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LUNG Publication-derived

Generation of Lung Bud Organoids (LBOs) from Human Pluripotent Stem Cells

Source Chen et al., 2017 · Columbia Center for Human Development, 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 ⏱ 170 days 📋 7 phases 🧫 Human ESC (RUES2), Human iPSC

Abstract

This protocol describes the generation of three-dimensional lung bud organoids (LBOs) from human pluripotent stem cells that recapitulate lung development through branching airway and early alveolar structures. LBOs can be cultured in vitro in Matrigel or xenotransplanted to model normal lung development and disease states including respiratory syncytial virus infection and fibrotic lung disease.

Cell source
Human ESC (RUES2), Human iPSC
Application
Developmental study, Disease modeling (RSV infection, pulmonary fibrosis)

Protocol overview

23 steps across 7 phases

hPSC Maintenance and Endoderm Induction Day 0 to Day 4
  1. 1 hPSC Culture on MEF Feeders
  2. 2 MEF Depletion
  3. 3 Primitive Streak and Embryoid Body Induction
  4. 4 Endoderm Induction
Anterior Foregut Endoderm (AFE) Specification Day 4 to Day 6
  1. 5 Dissociation and Plating
  2. 6 Anteriorization Step 1
  3. 7 Anteriorization Step 2
Lung Bud Organoid (LBO) Formation in Suspension Day 6 to Day 20–25
  1. 8 Ventralization and Clump Formation
  2. 9 LBO Suspension Culture and Expansion
  3. 10 LBO Characterization (Optional)
Matrigel Embedding and Branching Morphogenesis Day 20–25 to Day 70+
  1. 11 Matrigel Embedding
  2. 12 Branching Culture Setup
  3. 13 Branching Morphogenesis Culture
  4. 14 Long-Term Culture to Day 170+
Xenotransplantation (Optional) Day 20–25 onwards
  1. 15 LBO Preparation for Transplantation
  2. 16 Kidney Capsule Transplantation
Disease Modeling: RSV Infection Day 170 (or any mature timepoint)
  1. 17 RSV Preparation
  2. 18 RSV Infection of LBOs
  3. 19 RSV Detection and Imaging
Disease Modeling: HPS1 Pulmonary Fibrosis Day 15 to Day 50+
  1. 20 Generation of RUES2-HPS1 Cell Line
  2. 21 LBO Generation from RUES2-HPS1 Cells
  3. 22 Matrigel Culture and Fibrotic Phenotype Assessment
  4. 23 Hydroxyproline Measurement

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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