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LUNG Publication-derived

Generation of three-dimensional lung bud organoids from human pluripotent stem cells

Source Chen et al., 2017 · Columbia University Medical Center · 10.1038/ncb3510

👤 Ya-Wen Chen, Sarah Xuelian Huang, Ana Luisa Rodrigues Toste de Carvalho, Siu-Hong Ho, Mohammad Naimul Islam, Stefano Volpi, Luigi D Notarangelo, Michael Ciancanelli, Jean-Laurent Casanova, Jahar Bhattacharya, Alice F. Liang, Laura M Palermo, Matteo Porotto, Anne Moscona, HansWillem Snoeck ⏱ 210 days 📋 14 phases 🧫 Human ESC (RUES2) and Human iPSC

Abstract

This protocol describes the generation of lung bud organoids (LBOs) from human pluripotent stem cells (hPSCs) that recapitulate lung development and branching morphogenesis. LBOs can be cultured in three dimensions in Matrigel to reach developmental stages equivalent to the second trimester of human gestation, and can be used to model infectious diseases such as RSV bronchiolitis and genetic fibrotic lung disease.

Cell source
Human ESC (RUES2) and Human iPSC
Application
Lung development modeling and disease modeling (respiratory syncytial virus infection, pulmonary fibrosis)

Protocol overview

66 steps across 14 phases

hPSC Maintenance Ongoing
  1. 1 Plate mouse embryonic fibroblasts (MEFs)
  2. 2 Culture hPSCs on MEF feeders
  3. 3 Passage hPSCs
  4. 4 Monitor cell quality
Endoderm Induction Days 0–2
  1. 1 Deplete MEF feeders
  2. 2 Induce primitive streak and embryoid bodies
  3. 3 Switch to endoderm induction media
  4. 4 Verify endoderm yield
Anterior Foregut Endoderm Induction Days 3–4
  1. 1 Dissociate embryoid bodies
  2. 2 Plate on fibronectin
  3. 3 Incubate in Anteriorization media-1
  4. 4 Switch to Anteriorization media-2
Lung Bud Organoid Formation (d6–d25) Days 6–25
  1. 1 Treat with Ventralization media (Branching media)
  2. 2 Suspend lung bud organoid clumps
  3. 3 Maintain LBOs in suspension
Branching Morphogenesis in Matrigel (d25–d170) Days 25–170
  1. 1 Embed LBOs in Matrigel
  2. 2 Add Branching media
  3. 3 Culture with media changes
In Vivo Xenotransplantation (optional) At d20–d25 post-transplantation, continuing through d210
  1. 1 Prepare LBO cells for transplantation
  2. 2 Transplant under kidney capsule
  3. 3 Harvest grafts at time points
RSV Infection Studies (optional) Day 170 of organoid culture
  1. 1 Prepare recombinant RFP-expressing RSV
  2. 2 Determine viral titer
  3. 3 Infect d170 LBO cultures
  4. 4 Wash and maintain cultures
  5. 5 Collect samples at time points
HPS1 Mutation Studies (optional) Days 0–50 of organoid differentiation
  1. 1 Generate RUES2-HPS1 line via CRISPR-Cas9
  2. 2 Sort and select edited cells
  3. 3 Pick and expand colonies
  4. 4 Generate LBOs from RUES2-HPS1 cells
Immunofluorescence Staining and Analysis Any stage after cell differentiation
  1. 1 Embed samples in OCT
  2. 2 Section tissue
  3. 3 Fix sections
  4. 4 Permeabilize and block
  5. 5 Incubate primary antibodies
  6. 6 Wash and apply secondary antibodies
  7. 7 Final wash and mount
  8. 8 Image sections
  9. 9 Quantify immunofluorescence
Flow Cytometry Analysis Any stage after cell differentiation
  1. 1 Dissociate LBOs
  2. 2 Stain with FACS antibody
  3. 3 Sort EPCAM+ and EPCAM- cells
RNA Sequencing and Gene Expression Analysis Any stage after cell differentiation
  1. 1 Extract and quality-check RNA
  2. 2 Enrich mRNA and prepare libraries
  3. 3 Sequence on Illumina HiSeq2000
  4. 4 Process sequencing data
  5. 5 Map reads to reference genome
  6. 6 Quantify gene expression
  7. 7 Test for differentially expressed genes
  8. 8 Compare to KeyGenes database
Transmission Electron Microscopy After organoid culture reaches desired stage
  1. 1 Fix samples with glutaraldehyde
  2. 2 Post-fix with osmium tetroxide
  3. 3 Process and embed in resin
  4. 4 Cut semi-thin sections for quality control
  5. 5 Cut and stain ultrathin sections
  6. 6 Image on transmission electron microscope
Surfactant Protein B Uptake Assay Day 170 of organoid culture
  1. 1 Prepare organoids for live imaging
  2. 2 Load SFTPB-BODIPY
  3. 3 Perform time-lapse microscopy
  4. 4 Quantify SFTPB-BODIPY uptake
Extracellular Matrix Quantification (Hydroxyproline Assay) Days 15–50 of organoid culture
  1. 1 Homogenize samples
  2. 2 Hydrolyze samples
  3. 3 Dry hydrolyzates
  4. 4 Oxidize hydroxyproline
  5. 5 Color development
  6. 6 Quantify hydroxyproline

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Chen et al., 2017. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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