We sincerely thank members of the Wilson, Kotton, and Hawkins laboratories for their helpful discussions. We are also very grateful to Greg Miller (CReM Laboratory Manager) and Marianne James (iPSC Core Manager) for their invaluable support. We thank Brian Tilton and the BUMC Flow Cytometry Core for their technical assistance with cell sorting (supported by NIH grant #1S10OD021587-01A1). This work was supported by a CJ Martin Early Career Fellowship from the Australian National Health and Medical Research Council awarded to RBW; NIH grant F30HL147426 to KMA; an I.M. Rosenzweig Junior Investigator Award from The Pulmonary Fibrosis Foundation and an Integrated Pilot Grant Award through Boston University Clinical &#38; Translational Science Institute (1UL1TR001430) to KDA; Swiss National Science Foundation (P2ELP3_191217) to ABA; NIH grants U01HL148692, U01HL134745, U01HL134766 and R01HL095993 to DNK; and NIH grants U01TR001810, R01DK101510, and R01DK117940 awarded to AAW; iPSC maintenance, banking, and sharing are supported by NIH grant NO1 75N92020C00005. Figure 1 was created using Biorender.com.

All experiments involving the differentiation of human iPSC lines were performed in compliance with the Institutional Review Board of Boston University (protocol H33122). The dermal fibroblasts, procured for reprogramming to iPSCs, were obtained from a donor with written informed consent, under the approval of the Human Research Protection Office of Washington University School of Medicine, St. Louis, MO. Reprogrammed iPSCs were generated at the Center for Regenerative Medicine at Boston University and Boston Medical Center, Boston, MA.
1. Alveolosphere dissociation
<ol>
	<li>Prepare complete serum-free differentiation media (cSFDM) as per the composition mentioned in Table 1.</li>
	<li>Prepare CK + DCI media in the prepared cSFDM base as per Table 2.</li>
	<li>Thaw 2D (human embryonic stem cell-qualified) and/or 3D (growth-factor reduced) matrix on ice as required for the experimental needs.</li>
	<li>Aspirate all the CK + DCI medium using a pipette or aspirating pipette with vacuum from the 3D matrix droplets containing alveolospheres, derived from directed differentiation19, in a 12-well plate.</li>
	<li>Add 1 mL of dispase (2 mg/mL) per droplet. Gently pipette the droplet into the dispase using a P1000 pipette. Incubate at 37 &#176;C for 1 h, pipetting up and down once after 30 min.</li>
	<li>Transfer the dissociated organoids (from Step 1.5) from one matrix droplet in the dispase to a 15 mL conical tube. To wash, add 10 mL of Iscove&#39;s Modified Dulbecco&#39;s Medium (IMDM, see Table of Materials).</li>
	<li>Centrifuge at 300 x g for 5 min at room temperature. Aspirate the supernatant using a pipette or aspirating pipette with vacuum, leaving as little supernatant as possible. 
	NOTE: It is important to remove all dispase as any remaining dispase may dissolve the matrix that the cells will subsequently be seeded into. If a clear haze is seen above the pellet, the dispase has not completely dissolved the matrix, and more dispase can be added to the pellet for another 20-30 min at 37 &#176;C.</li>
	<li>Resuspend the cells in 1 mL of 0.05% trypsin per droplet and transfer back to the 12-well plate. Incubate at 37 &#176;C for 12-15 min. Observe the dissociation under a microscope. Avoid over-pipetting the cells at this stage. 
	NOTE: At the end of incubation, the cells need to achieve a single-cell suspension after pipetting 3-5 times with a P1000 pipette. For passaging iAT2s to ALI (Step 3), the trypsinization time needs to be minimized (maximum 12 min), such that the cells are in 2-3-cell clumps rather than single-cell suspension when ready for plating onto the cell culture insert.</li>
	<li>Stop the action of trypsin with an equal volume of FBS-containing medium (10% ES-qualified FBS in DMEM). Centrifuge at 300 x g for 5 min at room temperature.</li>
	<li>Wash the cells with 10 mL of IMDM. Centrifuge at 300 x g for 5 min at room temperature.</li>
	<li>Resuspend the cells in an appropriate volume for counting, and then count the cells using a hemocytometer (see Table of Materials). 
	NOTE: From one confluent 50 &#181;L matrix droplet seeded at 400 cells/&#181;L, the expected yield is 500,000 to 1.5 x 106 cells per droplet.</li>
	<li>Use the single-cell suspension of iAT2 cells to generate alveolospheres by plating in the 3D matrix (Step 2) and/or plating on cell culture inserts for ALI culture (Step 3).</li>
</ol>
2. 3D plating of iAT2s
<ol>
	<li>After counting (Step 1.11), determine the number of desired cells to replate in the 3D matrix (400 cells/&#181;L of the matrix with 50-100 &#181;L of 3D matrix droplets per well of a 12-well plate). Centrifuge the cells at 300 x g for 5 min at room temperature. Remove as much supernatant as possible using a pipette.</li>
	<li>Resuspend the cells in the 3D matrix. Resuspend quickly and on ice, if needed, to prevent the matrix from polymerizing (which occurs when warm).</li>
	<li>Use a P200 pipette to dispense one 3D matrix droplet per well into a pre-warmed 12-well plate. Pipette carefully to avoid creating bubbles in the matrix droplet. Do not allow the cell suspension to settle while dispensing multiple droplets.</li>
	<li>Place the plate in a 37 &#176;C incubator for 20-30 min to allow the matrix droplets to polymerize.</li>
	<li>Add 1 mL of CK + DCI + 10 &#181;M of Y-27632 medium (see Table of Materials) per well to cover the matrix droplet.</li>
	<li>After 72 h, change the medium to CK + DCI without 10 &#181;M of Y-27632.</li>
	<li>Replace the medium with fresh CK + DCI every 48-72 h. 
	&#8203;NOTE: iAT2s will typically need to be passaged approximately every 10-14 days, depending on cell line and plating density.</li>
</ol>
3. Passaging of iAT2s to ALI
<ol>
	<li>Prepare freshly coated 6.5 mm cell culture inserts 1 h before use by diluting the 2D matrix in Dulbecco&#39;s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12) to working solution as per manufacturer&#39;s instructions for the 2D matrix (see Table of Materials). Then, add 100 &#181;L of the diluted matrix per 6.5 mm cell culture insert. Allow the matrix to polymerize in a 37 &#176;C incubator for 30 min or at room temperature for 1 h.</li>
	<li>Aspirate the excess matrix from the cell culture inserts using a pipette or aspirating pipette with vacuum and rinse once with DMEM/F12. Aspirate this wash immediately before adding the cells.</li>
	<li>After counting, determine the number of required cells to seed the desired number of cells in the culture inserts (520,000 live cells/cm2, equivalent to 172,000 live cells per 6.5 mm cell culture insert). Centrifuge the cells at 300 x g for 5 min at room temperature. Remove as much supernatant as possible.</li>
	<li>Resuspend the cells in the appropriate volume to seed with 100 &#181;L of CK + DCI + 10 &#181;M Y-27632 per cell culture insert (cell suspension should be 1,720 cells/&#181;L ). Add 100 &#181;L to the apical compartment of the cell culture insert. Gently agitate the plate in a cross-pattern to ensure an even distribution of cells across the cell culture insert, and confirm this by checking under a microscope at 4x objective. 
	NOTE: Seeding density is critical and may require optimization, ranging from 160,000-300,000 cells per 6.5 mm cell culture insert. Calcein green (see Table of Materials) cell-permeable dye may be added to the cells and viewed on an inverted fluorescent microscope to visualize the cells after seeding.</li>
	<li>Add 500 &#181;L of CK + DCI + 10 &#181;M of Y-27632 to the basolateral compartment of each cell culture insert.</li>
	<li>Aspirate apical CK + DCI + 10 &#181;M of Y-27632 medium 48 h after seeding using a pipette to initiate ALI (known as &#34;air-lift&#34;). 
	NOTE: The cells should be 100% confluent at this point. If the cells are not 100% confluent, Steps 3.7-3.8 should still be performed at the indicated time points; confluent cell monolayers may take longer to form.</li>
	<li>Change the basolateral CK + DCI + 10 &#181;M of Y-27632 medium after 72 h to CK + DCI without 10 &#181;M Y-27632. 
	NOTE: There should be minimal if any, &#34;leak&#34; of medium to the apical side. However, if this does occur in the first few days, continue to aspirate the apical side daily until there is no further leakage.</li>
	<li>Replace the basolateral medium with fresh CK + DCI every 48-72 h. 
	NOTE: The iAT2s used in ALI cultures are maximally matured at 5-14 days post-plating.</li>
	<li>Maintain the cells with careful monitoring for up to 28 days post-plating, if needed, for more prolonged experimentation. 
	NOTE: Visible signs of ALI failure, such as peeling of the cells away from the edge of the insert or formation of holes in the monolayer, may be observed after an overextended time in culture, at which point the ALI is no longer usable for experiments.</li>
</ol>

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The authors have nothing to disclose.

AT2s maintain lung homeostasis, and dysfunction of these key alveolar cells can both cause and result from various lung diseases. Due to the difficulty of accessing and isolating primary human AT2s, iAT2s are generated. By applying the directed differentiation methods described elsewhere25 and the expansion and cell seeding described here, iPSCs generated from any individual can be differentiated into robustly self-renewing iAT2s, thus providing patient-specific cells for biomedical research, including basic biomedical research developmental studies, disease modeling, cell-based therapies, or drug screens. The present protocol details a method for dissociating iAT2s to a single-cell suspension, which can then be replated in 3D Matrigel to generate alveolospheres for cell expansion or replated in 2D ALI culture for further experiments.
The protocol has several critical steps to ensure successful passaging and replating in both 3D and ALI cultures. Mechanical trituration must be minimized, as excessive pipetting can decrease cell viability. In addition, the seeding density of iAT2s in both 3D and 2D ALI cultures is critical; for 3D culture, in general, 400 cells/&#181;L of 3D Matrigel is optimal for most iPSC lines. However, a range of densities from 100-500 cells/&#181;L has, at times, been successful, and optimizing the density may be required for different cell lines. For ALI culture, optimizing the seeding density of iAT2s on the cell culture inserts is also essential to achieve a confluent monolayer of cells 48 h after seeding (i.e., on the day of air-lift). Some iAT2 lines require more cells per insert; thus, if troubleshooting is required, a range from 160,000-300,000 cells/insert for 24-well cell culture inserts is recommended. 3D cultures can also be scaled to 24- and 48-well plates by maintaining the seeding density at 400 cells/&#181;L of 3D matrix and reducing matrix droplet size to 25 &#181;L and 20 &#181;L, respectively. ALI cultures can be scaled to 96-well cell culture inserts by coating each insert with 30 &#181;L of the matrix, plating 80,000 cells in 30 &#181;L per insert, and feeding with 150 &#181;L of basolateral media per well. Seeding density and matrix and media volumes need to be scaled and optimized accordingly for other plate formats.
A limitation of this protocol is that the cells used for ALI plating have to be sufficiently purified to be NKX2-1+ and SFTPC+19,20,21,23 to form iAT2 ALIs, and ALI culture formation may vary from line to line. In addition, this protocol allows expansion and generation of AT2-only cultures, providing a reductionist model system based on a single key alveolar cell type. Importantly, other relevant alveolar cell types, such as alveolar type 1 cells, were missing from these platforms. Other groups have had success culturing primary human AT2s as spheroids or organotypic cultures with or without fibroblasts1,14,15,16,17; however, primary human AT2s tend to lose their expression of key surfactants when cultured in normal 2D culture without ALI conditions26. Furthermore, recent work has shown that iAT2s express higher proliferative markers and lower AT2 maturation markers than fresh, uncultured human primary AT2s27. Despite these differences between iAT2s and fresh primary human AT2s, we found that even cultured primary AT2s displayed transcriptomic differences from fresh, uncultured primary AT2s27, and thus conclude that these in vitro models have various strengths and limitations for modeling in vivo lung biology.
The iAT2 platform can be applied to study genetic mutations from patient-derived iPSCs19,20. The system could also be substantially scaled up to accommodate high throughput drug screening. In addition, iAT2 ALIs are suitable for environmental exposures such as viral or bacterial infection or exposure to cigarette smoke, e-cigarette vapor, or other aerosols23. In summary, this protocol for generating both 3D and 2D ALI cultures of iAT2s allows for long-term culture of a disease-relevant cell type and provides a physiological, scalable platform that enables the usage of these cells in many applications.

In the lung, the airway and alveolar epithelial cells are the first to encounter inhaled environmental exposures, including pathogens transmitted via inhaled aerosols and noxious stimuli such as cigarette smoke. Type 2 alveolar epithelial cells (AT2s) are essential in maintaining lung homeostasis as they are the facultative progenitors of the distal lung epithelium, produce surfactants to relieve alveolar surface tension, and mount the innate immune response to inhaled exposures1,2. However, AT2 dysfunction can result from lung injuries or mutations in genes that are selectively expressed in AT2s, such as SFTPC, SFTPB, and ABCA33,4,5. Previous approaches for studying these genetic mutations have relied on mouse models6 or engineered, mutation-containing vectors introduced into immortalized cell lines7. Therefore, platforms that can model the effects of genetic and environmental perturbations on AT2s in the physiologically relevant cell types and in a productive in vitro system are needed to understand further the role AT2s play in health and disease.
In terms of modeling airborne exposures, air-liquid interface (ALI) cultures of primary airway epithelial cells have been successfully utilized to reveal key molecular responses to cigarette smoke8 and to model airway infection9,10. A comparable ALI culture system for the alveolar epithelium, a key site of infection or injury in disease, is much less developed compared to the airway epithelial model system. Immortalized cell lines have been cultured at ALI as a proxy for the alveolar epithelium11,12, but these cell lines are transcriptomically distinct from primary alveolar epithelial cells13 and lack key cellular machinery, such as the ability to secrete surfactants or form tight junctions at ALI12. Primary human AT2s can be cultured in 3D spheres in the presence of fibroblasts1,14 but are subject to limitations, including the limited accessibility from explant lungs and their tendency to senesce or lose cellular phenotype in most cultures to date. These characteristics present barriers to the widespread adoption of primary AT2 in vitro studies, although recent progress has been made in optimizing feeder-free 3D cultures for the expansion of primary AT2s15,16,17,18.
Directed differentiation protocols have been developed to recapitulate in vivo developmental milestones to generate human induced pluripotent stem cell (iPSC)-derived type 2 alveolar epithelial cells (iAT2s)19. iAT2s grow as self-renewing spheres in 3D serum-free culture in a defined medium containing CHIR99021, KGF, dexamethasone, cAMP, and 3-isobutyl-1-methylxanthine (IBMX), termed &#34;CK + DCI&#34;19, in the absence of fibroblast feeders and can be cultured for &#62;20 passages20,21. Moreover, iAT2s share a transcriptomic program with human adult primary AT2s, form lamellar bodies, and produce and package surfactant19,21,22. This protocol details the serial passaging of iAT2s by dissociating the cells to a single-cell suspension. At this point, iAT2s can be replated and expanded further in 3D culture or plated in 2D ALI culture23. These methods can be used to study the intrinsic biology of AT2s in homeostasis and disease20,22 and to interrogate the effects of compounds or stimuli in a scalable, physiologically relevant platform23,24, as has previously been shown.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>3-Isobutyl-1-methylxanthine (IBMX)</td>
			<td>Sigma</td>
			<td>I5879</td>
			<td>For CKDCI</td>
		</tr>
		<tr>
			<td>12 Well Cell Culture Plate</td>
			<td>Corning</td>
			<td>3513</td>
			<td>Plates for culturing</td>
		</tr>
		<tr>
			<td>1-Thioglycerol (MTG)</td>
			<td>M6145</td>
			<td>Sigma</td>
			<td>For CKDCI</td>
		</tr>
		<tr>
			<td>2D Matrigel (matrix) - Matrigel hESC-Qualified Matrix</td>
			<td>Corning</td>
			<td>8774552</td>
			<td>To coat ALI Transwells</td>
		</tr>
		<tr>
			<td>3D Matrigel (matrix) - Growth Factor Reduced Matrigel</td>
			<td>Corning</td>
			<td>356230</td>
			<td>To grow iAT2 spheres in</td>
		</tr>
		<tr>
			<td>8-bromoadenosine 30,50-cyclic monophosphate sodium salt (cAMP)</td>
			<td>Sigma</td>
			<td>B7880</td>
			<td>For CKDCI</td>
		</tr>
		<tr>
			<td>Ascorbic Acid</td>
			<td>A4544</td>
			<td>Sigma</td>
			<td>For CKDCI</td>
		</tr>
		<tr>
			<td>B27 w/out retinoic acid</td>
			<td>Life Technologies</td>
			<td>12587-010</td>
			<td>For CKDCI</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA) (7.5%)</td>
			<td>Thermo Fisher</td>
			<td>15260037</td>
			<td>For CKDCI</td>
		</tr>
		<tr>
			<td>Calcein blue</td>
			<td>Life Technologies</td>
			<td>C1429</td>
			<td>For live/dead discrimination in flow cytometry</td>
		</tr>
		<tr>
			<td>Calcein green</td>
			<td>Life Technologies</td>
			<td>C1430</td>
			<td>Optional visualisation of cells on cell culture insert</td>
		</tr>
		<tr>
			<td>Cell culture inserts - Costar 6.5 mm Clear Transwells with 0.4 &#181;m pore size</td>
			<td>Millipore-Sigma</td>
			<td>CLS3470-48EA</td>
			<td>ALI transwells</td>
		</tr>
		<tr>
			<td>CHIR99021</td>
			<td>Tocris</td>
			<td>4423</td>
			<td>For CKDCI</td>
		</tr>
		<tr>
			<td>Dexamethasone</td>
			<td>Sigma</td>
			<td>D4902</td>
			<td>For CKDCI</td>
		</tr>
		<tr>
			<td>Dispase (2 mg/mL)</td>
			<td>Thermo Fisher</td>
			<td>354235</td>
			<td>To dissolve matrigel</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td>Thermo Fisher</td>
			<td>11995</td>
			<td>To make trypsin inactivation media (10% FBS in DMEM)</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12)</td>
			<td>Thermo Fisher</td>
			<td>A4192002</td>
			<td>To wash</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s phosphate buffered saline (PBS)</td>
			<td>Thermo Fisher</td>
			<td>14190</td>
			<td>For flow cytometric analyses</td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS) (Hyclone charaterized)</td>
			<td>GE Healthcare Life Sciences</td>
			<td>SH30071.03</td>
			<td>To make trypsin inactivation media (10% FBS in DMEM)</td>
		</tr>
		<tr>
			<td>Glutamax</td>
			<td>Life Technologies</td>
			<td>35050-061</td>
			<td>For CKDCI</td>
		</tr>
		<tr>
			<td>Ham&#39;s F-12 Nutrient Mixture (F12)</td>
			<td>Cellgro</td>
			<td>10-080-CV</td>
			<td>For CKDCI</td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>Fisher</td>
			<td>02-671-6</td>
			<td>For cell counting</td>
		</tr>
		<tr>
			<td>Invivogen&#160;Primocin 1 G (50 mg/mL)</td>
			<td>Fisher Scientific</td>
			<td>NC9392943</td>
			<td>For CKDCI</td>
		</tr>
		<tr>
			<td>Iscove&#39;s Modified Dulbecco&#39;s Medium (IMDM)</td>
			<td>Thermo Fisher</td>
			<td>12440053</td>
			<td>For CKDCI</td>
		</tr>
		<tr>
			<td>Millicell ERS-2 Voltohmeter</td>
			<td>Millipore</td>
			<td>MERS00002</td>
			<td>To measure trans-epithlial electrical resistance</td>
		</tr>
		<tr>
			<td>N2 supplement</td>
			<td>Life Technologies</td>
			<td>17502-048</td>
			<td>For CKDCI</td>
		</tr>
		<tr>
			<td>Recombinant human KGF</td>
			<td>R&#38;D Systems</td>
			<td>251-KG-010</td>
			<td>For CKDCI</td>
		</tr>
		<tr>
			<td>Retinoic acid</td>
			<td>Sigma</td>
			<td>R2625</td>
			<td>For CKDCI</td>
		</tr>
		<tr>
			<td>Trypan blue</td>
			<td>Thermo Fisher</td>
			<td>15250061</td>
			<td>For cell count during passaging</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.05%)</td>
			<td>Gibco</td>
			<td>25-300-062</td>
			<td>To dissociate iAT2 spheres</td>
		</tr>
		<tr>
			<td>Y-27632 dihydrochloride</td>
			<td>Tocris</td>
			<td>1254</td>
			<td>Add to cells after passaging</td>
		</tr>
	</tbody>
</table>

generating 3d spheres and 2d air-liquid interface cultures of human induced pluripotent stem cell-derived type 2 alveolar epithelial cells

In the lung, the alveolar epithelium is a physical barrier from environmental stimuli and plays an essential role in homeostasis and disease. Type 2 alveolar epithelial cells (AT2s) are the facultative progenitors of the distal lung epithelium. Dysfunction and injury of AT2s can result from and contribute to various lung diseases. Improved understanding of AT2 biology is, thus, critical for understanding lung biology and disease; however, primary human AT2s are generally difficult to isolate and limited in supply. To overcome these limitations, human induced pluripotent stem cell (iPSC)-derived type 2 alveolar epithelial cells (iAT2s) can be generated through a directed differentiation protocol that recapitulates in vivo lung development. iAT2s grow in feeder-free conditions, share a transcriptomic program with human adult primary AT2s, and execute key functions of AT2s such as production, packaging, and secretion of surfactant. This protocol details the methods for maintaining self-renewing iAT2s through serial passaging in three-dimensional (3D) culture or adapting iAT2s to air-liquid interface (ALI) culture. A single-cell suspension of iAT2s is generated before plating in 3D solubilized basement membrane matrix (hereafter referred to as &#34;matrix&#34;), where they self-assemble into monolayered epithelial spheres. iAT2s in 3D culture can be serially dissociated into single-cell suspensions to be passaged or plated in 2D ALI culture. In ALI culture, iAT2s form a polarized monolayer with the apical surface exposed to air, making this platform readily amenable to environmental exposures. Hence, this protocol generates an inexhaustible supply of iAT2s, producing upwards of 1 x 1030 cells per input cell over 15 passages while maintaining the AT2 program indicated by SFTPCtdTomato expression. The resulting cells represent a reproducible and relevant platform that can be applied to study genetic mutations, model environmental exposures, or screen drugs.

The iAT2s were passaged to a single-cell suspension and then replated in 3D matrix or in 2D on cell culture inserts for ALI culture (Figure 1). After single-cell dissociation, the iAT2s were analyzed by flow cytometry. Briefly, the cells resuspended in 1% fetal bovine serum (FBS) in phosphate-buffered saline (PBS) with calcein blue viability dye (1:1000) were analyzed on a flow cytometer, gating for non-fragments, singlets, and tdTomato. Here, as an example, is the SPC2 line (SPC2-ST-B2 clone20), which has a tdTomato reporter targeted to the endogenous SFTPC locus for ease of visualization and tracking of the AT2 program over time in culture (Figure 2A). The iAT2 SFTPC-tdTomato expression was maintained when replated in the 3D matrix as spheres (Figure 2B) and when plated on cell culture inserts (Figure 2C). Trans-epithelial electrical resistance (TEER) can be measured to determine the integrity of the ALI culture (Figure 2D). To measure TEER, a voltohmmeter was used (see Table of Materials), with 100 &#181;L of CK + DCI medium added to the apical chamber. The cell culture inserts coated with Matrigel in the absence of seeded cells were treated as blanks. Readings were taken at three locations in every well.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63875/63875fig01v2.jpg" /> 
Figure 1: Schematic representation of the protocol workflow. <a href="https://www.jove.com/files/ftp_upload/63875/63875fig01largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63875/63875fig02.jpg" /> 
Figure 2: Representative results of the protocol. (A) Representative flow cytometry results for SFTPC-tdTomato in human induced pluripotent stem cell-derived type 2 alveolar epithelial-like cells (iAT2s) cultured in 3D. The negative control shown is non-lung endoderm (CD47lo). (B) Representative live-cell imaging of iAT2s cultured in 3D at various days post passage (dpp) (SFTPC-tdTomato, scale bar = 50 &#181;m). (C) Representative live-cell imaging of iAT2s plated at air-liquid interface (SFTPC-tdTomato, scale bar = 500 nm). (D) Representative trans-epithelial electrical resistance of iAT2s plated at the air-liquid interface. n = 3, error bars indicate standard deviation. <a href="https://www.jove.com/files/ftp_upload/63875/63875fig02large.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>Reagents</td>
			<td>Volume for 500 mL</td>
			<td>Final concentration</td>
		</tr>
		<tr>
			<td>Iscove&#39;s Modified Dulbecco&#39;s Medium (IMDM)</td>
			<td>375 mL</td>
			<td>75%</td>
		</tr>
		<tr>
			<td>Ham&#39;s F-12 Nutrient Mixture (F12)</td>
			<td>125 mL</td>
			<td>25%</td>
		</tr>
		<tr>
			<td>B-27 (with RA) supplement</td>
			<td>5 mL</td>
			<td>1%</td>
		</tr>
		<tr>
			<td>N-2 supplement</td>
			<td>2.5 mL</td>
			<td>0.50%</td>
		</tr>
		<tr>
			<td>BSA (7.5% stock)</td>
			<td>3.3 mL</td>
			<td>0.05%</td>
		</tr>
		<tr>
			<td>Primocin (50 mg/mL stock)</td>
			<td>1 mL</td>
			<td>100 &#181;g/mL</td>
		</tr>
		<tr>
			<td>Glutamax (100x stock)</td>
			<td>5 mL</td>
			<td>1x</td>
		</tr>
		<tr>
			<td>Ascorbic Acid (50 mg/mL stock)</td>
			<td>500 &#181;L</td>
			<td>50 &#181;g/mL</td>
		</tr>
		<tr>
			<td>1-Thioglycerol (MTG) (from 26 &#181;L in 2 mL IMDM)</td>
			<td>1.5 mL</td>
			<td>4.5 x 10-4 M</td>
		</tr>
	</tbody>
</table>
Table 1: Composition of the complete serum-free differentiation media (cSFDM).
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reagent</td>
			<td>Final concentration</td>
		</tr>
		<tr>
			<td>CHIR99021</td>
			<td>3 &#181;M</td>
		</tr>
		<tr>
			<td>rhKGF</td>
			<td>10 ng/mL</td>
		</tr>
		<tr>
			<td>Dexamethasone</td>
			<td>50 nM</td>
		</tr>
		<tr>
			<td>8-bromoadenosine 30,50-cyclic monophosphate sodium salt (cAMP)</td>
			<td>0.1 mM</td>
		</tr>
		<tr>
			<td>3-Isobutyl-1-methylxanthine (IBMX)</td>
			<td>0.1 mM</td>
		</tr>
	</tbody>
</table>
Table 2: Compositions of the CK + DCI media.

Watch this Scientific Journal Video about Generating 3D Spheres and 2D Air-Liquid Interface Cultures of Human Induced Pluripotent Stem Cell-Derived Type 2 Alveolar Epithelial Cells at JoVE.com

Generating 3D Spheres and 2D Air-Liquid Interface Cultures of Human Induced Pluripotent Stem Cell-Derived Type 2 Alveolar Epithelial Cells

The present protocol describes human induced pluripotent stem cell-derived type 2 alveolar epithelial-like cells (iAT2s). These cells can be cultured as self-renewing spheres in 3D culture or adapted to air-liquid interface (ALI) culture.

In the lung, the alveolar epithelium is a physical barrier from environmental stimuli and plays an essential role in homeostasis and disease. Type 2 ...

generating-3d-spheres-2d-air-liquid-interface-cultures-human-induced

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Developmental Biology

4.5K Views.  Boston University and Boston Medical Center. The present protocol describes human induced pluripotent stem cell-derived type 2 alveolar epithelial-like cells (iAT2s). These cells can be cultured as self-renewing spheres in 3D culture or adapted to air-liquid interface (ALI) culture.

Video: Generating 3D Spheres and 2D Air-Liquid Interface Cultures of Human Induced Pluripotent Stem Cell-Derived Type 2 Alveolar Epithelial Cells

Cultivate Primary Nasal Epithelial Cells from Children and Reprogram into Induced Pluripotent Stem Cells

Nasal epithelial cells (NECs) are the part of the airways that respond to air pollutants and are the first cells infected with respiratory viruses. They are also involved in many airway diseases through their innate immune response and interaction with immune and airway stromal cells. NECs are of particular interest for studies in children due to their accessibility during clinical visits. Human induced pluripotent stem cells (iPSCs) have been generated from multiple cell types and are a powerful tool for modeling human development and disease, as well as for their potential applications in regenerative medicine. This is the first protocol to lay out methods for successful generation of iPSCs from NECs derived from pediatric participants for research purposes. It describes how to obtain nasal epithelial cells from children, how to generate primary NEC cultures from these samples, and how to reprogram primary NECs into well-characterized iPSCs. Nasal mucosa samples are useful in epidemiological studies related to the effects of air pollution in children, and provide an important tool for studying airway disease. Primary nasal cells and iPSCs derived from them can be a tool for providing unlimited material for patient-specific research in diverse areas of airway epithelial biology, including asthma and COPD research.

This publication demonstrates methods for successful sampling and culture of nasal epithelial mucosa from children, and reprogramming these cells to induced Pluripotent Stem Cells (iPSCs).

Nasal epithelial cells (NECs) are the part of the airways that respond to air pollutants and are the first cells infected with respiratory viruses. They ...

Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing a detailed and thorough protocol is to clearly demonstrate each step and to make this readily available to researchers in the field. This protocol results in a homogenous layer of RPE with minimal or no manual dissection needed. The method presented here has been shown to be effective for induced pluripotent stem cells (iPSC) and human embryonic stem cells. Additionally, we describe methods for cryopreservation of intermediate cell banks that allow long-term storage. RPE generated using this protocol might be useful for iPSC disease-in-a-dish modeling or clinical application.

This protocol describes how to produce retinal pigment epithelial cells (RPE) from pluripotent stem cells. The method uses a combination of growth factors and small molecules to direct the differentiation of stem cells into immature RPE in fourteen days and mature, functional RPE after three months.

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing ...

Generation of Standardized and Reproducible Forebrain-type Cerebral Organoids from Human Induced Pluripotent Stem Cells

The human cortex is highly expanded and exhibits a complex structure with specific functional areas, providing higher brain function, such as cognition. Efforts to study human cerebral cortex development have been limited by the availability of model systems. Translating results from rodent studies to the human system is restricted by species differences and studies on human primary tissues are hampered by a lack of tissue availability as well as ethical concerns. Recent development in human pluripotent stem cell (PSC) technology include the generation of three-dimensional (3D) self-organizing organotypic culture systems, which mimic to a certain extent human-specific brain development in vitro. Currently, various protocols are available for the generation of either whole brain or brain-region specific organoids. The method for the generation of homogeneous and reproducible forebrain-type organoids from induced PSC (iPSC), which we previously established and describe here, combines the intrinsic ability of PSC to self-organize with guided differentiation towards the anterior neuroectodermal lineage and matrix embedding to support the formation of a continuous neuroepithelium. More specifically, this protocol involves: (1) the generation of iPSC aggregates, including the conversion of iPSC colonies to a confluent monolayer culture; (2) the induction of anterior neuroectoderm; (3) the embedding of neuroectodermal aggregates in a matrix scaffold; (4) the generation of forebrain-type organoids from neuroectodermal aggregates; and (5) the fixation and validation of forebrain-type organoids. As such, this protocol provides an easily applicable system for the generation of standardized and reproducible iPSC-derived cortical tissue structures in vitro.

Cerebral organoids represent a new model system to investigate early human brain development in vitro. This article provides the detailed methodology to efficiently generate homogeneous dorsal forebrain-type organoids from human induced pluripotent stem cells including critical characterization and validation steps.

The human cortex is highly expanded and exhibits a complex structure with specific functional areas, providing higher brain function, such as cognition. ...

Using Human Induced Pluripotent Stem Cell-derived Hepatocyte-like Cells for Drug Discovery

The ability to differentiate human induced pluripotent stem cells (iPSCs) into hepatocyte-like cells (HLCs) provides new opportunities to study inborn errors in hepatic metabolism. However, to provide a platform that supports the identification of small molecules that can potentially be used to treat liver disease, the procedure requires a culture format that is compatible with screening thousands of compounds. Here, we describe a protocol using completely defined culture conditions, which allow the reproducible differentiation of human iPSCs to hepatocyte-like cells in 96-well tissue culture plates. We also provide an example of using the platform to screen compounds for their ability to lower Apolipoprotein B (APOB) produced from iPSC-derived hepatocytes generated from a familial hypercholesterolemia patient. The availability of a platform that is compatible with drug discovery should allow researchers to identify novel therapeutics for diseases that affect the liver.

The protocol presented here describes a platform for identifying small molecules for the treatment of liver disease. A step-by-step description is presented detailing how to differentiate iPSCs into cells with hepatocyte characteristics in 96-well plates, and to use the cells to screen for small molecules with potential therapeutic activity.

The ability to differentiate human induced pluripotent stem cells (iPSCs) into hepatocyte-like cells (HLCs) provides new opportunities to study inborn ...

A Familial Hypercholesterolemia Human Liver Chimeric Mouse Model Using Induced Pluripotent Stem Cell-derived Hepatocytes

Familial hypercholesterolemia (FH) is mostly caused by low-density lipoprotein receptor (LDLR) mutations and results in an increased risk of early-onset cardiovascular disease due to marked elevation of LDL cholesterol (LDL-C) in blood. Statins are the first line of lipid-lowering drugs for treating FH and other types of hypercholesterolemia, but new approaches are emerging, in particular PCSK9 antibodies, which are now being tested in clinical trials. To explore novel therapeutic approaches for FH, either new drugs or new formulations, we need appropriate in vivo models. However, differences in the lipid metabolic profiles compared to humans are a key problem of the available animal models of FH. To address this issue, we have generated a human liver chimeric mouse model using FH induced pluripotent stem cell (iPSC)-derived hepatocytes (iHeps). We used Ldlr-/-/Rag2-/-/Il2rg-/- (LRG) mice to avoid immune rejection of transplanted human cells and to assess the effect of LDLR-deficient iHeps in an LDLR null background. Transplanted FH iHeps could repopulate 5-10% of the LRG mouse liver based on human albumin staining. Moreover, the engrafted iHeps responded to lipid-lowering drugs and recapitulated clinical observations of increased efficacy of PCSK9 antibodies compared to statins. Our human liver chimeric model could thus be useful for preclinical testing of new therapies to FH. Using the same protocol, similar human liver chimeric mice for other FH genetic variants, or mutations corresponding to other inherited liver diseases, may also be generated.

Here, we present a protocol to generate a human liver chimeric mouse model of familial hypercholesterolemia using human induced pluripotent stem cell-derived hepatocytes. This is a valuable model for testing new therapies for hypercholesterolemia.

Familial hypercholesterolemia (FH) is mostly caused by low-density lipoprotein receptor (LDLR) mutations and results in an increased risk of early-onset ...

Microfluidic Assay for the Assessment of Leukocyte Adhesion to Human Induced Pluripotent Stem Cell-derived Endothelial Cells (hiPSC-ECs)

Endothelial cells (ECs) are essential for the regulation of inflammatory responses by either limiting or facilitating leukocyte recruitment into affected tissues via a well-characterized cascade of pro-adhesive receptors which are upregulated on the leukocyte cell surface upon the inflammatory trigger. Inflammatory responses differ between individuals in the population and the genetic background can contribute to these differences. Human induced pluripotent stem cells (hiPSCs) have been shown to be a reliable source of ECs (hiPSC-ECs), thus representing an unlimited source of cells that capture the genetic identity and any genetic variants or mutations of the donor. hiPSC-ECs can therefore be used for modeling inflammatory responses in donor-specific cells. Inflammatory responses can be modeled by determining leukocyte adhesion to the hiPSC-ECs under physiological flow. This step-by-step protocol provides a detailed description of the experimental setup and data analysis for the assessment of inflammatory responses in hiPSC-ECs and the analysis of leukocyte adhesion under physiological flow.

Microfluidic Assay for the Assessment of Leukocyte Adhesion to Human Induced Pluripotent Stem Cell-derived Endothelial Cells hiPSC-ECs

This step-by-step protocol provides a detailed description of the experimental setup and data analysis for the assessment of inflammatory responses in hiPSC-ECs and the analysis of leukocyte adhesion under physiological flow.

Endothelial cells (ECs) are essential for the regulation of inflammatory responses by either limiting or facilitating leukocyte recruitment into affected ...

Generation of 3D Skin Organoid from Cord Blood-derived Induced Pluripotent Stem Cells

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily functions. Biomimetics is the imitation of the models, systems, and elements of nature for the purpose of solving complex human problems1. Skin biomimetics is a useful tool for in vitro disease research and in vivo regenerative medicine. Human induced pluripotent stem cells (iPSCs) have the characteristic of unlimited proliferation and the ability of differentiation to three germ layers. Human iPSCs are generated from various primary cells, such as blood cells, keratinocytes, fibroblasts, and more. Among them, cord blood mononuclear cells (CBMCs) have emerged as an alternative cell source from the perspective of allogeneic regenerative medicine. CBMCs are useful in regenerative medicine because human leukocyte antigen (HLA) typing is essential to the cell banking system. We provide a method for the differentiation of CBMC-iPSCs into keratinocytes and fibroblasts and for generation of a 3D skin organoid. CBMC-iPSC-derived keratinocytes and fibroblasts have characteristics similar to a primary cell line. The 3D skin organoids are generated by overlaying an epidermal layer onto a dermal layer. By transplanting this 3D skin organoid, a humanized mice model is generated. This study shows that a 3D human iPSC-derived skin organoid may be a novel, alternative tool for dermatologic research in vitro and in vivo.

We propose a protocol that shows how to differentiate induced pluripotent stem cell-derived keratinocytes and fibroblasts and generate a 3D skin organoid, using these keratinocytes and fibroblasts. This protocol contains an additional step of generating a humanized mice model. The technique presented here will improve dermatologic research.

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give rise to multiple cell types, including the myotome (MYO), sclerotome (SCL), dermatome (D), and syndetome (SYN), which in turn develop into skeletal muscle, axial skeleton, dorsal dermis, and axial tendon/ligament, respectively. Therefore, the generation of SMs and their derivatives from human induced pluripotent stem cells (iPSCs) is critical to obtain pluripotent stem cells (PSCs) for application in regenerative medicine and for disease research in the field of orthopedic surgery. Although the induction protocols for MYO and SCL from PSCs have been previously reported by several researchers, no study has yet demonstrated the induction of SYN and D from iPSCs. Therefore, efficient induction of fully competent SMs remains a major challenge. Here, we recapitulate human SM patterning with human iPSCs in vitro by mimicking the signaling environment during chick/mouse SM development, and report on methods of systematic induction of SM derivatives (MYO, SCL, D, and SYN) from human iPSCs under chemically defined conditions through the presomitic mesoderm (PSM) and SM states. Knowledge regarding chick/mouse&#160;SM development was successfully applied to the induction of SMs with human iPSCs. This method could be a novel tool for studying human somitogenesis and patterning without the use of embryos and for cell-based therapy and disease modeling.

We present here a protocol for the differentiation of human induced pluripotent stem cells into each somite derivative (myotome, sclerotome, dermatome, and syndetome) in chemically defined conditions, which has applications in future disease modeling and cell-based therapies in orthopedic surgery.

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...

Controlled Cortical Impact Model of Mouse Brain Injury with Therapeutic Transplantation of Human Induced Pluripotent Stem Cell-Derived Neural Cells

Traumatic brain injury (TBI) is a leading cause of morbidity and mortality worldwide. Disease pathology due to TBI progresses from the primary mechanical insult to secondary injury processes, including apoptosis and inflammation. Animal modeling has been valuable in the search to unravel injury mechanisms and evaluate potential neuroprotective therapies. This protocol describes the controlled cortical impact (CCI) model of focal, open-head TBI. Specifically, parameters for producing a mild unilateral cortical injury are described. Behavioral consequences of CCI are analyzed using the adhesive tape removal test of bilateral sensorimotor integration. Regarding experimental therapy for TBI pathology, this protocol also illustrates a process for transplanting cultured cells into the brain. Neural cell cultures derived from human induced pluripotent stem cells (hiPSCs) were chosen for their potential to show superior functional restoration in human TBI patients. Chronic survival of hiPSCs in the host mouse brain tissue is detected using a modified DAB immunohistochemical process.

This protocol demonstrates methodologies for a mouse model of open-skull traumatic brain injury and transplantation of cultured human induced pluripotent stem cell-derived cells into the injury site. Behavioral and histologic tests of outcomes from these procedures are also described in brief.

Traumatic brain injury (TBI) is a leading cause of morbidity and mortality worldwide. Disease pathology due to TBI progresses from the primary mechanical ...

In vitro Neuromuscular Junction Induced from Human Induced Pluripotent Stem Cells

The neuromuscular junction (NMJ) is a specialized synapse that transmits action potentials from the motor neuron to skeletal muscle for mechanical movement. The architecture of the NMJ structure influences the functions of the neuron, the muscle and the mutual interaction. Previous studies have reported many strategies by co-culturing the motor neurons and myotubes to generate NMJ in vitro with complex induction process and long culture period but have struggled to recapitulate mature NMJ morphology and function. Our in vitro NMJ induction system is constructed by differentiating human iPSC in a single culture dish. By switching the myogenic and neurogenic induction medium for induction, the resulting NMJ contained pre- and post- synaptic components, including motor neurons, skeletal muscle and Schwann cells in the one month culture. The functional assay of NMJ also showed that the myotubes contraction can be triggered by Ca++ then inhibited by curare, an acetylcholine receptor (AChR) inhibitor, in which the stimulating signal is transmitted through NMJ. This simple and robust approach successfully derived the complex structure of NMJ with functional connectivity. This in vitro human NMJ, with its integrated structures and function, has promising potential for studying pathological mechanisms and compound screening.

Here we provide a protocol to generate in vitro NMJs from human induced pluripotent stem cells (iPSCs). This method can induce NMJs with mature morphology and function in 1 month in a single well. The resulting NMJs could potentially be used to model related diseases, to study pathological mechanisms or to screen drug compounds for therapy.