This research was supported by the California Institute for Regenerative Medicine (CIRM)&#160;&#160;(DISC2-COVID19-12022).

This study protocol was approved by the Institutional Review Board of UCSD&#39;s Human Research Protections Program (181180).
1. Definitive endoderm induction from induced pluripotent stem cells (Day 1 - 3)
<ol>
	<li>Slowly thaw growth factor reduced (GFR)-basement membrane (BM) matrix medium on ice 30 minutes prior to use. In cold DMEM/F12, mixture, dilute the GFR BM matrix medium 1:1 such that it constitutes 50% of this medium. Place P1000 pipette tips in the freezer to chill prior to use.</li>
	<li>Coat each well of a 12-well plate with 500 &#181;L of 50% GFR-basement membrane matrix medium prepared in ice-cold DMEM/F12. Once the desired number of wells are coated, remove any excess medium mixture and/or bubbles from wells and place the plate on ice or refrigerator at 4 &#176;C for 20 min to set. Then, move the plate to the incubator at 37 &#176;C overnight to gel and dry.</li>
	<li>Once hiPSCs reach 70-90% confluency, add 10 &#181;M of Rho-associated kinase (ROCK)&#160;Inhibitor Y-27632 an hour prior to dissociation. Aspirate off the media and wash once with Phosphate buffered saline (PBS). Dissociate hiPSCs by adding cell detachment medium (0.5 mL/ well of a 12-well plate) and incubate for 20 min at 37 &#176;C in a 5% CO2 incubator.</li>
	<li>Remove plates from the incubator and add 0.5 mL/12-well of stem cell passaging medium (Table 1) to the wells; gently triturate&#160;cells using a P1000 tip to obtain single-cell suspension. Transfer dissociated cells into a 15 mL conical centrifuge tube; centrifuge for 5 min at 300 x g.</li>
	<li>Aspirate off the medium and resuspend the cell pellet with 1 mL of mTeSR Plus media supplemented with 10 &#181;M ROCK inhibitor (Y-27632). Perform a cell count. Add 2.0 x 105&#160;hiPSCs in 1 mL of mTeSR supplemented with ROCK Inhibitor Y-27632 per well of a 12-well GFR-basement membrane medium coated plate. Incubate at 37 &#176;C overnight. 
	NOTE: Cell seeding number must be optimized per cell line. 24 h after plating, wells should be 50%-70% confluent.</li>
	<li>On day 1, aspirate off the mTeSR Plus and add Definitive Endoderm (DE) induction media (Table 1) supplemented with 100 ng/mL of human activin A and 5 &#181;M of GSK3&#946; inhibitor/Wnt activator CHIR99021. 
	&#8203;NOTE: DE media with GSK3&#946; inhibitor/Wnt activator CHIR99021 should be removed within 20-24 h of day 1 DE induction for successful differentiation.</li>
	<li>On day 2 and day 3, change to DE induction media supplemented with 100 ng/mL of activin A only. 
	&#8203;NOTE: DE differentiation should not exceed a total of 72 h, or efficacy will decrease. On day 4, if large cell die-off is observed, decrease total DE media exposure time by 6-12 h.</li>
	<li>To analyze DE efficiency, confirm greater than 90% CXCR4 and/or cKit expression via flow cytometry or immunofluorescence analysis of FOXA2 and/or SOX17 (Figure 2a).</li>
</ol>
2. Anterior foregut endoderm (AFE) induction (Day 4 - 6)
<ol>
	<li>On day 4, change media to serum free basal medium (SFBM) (Table 1) supplemented with 10 &#181;M SB431542 and 2 &#181;M Dorsomorphin for AFE induction. Change AFE media daily for 3 days (day 4, day 5, and day 6).</li>
	<li>To analyze AFE efficiency, confirm robust expression of SOX2, TBX1, and FOXA2 via immunofluorescence staining (Figure 2b).</li>
</ol>
3. Lung progenitor cell (LPC) differentiation (Day 7 - 16)
<ol>
	<li>On day 7, thaw GFR-basement membrane matrix medium on ice. Aspirate off the AFE media and wash well with 1x PBS. Add 1 mL of cell detachment solution and incubate for 10 min at 37 &#176;C.</li>
	<li>Add 1 mL of quenching medium (2% FBS in DMEM/F12) to the wells containing cell detachment solution. Keep cells as aggregates by pipetting up and down gently. Make sure all cells are dislodged and transferred into a 15 mL conical centrifuge tube. Centrifuge for 5 min at 300 x g.</li>
	<li>Remove the supernatant and resuspend the cell pellet in Quenching medium&#160;supplemented with 10 ng/mL of human recombinant bone morphogenic protein-4 (BMP4), 0.1 &#181;M of all-trans retinoic acid (RA), 3 &#181;M of GSK3&#946; inhibitor/Wnt activator CHIR99021, and 10 &#181;M of Rock Inhibitor Y-27632.</li>
	<li>Perform a cell count. Add 2.5 x 105 cells to 100 &#181;L of cold GFR-basement membrane matrix medium, mix well, and place droplet into a well of a 12-well plate. Incubate the plate at 37 &#176;C for 30-60 min to allow the medium to polymerize. Add 1 mL of LPC media supplemented with 10 &#181;M of ROCK Inhibitor Y-27632 per well ensuring the medium drop is fully submerged and incubate at 37 &#176;C overnight.</li>
	<li>On day 8, 24 h after LPC induction, change LPC medium to remove ROCK Inhibitor Y-27632. Change the LPC medium every other day for a total of 9-11 days. 
	NOTE: If the medium becomes yellow within 24 h, change medium every day.</li>
	<li>To analyze LPC efficiency, confirm robust expression of the intracellular transcription factor NKX2-1 or perform flow cytometry for surface markers CD47hi/CD26low15 or CPM18 (Figure 2c). Grossly, the LPC spheroids should be round and transparent (Figure 2c). 
	&#8203;NOTE: Do not proceed with lung organoid differentiation if efficiency of NKX2-1 is below 30%.</li>
</ol>
4. 3D lung organoid induction (Day 16 - 22)
<ol>
	<li>On day 17, thaw GFR-basement membrane matrix medium on ice. Aspirate the LPC induction medium. Then add 2 &#181;g/mL of dispase (1&#160;mL) to the well and resuspend the medium/dispase mixture with a P1000 pipette. Incubate at&#160;37 &#176;C for 15 min. Triturate the mixture again and incubate at&#160;37 &#176;C for another 15 min.</li>
	<li>Transfer the dispase and cells into a 15 mL conical centrifuge tube. Use chilled PBS (2-3 mL) to wash the well and resuspend the dispase/cell mixture. Centrifuge for 5 min at 400 x g. Manually remove the supernatant, being careful not to distub the medium/cell pellet layer. Repeat the chilled PBS wash, and&#160;centrifuge the conical centrifuge tube for another 5 min at 400 x g.</li>
	<li>Manually remove the supernatant, and then add 2 mL of trypsin- based dissociation solution to the conical centrifuge tube. Incubate at 37 &#176;C for 12 min.</li>
	<li>After incubation, resuspend cells with a P1000 pipette tip. Then add an equal volume of quenching media to the conical centrifuge tube and spin down at 400 x g for 5 min. Aspirate the supernatant and resuspend cells&#160; in quenching medium + 10 &#181;M of ROCK Inhibitor Y-27632. 
	NOTE: Successful lung organoid induction occurs when cells are embedded as aggregates, not single cells, adjust pipetting accordingly.</li>
	<li>Perform a cell count. Calculate the volume needed to obtain 5.0-8.0 x 104 cells per well. Aliquot the LPC cell aggregates into 1.5 mL microcentrifuge tubes and centrifuge for 5 min at 400 x g. Remove excess supernatant, being careful to not agitate the cell pellet. Leave only 10 &#181;L of residual media.</li>
	<li>Re-suspend the cell pellet in 200 &#181;L of cold GFR-basement membrane matrix medium and add to cell culture membrane inserts (6.5 mm diameter, 0.4 &#181;m pore, polyester membrane). Incubate the plate at 37 &#176;C for 30-60 min to allow GFR-basement membrane matrix medium to polymerize.</li>
	<li>Add 1 mL of 3D organoid induction medium (Table 1) supplemented with&#160;fibroblast growth factor-7&#160;(FGF7) (10 ng/mL), FGF10 (10 ng/mL), GSK3&#946; inhibitor/Wnt activator CHIR (3 &#181;M), epidermal growth factor (EGF) (10 ng/mL), and 10 &#181;M of ROCK&#160;Inhibitor Y-27632 to the basolateral chamber of the membrane insert. Change the medium every other day for 6 days.</li>
</ol>
5. 3D Lung organoid branching (Day 23 - 28)
<ol>
	<li>On day 23 change to 3D branching medium (Table 1) supplemented with FGF7 (10 ng/mL), FGF10 (10 ng/mL), GSK3&#946; inhibitor/Wnt activator CHIR99021 (3 &#181;M), RA (0.1 &#181;M), EGF (10 ng/mL), and vascular endothelial growth factor (VEGF) / placental growth factor (PlGF)&#160;(10 ng/mL). Change the medium every other day for 6 days. 
	NOTE: At day 6 of 3D branching differentiation, there should be multiple branching organoids (Figure 2).</li>
</ol>
6. 3D lung organoid maturation (Day 29 - 34)
<ol>
	<li>On day 29, change to 3D maturation medium (Table 1), which is the same as 3D branching medium but with the addition of dexamethasone (50 nM), cAMP (100 &#181;M), and and 3-isobutyl-1-methylxanthine (IBMX), a phosphodiesterase inhibitor also termed isobutylxanthine&#160;(100 &#181;M). Change the medium every other day for 6 days. 
	&#8203;NOTE: Within 24 h after 3D maturation, the branching organoids should expand and change into transparent spheres.</li>
</ol>
7. 3D Lung organoid immunocytochemistry
<ol>
	<li>For 3D whole lung organoid analysis, fix GFR-basement membrane matrix medium in the membrane inserts with 4% paraformaldehyde (PFA) for 1 h at 4 &#176;C. Embed in paraffin wax and mount onto slides per standard published protocols.</li>
	<li>Perform antigen retrieval prior to staining. Airway markers include KRT5, MUC5AC, and SCGB3A2. Alveolar markers include SP-C, SP-B, HTII-280, HTI-56, and HOPX (Figure 3).</li>
</ol>
8. Removal of whole lung organoids from GFR-basement membrane matrix medium for passage, FACS, or cryopreservation
<ol>
	<li>To dissociate the organoids from the GFR-basement membrane matrix medium, remove media from the basal chamber and add 2 &#181;g/mL of dispase (1 mL) in the apical chamber.</li>
	<li>Gently triturate the medium /dispase mixture with a P1000 pipette and place in the incubator for 15 min. Gently triturate the mixture again and incubate for another 15 min.</li>
	<li>Add 1 mL of chilled PBS (4 &#176;C) and transfer organoids with the matrix medium into a 15 mL conical centrifuge tube. Spin at 400 x g for 5 min. Remove the supernatant carefully, not to disturb the cell pellet.</li>
	<li>Wash once more with 1 mL chilled PBS and spin down at 400 x g for 5 min. Remove the supernatant carefully, not to disturb the medium/cell mixture.</li>
	<li>Add 1 mL of cell detachment solution to the conical centrifuge tube, gently triturate resuspend the GFR-Basement membrane matrix medium/cell mixture. Place in the incubator for 12 min for passaging cells as aggregates or for cryopreservation), or 20 min for single cell suspension.</li>
	<li>Add equal volume of quenching media and spin down at 400 x g for 5 min. Resuspend in quenching medium + 10 &#181;M of ROCK Inhibitor Y-27632. 
	NOTE: At this step, no residual basement membrane medium should be seen in the tube. If residual medium remains, repeat steps 8.5 and 8.6.</li>
	<li>Perform a cell count. Calculate the volume needed for downstream applications.</li>
</ol>

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The authors have nothing to disclose.

The successful differentiation of 3D whole lung organoids (WLO) relies on a multi-step, 6-week protocol with attention to detail, including time of exposure to growth factors and small molecules, cellular density after passaging, and the quality of hiPSCs. For troubleshooting, see Table 2. hiPSCs should be approximately 70%-80% confluent, with less than 5% spontaneous differentiation prior to dissociation. This protocol calls for &#34;mTeSR plus&#34; medium; however,&#160; plain &#34;mTeSR&#34; medium&#1...

The lung is a complicated, heterogeneous, dynamic organ that develops in six distinct stages - embryonic, pseudoglandular, canalicular, saccular, alveolar, and microvascular maturation1,2. The latter two phases occur pre and postnatally in human development while the first four stages occur exclusively during fetal development unless preterm birth occurs3. The embryonic phase begins in the endodermal germ layer and concludes with the budding of the trachea and lung buds. Lung development occurs in part via signaling between the epithelial and mesenchymal cells4. These interactions result in lung branching, proliferation, cellular fate determination and cellular differentiation of the developing lung. The lung is divided into conducting zones (trachea to the terminal bronchioles) and respiratory zones (respiratory bronchioles to the alveoli). Each zone contains unique epithelial cell types; including basal, secretory, ciliated, brush, neuroendocrine, and ionocyte cells in the conducting airway5, followed by alveolar type I and II cells in the respiratory epithelium6. Much is still unknown about the development and response to injury of the various cell types. iPSC derived lung organoid models enable the study of mechanisms that drive human lung development, the effects of genetic mutations on pulmonary function, and the response of both the epithelium and mesenchyme to infectious agents without the need for primary human lung tissue.
Markers corresponding to the various stages of embryonic differentiation include CXCR4, cKit, FOXA2, and SOX17 for definitive endoderm (DE)7, FOXA2, TBX1, and SOX2 for anterior foregut endoderm (AFE)8, and NKX2-1 for early lung progenitor cells9. In embryonic lung development, the foregut divides into the dorsal esophagus and ventral trachea. The buds of the right and left lungs appear as two independent outpouchings around the tracheal bud10. During branching morphogenesis, the mesenchyme surrounding the epithelium produces elastic tissue, smooth muscle, cartilage, and vasculature11. The interaction between the epithelium and mesenchyme is essential for normal lung development. This includes the secretion of FGF1012 by the mesenchyme and SHH13 produced by the epithelium.
Here, we describe a protocol for the directed differentiation of hiPSCs into three-dimensional (3D) whole lung organoids (WLO). While there are similar approaches that incorporate isolation of lung progenitor cells via sorting at the LPC stage to make alveolar-like14,15 (distal) organoids or airway16 (proximal) organoids, or generate ventral-anterior foregut spheroids to make human lung organoids expressing alveolar-cell and mesenchymal markers and bud tip progenitor organoids17, the strength of this method is the inclusion of both lung epithelial and mesenchymal cell types to pattern and orchestrate lung branching morphogenesis, maturation, and expansion in vitro.
This protocol uses small molecules and growth factors to direct the differentiation of pluripotent stem cells through definitive endoderm, anterior foregut endoderm, and lung progenitor cells. These cells are then induced into 3D whole lung organoids through important developmental steps, including branching and maturation. The summary of the differentiation protocol is shown in Figure 1a with representative brightfield images of endodermal and organoid differentiation shown in Figure 1b. Figure 1c,d show the gene expression details of endodermal differentiation as well as the gene expression of both the proximal and distal populations of lung epithelial cells after completing the differentiation.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Cell Culture</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>12 well plates</td>
			<td>Corning</td>
			<td>3512</td>
			<td></td>
		</tr>
		<tr>
			<td>12-well inserts, 0.4um, translucent</td>
			<td>VWR</td>
			<td>10769-208</td>
			<td></td>
		</tr>
		<tr>
			<td>2-mercaptoethanol</td>
			<td>Sigma-Aldrich</td>
			<td>M3148</td>
			<td></td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>Innovative Cell Tech</td>
			<td>AT104</td>
			<td></td>
		</tr>
		<tr>
			<td>ascorbic acid</td>
			<td>Sigma</td>
			<td>A4544</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 without retinoic acid</td>
			<td>ThermoFisher</td>
			<td>12587010</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA) Fraction V, 7.5% solution</td>
			<td>Gibco</td>
			<td>15260-037</td>
			<td></td>
		</tr>
		<tr>
			<td>Dispase</td>
			<td>StemCellTech</td>
			<td>7913</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Gibco</td>
			<td>10565042</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Gibco</td>
			<td>10082139</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamax</td>
			<td>Life Technologies</td>
			<td>35050061</td>
			<td></td>
		</tr>
		<tr>
			<td>Ham&#8217;s F12</td>
			<td>Invitrogen</td>
			<td>11765-054</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Gibco</td>
			<td>15630-080</td>
			<td></td>
		</tr>
		<tr>
			<td>Iscove&#8217;s Modified Dulbecco&#8217;s Medium (IMDM) + Glutamax</td>
			<td>Invitrogen</td>
			<td>31980030</td>
			<td></td>
		</tr>
		<tr>
			<td>Knockout Serum Replacement (KSR)</td>
			<td>Life Technologies</td>
			<td>10828028</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>Corning</td>
			<td>354230</td>
			<td></td>
		</tr>
		<tr>
			<td>Monothioglycerol</td>
			<td>Sigma</td>
			<td>M6145</td>
			<td></td>
		</tr>
		<tr>
			<td>mTeSR plus Kit (10/case)</td>
			<td>Stem Cell Tech</td>
			<td>5825</td>
			<td></td>
		</tr>
		<tr>
			<td>N2</td>
			<td>ThermoFisher</td>
			<td>17502048</td>
			<td></td>
		</tr>
		<tr>
			<td>NEAA</td>
			<td>Life Technologies</td>
			<td>11140050</td>
			<td></td>
		</tr>
		<tr>
			<td>Pen/strep</td>
			<td>Lonza</td>
			<td>17-602F</td>
			<td></td>
		</tr>
		<tr>
			<td>ReleSR</td>
			<td>Stem Cell Tech</td>
			<td>5872</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI1640 + Glutamax</td>
			<td>Life Technologies</td>
			<td>12633012</td>
			<td></td>
		</tr>
		<tr>
			<td>TrypLE</td>
			<td>Gibco</td>
			<td>12605-028</td>
			<td></td>
		</tr>
		<tr>
			<td>Y-27632 (Rock Inhibitor)</td>
			<td>R&#38;D Systems</td>
			<td>1254/1</td>
			<td></td>
		</tr>
		<tr>
			<td>Growth Factors/Small Molecules</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Activin A</td>
			<td>R&#38;D Systems</td>
			<td>338-AC</td>
			<td></td>
		</tr>
		<tr>
			<td>All-trans retinoic acid (RA)</td>
			<td>Sigma-Aldrich</td>
			<td>R2625</td>
			<td></td>
		</tr>
		<tr>
			<td>BMP4</td>
			<td>R&#38;D Systems</td>
			<td>314-BP/CF</td>
			<td></td>
		</tr>
		<tr>
			<td>Br-cAMP</td>
			<td>Sigma-Aldrich</td>
			<td>B5386</td>
			<td></td>
		</tr>
		<tr>
			<td>CHIR99021</td>
			<td>Abcam</td>
			<td>ab120890</td>
			<td></td>
		</tr>
		<tr>
			<td>Dexamethasone</td>
			<td>Sigma-Aldrich</td>
			<td>D4902</td>
			<td></td>
		</tr>
		<tr>
			<td>Dorsomorphin</td>
			<td>R&#38;D Systems</td>
			<td>3093</td>
			<td></td>
		</tr>
		<tr>
			<td>EGF</td>
			<td>R&#38;D Systems</td>
			<td>236-EG</td>
			<td></td>
		</tr>
		<tr>
			<td>FGF10</td>
			<td>R&#38;D Systems</td>
			<td>345-FG/CF</td>
			<td></td>
		</tr>
		<tr>
			<td>FGF7</td>
			<td>R&#38;D Systems</td>
			<td>251-KG/CF</td>
			<td></td>
		</tr>
		<tr>
			<td>IBMX (3-Isobtyl-1-methylxanthine)</td>
			<td>Sigma-Aldrich</td>
			<td>I5879</td>
			<td></td>
		</tr>
		<tr>
			<td>SB431542</td>
			<td>R&#38;D Systems</td>
			<td>1614</td>
			<td></td>
		</tr>
		<tr>
			<td>VEGF/PIGF</td>
			<td>R&#38;D Systems</td>
			<td>297-VP/CF</td>
			<td></td>
		</tr>
		<tr>
			<td>Primary antibodies</td>
			<td></td>
			<td></td>
			<td>Dilution rate</td>
		</tr>
		<tr>
			<td>CXCR4-PE</td>
			<td>R&#38;D Systems</td>
			<td>FAB170P</td>
			<td>1:200 (F)</td>
		</tr>
		<tr>
			<td>HOPX</td>
			<td>Santa Cruz Biotech</td>
			<td>sc-398703</td>
			<td>0.180555556</td>
		</tr>
		<tr>
			<td>HTII-280</td>
			<td>Terrace Biotech</td>
			<td>TB-27AHT2-280</td>
			<td>0.145833333</td>
		</tr>
		<tr>
			<td>KRT5</td>
			<td>Abcam</td>
			<td>ab52635</td>
			<td>0.180555556</td>
		</tr>
		<tr>
			<td>NKX2-1</td>
			<td>Abcam</td>
			<td>ab76013</td>
			<td>0.25</td>
		</tr>
		<tr>
			<td>NKX2-1-APC</td>
			<td>LS-BIO</td>
			<td>LS-C264437</td>
			<td>1:1000 (F)</td>
		</tr>
		<tr>
			<td>proSPC</td>
			<td>Abcam</td>
			<td>ab40871</td>
			<td>0.215277778</td>
		</tr>
		<tr>
			<td>SCGB3A2</td>
			<td>Abcam</td>
			<td>ab181853</td>
			<td>0.25</td>
		</tr>
		<tr>
			<td>SOX2</td>
			<td>Invitrogen</td>
			<td>MA1-014</td>
			<td>0.180555556</td>
		</tr>
		<tr>
			<td>SOX9</td>
			<td>R&#38;D Systems</td>
			<td>AF3075</td>
			<td>0.180555556</td>
		</tr>
		<tr>
			<td>SPB (mature)</td>
			<td>7 Hills</td>
			<td>48604</td>
			<td>1: 1500 (F) 1:500 (W)a</td>
		</tr>
		<tr>
			<td>SPC (mature)</td>
			<td>LS Bio</td>
			<td>LS-B9161</td>
			<td>1:100 (F); 1:500 (W) a</td>
		</tr>
	</tbody>
</table>

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.

24 hours after plating, day 1, iPSCs should be 50%-90% confluent. On day 2, DE should be 90%-95% confluent. During DE induction, it is common to observe significant cell death on day 4 but attached cells will retain a compact cobblestone morphology (Figure 2b). Discontinue differentiation if the majority of adherent cells detach and consider shortening exposure to DE media with activin A by 6-12 h. During AFE induction, cell death is minimal, and cells remain adherent, but will appear smalle...

Watch this Scientific Journal Video about Generation of 3D Whole Lung Organoids from Induced Pluripotent Stem Cells for Modeling Lung Developmental Biology and Disease at JoVE.com

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

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 ...

generation-3d-whole-lung-organoids-from-induced-pluripotent-stem

Research

JoVE Journal

Developmental Biology

7.7K Views.  University of California, San Diego School of Medicine. 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.

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

Generation of Induced-pluripotent Stem Cells Using Fibroblast-like Synoviocytes Isolated from Joints of Rheumatoid Arthritis Patients

Mature somatic cells can be reversed into a pluripotent stem cell-like state using a defined set of reprogramming factors. Numerous studies have generated induced-Pluripotent Stem Cells (iPSCs) from various somatic cell types by transducing four Yamanaka transcription factors: Oct4, Sox2, Klf4 and c-Myc. The study of iPSCs remains at the cutting edge of biological and clinical research. In particular, patient-specific iPSCs can be used as a pioneering tool for the study of disease pathobiology, since iPSCs can be induced from the tissue of any individual. Rheumatoid arthritis (RA) is a chronic inflammatory disease, classified by the destruction of cartilage and bone structure in the joint. Synovial hyperplasia is one of the major reasons or symptoms that lead to these results in RA. Fibroblast-like Synoviocytes (FLSs) are the main component cells in the hyperplastic synovium. FLSs in the joint limitlessly proliferate, eventually invading the adjacent cartilage and bone. Currently, the hyperplastic synovium can be removed only by a surgical procedure. The removed synovium is used for RA research as a material that reflects the inflammatory condition of the joint. As a major player in the pathogenesis of RA, FLSs can be used as a material to generate and investigate the iPSCs of RA patients. In this study, we used the FLSs of a RA patient to generate iPSCs. Using a lentiviral system, we discovered that FLSs can generate RA patient-specific iPSC. The iPSCs generated from FLSs can be further used as a tool to study the pathophysiology of RA in the future.

Here we describe a protocol for generating human induced-pluripotent stem cells from patient-derived fibroblast-like synoviocytes, using a lentiviral system without feeder cells.

Mature somatic cells can be reversed into a pluripotent stem cell-like state using a defined set of reprogramming factors. Numerous studies have generated ...

Generation of Induced Pluripotent Stem Cells from Human Melanoma Tumor-infiltrating Lymphocytes

Adoptive transfer of ex vivo expanded autologous tumor-infiltrating lymphocytes (TILs) can mediate durable and complete responses in significant subsets of patients with metastatic melanoma. Major obstacles of this approach are the reduced viability of transferred T cells, caused by telomere shortening, and the limited number of TILs obtained from patients. Less-differentiated T cells with long telomeres would be an ideal T cell subset for adoptive T cell therapy;however, generating large numbers of these less-differentiated T cells is problematic. This limitation of adoptive T cell therapy can be theoretically overcome by using induced pluripotent stem cells (iPSCs) that self-renew, maintain pluripotency, have elongated telomeres, and provide an unlimited source of autologous T cells for immunotherapy. Here, we present a protocol to generate iPSCs using Sendai virus vectors for the transduction of reprogramming factors into TILs. This protocol generates fully reprogrammed, vector-free clones. These TIL-derived iPSCs might be able to generate less-differentiated patient- and tumor-specific T cells for adoptive T cell therapy.

The goal of this protocol is to show the protocol for reprogramming melanoma tumor-infiltrating lymphocytes into induced pluripotent stem cells.

Adoptive transfer of ex vivo expanded autologous tumor-infiltrating lymphocytes (TILs) can mediate durable and complete responses in significant subsets ...

Induced Pluripotent Stem Cell Generation from Blood Cells Using Sendai Virus and Centrifugation

The recent development of human induced pluripotent stem cells (hiPSCs) proved that mature somatic cells can return to an undifferentiated, pluripotent state. Now, reprogramming is done with various types of adult somatic cells: keratinocytes, urine cells, fibroblasts, etc. Early experiments were usually done with dermal fibroblasts. However, this required an invasive surgical procedure to obtain fibroblasts from the patients. Therefore, suspension cells, such as blood and urine cells, were considered ideal for reprogramming because of the convenience of obtaining the primary cells. Here, we report an efficient protocol for iPSC generation from peripheral blood mononuclear cells (PBMCs). By plating the transduced PBMCs serially to a new, matrix-coated plate using centrifugation, this protocol can easily provide iPSC colonies. This method is also applicable to umbilical cord blood mononuclear cells (CBMCs). This study presents a simple and efficient protocol for the reprogramming of PBMCs and CBMCs.

We propose a protocol for reprogramming peripheral blood mononuclear cells (PBMCs) into induced pluripotent stem cells (iPSCs). By plating the transduced blood cells onto matrix-coated plates with centrifugation, iPSCs are successfully induced from floating cells. This technique suggests a simple and effective reprogramming protocol for cells such as PBMCs and CBMCs.

The recent development of human induced pluripotent stem cells (hiPSCs) proved that mature somatic cells can return to an undifferentiated, pluripotent ...

Generation of Integration-free Induced Pluripotent Stem Cells from Human Peripheral Blood Mononuclear Cells Using Episomal Vectors

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal Vectors (EV) to generate integration-free iPSCs from peripheral blood mononuclear cells (PB MNCs). The episomal vectors used are DNA plasmids incorporated with oriP and EBNA1 elements from the Epstein-Barr (EB) virus, which&#160;allow for replication and long-term retainment of plasmids in mammalian cells, respectively. With further optimization, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood. Two critical factors for achieving high reprogramming efficiencies&#160;are: 1) the use of a 2A &#34;self-cleavage&#34; peptide to link OCT4 and SOX2, thus achieving equimolar expression of the two factors; 2) the use of two vectors to express MYC and KLF4 individually. Here we describe a step-by-step protocol for generating integration-free iPSCs from adult peripheral blood samples. The generated iPSCs are integration-free as residual episomal plasmids are undetectable after five passages. Although the reprogramming efficiency is comparable to that of Sendai Virus (SV) vectors, EV plasmids are considerably more economical than the commercially available SV vectors. This affordable EV reprogramming system holds potential for clinical applications in regenerative medicine and provides an approach for the direct reprogramming of PB MNCs to integration-free mesenchymal stem cells, neural stem cells, etc.

This protocol describes a detailed method for efficient generation of integration-free iPSCs from human adult peripheral blood cells. With the use of four oriP/EBNA-based episomal vectors to express the reprogramming factors, KLF4, MYC, BCL-XL, or OCT4 and SOX2, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood.

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal ...

Chondrogenic Pellet Formation from Cord Blood-derived Induced Pluripotent Stem Cells

Human articular cartilage lacks the ability to repair itself. Cartilage degeneration is thus treated not by curative but by conservative treatments. Currently, efforts are being made to regenerate damaged cartilage with ex vivo expanded chondrocytes or bone marrow-derived mesenchymal stem cells (BMSCs). However, the restricted viability and instability of these cells limit their application in cartilage reconstruction. Human induced pluripotent stem cells (hiPSCs) have received scientific attention as a new alternative for regenerative applications. With unlimited self-renewal ability and multipotency, hiPSCs have been highlighted as a new replacement cell source for cartilage repair. However, obtaining a high quantity of high-quality chondrogenic pellets is a major challenge to their clinical application. In this study, we used embryoid body (EB)-derived outgrowth cells for chondrogenic differentiation. Successful chondrogenesis was confirmed by PCR and staining with alcian blue, toluidine blue, and antibodies against collagen types I and II (COL1A1 and COL2A1, respectively). We provide a detailed method for the differentiation of cord blood mononuclear cell-derived iPSCs (CBMC-hiPSCs) into chondrogenic pellets.

Here, we propose a protocol for chondrogenic differentiation from cord blood mononuclear cell-derived human induced pluripotent stem cells.

Human articular cartilage lacks the ability to repair itself. Cartilage degeneration is thus treated not by curative but by conservative treatments. ...

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. ...

Partial Lobular Hepatectomy: A Surgical Model for Morphologic Liver Regeneration

Morphological organ regeneration following acute tissue loss is common among lower vertebrates, but is rarely observed in mammalian postnatal life. Adult liver regeneration after 70% partial hepatectomy results in hepatocyte hypertrophy with some replication in remaining lobes with restoration of metabolic activity, but with permanent loss of the injured lobe&#39;s morphology and architecture. Here, we detail a new surgical method in the neonate that leaves a physiologic environment conducive to regeneration. This model involves amputation of the left lobe apex and a subsequent conservative management regimen, and lacks the necessity for ligation of major liver vessels or chemical injury, leaving a physiologic environment where regeneration may occur. We extend this protocol to amputations on juvenile (P7-14) mice, during which the injured liver transitions from organ regeneration to compensatory growth by hypertrophy. The presented, brief 30 min protocol provides a framework to study the mechanisms of regeneration, its age-associated decline in mammals, and the characterization of putative hepatic stem or progenitors.

Here, we present a new method for partial resection of the left hepatic lobe in neonatal (day 0) mice. This new protocol is suitable for studying acute liver injury and injury response in the neonatal setting.

Morphological organ regeneration following acute tissue loss is common among lower vertebrates, but is rarely observed in mammalian postnatal life. Adult ...

Synaptic Microcircuit Modeling with 3D Cocultures of Astrocytes and Neurons from Human Pluripotent Stem Cells

A barrier to our understanding of how various cell types and signals contribute to synaptic circuit function is the lack of relevant models for studying the human brain. One emerging technology to address this issue is the use of three dimensional (3D) neural cell cultures, termed &#39;organoids&#39; or &#39;spheroids&#39;, for long term preservation of intercellular interactions including extracellular adhesion molecules. However, these culture systems are time consuming and not systematically generated. Here, we detail a method to rapidly and consistently produce 3D cocultures of neurons and astrocytes from human pluripotent stem cells. First, pre-differentiated astrocytes and neuronal progenitors are dissociated and counted. Next, cells are combined in sphere-forming dishes with a Rho-Kinase inhibitor and at specific ratios to produce spheres of reproducible size. After several weeks of culture as floating spheres, cocultures (&#39;asteroids&#39;) are finally sectioned for immunostaining or plated upon multielectrode arrays to measure synaptic density and strength. In general, it is expected that this protocol will yield 3D neural spheres that display mature cell-type restricted markers, form functional synapses, and exhibit spontaneous synaptic network burst activity. Together, this system permits drug screening and investigations into mechanisms of disease in a more suitable model compared to monolayer cultures.

In this protocol, we aim to describe a reproducible method for combining dissociated human pluripotent stem cell derived neurons and astrocytes together into 3D sphere cocultures, maintaining these spheres in free floating conditions, and subsequently measuring synaptic circuit activity of the spheres with immunoanalysis and multielectrode array recordings.

A barrier to our understanding of how various cell types and signals contribute to synaptic circuit function is the lack of relevant models for studying ...

RNA-based Reprogramming of Human Primary Fibroblasts into Induced Pluripotent Stem Cells

Induced pluripotent stem cells (iPSCs) have proven to be a valuable tool to study human development and disease. Further advancing iPSCs as a regenerative therapeutic requires a safe, robust, and expedient reprogramming protocol. Here, we present a clinically relevant, step-by-step protocol for the extremely high-efficiency reprogramming of human dermal fibroblasts into iPSCs using a non-integrating approach. The core of the protocol consists of expressing pluripotency factors (SOX2, KLF4, cMYC, LIN28A, NANOG, OCT4-MyoD fusion) from in vitro transcribed messenger RNAs synthesized with modified nucleotides (modified mRNAs). The reprogramming modified mRNAs are transfected into primary fibroblasts every 48 h together with mature embryonic stem cell-specific microRNA-367/302 mimics for two weeks. The resulting iPSC colonies can then be isolated and directly expanded in feeder-free conditions. To maximize efficiency and consistency of our reprogramming protocol across fibroblast samples, we have optimized various parameters including the RNA transfection regimen, timing of transfections, culture conditions, and seeding densities. Importantly, our method generates high-quality iPSCs from most fibroblast sources, including difficult-to-reprogram diseased, aged, and/or senescent samples.

Here we describe a clinically relevant, high-efficiency, feeder-free method to reprogram human primary fibroblasts into induced pluripotent stem cells using modified mRNAs encoding reprogramming factors and mature microRNA-367/302 mimics. Also included are methods to assess reprogramming efficiency, expand clonal iPSC colonies, and confirm expression of the pluripotency marker TRA-1-60.

Induced pluripotent stem cells (iPSCs) have proven to be a valuable tool to study human development and disease. Further advancing iPSCs as a regenerative ...

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 ...