We wish to thank Dr. Rossant and Dr. Bilodeau for the Nkx2-1mcherry ESC used in experiments depicted in Figures 1-3. FACS was performed in The SickKids-UHN Flow Cytometry Facility. This work was supported by operating grants from the Canadian Institutes for Health Research and an infrastructure grant (CSCCD) from the Canadian Foundation of Innovation.

Animal experiments were carried out in accordance with the Animal Care Committee guidelines of the Hospital for Sick Children Research Institute.
1. Scaffold Preparation
<ol>
	<li>Decellularization of lungs
	<ol>
		<li>Euthanize adult Wistar rats using CO2 chamber. Place animal in the chamber and start 100% CO2 exposure at a fill rate of 10-30% of chamber volume per minute.
		<ol>
			<li>Observe animal for unconsciousness; this will occur after approximately 2-3 min. If unconsciousness does not occur in this time period, check fill rate and chamber seal. Following unconsciousness, observe animal for faded eye color and lack of respiration. If both are observed, maintain CO2 filling for 1-2 min and then remove animal from chamber for procedure.</li>
		</ol>
		</li>
		<li>Secure animal to dissecting surface by fixing forepaws and hind legs, and spray down the chest and abdomen area with 70% ethanol. Access the heart and lungs by opening the thoracic cavity using a vertical incision along the sternum.</li>
		<li>Make small incisions through diaphragm first to cause the lungs to retract, reducing the chance of puncturing the lungs. Ligate the inferior vena cava with a suture and place a small incision in the left atrium with small dissecting scissors.</li>
		<li>Using a prepared 10 ml syringe with a 25 G needle filled with heparinized Hank&#8217;s balanced salt solution (HBSS) (10 U/ml heparin in HBSS), start lung perfusion by inserting the needle into the right ventricle to push the buffer through the pulmonary circulation (rate of 2 ml/min). Continue this procedure until the lungs turn white and the fluid flowing from the left atrium runs clear.</li>
		<li>Following perfusion, expose the trachea and cannulate with a plastic catheter near the thyroid cartilage and secure in place with a suture.</li>
		<li>Setup a gravity perfusion system by fixing a 10 ml syringe to a retort stand and clamp. Remove and discard syringe plunger. Secure the maximum filling point on syringe barrel at 20 cm above the lungs. Attach a two-way stopcock to the end of the syringe, and a long plastic tubing to the other end of the stopcock for delivering decellularization solution to the cannulated trachea. Pour solution into syringe and allow solution to fill attached plastic tubing and catheter.
		<ol>
			<li>Lavage the lungs by filling to total lung capacity (approximately 12 ml) for 1 min and remove the plastic catheter from the trachea to allow the fluid to flow out of the lungs. Do not fill syringe more than 10 ml when lavaging lungs to keep pressure below 20 cm of H2O.</li>
		</ol>
		</li>
		<li>Repeat lavage of lungs eight times with decellularization solution, followed by 10 rinses with phosphate-buffered saline (PBS).</li>
		<li>Dissect the trachea and lungs free from the neck and chest cavity and remove from the animal. Keep tissue in cold PBS at 4 &#176;C until preparation for vibratome sectioning.</li>
	</ol>
	</li>
	<li>Thick section generation
	<ol>
		<li>Prepare approximately 15 ml of 2% and 4% (w/v) agarose, and enough 6% (w/v) agarose for embedding all lobes into small rectangular blocks. Dissolve low melting point agarose powder in PBS by microwaving. Transfer the agarose to 50 ml tubes on a heat block and maintain temperature above 40 &#176;C to avoid gelling.</li>
		<li>Dissect decellularized lung at the end of each lobar bronchus to detach each lobe (cranial, middle, accessory, and caudal right lobes and the left lobe) using small scissors. Pat dry each lobe using absorbent sheets to remove excess PBS and place inside 2% (w/v) agarose while on the heating block.</li>
		<li>Remove each lobe after 5 min of coating in agarose, place in a Petri dish and allow the surface to gel for 1 min on a cold plate.</li>
		<li>Gently place lobes back into 4% (w/v) agarose, cool after 5 min, and repeat coating once more with the 6% (w/v) agarose.</li>
		<li>After sequential coating of each lobe, embed each lobe separately in 6% (w/v) agarose using metal base molds with at least 3 mm of agarose surrounding the tissue from the edges. Orient each lobe using forceps by positioning the lobe&#39;s largest flat edge at the surface of metal mold facing the experimenter. This edge will be the side fixed to the specimen plate for vibratome sectioning.</li>
		<li>Allow blocks to gel on cold plate for at least 30 min prior to sectioning with vibratome. Store blocks in a humidified chamber for up to 12 hr at 4 &#176;C prior to vibratome sectioning.</li>
		<li>Setup the vibratome by filling the sectioning chamber with cold PBS12. Maintain cold temperature throughout sectioning with the surrounding ice bath. Remove blocks from metal molds and use a razor blade to trim down excess agarose surrounding lobes, while keeping approximately 3 mm from the edge of the tissue.</li>
		<li>Fix tissue to the center of specimen plate using adhesive, and submerge plate into the PBS-filled sectioning chamber. Setup sectioning boundaries on vibratome by selecting the following speed, amplitude, and thickness values respectively: 0.2 mm/sec, 1.85 mm, and 350 &#181;m. 
		Note: Both longitudinal and transverse sections of the lobe orientation are acceptable.</li>
		<li>Section each lobe completely. Manually cut sections free using small scissors if a section is not fully separated by blade at the end of the section sequence. Collect scaffold sections gently and keep in PBS on ice until the next step. 
		Note: Sections generated will include both the proximal and distal lung areas and both sources can be used for recellularization; however, most of the surface will encompass distal lung.</li>
	</ol>
	</li>
	<li>Decontamination of scaffold sections
	<ol>
		<li>Transfer the 350 micron thick scaffold sections from PBS to microcentrifuge tubes (up to 30 sections/tube) and treat with nuclease (90 U/ml in PBS) (see List of Materials) for 12 - 24 hr at RT, on a rotator.</li>
		<li>Following nuclease treatment, transfer sections using forceps to new microcentrifuge tubes and treat with antimicrobial solution (200 U/ml penicillin streptomycin and 25 &#181;g/ml amphotericin B in PBS) under sterile conditions for 6 hr at RT, on a rotator. 
		Note: Scaffolds can be stored in antimicrobial solution for up to one week at 4 &#176;C before use.</li>
		<li>Following the decontamination step with antimicrobial solution, rinse scaffolds twice with PBS under sterile conditions and transfer to serum free differentiation media (SFDM) prior to seeding with cells.</li>
	</ol>
	</li>
</ol>
2. Endodermal Cell Preparation
<ol>
	<li>Definitive Endoderm Induction
	<ol>
		<li>Maintain mouse ESC lines under feeder-free, serum free culture using 2i conditions13. Start endoderm induction by removing cells from adherent pluripotent culture by trypsinization.</li>
		<li>Resuspend cells in SFDM and seed at a density of 20,000 cells/ml in low adherent plates for three days without media change to allow for EB formation.</li>
		<li>After three days gently transfer the EBs into 50 ml conical tubes using a 10 ml pipette and allow them to collect at the bottom for 3 min at RT.</li>
		<li>Carefully aspirate media and add fresh SFDM media supplemented with 50 ng/ml activin A.</li>
		<li>Seed cells back onto the low adherent plates at a 1:2 density and culture for three additional days to achieve definitive endoderm differentiation.</li>
	</ol>
	</li>
	<li>Definitive endoderm enrichment
	<ol>
		<li>Collect day 6 EBs, dissociate with trypsin and label for c-KIT and CXCR4 expression using conjugated fluorescent antibodies14.</li>
		<li>Sort labeled cells using fluorescence-activated cell sorting (FACS) for expression of both markers to obtain an enriched definitive endoderm population15.</li>
	</ol>
	</li>
</ol>
3. Recellularization Setup
<ol>
	<li>Air-liquid interface culture setup
	<ol>
		<li>Transfer each decellularized scaffold section (step 1.2.9) from SFDM onto a hydrophobic floating membrane (8 &#181;m pore size) using sterile forceps. Ensure scaffold sections are spread evenly on membrane.</li>
		<li>Prepare 6- or 12-well plates by filling wells with 1 or 0.5 ml of SFDM, respectively. Gently place membranes into wells, allowing the membrane to float on top of media, creating an air-liquid culture setup.</li>
	</ol>
	</li>
	<li>Seeding of 3D scaffolds
	<ol>
		<li>Following FACS for definitive endoderm markers (step 2.2.2) count sorted cells using a hemocytometer, spin down at 400 x g for 5 min and resuspend in SFDM. Resuspend cells to obtain a volume containing approximately 100,000 cells/10 &#181;l/scaffold.</li>
		<li>To recellularize scaffolds, pipette 10 &#181;l of cells directly onto each prepared section from step 3.1.2.</li>
		<li>Replace SFDM media in cultures every 48 hr. Aspirate the old media by holding the plate at a slight incline to avoid disrupting the culture. Slowly add fresh SFDM to culture along the side of the well to prevent sinking of floating membrane.</li>
		<li>Maintain air-liquid cultures for up to 21 days to achieve differentiation of seeded cells in to mature airway epithelia. 
		Note: Recellularized sections can be processed for tissue staining and immunofluorescent (IF) microscopy at any time point during cell culture16.</li>
	</ol>
	</li>
</ol>

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There are no competing financial interests to declare.

The protocol described here generates mature ESC-derived airway epithelia using only natural lung scaffolds to direct differentiation with no other supplementation. This culture setup is defined, serum-free, inexpensive, and reproducible. No growth factor supplementation of base differentiation media is required. Previously published methods for generating stem cell-derived lung epithelial cells have used 2-dimensional strategies with growth factor supplementation to promote lineage restriction3,4,8,18,19. The technique described here is advantageous over these methods for several reasons, including: defined culture conditions, high differentiation efficiency to functional airway cells, limited contamination from other endodermal lineages (thyroid, liver, pancreas), and a 3D differentiation microenvironment. This technique yields similar results with mouse-derived ECM and can therefore be modified to generate decellularized scaffolds using mouse lungs. Rat-derived ESCs differentiated to definitive endoderm and seeded onto scaffolds using this protocol also differentiate to airway epithelia.
Decellularized scaffolds can be stored in decontamination solution at 4 &#176;C for up to 7 days prior to recellularization. Scaffolds kept for longer durations may be functional, however we have not tested to confirm this. To achieve efficient organization and differentiation it is required to enrich for definitive endoderm by FACS following induction with activin A. Non-endoderm cells in day 6 EBs often express pluripotency marker Oct4 and if seeded on scaffolds will continue to proliferate without organization and specification to the lung lineage. The seeding density can also be adjusted based on cell line-specificity to achieve complete recellularization of scaffolds.
A limitation of this protocol is in its inability to promote differentiation to alveolar epithelial cells. Although NKX2-1+/SOX9+ distal progenitor cells are detected after seven days of culture on scaffolds, this population is not present at day 14, and day 21. Markers of mature type I alveolar epithelial cells (AQP5) and type II alveolar epithelial cells (SFTPB, SFTPC) are not detected at any stage during scaffold culture. This may be due to a requirement for additional growth factor supplementation and submerged culture conditions to promote alveolar lineage specification, while as described here, matrix proteins and the remnant matrix-bound growth factors on decellularized scaffolds is sufficient to promote airway lineage differentiation.
The critical step for promoting differentiation of definitive endoderm to airway epithelial cells using lung scaffolds is optimization of the decellularization process. Cellular components must be completely removed while matrix components are preserved. Tissue staining for nuclear material using DAPI, ultra-structural analysis of scaffolds with scanning and transmission electron microscopy, and DNA assays can be used to confirm removal of all cellular components. ECM protein composition of scaffolds can be assessed using IF staining and immunoblot analysis for matrix proteins: collagen I, collagen IV, elastin, fibronectin, heparin sulfate proteoglycans, and laminin16.
These stem cell-derived lung epithelia can be used in disease modeling and drug-discovery platforms of airway-related pathologies such as cystic fibrosis. The potential of these cell-matrix constructs in regenerative applications such as tissue repair and cell therapy can be examined by in vivo transplantation of engineered constructs in various mouse models. Furthermore, this 3D in vitro method for airway differentiation can be easily adapted to study various parameters effecting lung lineage specification by manipulating growth factor availability and cell-matrix signaling of cultures during different stages of differentiation on scaffolds.

Directed differentiation of pluripotent cells to the lung lineage is dependent on precise signaling events in the microenvironment 1,2. Due to the dynamic nature of this process it has been challenging to mimic the precise events of lung organogenesis in vitro. Recent reports have used step-wise lineage restriction strategies with soluble growth factor supplementation of two-dimensional cultures to achieve lung differentiation3-8. In step-wise differentiation protocols, pluripotent cells, whether embryonic stem cells (ESC) or induced pluripotent stem cells, were first differentiated to the definitive endoderm germ layer. Endodermal cells were subsequently pushed to an anterior endoderm fate and thereafter to lung progenitor cells, as identified by the expression of homeodomain-containing transcription factor NKX2-1. These lung progenitors were further differentiated to proximal (airway) or distal (alveolar) lung epithelial cells with continued growth factor supplementation. Such 2-dimensional strategies have had some success in generating lung epithelial cells, however there are several limitations including unclear efficiencies, possible contamination from other endodermal lineages, lack of a 3-dimensional (3D) structure, and in some instances use of undefined cultures with serum supplementation. Culture of pluripotent or differentiated cells on decellularized lung scaffolds is increasingly used as an assay to assess the regenerative potential of seeded cells in forming lung epithelial structures3,5,6,8,9. Such reports culture seeded cells on scaffolds with continued growth factor or serum supplementation.
Lung development involves the division, migration, gene expression and differentiation of individual cells in response to environmental cues. The extra cellular matrix (ECM) is a latticework of glycoproteins that in addition to providing structural support, directs tissue morphogenesis by integrating and regulating these processes10,11. By using the lung ECM scaffold as a natural platform for endoderm culture to better mimic the in vivo lung developmental milieu, we have generated stem cell-derived airway epithelial cells in a defined 3D-culture setting with high efficiency and reproducibility.
Rat lung ECM scaffolds were generated by decellularization as well as mouse ESC-derived endodermal cells were generated and subsequently seeded onto these scaffolds. Dual expression of CXCR4 &#38; c-KIT proteins indicates a definitive endoderm cell identity and cells positive for both SOX2 &#38; NKX2-1 expression are identified as airway (proximal lung) progenitor cells. Definitive endoderm cells were cultured at air liquid interface (ALI) for up to three weeks to generate functional airway epithelial cells in vitro.
This protocol promotes lung lineage differentiation of definitive endoderm as early as 7 days, observed with the emergence of NKX2-1+/SOX2+ early proximal lung progenitors. By day 14 and 21 of culture mature airway epithelial cell populations emerge including ciliated (TUBB4A+), club (SCGB1A1+), and basal (TRP63+, KRT5+) cells with morphological and functional resemblance to native mouse airways. This protocol demonstrates the importance of the 3D-matrix microenvironment for achieving robust differentiation to airway epithelial cells.

<table><tbody><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Perfusion solution</td><td>Sigma</td><td>H0777</td><td>10 U/ml heparin</td></tr><tr><td>Perfusion solution</td><td>Gibco</td><td>14170112</td><td>dissolved in Hank&#039;s balanced salt solution (HBSS-)</td></tr><tr><td>Decellularization solution</td><td>BioShop</td><td>CHA003</td><td>8&amp;nbsp;mM CHAPS</td></tr><tr><td>Decellularization solution</td><td>Sigma</td><td>E9884</td><td>25&amp;nbsp;mM EDTA</td></tr><tr><td>Decellularization solution</td><td>BioShop</td><td>SOD002</td><td>1&amp;nbsp;M NaCl</td></tr><tr><td>Decellularization solution</td><td>Gibco</td><td>14190-144</td><td>dissolved in PBS</td></tr><tr><td>Benzonase nuclease</td><td>Novagen</td><td>70664-3</td><td>90&amp;nbsp;U/ml Benzonase nuclease&amp;nbsp;</td></tr><tr><td>Benzonase nuclease</td><td>Gibco</td><td>14190-144</td><td>diluted in PBS</td></tr><tr><td>Antimicrobial solution&amp;nbsp;</td><td>Gibco</td><td>15140</td><td>200&amp;nbsp;U/ml penicillin streptomycin</td></tr><tr><td>Antimicrobial solution&amp;nbsp;</td><td>Gibco</td><td>15290</td><td>25&amp;nbsp;&amp;mu;g/ml amphotericin B</td></tr><tr><td>Antimicrobial solution&amp;nbsp;</td><td>Gibco</td><td>14190-144</td><td>diluted in PBS</td></tr><tr><td>Trypsinization&amp;nbsp;</td><td>Gibco</td><td>12605-028</td><td>TrypLE</td></tr><tr><td>Serum free differentiation media (SFDM)</td><td>Gibco</td><td>IMDM 2440-053, F12 11765-054</td><td>3:1 ratio of IMDM and Ham&amp;rsquo;s modified F12 medium</td></tr><tr><td>Serum free differentiation media (SFDM)</td><td>Gibco</td><td>12587-010</td><td>B27 supplement (50x dilution)</td></tr><tr><td>Serum free differentiation media (SFDM)</td><td>Gibco</td><td>17502-048</td><td>N2 supplement (100x dilution)</td></tr><tr><td>Serum free differentiation media (SFDM)</td><td>Gibco</td><td>15260-037</td><td>0.05% (Fraction V) bovine serum albumin</td></tr><tr><td>Serum free differentiation media (SFDM)</td><td>Gibco</td><td>35050-061</td><td>200&amp;nbsp;mM Glutamax</td></tr><tr><td>Serum free differentiation media (SFDM)</td><td>Sigma</td><td>M6145</td><td>4&amp;nbsp;&amp;mu;M monothioglycerol</td></tr><tr><td>Serum free differentiation media (SFDM)</td><td>Sigma</td><td>A4403&amp;nbsp;</td><td>0.05&amp;nbsp;mg/ml ascobic acid</td></tr><tr><td>Endoderm induction</td><td>R&amp;amp;D</td><td>338-AC/CF</td><td>Activin A</td></tr><tr><td>&lt;strong&gt;Antibodies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>CDH1</td><td>BD Biosciences</td><td>610181</td><td>Mouse, non-conjugated, 1:100</td></tr><tr><td>C-KIT</td><td>BD Biosciences</td><td>558163</td><td>Rat, PE-Cy7, 1:100</td></tr><tr><td>CXCR4</td><td>BD Biosciences</td><td>558644</td><td>Rat, APC, 1:100</td></tr><tr><td>KRT5</td><td>Abcam</td><td>ab24647</td><td>Rabbit, non-conjugated, 1:1,000</td></tr><tr><td>NKX2-1</td><td>Abcam</td><td>ab76013</td><td>Rabbit, non-conjugated, 1:200</td></tr><tr><td>Laminin</td><td>Novus Biologicals</td><td>NB300-144</td><td>Rabbit, non-conjugated, 1:200</td></tr><tr><td>SCGB1A1</td><td>Santa Cruz</td><td>sc-9772&amp;nbsp;</td><td>Goat, non-conjugated, 1:1,000</td></tr><tr><td>SOX2</td><td>R&amp;amp;D Systems</td><td>AF2018&amp;nbsp;</td><td>Goat, non-conjugated, 1:400</td></tr><tr><td>TRP63</td><td>Santa Cruz</td><td>sc-8431</td><td>Mouse, non-conjugated, 1:200</td></tr><tr><td>TUBB4A</td><td>BioGenex</td><td>MU178-UC</td><td>Mouse, non-conjugated, 1:500</td></tr><tr><td>Goat IgG&amp;nbsp;</td><td>Invitrogen</td><td>A-11055</td><td>Donkey, Alexa Fluor 488, 1:200</td></tr><tr><td>Mouse IgG&amp;nbsp;</td><td>Invitrogen</td><td>A-21202</td><td>Donkey, Alexa Fluor 488, 1:200</td></tr><tr><td>Mouse IgG&amp;nbsp;</td><td>Invitrogen</td><td>A-31571</td><td>Donkey, Alexa Fluor 647, 1:200</td></tr><tr><td>Rabbit IgG</td><td>Invitrogen</td><td>A-21206</td><td>Donkey, Alexa Fluor 488, 1:200</td></tr><tr><td>Rabbit IgG&amp;nbsp;</td><td>Invitrogen</td><td>A-31573</td><td>Donkey, Alexa Fluor 647, 1:200</td></tr><tr><td>&lt;strong&gt;Other Materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Low adherent plates</td><td>Nunc</td><td>Z721050&amp;nbsp;</td><td>Low cell binding plates,&amp;nbsp; 6 wells&amp;nbsp;&amp;nbsp;</td></tr><tr><td>Air-liquid interface membranes&amp;nbsp;</td><td>Whatman</td><td>110614</td><td>Hydrophobic Nucleopore membrane, 8&amp;nbsp;&amp;mu;m pore size</td></tr><tr><td>Vibratome</td><td>Leica</td><td>VT1200S&amp;nbsp;</td><td>Leica Vibratome</td></tr><tr><td>Tissue Adhesive</td><td>Ted Pella</td><td>10033</td><td>Pelco tissue adhesive</td></tr></tbody></table>

generation of esc-derived mouse airway epithelial cells using decellularized lung scaffolds

Lung lineage differentiation requires integration of complex environmental cues that include growth factor signaling, cell-cell interactions and cell-matrix interactions. Due to this complexity, recapitulation of lung development in vitro to promote differentiation of stem cells to lung epithelial cells has been challenging. In this protocol, decellularized lung scaffolds are used to mimic the 3-dimensional environment of the lung and generate stem cell-derived airway epithelial cells. Mouse embryonic stem cell are first differentiated to the endoderm lineage using an embryoid body (EB) culture method with activin A. Endoderm cells are then seeded onto decellularized scaffolds and cultured at air-liquid interface for up to 21 days. This technique promotes differentiation of seeded cells to functional airway epithelial cells (ciliated cells, club cells, and basal cells) without additional growth factor supplementation. This culture setup is defined, serum-free, inexpensive, and reproducible. Although there is limited contamination from non-lung endoderm lineages in culture, this protocol only generates airway epithelial populations and does not give rise to alveolar epithelial cells. Airway epithelia generated with this protocol can be used to study cell-matrix interactions during lung organogenesis and for disease modeling or drug-discovery platforms of airway-related pathologies such as cystic fibrosis.

As outlined in this protocol, robust differentiation of definitive endoderm to mature airway epithelial cells can be achieved using extended culture of seeded cells on decellularized lung scaffold sections. It is recommended that decellularized scaffolds be characterized to ensure (1) host cells are completely removed, and (2) extracellular matrix proteins are preserved prior to using scaffolds for differentiation. Decellularization can be assessed using tissue staining with hematoxylin and eosin (H&#38;E) and 4&#39;,6-diamidino-2-phenylindole (DAPI), as illustrated in Figure 1A. High-magnification scanning electron microscope (SEM) analysis of scaffolds confirms the absence of host cells and intact matrix architecture, as illustrated in Figure 1B. Additional characterization of the remaining scaffold by tensile testing and immunostaining for matrix proteins can be carried out to ensure preservation of the ECM following decellularization16.
Non-adherent culture of ESCs in SFDM media for six days results in the formation of cell aggregates (EBs) as illustrated in Figure 2A. Three days of activin A treatment results in definitive endoderm induction. Lineage restriction of ESC to definitive endoderm can be confirmed by upregulation and expression of endoderm lineage-associated genes and proteins17, and cannot be asserted morphologically. Endoderm-induction efficiency ranges from 50-70% depending on the ESC line used. Experiments were carried out in mouse ESC lines: R1 (Nkx2-1mCherry), G4 (dsRed-MST), and 129/Ola (Bry-GFP/Foxa2-hCD4), while all data illustrated in this manuscript is using the R1 cell line. The double positive CXCR4+/c-KIT+ definitive endoderm population is sorted as illustrated in Figure 2B, seeded onto 3D lung scaffolds, and cultured for up to 21 days at air-liquid interface in SFDM. It is important to sort and enrich for definitive endoderm as limited organization and differentiation is achieved with culture of unsorted cells from heterogeneous day 6 EBs as illustrated in Figure 2C. Structures formed by sorted cells on scaffolds are reminiscent of developing lung (embryonic day 13.5) as illustrated in Figure 2C-D. In our experience, a seeding density of 100,000 sorted cells/scaffold has yielded the best scaffold repopulation.
Differentiation to the lung lineage is observed as early as 7 days following scaffold culture. Airway epithelial progenitor cells positive for NKX2-1 and SOX2 are detected using IF confocal analysis as illustrated in Figure 3A. SOX2 positive, NKX2-1 negative cells are likely pluripotent endodermal cells that have not differentiated to the lung lineage. With longer culture these cells may start to express NKX2-1 or undergo apoptosis without adequate support in the microenvironment. IF analysis of day 21 cultures reveals a mature airway epithelial population containing TUBB4A+ ciliated cells, SCGB1A1+ club cells, and TRP63+/KRT5+ basal cells as illustrated in Figure 3B. SEM analysis of the surface of day 21 cultures reveals morphological similarity of stem cell-derived cultures to mouse airways.
Both isolated matrix proteins (collagen I, collagen IV, fibronectin, laminin) and decellularized rat kidney scaffolds were unable to promote lung-lineage differentiation when seeded with definitive endoderm and cultured under the same conditions as described above16. This demonstrates that the 3D microenvironment created by lung-derived scaffolds is distinct from kidney-derived scaffolds in its ability to promote lung-lineage differentiation of seeded endodermal cells.
<img alt="Figure 1" src="/files/ftp_upload/54019/54019fig1.jpg" /> 
Figure 1. Decellularization technique removes cellular components and preserves ECM. (A) H&#38;E (top panel) and DAPI (lower panel) staining of natural and decellularized lung tissue showing the absence of nuclei following decellularization. Right panel shows examples of tissue sections from lungs without optimal decellularization. Arrowheads show remaining nuclei on scaffolds due to incomplete decellularization, highlighting the importance of optimizing technique before recellularization. Scale bar = 25 &#181;m. (B) SEM images of natural and decellularized lung, showing absence of cells and preservation of matrix architecture on decellularized scaffolds. Scale bar = 10 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54019/54019fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54019/54019fig2.jpg" /> 
Figure 2. Sorted ESC-derived definitive endoderm results in optimal recellularization. (A) Day 6 embryoid bodies (EBs) following three days of activin A treatment for promoting definitive endoderm differentiation. (B) The EBs are sorted by FACS for dual expression of surface markers CXCR4 and c-KIT to isolate the definitive endoderm population. This population will be seeded on decellularized scaffolds. (C) H&#38;E staining of recellularized scaffolds at day 7 and day 21 of culture. Left panel represents organized, airway-like structures following seeding with sorted cells, and right panel represents disorganized, highly proliferative unsorted cells on scaffolds highlighting the importance of sorting of cells prior to recellularization. Scale bar = 30 &#181;m. (D) H&#38;E staining of embryonic day 13.5 mouse lungs. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54019/54019fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54019/54019fig3.jpg" /> 
Figure 3. Extended culture of definitive endoderm on natural lung scaffolds directs differentiation to airway epithelial cells. (A) Proximal lung progenitor cells (NKX2-1+/SOX2+) are detected following seven days of culture on decellularized scaffolds. Scale bar = 15 &#181;m. (B) Left image shows mature pseudostratified epithelial cell (CDH1+) structures lined by basement membrane protein laminin on the basal side of structures. Center and right image show mature airway epithelia complete with ciliated cells (TUBB4A+), club cells (SCGB1A1+) and basal cells (KRT5+/TRP63+). Left image scale bar = 30 &#181;m; center and right image scale bar = 15 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54019/54019fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Generation of ESC-derived Mouse Airway Epithelial Cells Using Decellularized Lung Scaffolds at JoVE.com

Generation of ESC-derived Mouse Airway Epithelial Cells Using Decellularized Lung Scaffolds

This protocol efficiently directs mouse embryonic stem cell-derived definitive endoderm to mature airway epithelial cells. This differentiation technique uses 3-dimensional decellularized lung scaffolds to direct lung lineage specification, in a defined, serum-free culture setting.

Lung lineage differentiation requires integration of complex environmental cues that include growth factor signaling, cell-cell interactions and ...

generation-esc-derived-mouse-airway-epithelial-cells-using

Ateneo de Manila University

Research

JoVE Journal

Bioengineering

8.7K Views.  Hospital for Sick Children. This protocol efficiently directs mouse embryonic stem cell-derived definitive endoderm to mature airway epithelial cells. This differentiation technique uses 3-dimensional decellularized lung scaffolds to direct lung lineage specification, in a defined, serum-free culture setting.

Video: Generation of ESC-derived Mouse Airway Epithelial Cells Using Decellularized Lung Scaffolds

Procedure for Lung Engineering

Lung tissue, including lung cancer and chronic lung diseases such as chronic obstructive pulmonary disease, cumulatively account for some 280,000 deaths annually; chronic obstructive pulmonary disease is currently the fourth leading cause of death in the United States1. Contributing to this mortality is the fact that lungs do not generally repair or regenerate beyond the microscopic, cellular level. Therefore, lung tissue that is damaged by degeneration or infection, or lung tissue that is surgically resected is not functionally replaced in vivo. To explore whether lung tissue can be generated in vitro, we treated lungs from adult rats using a procedure that removes cellular components to produce an acellular lung extracellular matrix scaffold. This scaffold retains the hierarchical branching structures of airways and vasculature, as well as a largely intact basement membrane, which comprises collagen IV, laminin, and fibronectin. The scaffold is mounted in a bioreactor designed to mimic critical aspects of lung physiology, such as negative pressure ventilation and pulsatile vascular perfusion. By culturing pulmonary epithelium and vascular endothelium within the bioreactor-mounted scaffold, we are able to generate lung tissue that is phenotypically comparable to native lung tissue and that is able to participate in gas exchange for short time intervals (45-120 minutes). These results are encouraging, and suggest that repopulation of lung matrix is a viable strategy for lung regeneration. This possibility presents an opportunity not only to work toward increasing the supply of lung tissue for transplantation, but also to study respiratory cell and molecular biology in vitro for longer time periods and in a more accurate microenvironment than has previously been possible.

We have developed a decellularized lung extracellular matrix and novel biomimetic bioreactor that can be used to generate functional lung tissue. By seeding cells into the matrix and culturing in the bioreactor, we generate tissue that demonstrates effective gas exchange when transplanted in vivo for short periods of time.

Lung tissue, including lung cancer and chronic lung diseases such as chronic obstructive pulmonary disease, cumulatively account for some 280,000 deaths ...

Nonhuman Primate Lung Decellularization and Recellularization Using a Specialized Large-organ Bioreactor

There are an insufficient number of lungs available to meet current and future organ transplantation needs. Bioartificial tissue regeneration is an attractive alternative to classic organ transplantation. This technology utilizes an organ&#39;s natural biological extracellular matrix (ECM) as a scaffold onto which autologous or stem/progenitor cells may be seeded and cultured in such a way that facilitates regeneration of the original tissue. The natural ECM is isolated by a process called decellularization. Decellularization is accomplished by treating tissues with a series of detergents, salts, and enzymes to achieve effective removal of cellular material while leaving the ECM intact. Studies conducted utilizing decellularization and subsequent recellularization of rodent lungs demonstrated marginal success in generating pulmonary-like tissue which is capable of gas exchange in vivo. While offering essential proof-of-concept, rodent models are not directly translatable to human use. Nonhuman primates (NHP) offer a more suitable model in which to investigate the use of bioartificial organ production for eventual clinical use.
The protocols for achieving complete decellularization of lungs acquired from the NHP rhesus macaque are presented. The resulting acellular lungs can be seeded with a variety of cells including mesenchymal stem cells and endothelial cells. The manuscript also describes the development of a bioreactor system in which cell-seeded macaque lungs can be cultured under conditions of mechanical stretch and strain provided by negative pressure ventilation as well as pulsatile perfusion through the vasculature; these forces are known to direct differentiation along pulmonary and endothelial lineages, respectively. Representative results of decellularization and cell seeding are provided.

Whole-organ decellularization produces natural biological scaffolds that may be used for regenerative medicine. The description of a nonhuman primate model of lung regeneration in which whole lungs are decellularized and then seeded with adult stem cells and endothelial cells in a bioreactor that facilitates vascular circulation and liquid media ventilation is presented.

There are an insufficient number of lungs available to meet current and future organ transplantation needs. Bioartificial tissue regeneration is an ...

Decellularization of the Murine Cardiopulmonary Complex

We present here a decellularization protocol for mouse heart and lungs. It produces structural ECM scaffolds that can be used to analyze ECM topology and composition. It is based on a microsurgical procedure designed to catheterize the trachea and aorta of a euthanized mouse to perfuse the heart and lungs with decellularizing agents. The decellularized cardiopulmonary complex can subsequently be immunostained to reveal the location of structural ECM proteins. The whole procedure can be completed in 4 days.
The ECM scaffolds resulting from this protocol are free of dimensional distortions. The absence of cells enables structural examination of ECM structures down to submicron resolution in 3D. This protocol can be applied to healthy and diseased tissue from mice as young as 4-weeks old, including mouse models of fibrosis and cancer, opening the way to determine ECM remodeling associated with cardiopulmonary disease.

This protocol aims to decellularize the heart and lungs of mice. The resulting extracellular matrix (ECM) scaffolds can be immunostained and imaged to map the location and topology of their components.

We present here a decellularization protocol for mouse heart and lungs. It produces structural ECM scaffolds that can be used to analyze ECM topology and ...

Cancer Research

Production, Characterization and Potential Uses of a 3D Tissue-engineered Human Esophageal Mucosal Model

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore esophageal adenocarcinoma generally has a poor prognosis, with little improvement in survival rates in recent years. These are difficult conditions to study and there has been a lack of suitable experimental platforms to investigate disorders of the esophageal mucosa.
A model of the human esophageal mucosa has been developed in the MacNeil laboratory which, unlike conventional 2D cell culture systems, recapitulates the cell-cell and cell-matrix interactions present in vivo and produces a mature, stratified epithelium similar to that of the normal human esophagus. Briefly, the model utilizes non-transformed normal primary human esophageal fibroblasts and epithelial cells grown within a porcine-derived acellular esophageal scaffold. Immunohistochemical characterization of this model by CK4, CK14, Ki67 and involucrin staining demonstrates appropriate recapitulation of the histology of the normal human esophageal mucosa.
This model provides a robust, biologically relevant experimental model of the human esophageal mucosa. It can easily be manipulated to investigate a number of research questions including the effectiveness of pharmacological agents and the impact of exposure to environmental factors such as alcohol, toxins, high temperature or gastro-esophageal refluxate components. The model also facilitates extended culture periods not achievable with conventional 2D cell culture, enabling, inter alia, the study of the impact of repeated exposure of a mature epithelium to the agent of interest for up to 20 days. Furthermore, a variety of cell lines, such as those derived from esophageal tumors or Barrett&#8217;s Metaplasia, can be incorporated into the model to investigate processes such as tumor invasion and drug responsiveness in a more biologically relevant environment.

This manuscript describes the production, characterization and potential uses of a tissue engineered 3D esophageal construct prepared from normal primary human esophageal fibroblast and squamous epithelial cells seeded within a de-cellularized porcine scaffold. The results demonstrate the formation of a mature stratified epithelium similar to the normal human esophagus.

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore ...

Generation of 3D Whole Lung Organoids from Induced Pluripotent Stem Cells for Modeling Lung Developmental Biology and Disease

Human lung development and disease has been difficult to study due to the lack of biologically relevant in vitro model systems. Human induced pluripotent stem cells (hiPSCs) can be differentiated stepwise into 3D multicellular lung organoids, made of both epithelial and mesenchymal cell populations. We recapitulate embryonic developmental cues by temporally introducing a variety of growth factors and small molecules to efficiently generate definitive endoderm, anterior foregut endoderm, and subsequently lung progenitor cells. These cells are then embedded in growth factor reduced (GFR)-basement membrane matrix medium, allowing them to spontaneously develop into 3D lung organoids in response to external growth factors. These whole lung organoids (WLO) undergo early lung developmental stages including branching morphogenesis and maturation after exposure to dexamethasone, cyclic AMP and isobutylxanthine. WLOs possess airway epithelial cells expressing the markers KRT5 (basal), SCGB3A2 (club) and MUC5AC (goblet) as well as alveolar epithelial cells expressing HOPX (alveolar type I) and SP-C (alveolar type II). Mesenchymal cells are also present, including smooth muscle actin (SMA), and platelet-derived growth factor receptor A (PDGFR&#945;). iPSC derived WLOs can be maintained in 3D culture conditions for many months and can be sorted for surface markers to purify a specific cell population. iPSC derived WLOs can also be utilized to study human lung development, including signaling between the lung epithelium and mesenchyme, to model genetic mutations on human lung cell function and development, and to determine the cytotoxicity of infective agents.

The article describes step wise directed differentiation of induced pluripotent stem cells to three-dimensional whole lung organoids containing both proximal and distal epithelial lung cells along with mesenchyme.

Human lung development and disease has been difficult to study due to the lack of biologically relevant in vitro model systems. Human induced pluripotent ...

Developmental Biology

Generation of Airway Epithelial Cell Air-Liquid Interface Cultures from Human Pluripotent Stem Cells

Diseases of the conducting airway such as asthma, cystic fibrosis (CF), primary ciliary dyskinesia (PCD), and viral respiratory infections are major causes of morbidity and mortality worldwide. In vitro platforms using human bronchial epithelial cells (HBECs) have been instrumental to our understanding of the airway epithelium in health and disease. Access to HBECs from individuals with rare genetic diseases or rare mutations is a bottleneck in lung research.
Induced pluripotent stem cells (iPSCs) are readily generated by "reprogramming" somatic cells and retain the unique genetic background of the individual donor. Recent advances allow for the directed differentiation of iPSCs to lung epithelial progenitor cells, alveolar type 2 cells, as well as the cells of the conducting airway epithelium via basal cells, the major airway stem cells.
Here we outline a protocol for the maintenance and expansion of iPSC-derived airway basal cells (hereafter iBCs) as well as their trilineage differentiation in air-liquid interface (ALI) cultures. iBCs are maintained and expanded as epithelial spheres suspended in droplets of extracellular matrix cultured in a primary basal cell medium supplemented with inhibitors of TGF-&#223; and BMP signaling pathways. iBCs within these epithelial spheres express key basal markers TP63 and NGFR, can be purified by fluorescence activated cell sorting (FACS), and when plated on porous membranes in standard ALI culture conditions, differentiate into a functional airway epithelium. ALI cultures derived from healthy donors are composed of basal, secretory and multiciliated cells and demonstrate epithelial barrier integrity, motile cilia, and mucus secretion. Cultures derived from individuals with CF or PCD recapitulate the dysfunctional CFTR-mediated chloride transport or immotile cilia, the respective disease-causing epithelial defects.
Here, we present a protocol for the generation of human cells that can be applied for modeling and understanding airway diseases.

Recent advances in human induced pluripotent stem cell differentiation protocols allow for the stepwise derivation of organ-specific cell types. Here, we provide detailed steps for the maintenance and expansion of iPSC-derived airway basal cells and their differentiation into a mucociliary epithelium in air-liquid interface cultures.

Diseases of the conducting airway such as asthma, cystic fibrosis (CF), primary ciliary dyskinesia (PCD), and viral respiratory infections are major ...

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

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.

Author Spotlight: Modeling Human Airway Remodeling and Viral Responses Using Isogenic Epithelial, Endothelial, and Immune Cells

This article describes the generation of a complex, multi-cellular airway barrier model composed of induced pluripotent stem cell (iPSC)-derived lung epithelium, mesenchyme, endothelial cells, and macrophages in an air-liquid interface culture.

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

Engineered Lung Tissues Prepared from Decellularized Lung Slices

There is a need for improved 3-dimensional (3D) lung models that recapitulate the architectural and cellular complexity of the native lung alveolus ex vivo. Recently developed organoid models have facilitated the expansion and study of lung epithelial progenitors in vitro, but these platforms typically rely on mouse tumor-derived matrix and/or serum, and incorporate just one or two cellular lineages. Here, we describe a protocol for generating engineered lung tissues (ELTs) based on the multi-lineage recellularization of decellularized precision-cut lung slices (PCLS). ELTs contain alveolar-like structures comprising alveolar epithelium, mesenchyme, and endothelium, within an extracellular matrix (ECM) substrate closely resembling that of native lung. To generate the tissues, rat lungs are inflated with agarose, sliced into 450 &#181;m-thick slices, cut into strips, and decellularized. The resulting acellular ECM scaffolds are then reseeded with primary endothelial cells, fibroblasts, and alveolar epithelial type 2 cells (AEC2s). AEC2s can be maintained in ELT culture for at least 7 days with a serum-free, chemically-defined growth medium. Throughout the tissue preparation and culture process, the slices are clipped into a cassette system that facilitates handling and standardized cell seeding of multiple ELTs in parallel. These ELTs represent an organotypic culture platform that should facilitate investigations of cell-cell and cell-matrix interactions within the alveolus as well as biochemical signals regulating AEC2s and their niche.

This protocol describes a method to generate reproducible, small-scale engineered lung tissues, by repopulating decellularized precision-cut lung slices with alveolar epithelial type 2 cells, fibroblasts, and endothelial cells.

There is a need for improved 3-dimensional (3D) lung models that recapitulate the architectural and cellular complexity of the native lung alveolus ex ...

Imaging-Guided Bioreactor for Generating Bioengineered Airway Tissue

Repeated injury to airway tissue can impair lung function and cause chronic lung disease, such as chronic obstructive pulmonary disease. Advances in regenerative medicine and bioreactor technologies offer opportunities to produce lab-grown functional tissue and organ constructs that can be used to screen drugs, model disease, and engineer tissue replacements. Here, a miniaturized bioreactor coupled with an imaging modality that allows in situ visualization of the inner lumen of explanted rat trachea during in vitro tissue manipulation and&#160;culture is described. Using this bioreactor, the protocol demonstrates imaging-guided selective removal of endogenous cellular components while preserving the intrinsic biochemical features and ultrastructure of the airway tissue matrix. Furthermore, the delivery, uniform distribution, and subsequent prolonged culture of exogenous cells on the decellularized airway lumen with optical monitoring in situ are shown. The results highlight that the imaging-guided bioreactor can potentially be used to facilitate the generation of functional in vitro airway tissues.

The protocol describes an imaging-enabled bioreactor that allows the selective removal of the endogenous epithelium from the rat trachea and homogenous distribution of exogenous cells on the lumen surface, followed by long-term in vitro culture of the cell-tissue construct.

Repeated injury to airway tissue can impair lung function and cause chronic lung disease, such as chronic obstructive pulmonary disease. Advances in ...

Transplantation of Bioengineered Lung Using Decellularized Mouse Lungs and Primary Human Endothelial Cells

Lung transplantation is a critical treatment for patients with end-stage lung diseases like idiopathic pulmonary fibrosis, but challenges such as donor shortages and posttransplant complications persist. Bioengineered lungs, integrating patient-specific cells into decellularized animal scaffolds, present a promising alternative. Despite progress in using bioengineered lungs in animal models, functionality and structure remain immature. This protocol addresses a critical barrier in organ bioengineering: the need for a cost-effective experimental platform. By using mouse models instead of larger animals like rats or swine, researchers can significantly reduce the resources required for each experiment, accelerating research progress. 
The protocol outlines a detailed procedure for lung bioengineering using mouse heart-lung blocks and human primary cells, focusing on isolation strategy for the mouse heart-lung block, decellularization, bioreactor setup, perfusion-based organ culture, and orthotopic transplantation of bioengineered lungs. This mouse-scale platform not only reduces experimental costs but also provides a viable framework for optimizing cell types and numbers for recellularization, testing different cell types using histological and molecular methods, and ensuring blood flow post-transplantation. The method holds potential for broad applications, including studying cell interactions in three-dimensional culture conditions, cell-matrix interactions, and ex vivo cancer modeling, thereby advancing the field of organ bioengineering.

This paper describes how to create bioengineered mouse lungs using decellularization and recellularization methods. It also details subsequent orthotopic lung transplantation.

Lung transplantation is a critical treatment for patients with end-stage lung diseases like idiopathic pulmonary fibrosis, but challenges such as donor ...