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LUNG Publication-derived

Reversal of Surfactant Protein B Deficiency in Patient-Specific Human Induced Pluripotent Stem Cell-Derived Lung Organoids by Gene Therapy

Source Leibel et al., 2019 · University of California, San Diego; Rady Children's Hospital; Sanford Burnham Prebys Medical Discovery Institute · 10.1038/s41598-019-49696-8

👤 Sandra Lawrynowicz Leibel, Alicia Winquist, Irene Tseu, Jinxia Wang, Daochun Luo, Sharareh Shojaie, Neal Nathan, Evan Snyder, Martin Post ⏱ 40 days 📋 6 phases 🧫 Patient-Derived iPSC (SFTPB deficiency - p.Pro133GlnfsTer95)

Abstract

This protocol describes the generation of patient-specific iPSC-derived lung organoids containing both epithelial and mesenchymal cell populations from a patient with surfactant protein B (SFTPB) deficiency, followed by lentiviral correction of the mutation and characterization of functional alveolar type II cells capable of producing and secreting surfactant.

Cell source
Patient-Derived iPSC (SFTPB deficiency - p.Pro133GlnfsTer95)
Application
Disease modeling and gene therapy validation

Protocol overview

27 steps across 6 phases

Derivation of iPSC from patient fibroblasts Day 0-28
  1. 1 Isolate and expand fibroblasts
  2. 2 Reprogramme fibroblasts to iPSCs using Sendai virus
  3. 3 Culture iPSC colonies on feeder layer
  4. 4 Characterize and authenticate iPSCs
  5. 5 Verify SFTPB mutation by genomic sequencing and restriction digestion
Lentiviral infection with wild-type SFTPB gene Day 0-14+
  1. 1 Generate lentiviral particles
  2. 2 Harvest and concentrate lentiviral supernatant
  3. 3 Infect iPSCs with lentiviral vector
  4. 4 Select transduced cells with puromycin
  5. 5 Verify SFTPB transgene expression by qPCR and immunoblotting
Directed differentiation to definitive endoderm (DE) and anterior foregut endoderm (AFE) Day 0-8
  1. 1 Prepare iPSCs for differentiation
  2. 2 Induce definitive endoderm (DE) with Activin A
  3. 3 Induce anterior foregut endoderm (AFE)
Differentiation to lung progenitor cells (LPC) and characterization Day 8-18
  1. 1 Induce lung progenitor cells (LPC)
  2. 2 Characterize LPC monolayers by immunostaining
  3. 3 Verify GFP and SFTPB expression in transduced LPC cells
3D lung organoid differentiation and maturation Day 18-40
  1. 1 Prepare and seed LPC cells into 3D Matrigel
  2. 2 3D Step 1 culture: branching morphogenesis (Days 18-25)
  3. 3 3D Step 2 culture: alveolar specification (Days 25-33)
  4. 4 3D Step 3 culture: epithelial maturation (Days 33-40)
  5. 5 Harvest and process day 40 organoids
Characterization of lung organoids Day 40
  1. 1 Immunofluorescence staining of organoid sections
  2. 2 RNA extraction and qRT-PCR analysis
  3. 3 Protein expression analysis by Western blotting
  4. 4 Flow cytometry analysis of organoid cells
  5. 5 Transmission electron microscopy (TEM) of lamellar bodies
  6. 6 Surfactant secretion analysis by tandem mass spectrometry (MS/MS)

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Leibel et al., 2019. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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