key: cord-0029627-f4jeb3s0 authors: Han, Le; Zhao, Shanshan; Yu, Fuxun; Rong, Zhili; Lin, Ying; Chen, Yong title: Generation of human embryonic stem cell-derived lung organoids date: 2022-04-04 journal: STAR Protoc DOI: 10.1016/j.xpro.2022.101270 sha: f607aab9705a05f999515cbfa1edf6bdad1e98de doc_id: 29627 cord_uid: f4jeb3s0 This protocol describes how to generate lung organoids from human embryonic stem cells. Lung organoids form by self-assembly in Matrigel and contain lung epithelial cell types. The protocol presented in this study is simple and only uses 6 cytokines or small molecules. This protocol provides a promising tool to study human lung development, drug screening, regeneration, and disease modeling in vitro. For complete details on the use and execution of this protocol, please refer to Chen et al. (2018). This protocol describes how to generate lung organoids from human embryonic stem cells. Lung organoids form by self-assembly in Matrigel and contain lung epithelial cell types. The protocol presented in this study is simple and only uses 6 cytokines or small molecules. This protocol provides a promising tool to study human lung development, drug screening, regeneration, and disease modeling in vitro. For complete details on the use and execution of this protocol, please refer to Chen et al. (2018) . Planning lung organoids differentiation timings To date, the technology to generate human embryonic stem cell (hESC)-derived lung cells have changed from traditional two-dimensional (2D) monolayer cell-based differentiation to threedimensional (3D) organoid differentiation (Chen et al., 2017 (Chen et al., , 2018 Dye et al., 2015; Kim et al., 2021; Li et al., 2021) . Organoid models possess structural organization similar to the native organ and cell types from multiple germ layers, making them a physiologically complex model to investigate organ developmental processes, drug screening, regeneration and disease modeling in vitro (Chen et al., 2020 Clevers, 2016; Kim et al., 2021; Wang et al., 2021) . However, current protocols use more cytokines/small molecules and require longer time to generate hESC-derived lung organoids (HLOs) (Chen et al., 2017; Dye et al., 2015 Dye et al., , 2016 Strikoudis et al., 2019; Wang et al., 2020) . The present study provides a simple protocol that uses less cytokines and small molecules to generate HLOs. Timing: 30-60 min Note: Thaw stock solution of Matrigel (hESC-qualified Matrix, BD Bioscience, Cat#354277) on ice following the operation manual. Small aliquots (150 mL) can be stored at À80 C for 6 months. 2. Dilute the thawed Matrigel (150 mL) in ice-cold DMEM/F12 (12 mL). Diluted Matrigel can be stored at 4 C for 2 weeks. 3. For each well of a 6 well plate, add 1 mL of the diluted Matrigel. 4. Following Matrigel coating, incubate the plates in a 37 C/5% CO2 cell culture incubator for 30 min. CRITICAL: Ensure that the diluted Matrigel solution covers the whole surface. Do not let the diluted Matrigel evaporate. To this purpose, we recommend preparing the Matrigelcoated plate soon before use (30 min -4 h). Timing: 2 h Note: All procedures should be performed under sterile conditions and done in a Biosafety Level 2 (BSL2) tissue culture hood. All reconstituted media should be used within 2 weeks following preparation. If needed, reconstituted medium can be stored at À20 C for 6 months or at À80 C for long-term storage. Do not freeze again after thawing. 5. hESCs medium. a. Add 100 mL mTeSR1 TM 5 3 supplement to 400 mL mTeSR1 TM basal medium. 6. hESCs-derived lung organoids differentiation medium. a. For definitive endoderm differentiation: i. Prepare definitive endoderm differentiation medium by adding 13 penicillin streptomycin (PS), 100 ng/ mL Activin A, 2 mM CHIR99021, 13 MEM Non-Essential Amino Acids Solution (NEAA), 13 GlutaMax, and 0.5% BSA to 100 mL RPMI 1640 medium. b. For anterior foregut endoderm differentiation: i. Prepare anterior foregut endoderm differentiation medium by mixing 100 mL Advanced-DMEM/F12, 200 ng/ mL noggin, 10 mM SB431542, 2 mM CHIR99021, 500 ng/ mL FGF4, 10 mM HEPES, 13 NEAA, 23 B27, 13 N2, 13 GlutaMAX, and 13 PS. c. For lung organoid differentiation: i. Prepare lung organoid differentiation medium by mixing 100 mL Advanced-DMEM/F12, 1% FBS, 13 NEAA, 13 GlutaMAX and 13 PS. d. For anterior foregut endoderm cell dissociation: i. Accutase with 10 mM Y-27632 was used for anterior foregut endoderm cell dissociation. ii. For each well of a 6 well plate, add 1 mL of the cell dissociation solution. This step describes how to prepare hESCs from colony to single-cell suspension which will be used for differentiation into definitive endoderm cells. This protocol requires a starting population of hESC colonies in a 6 well plate format to be in 75%-85% confluency. This protocol was adapted from previous reports (Chen et al., 2018; Meseguer-Ripolles et al., 2021) . 1. Aspirate the medium from hESCs at 75%-85% confluency ( Figures 1A and 1B ). 2. Wash hESCs with 2 mL of 1 3 room temperature DPBS (without Ca2 + /Mg2 + ) per well of a 6 well plate. 3. Aspirate the DPBS from the well. 4. Add 1 mL of room temperature Accutase containing Y-27632 (10 mM) per well. 5. Leave the cultures in a 37 C/5% CO 2 cell culture incubator for 3-6 min to dissociate into single cells. Note: Incubation time can vary depending on the extent of cell confluency or cell lines used (e.g., H1 hESCs). The use of Y-27632 (Rho-associated kinase inhibitor) enhances cell survival. 6. 3 min later, check the hESC colonies under the microscope, the cell-cell interactions should become loose and cells within the colonies should become more refringent (cells should not be floating) ( Figure 1C ). All reconstituted medium should be used within 2 weeks following preparation. 7. Carefully aspirate the Accutase solution without disturbing the cells and immediately add 1 mL of pre-warmed mTeSR1 TM with 10 mM Y-27632 per well into a 6 well plate. Note: Proper time of Accutase incubation (3-6 min) will not make cells float, check cells under the microscope every 2 min, the cell-cell interactions will become more relaxed and cells will be easily detached by pipetting medium over the cells ( Figure 1C ). A longer time of Accutase incubation (8-15 min) will make cells floating. 8. Use 1 mL tips to gently pipette up and down to detach the cells from the well into the medium. 9. Carefully collect the medium with cells into a 15 mL centrifuge tube. 10. Centrifuge cells at 200 g for 5 min at room temperature. 11. Aspirate the supernatant carefully without disturbing the cell pellet. 12. Carefully aspirate the Matrigel without damaging the Matrigel-coated surface. 13. Add 6-12 mL of pre-warmed mTeSR1 TM with 10 mM Y-27632 to cells and gently pipette up and down to ensure a homogeneous single cell solution. 14. Count cells using an automatic cell counter using trypan blue to exclude dead cells. 15. Seed cells at a density of 1.0-2.0 3 10 5 cells/cm 2 into the Matrigel coated well using mTeSR1 TM with 10 mM Y-27632 to a final volume of 2 mL per well in a 6 well plate. CRITICAL: Gently move the plate several times back and forth and left to right to ensure an even distribution of the cell suspension. 16. Incubate the plate in a 37 C/5% CO 2 cell culture incubator overnight (12-16 h) to start differentiation into definitive endoderm cells the day after. Timing: day 1, 41 days, 1 h daily This step describes how to generate hESC-derived lung organoids by following embryonic lung developmental cues by temporarily introducing a variety of growth factors and small molecules to efficiently generate the definitive endoderm (DE) stage, anterior foregut endoderm (AFE) stage, lung progenitor stage and finally lung organoids (Figures 2A and 2B ). This protocol generates lung organoids containing lung proximal and distal cell types, such as ciliated cells (ACE-TUB+), basal cells (P63+), club cells (SCGB1A1+), alveolar type I like cells (AT1, HOPX+) and alveolar type II like cells (AT2, SPC+/SOX9+). This protocol was adapted from a published paper (Chen et al., 2018) . 17. After overnight growth when hESCs confluency reaches 95%, aspirate the medium from hESCs. Troubleshooting 1, Troubleshooting 2. 18. Wash hESCs with 2 mL of room temperature RPMI 1640 medium per well in a 6 well plate then aspirate the RPMI 1640 medium from the well. CRITICAL: Remove the mTeSR1 TM complete medium from hESCs and gently wash hESCs with room temperature RPMI 1640 medium. 19. Add 2 mL per well of definitive endoderm differentiation medium. Incubate cultures at 37 C/5% CO 2 . Replenish the definitive endoderm differentiation medium every 24 h for 3 days. 20. On day 3, aspirate the medium from cultures, wash the cultures with 2 mL of room temperature RPMI 1640 medium per well in the 6 well plate then aspirate the RPMI 1640 medium from the well. Troubleshooting 3. Note: Cultures can be collected to analyze marker of definitive endoderm (FOXA2+, SOX17+) ( Figure 2D ). 21. Add 2 mL per well of anterior foregut endoderm differentiation medium. Incubate cultures at 37 C/ 5% CO2. Replenish the anterior foregut endoderm differentiation medium every 24 h for 4 days. 22. On day 7, aspirate the medium from cultures, wash the cultures with 2 mL of 1 3 room temperature DPBS (without Ca2+/Mg2+) per well in the 6 well plate. a. Aspirate DPBS from the well. b. Add 1 mL of room temperature Accutase containing Y-27632 (10 mM) per well and leave the cultures in 37 C/5% CO2 cell culture incubator for 2-4 min. c. Check the cells under the microscope after 2 min, the cell-cell interactions should become looser and cells should become more refringent (cells should not be floating). Note: Proper time of Accutase incubation (2-4 min) will not make cells float, check cells under microscope every 2 min, cell-cell interactions will become more relaxed and cells will be easy detached by following pipetting medium over the cells. A longer time of Accutase incubation (8-15 min) will make cells float. d. Aspirate the Accutase carefully and immediately add 1 mL of pre-warmed lung organoids differentiation medium with 10 mM Y-27632 per well in the 6 well plate. e. Use a 1 mL tips to gently pipette up and down to detach the cells from the well into the medium. f. Carefully collect the medium with cells into a 1.5 mL sterile eppendorf tube. Count cells using an automatic cell counter using trypan blue to exclude death cells. g. Centrifuge cells at 200 g for 5 min at room temperature. h. Aspirate the supernatant carefully without disturbing the cell pellet. Note: Use a 20 mL tip to further aspirate the remaining supernatant without disturbing the cell pellet (cells will be embedded in 3D Matrigel, so residual culture medium will affect the solidifying of the 3D Matrigel). Then, tap the cell pellet to ensure that there are no cell clumps. i. Put the eppendorf tube on ice for 1-3 min. j. Add 100-200 mL ice-cold 3D Matrigel (stock solution, 2.0-4.0 3 10 5 cells/25 mL ice-cold 3D Matrigel) to cells and gently pipette up and down several times to ensure an even distribution of the cell suspension. Note: This procedure will be used to induce anterior foregut endoderm cells to lung progenitors in 3D Matrigel. To avoid 3D Matrigel evaporation, this procedure should be carried out on ice. Pipette with care to avoid making bubbles. CRITICAL: 3D Matrigel should be stored at À80 C and thawed on ice overnight before use. k. Plate cells in 3D Matrigel (stock solution) using a 100 mL tip to estimate 20-30 mL of droplets ( Figure 3A ). Note: Pre-cold tips are required. To estimate 20-30 mL of droplets into a 6 well plate (e.g., 6-10 droplets in a well of 6 well plate) ( Figure 3A ). l. Place the plate in tissue culture incubator for 15-30 min until Matrigel droplets have solidified ( Figure 3A ). m. Add 3-5 mL of pre-warmed lung organoids differentiation medium per well into a 6 well plate, ensuring that the medium completely covers the Matrigel droplet. experience, cells will first form small cell clumps 2-4 days later, and gradually form organoids by re-embedding every 5-10 days. ii. Pick up the entire droplet with a serum pipette and move it to a 1.5 mL sterile eppendorf tube and remove all media using 200 mL or 1 mL tips ( Figures 3B and 4) . Note: Centrifuge the cultures at 200 g for 5 min at room temperature will be easy to remove most of the media, then use a 20 mL tip to further aspirate residual culture medium (30-80 mL residual medium usually), residual culture medium will affect the solidification of 3D Matrigel (Figure 4 ). iii. Gently pipette up and down to cut the organoids and the old Matrigel using 20 mL tips, then on ice for 1-2 min to make it cold before re-embedding into new 3D Matrigel (Figure 4 ). Note: To make even distribution of the organoids in the re-embedding procedure, old 3D Matrigel droplets containing organoids were mechanically cut into small clumps before reembedding with new 3D Matrigel by using a 20 mL tip to gently pipette up and down. Pipette with care to avoid making bubbles ( Figure 4 ). iv. Transfer 100-200 mL of ice-cold fresh 3D Matrigel to the eppendorf tube, mix well (Figure 4) . CRITICAL: To avoid 3D Matrigel evaporation, this procedure should be carried out on ice. Pipette with care to avoid making bubbles. v. Plate re-embedded organoids in a 6 well plate (Figure 4) . vi. Place the plate in tissue culture incubator for 15-30 min until Matrigel droplets have solidified. vii. Add 3-5 mL of pre-warmed lung organoids differentiation medium per well into a 6 well plate, ensuring that the medium completely covers the Matrigel droplet. Incubate cultures at 37 C/5% CO2. Replenish the lung organoids differentiation medium every 48-72 h. CRITICAL: Keep the 3D Matrigel on ice throughout the procedure to avoid its polymerization inside the eppendorf tube. Note: Pipette with care to avoid making bubbles. If there are bubbles, centrifuge the eppendorf tube at 200 g at 4 C for 5 min and carefully remove bubbles with 200 mL tips. Then, mix organoids and 3D Matrigel again to make an even distribution of organoids. This protocol generates hESC-derived lung organoids by following embryonic lung developmental cues by temporarily introducing a variety of growth factors and small molecules to efficiently generate the definitive endoderm stage, anterior foregut endoderm stage, lung progenitor stage and finally lung organoids (Figure 2A ). Anterior foregut endoderm cells are encapsulated in a 3D extracellular matrix to provide a 3D growth environment to generate lung organoids (Figures 2A and 2B ). Using quantitative real-time PCR, we checked the expression of various lineage markers at day (D) 0, 3, 21, 31, and 41 along with differentiation. SOX17 is a DE marker, and it was highly expressed at D3. NKX2.1 and P63 are lung progenitor and stem cell markers and are highly expressed at D21 and D31. The markers for more differentiated cell types appeared later at D41, including SCGB1A1 (club cells), SPC (AT2 cells), and HOPX (AT1 cells) ( Figure 2C ). HLOs contain lung proximal and distal cell types, such as lung progenitor (NKX2.1+), ciliated cells (ACE-TUB+), basal cells (P63+), distal progenitor like cells (SOX9+) and alveolar type II like cells (AT2, SPC+) ( Figures 2D and 2E ). Long-term transplanted D21 HLOs possess AT2-like cells (SOX9+/SPC+) and BP (bipotent progenitor, PDPN+/SPC+/SOX9+) like cells (Figures 5A and 5B ). If using different cell lines such as induced pluripotent stem cells (iPSCs), optimization of cell seeding, prior to differentiation, may be required. To optimize the cell density, try adding or reducing 1 3 10 5 cells/cm 2 at a time was recommended. Single cell detachment is not achieved following the Accutase incubation time (step 17). Ensure Accutase contains Y-27632 (10 mM), check if the cultures have been dissociated into single cells, observe under the microscope, and increase the incubation time if necessary ( Figure 1C ). Following single cell seeding, if cell distribution is not homogeneous, it can lead to a heterogenous differentiation (expression of definitive endoderm markers less than 80% by FACS or immunofluorescence analysis) (step 17). Undifferentiated hESCs are required for effective definitive endoderm differentiation, more information regarding the maintenance of hESCs with mTeSR TM 1 can be found at the STEMCELL Technologies website. To ensure that the single cell suspension is evenly dispersed across the well gently agitate the plate back and forth and side-to-side multiple times (> 10 times). Problem 3 No more than 50% cells left after definitive endoderm differentiation (step 20). Adding 1 3 10 5 cells/cm 2 at a time to make the cell confluency reach 95% before definitive endoderm differentiation. Cell death after seeding cells in 3D Matrigel (step 22-m). Ensure Accutase contains Y-27632 (10 mM) when used for anterior foregut endoderm cell dissociation, and reduce the incubation time. Minimize the operation time of 3D culture as much as possible. No organoids formation after seeding cells in 3D Matrigel (step 22-m). Use proper volume of ice-cold 3D Matrigel (reduce 50 mL a time) and gently pipette up and down several times to ensure an even distribution of the cell suspension. Check cultures every 2-3 days under the microscope. Based on our experiences, cells will first form small cell clumps 2-4 days later, and gradually form organoids after re-embedding every 5-10 days. Lead contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Yong Chen, gene_cell@hotmail.com. This study did not generate new unique regents. Data and code availability This study did not generate datasets/code. Modeling sporadic Alzheimer's disease in human brain organoids under serum exposure Lung development: AT1 and AT2 property Long-term engraftment promotes differentiation of alveolar epithelial cells from human embryonic stem cell derived lung organoids A three-dimensional model of human lung development and disease from pluripotent stem cells Modeling development and disease with organoids A bioengineered niche promotes in vivo engraftment and maturation of pluripotent stem cell derived human lung organoids In vitro generation of human pluripotent stem cell derived lung organoids Human pluripotent stem-cellderived alveolar organoids for modeling pulmonary fibrosis and drug testing SOX9 inactivation affects the proliferation and differentiation of human lung organoids Protocol for automated production of human stem cell derived liver spheres Modeling of fibrotic lung disease using 3D organoids derived from human pluripotent stem cells ApoE-isoform-dependent SARS-CoV-2 neurotropism and cellular response Differentiation of human airwayorganoids from induced pluripotent stem cells (iPSCs) The authors declare no competing interests.