Name: iPSC-Derived Epithelial, Mesenchymal, Endothelial, and Immune Cell Co-Culture to Model Airway Barrier Integrity in Lung Health and Disease (Video) | JoVE Journal
Uploaded: 2024-12-06T13:40:00.000+00:00
Duration: 6 min 27 s
Description: Learn to build a multi-cellular airway barrier model using iPSC-derived epithelial, mesenchymal, endothelial, and immune cells in air-liquid interface culture. This method enables study of airway remodeling and toxin responses.

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5 minutes

This research was supported by CIRM (DISC2COVID19-12022).

Airway Model for Studying Lung Barrier Integrity, Disease, and Drug Discovery

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</ol>

The authors have nothing to disclose.

The development and implementation of a model of the blood-air barrier in the large airways for studying viral infections and other toxins require meticulous attention to detail to ensure the successful differentiation and function of the various cell types involved. This discussion will address key factors for successful differentiation, potential challenges, alternative applications, and implications for studying human diseases. 
To ensure a successful differentiation, attention to the type of cell culture insert and pore size is important. Large pores should be used to ensure cell-cell communication through the layers of ECM. Ensuring the optimal seeding density for induced pluripotent stem cell (iPSC) lines is critical. Proper density facilitates uniform differentiation and prevents the formation of unwanted cell clumps, leading to a more homogenous and functional epithelial layer. To maintain a healthy and functional epithelial layer, washing the apical surface before adding any compounds, toxins, or viruses is important. This helps remove accumulated mucus, dead cells, and other debris, thereby promoting a stable and viable culture environment and allowing access to the epithelial cells.
For the endothelial component, it is essential to ensure that the cells are properly sorted and homogeneous. This can be achieved through techniques such as FACS, which ensures that only the desired endothelial cells are used, thereby maintaining consistency and function.
For the macrophage component, despite adhering to plastic easily, macrophages do not adhere well to apical epithelial cells. Increasing the number of macrophages may be beneficial to ensure a large population of macrophages in the iAirways. Adherent macrophages are better positioned to interact with pathogens and other immune cells, thus enhancing the model's relevance. If the study of endothelial immune signaling is preferred, the macrophages can be seeded in the basolateral side of the cell culture inserts in the media to mimic the circulating monocyte population22. 
The iAirway model has several other significant applications, including studying lung development of the airway. The model can be used to study the intricate processes involved in lung development, which involves multi-cellular signaling between different populations of cells, providing insights into normal and pathological conditions. To study alveolar development and disease, airway organoids can be substituted for distal lung organoids (DLOs)23,24 This allows researchers to study the distal lung, including alveolar development and function. This is particularly useful for generating and studying alveolar macrophages, which play a key role in lung immunity.
Developing a reliable and reproducible model of the blood-air barrier in large airways comes with several challenges. Lung progenitor cells must express greater than 50% NKX2-1 for successful differentiation. If the yield is not reached, the ALI cultures may harbor more mesenchyme or be contaminated with non-lung epithelial cells. Maintaining sterility during the plating of endothelial cells is a significant challenge since they are cultured on the basolateral side of the cell culture insert. This prevents the plate lid from closing the sterile environment, so other plates must be used during the seeding of endothelial cells. Contaminants can disrupt the culture and compromise the integrity of the model. Most ALI cultures are incubated for a minimum of 3 weeks to generate most of the airway cells, including ciliated cells. Most protocols seed basal cells, and thus must allow time for signaling mechanisms to differentiate the other airway cells. The current protocol dissociates airway organoids, which already contain many of the airway cells, albeit at lower quantities compared to mature HBEC-derived ALI. Few ciliated cells are seen along with epithelial and mesenchymal cell types (Figure 2D). To generate more ciliated cells, DAPT can be added to the airway organoid cultures and/or the ALI media25. This platform has also only tested the short-term viability of the apical and basolateral cells after air-lift, when the basolateral media is changed to 1:1 epithelial and endothelial media. More research needs to be done to test the viability over a span of weeks, which may increase the complexity of the cells in ALI. 
The blood-air barrier model has profound implications for studying human diseases and developing therapeutic interventions. The model can be used to screen and optimize therapeutic agents, including antiviral drugs, by providing a physiologically relevant environment that mimics human airways, especially with the exposure of the airway cells to air. Gene knockout techniques can be employed to study specific mechanisms of barrier dysfunction and host-pathogen interactions. The model offers a platform to study various aspects of human respiratory diseases, from viral infections to chronic conditions like asthma and COPD. This understanding can lead to the development of more effective and targeted therapies.
In conclusion, the blood-air barrier model of the large airway (iAirway) represents a powerful tool for studying barrier dysfunction due to viral infections and other respiratory diseases. Ensuring successful differentiation of cell types, addressing developmental challenges, and exploring alternative applications will enhance the utility and accuracy of this model. Its application in therapeutic targeting and gene knockout studies underscores its potential in advancing our understanding and treatment of human respiratory diseases.

The blood-air barrier in the large airways includes the trachea, bronchi, and bronchioles. It plays a crucial role in maintaining respiratory health and is made up of the airway epithelium, basement membrane, blood vessels and endothelial cells, and immune cells. The primary epithelial cells in the airway encompass basal cells, club cells, ciliated cells, and goblet cells. Basal cells, acting as the stem cells of the airway epithelium, are multipotent progenitors with high proliferative and self-renewal capabilities, giving rise to mature airway epithelial cells1. Club cells are non-ciliated, secretory cells that contribute to the maintenance of the airway lining by secreting protective proteins and surfactants2. Goblet cells, located in the lumen and in the submucosal glands, secrete mucins to trap debris and safeguard the airway3. Ciliated cells are integral to the mucociliary escalator mechanism, preventing the accumulation of harmful microorganisms4. The basement membrane is made up of an extracellular matrix, which provides structural support5. The trachea and the rest of the airway are surrounded by a rich network of blood vessels, which are lined with endothelial cells that play a vital role in supporting tracheal function by supplying nutrients and oxygen, removing waste, regulating inflammation, and contributing to tissue repair and angiogenesis6. Finally, airway macrophages are tissue-specific immune cells, essential for protecting the respiratory system from infections, clearing inhaled particles, and maintaining a balanced immune response7.
The coordinated actions of epithelial, mesenchymal, endothelial cells, and macrophage cells are critical for an effective immune response to pathogens in the airway8. Epithelial cells form the first line of defense against viral infections by acting as a physical barrier, with tight junctions restricting the passage of harmful substances. The coordinated action of ciliated cells and goblet cells helps to trap and remove inhaled particles, pathogens, and debris4. Additionally, airway epithelial cells produce cytokines and chemokines to recruit immune cells9. Endothelial cells maintain vascular integrity, preventing the spread of viral particles through the bloodstream, upregulate adhesion molecules (VCAM-1) to facilitate immune cell adhesion and produce pro-inflammatory cytokines to recruit immune cells from the bloodstream to the site of infection10. Airway macrophages engulf and digest viral particles, infected cells, and debris, present viral antigens to T cells, and produce cytokines to activate and recruit other immune cells, along with type I interferons to inhibit viral replication11. The coordinated actions of epithelial, mesenchymal, endothelial, and macrophage cells create a robust and dynamic defense system that protects the airway from viral infections and maintains respiratory health.
Understanding the dynamic interactions among various cell types in the human lung is crucial for comprehending the lung&#39;s response to viral infections, inflammatory diseases, and drug delivery. In vitro co-cultures allow for the study of cell-cell signaling between the epithelium, endothelial cells, and innate immune cells12. We developed the first authentic multi-cell type lung model derived from patient-specific hiPSCs13. This incorporates both epithelial and mesenchymal cell populations, formed in a 3D orientation. Subsequently, the lung progenitor cells can be differentiated into an &#34;airway organoid&#34;14, cultured onto sterile cell culture inserts, and exposed to an air-liquid interface (ALI), replicating the conditions of the human airway15,16,17. iPSC-derived endothelial cells are cultured on the basolateral side of the membrane, mimicking their orientation in the human airway, situated below the epithelial/mesenchymal layer in the basement membrane. Finally, iPSC-derived macrophages are added to the apical side of the membrane, interacting with epithelial cells and awaiting activation signals (Figure 1A). This model accurately reproduces the biology and function of the airway. We posit that hiPSC-derived, patient-specific, authentic multi-cell type iAirway cultures are best suited to elucidate the intrinsic, acute response of the airway barrier and pathogens, including viral infections. For instance, this model can be used to (1) study viral entry and replication, (2) investigate the initial immune response by epithelial and tissue-specific immune cells, (3) examine barrier integrity and function, (4) test the efficacy of therapeutic agents, and (5) study cellular and molecular mechanisms of pathogenesis in a patient-specific model.
This article describes a detailed protocol for preparing multi-cellular lung co-cultures to study cellular responses to viral infections.

<table><tbody><tr><td>12 well plates</td><td>Corning</td><td>3512</td><td></td></tr><tr><td>12-well inserts, 0.4 &amp;micro;m, translucent&amp;nbsp;</td><td>VWR</td><td>10769-208</td><td></td></tr><tr><td>2-mercaptoethanol&amp;nbsp;</td><td>Sigma-Aldrich</td><td>M3148</td><td></td></tr><tr><td>Accutase</td><td>Innovative Cell Tech</td><td>AT104</td><td></td></tr><tr><td>Activin A</td><td>R&amp;amp;D Systems</td><td>338-AC</td><td></td></tr><tr><td>All-trans retinoic acid (RA)&amp;nbsp;</td><td>Sigma-Aldrich</td><td>R2625</td><td></td></tr><tr><td>ascorbic acid</td><td>Sigma</td><td>A4544</td><td></td></tr><tr><td>B27 without retinoic acid&amp;nbsp;</td><td>ThermoFisher</td><td>12587010</td><td></td></tr><tr><td>BMP4&amp;nbsp;</td><td>R&amp;amp;D Systems</td><td>314-BP/CF</td><td></td></tr><tr><td>Bovine serum albumin (BSA) Fraction V, 7.5% solution&amp;nbsp;</td><td>Gibco</td><td>15260-037</td><td></td></tr><tr><td>Br-cAMP</td><td>Sigma-Aldrich</td><td>B5386</td><td></td></tr><tr><td>CD 14 (FITC)</td><td>BioLegend</td><td>982502</td><td></td></tr><tr><td>CD 31 PECAM-1(APC)</td><td>R&amp;amp;D System</td><td>FAB3567A</td><td></td></tr><tr><td>CD 45 (PE)</td><td>BD Biosciences</td><td>560975</td><td></td></tr><tr><td>CD 68 (PE)</td><td>BioLegend</td><td>33808</td><td></td></tr><tr><td>CHIR99021&amp;nbsp;</td><td>Abcam</td><td>ab120890</td><td></td></tr><tr><td>CPM</td><td>Fujifilm</td><td>014-27501</td><td></td></tr><tr><td>Dexamethasone&amp;nbsp;</td><td>Sigma-Aldrich</td><td>D4902</td><td></td></tr><tr><td>Dispase&amp;nbsp;</td><td>StemCellTech</td><td>7913</td><td></td></tr><tr><td>DMEM/F12&amp;nbsp;</td><td>Gibco</td><td>10565042</td><td></td></tr><tr><td>Dorsomorphin</td><td>R&amp;amp;D Systems</td><td>3093</td><td></td></tr><tr><td>E-CAD/CD 324 (APC)</td><td>BioLegend</td><td>324107</td><td></td></tr><tr><td>EGF</td><td>R&amp;amp;D Systems</td><td>236-EG</td><td></td></tr><tr><td>EGM2 Medium</td><td>Lonza</td><td>CC-3162</td><td></td></tr><tr><td>EPCAM/CD 326 (APC)</td><td>BioLegend</td><td>324212</td><td></td></tr><tr><td>FBS&amp;nbsp;</td><td>Gibco</td><td>10082139</td><td></td></tr><tr><td>FGF10</td><td>R&amp;amp;D Systems</td><td>345-FG/CF</td><td></td></tr><tr><td>FGF7</td><td>R&amp;amp;D Systems</td><td>251-KG/CF</td><td></td></tr><tr><td>Fibronectin</td><td>Fisher</td><td>356008</td><td></td></tr><tr><td>Forskolin</td><td>Abcam</td><td>ab120058</td><td></td></tr><tr><td>Glutamax&amp;nbsp;</td><td>Life Technologies</td><td>35050061</td><td></td></tr><tr><td>Ham&amp;rsquo;s F12&amp;nbsp;</td><td>Invitrogen</td><td>11765-054</td><td></td></tr><tr><td>HEPES</td><td>Gibco</td><td>15630-080</td><td></td></tr><tr><td>IBMX (3-Isobtyl-1-methylxanthine)</td><td>Sigma-Aldrich</td><td>I5879</td><td></td></tr><tr><td>IL-3</td><td>Peprotech</td><td>200-03</td><td></td></tr><tr><td>Iscove&amp;rsquo;s Modified Dulbecco&amp;rsquo;s Medium (IMDM) + Glutamax&amp;nbsp;</td><td>Invitrogen</td><td>31980030</td><td></td></tr><tr><td>Knockout Serum Replacement (KSR)</td><td>Life Technologies</td><td>10828028</td><td></td></tr><tr><td>Matrigel</td><td>Corning</td><td>354230</td><td></td></tr><tr><td>M-CSF</td><td>Peprotech&amp;nbsp;</td><td>300-25</td><td></td></tr><tr><td>Monothioglycerol&amp;nbsp;</td><td>Sigma</td><td>M6145</td><td></td></tr><tr><td>mTeSR plus Kit (10/case)</td><td>Stem Cell Tech</td><td>5825</td><td></td></tr><tr><td>N2&amp;nbsp;</td><td>ThermoFisher</td><td>17502048</td><td></td></tr><tr><td>NEAA</td><td>Life Technologies</td><td>11140050</td><td></td></tr><tr><td>PBS</td><td>Gibco</td><td>10010023</td><td></td></tr><tr><td>Pen/strep</td><td>Lonza</td><td>17-602F</td><td></td></tr><tr><td>ReleSR</td><td>Stem Cell Tech</td><td>5872</td><td></td></tr><tr><td>RPMI1640 + Glutamax&amp;nbsp;</td><td>Life Technologies</td><td>12633012</td><td></td></tr><tr><td>SB431542&amp;nbsp;</td><td>R&amp;amp;D Systems</td><td>1614</td><td></td></tr><tr><td>SCF</td><td>PeproTech</td><td>300-07</td><td></td></tr><tr><td>SMA</td><td>Invitrogen</td><td>50-9760-80</td><td></td></tr><tr><td>STEMdiff APEL 2 Medium</td><td>STEMCELL Technologies</td><td>5275</td><td></td></tr><tr><td>TrypLE Express</td><td>Gibco</td><td>12605-028</td><td></td></tr><tr><td>VEGF165</td><td>Preprotech</td><td>100-20</td><td></td></tr><tr><td>Vimentin</td><td>Cell Signaling</td><td>5741S</td><td></td></tr><tr><td>Y-27632 (Rock Inhibitor)&amp;nbsp;</td><td>R&amp;amp;D Systems</td><td>1254/1</td><td></td></tr><tr><td>ZO-1</td><td>Invitrogen</td><td>339100</td><td></td></tr></tbody></table>

ipsc-derived epithelial, mesenchymal, endothelial, and immune cell co-culture to model airway barrier integrity in lung health and disease

iPSC-Derived Epithelial, Mesenchymal, Endothelial, and Immune Cell Co-Culture to Model Airway Barrier Integrity in Lung Health and Disease

Human lung tissue is composed of an interconnected network of epithelium, mesenchyme, endothelium, and immune cells from the upper airway of the nasopharynx to the smallest alveolar sac. Interactions between these cells are crucial in lung development and disease, acting as a barrier against harmful chemicals and pathogens. Current in vitro co-culture models utilize immortalized cell lines with different biological backgrounds, which may not accurately represent the cellular milieu or interactions of the lung. We differentiated human iPSCs into 3D lung organoids (containing both epithelium and mesenchyme), endothelial cells, and macrophages. These were co-cultured in an air-liquid interface (ALI) format to form an epithelial/mesenchymal apical barrier invested with macrophages and a basolateral endothelial barrier (iAirway). iPSC-derived iAirways showed a reduction in barrier integrity in response to infection with respiratory viruses and cigarette toxins. This multi-lineage lung co-culture system provides a platform for studying cellular interactions, signaling pathways, and molecular mechanisms underlying lung development, homeostasis, and disease progression. iAirways closely mimic human physiology and cellular interactions, can be generated from patient-derived iPSC&#39;s, and can be customized to include different cell types of the airway. Overall, iPSC-derived iAirway models offer a versatile and powerful tool for studying barrier integrity to better understand genetic drivers for disease, pathogen response, immune regulation, and drug discovery or repurposing in vitro, with the potential to advance our understanding and treatment of airway diseases.

There are multiple stages at which the differentiation of iPSC-derived airway organoids, endothelial cells, immune cells, and co-cultures can be assessed as successfully completed. Differentiations can be performed in different iPSC lines, and this protocol has been tested in at least five different lines. The protocol does need to be adapted to every new iPSC line, specifically by modifying and optimizing the seeding density.
The successful yield of iPSC-derived airway organoids can be assessed via brightfield and will have four characteristics: defined boundaries/edges, translucent/cystic morphology, ability to proliferate/expand, and lack of necrotic cells. Airway organoids that appear fuzzy do not increase in size over the 2 weeks of culture, and have necrosis should not be used for co-culture experiments. Organoids with a thick luminal layer on the periphery vs. the translucent/cystic organoids can still be used for subsequent experiments. However, the translucent/cystic airway organoids are composed of predominantly basal cells, whereas the thick dual membrane organoids are composed of predominantly club cells. Because basal cells proliferate and expand easily, they are more suited for air-liquid interphase culture and polarization. Other methods for assessing the successful yield of iPSC-derived lung organoids are flow-cytometry analysis at the lung progenitor stage (Day 16 of the differentiation) for surface marker CPM (Table 1), a successful differentiation yield &gt;50% CPM+ cells.
For the airway organoid dissociation and air-liquid interphase culture, ensure that all of the ECM polymer has been removed, or it will prevent the cells from expanding on the membrane. The cells should be confluent by Day 3 of culture, prior to air-lifting. Confirmation of cell types in the iAirways can be assessed by staining for respective cell type-specific markers: basal cells (p63+), club cells (SCGB1A1+), goblet cells (MUC5AC+), and ciliated cells (AcTub+). Characterization and hallmark lung cell type-specific markers are verified on LungMAP21. Mesenchyme can be detected with antibodies against vimentin or smooth muscle actin. To generate more ciliated cells, the cultures may require air-lifting for a longer period of time or the addition of DAPT.
The successful yield of iPSC-derived endothelial cells can be assessed via brightfield, showing a cobblestone-like morphology (Day 6 of the iPSC-derived endothelial differentiation). A successful iPSC-derived endothelial culture will contain &gt;50% CD31+ cells, and enough cells should be generated so that after sorting, the yield should be more than 500,000 cells. Cells can be passaged or cryopreserved, and the CD31 marker should be tested via flow with each passage or new culture. Cells should not be passaged beyond P4. For reference, see iPSC-derived endothelial cells pre/post CD31 sort (Figure 1C). Endothelial cells will be flat with a large cell body with elongated and spindle-shaped morphology.
For macrophage differentiation, embryoid bodies should remain at the bottom of the ULA well, and only one EB should be in each well. They should show cystic changes by Day 5-7. If they do not form an EB after centrifugation, then seed the iPSCs into the 96-well ULA plate without spinning. Increasing the number of iPSCs may be necessary. The successful yield of iPSC-derived immune cells is entirely dependent on the ability of embryoid bodies (EBs) to develop into cysts (Day 7 of differentiation). iPSC-derived immune EBs that do not develop cysts will not form stroma when plated onto gelatin and will not produce myeloid progenitors. After transfer to the gelatin-coated plate, the EBs should remain attached to the bottom of the plate. Movement of the plate should be done carefully so as not to dislodge them. Plates should be changed twice a week manually, and the suspended cells should be passaged into Mac-CM2 or cryopreserved. Additionally, after harvesting the suspended myeloid progenitors, they should adhere to the non-coated plastic flask, and generate bright cells under brightfield with vacuoles, indicating macrophages. The marker for monocytes, CD14+, should be &gt;90% in the suspended cells. The macrophage marker, CD68, should increase over time, with ~50% of macrophages expressing CD68+ after 2 weeks (Figure 1D). Adhesion efficiency should be determined for each cell line. We achieve an average of 20% adhesion efficiency. For polarization, M0 macrophages are polarized to either M1 (iPSC-M1) or M2 (iPSC-M2) macrophages for 48 h in differentiation medium II supplemented with different stimuli: 100 ng/mL LPS and 20 ng/mL IFN-g for M1 polarization or 50 ng/mL IL-4 and 20 ng/mL IL-13 for M2 polarization. The marker for M1 polarization is CD80+, and M2 polarization is CD206+.
Successful generation of the triple co-culture can be assessed via immunofluorescence for epithelial tight junction markers EPCAM, ECAD, or ZO-1, or endothelial tight junction marker CD31 (Figure 2C, Table 1). The functionality of the triple co-culture can also be assessed via trans-electrical epithelial/endothelial resistance (TEER). Well-assembled airway organoid and endothelial co-cultures will have a greater electrical resistance, indicating optimal cell communication and connection. Environmental insults, such as e-cigarettes or viral challenge, will compromise the integrity of the epithelial and endothelial barrier, resulting in reduced electrical resistance of the co-culture (Figure 3A,B) and disorganized tight junctions (Figure 3C).
<img alt="iPSC differentiation diagram with tri-culture setup; microscopy, flow cytometry E-CAD, CD31 data." class="xfigimg" src="/files/ftp_upload/67247/67247fig01v2.jpg" data-altupdatedat="1788304762000" data-alt="Figure 1"> 
Figure 1: Differentiation and characterization of iPSC-derived airway epithelium, endothelium, and immune cultures. (A) Schematic of iPSC-directed differentiations for the assembly of the iAirway. (B) iPSC-derived airway epithelium organoids characterized by brightfield (left) and flow cytometry analysis (right) for EPCAM and ECAD. (C) iPSC-derived airway endothelium characterized by brightfield (left), immunofluorescence, and flow cytometry analysis (right) for CD31. The respective brightfield after CD31 sort is shown on the right. (D) iPSC-derived macrophages are characterized by brightfield (left) and flow cytometry analysis (right) for the pan hematopoietic marker CD45, monocyte maker CD14, and macrophage marker CD68. Scale bars: 100 µm. <a href="https://www.jove.com/files/ftp_upload/67247/67247fig01largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Tri-culture cell experiment diagram, showing epithelial, endothelial, and immune cell co-culture setup." class="xfigimg" src="/files/ftp_upload/67247/67247fig02.jpg" data-altupdatedat="1788304762000" data-alt="Figure 2"> 
Figure 2: Co-culture of iPSC-derived epithelial, mesenchymal, endothelial, and macrophage cells. (A) Schematic of the assembly of the iAirway. (B) Brightfield images of the respective epithelial and endothelial monoculture, the epithelial and endothelial bi-culture, and the epithelial, endothelial, and macrophage co-culture. Macrophages tagged with red cytotracker. Scale bars: 100 µm. (C) Immunofluorescent staining for tight junction marker ZO-1 on the apical side of the cell culture insert and CD31 on the basolateral side. Scale bars: 100 µm. (D) Cross-section of the cell culture insert with dissociated airway organoids on the apical side, 1-week post air-lift. The left panel with H&amp;E staining with the black arrow shows ciliated cells. The middle panel, with immunofluorescence imaging, shows the epithelial marker E-cadherin (ECAD). The right panel, with immunofluorescence imaging, shows mesenchymal markers such as vimentin (VIM) and smooth muscle actin (SMA). Scale bars: 100 µm. <a href="https://www.jove.com/files/ftp_upload/67247/67247fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Epithelial-endothelial co-culture TEER graph, microscopy images with ZO-1, DAPI, ECAD analysis." class="xfigimg" src="/files/ftp_upload/67247/67247fig03.jpg" data-altupdatedat="1788304762000" data-alt="Figure 3"> 
Figure 3: Changes in airway barrier function of the iAirways exposed to e-cigarettes and viruses. (A) Schematic of trans-electrical epithelial/endothelial resistance (TEER) measurement tool. (B) Bar graph of TEER after 7 days of menthol e-cigarette addition to media. Mean +/- 95% confidence interval. N = 5 biological replicates and 3 technical replicates; p-value &lt;0.001. (C) Immunofluorescent staining for tight junction protein ZO-1 before and after infection with SARS-CoV-2. Note the disordered layout of ZO-1 post-infection. Scale bars: 50 µm. <a href="https://www.jove.com/files/ftp_upload/67247/67247fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Antibody</td>
			<td>Application</td>
			<td>Dilution</td>
		</tr>
		<tr>
			<td>CD 31 PECAM-1(APC)</td>
			<td>flow cytometry (FC)</td>
			<td>10 μl in 100 ul buffer</td>
		</tr>
		<tr>
			<td>EPCAM/CD 326 (APC)</td>
			<td>FC</td>
			<td> 1:500</td>
		</tr>
		<tr>
			<td>E-CAD/CD 324 (APC)</td>
			<td>FC</td>
			<td>5 μl in 100 ul buffer</td>
		</tr>
		<tr>
			<td>CPM</td>
			<td>FC</td>
			<td> 1:200</td>
		</tr>
		<tr>
			<td>CD 45 (PE)</td>
			<td>FC</td>
			<td>5 μl in 100 ul buffer</td>
		</tr>
		<tr>
			<td>CD 14 (FITC)</td>
			<td>FC</td>
			<td>5 μl in 100 ul buffer</td>
		</tr>
		<tr>
			<td>CD 68 (PE)</td>
			<td>FC</td>
			<td>5 μl in 100 ul buffer</td>
		</tr>
		<tr>
			<td>ZO-1</td>
			<td>Immunofluorescence (IF)</td>
			<td> 1:300</td>
		</tr>
		<tr>
			<td>Vimentin</td>
			<td>IF</td>
			<td> 1:200</td>
		</tr>
		<tr>
			<td>SMA</td>
			<td>IF</td>
			<td> 1:100</td>
		</tr>
	</tbody>
</table>
Table 1: Antibodies used in FACS and Immunofluorescence.
Supplementary File 1: Media and buffer compositions. <a href="https://www.jove.com/files/ftp_upload/67247/Supplementary%20File%201.zip" target="_blank">Please click here to download this File.</a>

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Human lung tissue is composed of an interconnected network of epithelium, mesenchyme, endothelium, and immune cells from the upper airway of the ...

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JoVE Journal

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iPSC-Derived Epithelial, Mesenchymal, Endothelial, and Immune Cell Co-Culture to Model Airway Barrier Integrity in Lung Health and Disease (Video) | JoVE Journal

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