sabadell, Spain 5 Institut d’Investigacions Biomèdiques August Pi i Sunyer, 08036 Barcelona, Spain * Correspondence: jorge.otero@ub.edu † These authors contributed equally to this work. Abstract: One of the main limitations of in vitro studies on lung diseases is the difficulty of main- taining the type II phenotype of alveolar epithelial cells in culture. This fact has previously been related to the translocation of the mechanosensing Yes-associated protein (YAP) to the nuclei and Citation: Marhuenda, E.; Villarino, Rho signaling pathway. In this work, we aimed to culture and subculture primary alveolar type II A.; Narciso, M.L.; Camprubí-Rimblas, cells on extracellular matrix lung-derived hydrogels to assess their suitability for phenotype mainte- M.; Farré, R.; Gavara, N.; Artigas, A.; nance. Cells cultured on lung hydrogels formed monolayers and maintained type II phenotype for a Almendros, I.; Otero, J. Lung Extracellular Matrix Hydrogels longer time as compared with those conventionally cultured. Interestingly, cells successfully grew Enhance Preservation of Type II when they were subsequently cultured on a dish. Moreover, cells cultured on a plate showed the Phenotype in Primary Alveolar active form of the YAP protein and the formation of stress fibers and focal adhesions. The results of Epithelial Cells. Int. J. Mol. Sci. 2022, chemically inhibiting the Rho pathway strongly suggest that this is one of the mechanisms by which 23, 4888. https://doi.org/10.3390/ the hydrogel promotes type II phenotype maintenance. These results regarding protein expression ijms23094888 strongly suggest that the chemical and biophysical properties of the hydrogel have a considerable Academic Editors: Athanasios impact on the transition from ATII to ATI phenotypes. In conclusion, culturing primary alveolar G. Papavassiliou, Patrick C. Baer epithelial cells on lung ECM-derived hydrogels may facilitate the prolonged culturing of these cells, and Ralf Schubert and thus help in the research on lung diseases. Received: 27 March 2022 Keywords: extracellular matrix; hydrogels; alveolar cells; type II phenotype; YAP Accepted: 26 April 2022 Published: 28 April 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in 1. Introduction published maps and institutional affil- The fundamental questions on the precise mechanisms underlying alveolar epithelial iations. cells (AECs) damage and epithelium repair in relevant diseases, such as acute respiratory distress syndrome or chronic obstructive pulmonary disease, are still unsolved. Although it is well-known that the alveolar epithelium is repaired by the proliferation of type II AECs Copyright: © 2022 by the authors. (ATIIs), which differentiate into type I phenotype cells (ATIs), the involved mechanisms Licensee MDPI, Basel, Switzerland. are still poorly understood [1]. In fact, a limitation hampering translational studies in lung This article is an open access article diseases is the difficulty of maintaining the type II phenotype of primary AECs in vitro. distributed under the terms and Indeed, it is widely known that ATII-to-ATI transdifferentiation occurs very quickly in vitro; conditions of the Creative Commons thus, primary type II AECs neither adequately proliferate, nor can be subcultured under Attribution (CC BY) license (https:// conventional culture conditions [2]. creativecommons.org/licenses/by/ To extend the maintenance of the type II AECs proliferative phenotype in vitro, it 4.0/). was proposed to coat the culture plates with hydrogels, such as Matrigel, resembling the Int. J. Mol. Sci. 2022, 23, 4888. https://doi.org/10.3390/ijms23094888 https://www.mdpi.com/journal/ijms extracellular matrix (ECM) [3]. Moreover, pioneering studies on subculturing strateg for primary ATII cells [4] employed inhibitors of Rho kinases, as the connection betwe the activation of the Rho pathway and cell mechanosensing of the ECM Int. J. Mol. Sci. 2022, 23, 4888 well-established [5]. However, although the relationship between ATII 2 of 11 phenoty maintenance in vitro and culturing cells on ECM-like substrates was proposed th decades ago [6], the problem remains open, probably because no hydrogels derived fr lung ECM were extracellular available. matrix (ECM) [3]. Moreover, pioneering studies on subculturing strategies for Interestingly, primary ATII cells [4]based on an employed initial of inhibitors report for obtaining Rho kinases, hydrogels as the connection fromthe between the ECM activation of thelungs decellularized Rho pathway [7], weand cellrecently have mechanosensing of the described a ECM is well-established procedure for preparing [5]. such However, drogels byalthough the relationship exclusively using lungbetween ECM, ATII withphenotype no need to maintenance in vitro and add potentially toxic exter culturing cells on ECM-like substrates was proposed three decades ago [6], the problem cross-linkers [8]. If used as a culture substrate, this hydrogel, which realistically mim remains open, probably because no hydrogels derived from lung ECM were available. the native lung ECM, Interestingly, based oncould be particularly an initial well-suited report for obtaining hydrogelsfor providing from the ECM of a decel- physiomime microenvironment lularized lungs [7], wetohave primary AECs. recently Therefore, described we for a procedure hypothesized preparing such that a lungby hydrogels ECM-bas hydrogel exclusivelywould slowECM, using lung down withthe noATII-to-ATI transdifferentiation need to add potentially mediated by toxic external cross-linkers [8].the inh If used tion as aRho of the culture substrate, pathway, this hydrogel, resulting which realistically in a decrease in F-actinmimics the native lung polymerization and the f ECM, could be particularly well-suited for providing a physiomimetic mation of focal adhesions, as well as nuclear YAP activation [9–11]. microenvironment to primary AECs. Therefore, we hypothesized that a lung ECM-based hydrogel would slow down the ATII-to-ATI transdifferentiation mediated by the inhibition of the Rho pathway, 2.resulting Resultsin a decrease in F-actin polymerization and the formation of focal adhesions, as 2.1. wellPrimary Alveolar as nuclear Epithelial YAP activation Cells Form Monolayers on Lung-Derived Hydrogels [9–11]. Isolated AECs were grown either in lung-derived hydrogels or plates for four da 2. Results Cells were able 2.1. Primary to form Alveolar a monolayer Epithelial on a lung-derived Cells Form Monolayers hydrogel, on Lung-Derived as shown in bright-fi Hydrogels images in Figure Isolated AECs1. Differences were grown either in in thelung-derived morphology of the or hydrogels cells as for plates a function four days.of the su strate were also noticeable as shown by confocal images in Figure Cells were able to form a monolayer on a lung-derived hydrogel, as shown in bright-field 2. Throughout images incells culture, Figure on1.plates Differences in thetomorphology started show a moreof theflattened cells as a function shape of asthe substrate well as larger cy were also noticeable as shown by confocal images in Figure 2. Throughout plasms (cells cultured on plates presented, in general, sizes about 5 times larger the culture, cells than ce on plates started to show a more flattened shape as well as larger cytoplasms (cells cultured cultured on hydrogels), and the presence of vacuoles was noticeable. On the contra on plates presented, in general, sizes about 5 times larger than cells cultured on hydrogels), AECs and the cultured presence on hydrogels of vacuoles was showed noticeable.cuboidal shapes,AECs On the contrary, andcultured even their microvilli, a p on hydrogels notypical characteristic showed cuboidal shapes, of andATII evencells their[12], couldabe microvilli, distinguished. phenotypical Cells cultured characteristic of ATII on pla formed monolayers cells [12], faster (dayCells could be distinguished. 3 vs.cultured day 4).on plates formed monolayers faster (day 3 vs. day 4). Figure Figure 1. Ratprimary 1. Rat primary alveolar alveolar cellscells were were cultured cultured either oneither porcineonlung-derived porcine lung-derived hydrogel hydrogel (A) or on (A on a plate a plate (B).(B). Bright Bright field field imagesimages wereevery were taken taken24 every h from 24 dayh1.from day 1. x FOR PEER REVIEW 3 of 11 Int. J. Mol. Sci. 2022, 23, 4888 3 of 11 Figure 2. Three-dimensional representation of primary alveolar type II cells cultured for three days Figure 2. Three-dimensional representation on hydrogel of primary (A) and on a plate (B). Nucleialveolar type are stained II cells in blue cultured and actin for three cytoskeleton days in red. In the on hydrogel (A) and on a plate (B). Nuclei are stained in blue and actin cytoskeleton in red. In the case of the hydrogel-cultured cell, it can be distinguished by a cuboidal morphology and the presence case of the hydrogel-cultured of microvilli,cell, it can indicated be distinguished by arrows. bycell In the case of the a cultured cuboidal on amorphology and out plate, a more spread theand presence of microvilli, indicated by arrows. In the case of the cell cultured on a plate, a more spread wider cytoplasm can be observed showing increased size. out and wider cytoplasm2.2. can be observed Culture showingCells of Alveolar Epithelial increased size. Hydrogels Preserves the Expression of on Lung-Derived Type II Markers for Longer Periods 2.2. Culture of Alveolar Epithelial Cells The results ongenetic of the Lung-Derived expression Hydrogels Preserves of ATI and ATII typical the Expression markers of 2 to (from day 5) are shown Type II Markers for Longer Periods in Figure 3A. The gene expression of sftpc and sftpb decreased over time. There was a significant increase in ATII markers (surfactant proteins B and C) at earlier The results of the genetic times expression (day 2 and of ATI day 3) in cells andonATII cultured typicalhydrogels. lung-derived markersThere (from wasday 2 to no increase 5) are shown in Figure 3A. The gene expression of sftpc and sftpb decreased over time. on in ATI markers over time in cells cultured on hydrogels, in contrast to cells cultured plates, where the expression of pdpn and aqp5 increased with time (classical ATI markers). There was a significant increase in ATII markers (surfactant proteins B and C) at earlier These results show that the ATII phenotype and gene expression levels were maintained times (day 2 and day for 3) in cells longer cultured hydrogels. in lung-derived on lung-derived The resultshydrogels. There was from immunostaining are no in- in shown crease in ATI markers over Figure time 3B–E, in cells revealing thatcultured on hydrogels, the differential expression ofin contrast surfactant to cells protein cul- by C caused the substrate was noticeable not only at the gene expression tured on plates, where the expression of pdpn and aqp5 increased with time (classical ATI level, but also at the protein level. SPC expression was higher in cells cultured on lung hydrogels compared to that of markers). These results show that the ATII phenotype and gene expression levels were cells cultured on a plate, as shown by the immunostainings. Furthermore, culture time maintained for longer in lung-derived affected cells differently hydrogels. depending onThe results they the substrate from immunostaining were cultured on. Indeed, are cells shown in Figure 3B–E, revealing cultured on lung that thewere hydrogel differential expression able to maintain the SPCof surfactant expression overprotein C time, whereas cells cultured on a plate rapidly lost this ability. caused by the substrate was noticeable not only at the gene expression level, but also at the protein level. SPC expression was higher in cells cultured on lung hydrogels com- pared to that of cells cultured on a plate, as shown by the immunostainings. Furthermore, culture time affected cells differently depending on the substrate they were cultured on. Indeed, cells cultured on lung hydrogel were able to maintain the SPC expression over time, whereas cells cultured on a plate rapidly lost this ability. Int. Int. J. Mol. Sci. J. Mol. 2022,23, Sci.2022, 23,4888 x FOR PEER REVIEW 4 of 11 4 of 11 . Figure Figure3. 3. ATII cellscells ATII werewere cultured either either cultured on hydrogel (HG) or (HG) on hydrogel a tissueorculture plate a tissue (TCP).plate culture ATII (TCP). ATII typical typicalmarker surfactant marker proteins surfactant C (sftpc) proteins and B (sftpb) C (sftpc) and Band ATI typical (sftpb) and ATI marker aquaporin typical marker5 aquaporin (aqp5), 5 (aqp5), podoplanin (pdpn) were studied at different time points (from day 2 to day 5) by qPCR (A). Relative gene expression is shown. Expression of surfactant protein C (SPC) was studied using immunofluo- rescence on ATII cells cultured on hydrogel at day 2 (B) and day 4 (D) and on a plate at day 2 (C) and day 4 (E). * p < 0.05, *** p < 0.001. fluorescence on ATII cells cultured on hydrogel at day 2 (B) and day 4 (D) and on a plate at day 2 (C) and day 4 (E). * p < 0.05, *** p < 0.001. 2.3. Culture of Primary Alveolar Epithelial Cells in Lung-Derived Hydrogels Inhibits Type II-to-Type I Transdifferentiation by Altering the Hippo/Rho Pathway Int. J. Mol. Sci. 2022, 23, 4888 5 of 11 Since YAP is a key mechanotransduction protein, its expression was studied in cells cultured on hydrogels or plates 2.3. Culture for three of Primary Alveolardays (Figure Epithelial Cells in4A,B). In cells Lung-Derived cultured Hydrogels Inhibitson Typehydro- II-to-Type I Transdifferentiation by Altering the Hippo/Rho Pathway gels, the YAP protein was located mainly in the cytoplasm, which indicates that it was Since YAP is a key mechanotransduction protein, its expression was studied in cells being phosphorylated and subsequently degraded. In contrast, in cells cultured on plates, cultured on hydrogels or plates for three days (Figure 4A,B). In cells cultured on hydrogels, nuclear active YAPthewas observed. YAP protein Specifically, was located there mainly in the was awhich cytoplasm, two-fold indicatesincrease that it wasin the being amount of nuclear phosphorylated YAP when and cells were cultured subsequently degraded. onIn contrast, a plateincompared cells culturedto that of on plates, cells nuclear active YAP was observed. Specifically, there was a two-fold increase in the amount of cultured on hydrogels, indicating a higher transcriptional activity in the first group nuclear YAP when cells were cultured on a plate compared to that of cells cultured on (Figure 4C). hydrogels, indicating a higher transcriptional activity in the first group (Figure 4C). Figure 4. Alveolar type II cells were cultured on hydrogel (A) or on a plate (B) and stained for YAP protein (green). YAP nuclear and cytoplasmic expressions were quantified and expressed as Figure 4. Alveolar type II cells were cultured on hydrogel (A) or on a plate (B) and stained for YAP the nuclei/cytoplasm fluorescence ratio in both conditions hydrogel (HG) and tissue culture plate protein (green). YAP (TCP) nuclear and (C), *** cytoplasmic expressions were quantified and expressed as the p < 0.001. nuclei/cytoplasm fluorescence ratio in both conditions hydrogel (HG) and tissue culture plate (TCP) (C), *** p < 0.001. Owing to the role of focal adhesions (FAs) and the actin cytoskeleton in sensing extracellular matrix cues and transmitting them to the cell, the expressions of actin and paxillin, which is one of the proteins comprising FAs, were studied. Both were reported Owing to the role to beof focal adhesions implicated in the hippo(FAs) and pathway, by the actinitcytoskeleton inhibiting and promoting the in sensing YAP nuclearex- expression. tracellular matrix cues and In cells cultured forthem transmitting three days on lung-derived to the cell, the hydrogels, expressionsa pooroffocal adhesion actin and assembly was observed by the paxillin immunostaining. Moreover, no stress fibers were paxillin, which is one of the proteins comprising FAs, were studied. Both were reported formed as indicated by the phalloidin staining (Figure 5A). On the contrary, in cells that to be implicated in were the cultured hippo pathway, for three days by oninhibiting it and a plate, assembled promoting paxillin and stressthe YAP fibers werenuclear clearly expression. In cellsobserved cultured for 5B). (Figure three days The role on lung-derived of Rho, which is involved in hydrogels, the maturationa of poor focal focal ad- adhesions and YAP regulation [13,14] was studied by the use of the ROCK inhibitor (Y27632). The hesion assembly was observed by the paxillin immunostaining. Moreover, no stress fi- results show that it could play a role in the maintenance of the ATII phenotype, as reflected bers were formed asbyindicated an increase inby SPCthe phalloidinculture in conventional staining (Figure (p = 0.02) 5A). (Figure On thewith 5D) together contrary, a decreasein cells that were cultured in focal for three adhesion sizedays on a(Figure (p = 0.001) plate, assembled 5D), paxillin suggesting that and the use of HGsstress fibers as a substrate for AECs culture could be inhibiting the Rho pathway, and thus allowing for type II were clearly observed (Figure 5B). The role of Rho, which is involved in the maturation of phenotype maintenance. focal adhesions and YAP regulation [13,14] was studied by the use of the ROCK inhibitor (Y27632). The results show that it could play a role in the maintenance of the ATII phe- notype, as reflected by an increase in SPC in conventional culture (p = 0.02) (Figure 5D) together with a decrease in focal adhesion size (p = 0.001) (Figure 5D), suggesting that the use of HGs as a substrate for AECs culture could be inhibiting the Rho pathway, and thus allowing for type II phenotype maintenance. OR PEER REVIEW 6 of 11 Int. J. Mol. Sci. 2022, 23, 4888 6 of 11 Figure 5. Representative images of focal adhesions (FAs) in cells cultured on hydrogels (A) and plate Figure 5. Representative images of focal adhesions (FAs) in cells cultured on hydrogels (A) and (B). Red: phalloidin, green: paxillin, blue: nuclei. Quantification of the FA length (C) and the intensity plate (B). Red: phalloidin, green: paxillin, of surfactant blue: protein C (SPC) nuclei.type of alveolar Quantification of the FA II cultured on hydrogels for 3 length days with(C) and and (Y27632) the intensity of surfactant protein C (SPC) without (CTRL) of alveolar the addition typeinhibitor of the ROCK II cultured (D), * p