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LUNG Publication-derived

In vitro generation of human pluripotent stem cell derived lung organoids

Source Dye et al., 2015 · University of Michigan Medical School · 10.7554/elife.05098.001

👤 Briana R Dye, David R Hill, Michael AH Ferguson, Yu-Hwai Tsai, Melinda S Nagy, Rachel Dyal, James M Wells, Christopher N Mayhew, Roy Nattiv, Ophir D Klein, Eric S White, Gail H Deutsch, Jason R Spence ⏱ 100 days 📋 7 phases 🧫 Human ESC and Human iPSC

Abstract

This protocol describes the step-wise differentiation of human pluripotent stem cells (hESCs and iPSCs) into lung organoids (HLOs) through manipulation of developmental signaling pathways. HLOs consist of epithelial and mesenchymal compartments organized with structural features similar to native lung, including upper airway-like epithelium with basal and ciliated cells, and alveolar-like domains with bipotent progenitor cells. HLOs are transcriptionally similar to human fetal lung and serve as a model for studying lung development, maturation, and disease.

Cell source
Human ESC and Human iPSC
Application
Developmental study and disease modeling

Protocol overview

11 steps across 7 phases

hESC/iPSC Maintenance Ongoing prior to differentiation
  1. 1 Maintain hESCs on Matrigel in mTeSR1 medium
Differentiation into Definitive Endoderm Days 0-3
  1. 1 Initiate Activin A treatment
Anterior Foregut Endoderm Differentiation Days 4-7
  1. 1 Treat with NOG and SB431542 to generate anterior foregut
Generation of Ventral Anterior Foregut Spheroids Days 4-10
  1. 1 Add FGF4 and CHIR99021 to generate 3D spheroids
  2. 2 Embed spheroids in Matrigel droplets
HH Pathway Enhancement to Generate Lung Progenitors Days 4-10 (concurrent with spheroid generation)
  1. 1 Add Smoothened agonist (SAG) during spheroid formation phase
Lung Organoid Expansion and Maturation Days 10-100+
  1. 1 Supplement media with FGF10 for organoid expansion
  2. 2 Optional: Seed organoid spheroids onto acellular human lung matrix
Optional: Detailed Signaling Pathway Optimization (2D Monolayer Phase) Days 4-12 (in parallel, monolayer cultures only)
  1. 1 Test FGF2 dose response on foregut endoderm
  2. 2 Test FGF and HH pathway inhibition
  3. 3 Test SAG ± SU5402 on foregut monolayer

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Dye et al., 2015. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

This wording is awaiting legal review.

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