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

This protocol uses induced pluripotent stem cells to differentiate them into lung cells in order to model human lung disease and development in a dish. This protocol is significant because it's difficult to obtain and culture primary human lung tissue. The main advantages of differentiating lung cells from pluripotent stem cells is to have a constant supply of specific lung cells to study human lung development and disease.

These cells can be maintained in cell culture for long periods of time, can be cryopreserved for future applications, and can be genetically manipulated to study gene mutations. Demonstrating the procedure will be Rachel McVicar, a PhD candidate from my laboratory. Before starting the experiment, slowly thaw a growth factor reduced, or GFR, basement membrane matrix medium on ice, and dilute it in an equal volume of cold DMEM F12.

Place P1000 tips in the freezer to chill prior to use. Next, coat each well of a 12 well plate with 500 microliters of the 50%GFR basement membrane matrix medium, and remove any excess medium mixture and bubbles from the wells. Place the well plates on top of wet ice or a refrigerator at four degree Celsius to set for 20 minutes, then move the plate to the 37 degree Celsius incubator overnight.

When high PSEs reach 70%confluency, add 10 micromolar ROCK inhibitor Y27632 to human induced pluripotent stem cells, and wait an hour prior to dissociation. Aspirate the media, and wash the cells once with PVS. Dissociate IPSCs by adding 500 microliters of cell detachment medium per well, and incubate for 20 minutes at 37 degrees Celsius in a 5%carbon dioxide incubator.

After the incubation, add 500 microliters of stem cell passaging medium per well. Gently pipette cells using a P1000 tip to obtain a single cell suspension. Then transfer the dissociated cells into a 15 milliliter tube and centrifuge for five minutes at 300 times G.Following centrifugation, aspirate the medium, and resuspend the cell pellet with one milliliter of mTeSR Plus media supplemented with 10 micromolar ROCK inhibitor.

After counting the cells, add two times 10 to the fifth IPSCs to each well of a 12 well GFR medium coated plate, and incubate overnight at 37 degrees Celsius. On the next day, aspirate the mTeSR Plus before adding definitive endoderm, or DE, induction media. On days two and three, change the DE induction media.

On day four, begin AFE induction by replacing DE induction media with serum-free basal medium. Change AFE medium daily for three days before analyzing AFE efficiency at the end of day six. On day seven, thaw GFR basement membrane matrix medium on ice for later use.

Simultaneously, proceed with lung progenitor cell differentiation by aspirating the AFE media and washing the wells with PVS. Add one milliliter of cell detachment solution to the well and incubate for 10 minutes at 37 degrees Celsius. After incubation, add one milliliter of quenching media to the Wells containing cell detachment solution.

Keep cells as aggregates by gently pipetting up and down. Make sure all cells are dislodged, but for transferring them into a 15 milliliter conical tube for centrifugation at 300 times G for five minutes. Remove supernatant and resuspend the cell pellet in LPC induction media.

Count the cells, and add 2.5 times 10 to the fifth cells to 100 microliters of cold GFR basement membrane matrix medium, and mix well. Place a droplet into a well of a 12-well plate and incubate at 37 degree Celsius for 30 to 60 minutes. Next, add one milliliter of LPC media per well, ensuring that the medium drop is fully submerged and incubate overnight at 37 degrees Celsius.

On day eight, change LPC medium to remove ROCK inhibitor Y27632. Keep changing the media every other day and analyze LPC efficiency at the end of day 16. On day 17, wash wells and add 500 microliters of two microgram per milliliter dispase.

Pipette the dispase mixture with a P1000 pipette and incubate for 15 minute, then pipette the mixture and incubate for another 15 minutes. Following incubation, add one milliliter of the quenching media to the dispase containing wells, and transfer the cells into conical tubes. Centrifuge and resuspend the pellet in one milliliter of chilled PVS, then repeat the centrifugation.

Then resuspend the pellet in one milliliter of cell detachment medium. Incubate the cells at 37 degree Celsius for 12 minutes, then add an equal volume of quenching media and centrifuge again. Resuspend the pellet in one milliliter of quenching media and 10 micromolar ROCK inhibitor Y27632.

Perform a cell count to calculate the volume needed to obtain eight times 10 to the fourth cells per well. Then aliquot required volume of LPC cell aggregates into 1.5 milliliter micro centrifuge tubes, and centrifuge for five minutes at 300 times G.Remove excess supernatant without agitating the cell pellet, leaving 10 microliters of residual media. Resuspend the cell pellet in 200 microliters of cold GFR basement membrane matrix medium and transfer the cells to cell culture membrane inserts.

Incubate at 37 degrees Celsius for 30 to 60 minutes. After incubation, add one milliliter of 3D organoid induction medium to the basolateral chamber of the insert, and replace it with fresh medium every other day for six days. On day 23, switch the medium to 3D branching medium, then change the medium every other day for six days.

On day 29, change to 3D maturation medium, and continue replacing the medium every other day for the next six days. On day four of DE induction, attached cells display a compact cobblestone morphology. The definitive endoderm markers CXCR4 and SOX17 overlaid with nuclei was expressed, confirming endodermal differentiation.

Anterior foregut induction was confirmed on day seven with the morphology showing more tightly compacted cells, and the markers FOXA2 and SOX2 overlaid with nuclei. The generation of 3D lung progenitor cells was confirmed with 3D spheroids, and the endogenous expression of NKX2-1 GFP in a reporter cell line. After a three week differentiation into whole lung organoids, the lung markers were analyzed by immunocytochemistry.

Markers for branching morphogenesis SOX2 and SOX9 overlaid by nuclei was observed in the organoids. Proximal lung markers P63 and KRT5, both markers of basal cells, were successfully detected along with SCGB3A2, which is a marker for club cells. Distal lung markers induced Pro SPC and SPB markers for Alveolar Type II cells.

And HOPX markers of Alveolar Type I cells. Additionally, NKX2-1 and ZO1 were expressed overlaid with nuclei. The Mesenchyme lung cell markers PGFR Alpha, a marker for fibroblasts, co-expressed with SOX9, represented distal mesenchyme.

Vimentin was also expressed and was dispersed throughout the lung. Following this procedure, the lung organoids can be invested with induced pluripotent stem cell derived endothelial and immune cells. Lung development and disease occurs by a signaling between the epithelial and mesenchymal cell populations, but also from endothelial cells and macrophages.

A 3D lung tissue model system is clinically relevant to study human disease and therapeutics.