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LUNG Publication-derived

Surfactant Protein B Deficiency Alveolar Organoids

Full SOP with Academic or a plan

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

Authors: Sandra Lawrynowicz Leibel, Alicia Winquist, Irene Tseu, Jinxia Wang, Daochun Luo, Sharareh Shojaie, Neal Nathan, Evan Snyder, Martin Post Duration: 40 days 6 phases Cell source: 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

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

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Attribution

This SOP was written by Organthis based on the published method in Leibel et al., 2019. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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