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SKIN Publication-derived

Generation of iPSC-derived skin organoids from systemic sclerosis patients and screening for anti-fibrotic agents

Source Kim et al., 2022 · Catholic iPSC Research Center, College of Medicine, The Catholic University of Korea · 10.1186/s13287-022-02987-w

👤 Yena Kim, Yoojun Nam, Yeri Alice Rim, Ji Hyeon Ju ⏱ 30 days 📋 9 phases 🧫 Patient-Derived iPSC (Systemic Sclerosis)

Abstract

This protocol describes the generation of patient-derived iPSCs from PBMCs of systemic sclerosis patients using Sendai virus reprogramming, followed by differentiation into keratinocytes and fibroblasts. These cells are used to construct 3D skin organoids that recapitulate SSc pathology, enabling high-throughput screening of FDA-approved drugs to identify anti-fibrotic candidates.

Cell source
Patient-Derived iPSC (Systemic Sclerosis)
Application
Disease modeling and drug screening

Protocol overview

50 steps across 9 phases

PBMC isolation and iPSC generation Day 0–21
  1. 1 PBMC isolation via Ficoll gradient
  2. 2 PBMC culture before reprogramming
  3. 3 Sendai virus transduction
  4. 4 Transfer to vitronectin-coated plates
  5. 5 iPSC maintenance culture
iPSC characterization and pluripotency validation Day 5–25
  1. 1 iPSC expansion
  2. 2 Alkaline phosphatase staining
  3. 3 Immunocytochemical staining
  4. 4 Secondary antibody incubation and counterstaining
  5. 5 Teratoma formation assay
Differentiation of iPSCs into keratinocytes and fibroblasts Day 0–30
  1. 1 Embryoid body (EB) formation via hanging drop method
  2. 2 Keratinocyte differentiation: plating on collagen
  3. 3 Keratinocyte differentiation media (days 1–7)
  4. 4 Keratinocyte differentiation media (days 8–11)
  5. 5 Keratinocyte differentiation media (days 12–30)
  6. 6 Fibroblast differentiation: plating on Matrigel
  7. 7 Fibroblast differentiation media (days 1–3)
  8. 8 Fibroblast differentiation media (days 4–6) with BMP4
  9. 9 Fibroblast differentiation media (days 7–14)
  10. 10 Fibroblast passaging and plating (days 14 and 21)
Characterization of differentiated cells (gene and protein expression) Day 14–21
  1. 1 RNA isolation and cDNA synthesis
  2. 2 Quantitative RT-PCR (qRT-PCR)
  3. 3 Immunocytochemical staining of differentiated cells
  4. 4 Flow cytometric analysis
3D skin organoid generation Day 0–14
  1. 1 Preparation of fibroblast layer in collagen
  2. 2 Incubation of fibroblast layer
  3. 3 Seeding of keratinocytes
  4. 4 Transition to normal calcium medium (submerged culture)
  5. 5 Establishment of air–liquid interface
In vitro fibrosis assessment and functional assays Day 0–21
  1. 1 Proliferation assay of iPSC-derived fibroblasts
  2. 2 Hydroxyproline assay for collagen quantification
  3. 3 Western blot analysis of fibrosis markers
  4. 4 Phospho-kinase array analysis
  5. 5 Wound-scratch assay for fibroblast migration/proliferation
Organoid histology and xenografting Day 14–28 (in vitro); 2 weeks (in vivo)
  1. 1 Histological preparation of skin organoids
  2. 2 Hematoxylin and eosin (H&E) staining
  3. 3 Picrosirius Red (PSR) staining for collagen
  4. 4 Masson's trichrome staining
  5. 5 Xenografting of skin organoids onto immunodeficient mice
High-throughput drug screening and validation Day 0–7 (screening); variable (validation)
  1. 1 Primary drug screening: proliferation assay
  2. 2 Secondary screening: collagen quantification
  3. 3 Tertiary validation: TGF-β1 reversal assay
  4. 4 Hit drug validation: α-SMA suppression
  5. 5 Dose-response characterization
In vivo validation in bleomycin-induced SSc mouse model Day 0–21
  1. 1 Bleomycin-induced SSc model induction
  2. 2 Raloxifene treatment initiation
  3. 3 Concurrent bleomycin and raloxifene treatment
  4. 4 Tissue harvest and histological analysis
  5. 5 Western blot analysis of in vivo fibrosis markers
  6. 6 Gene expression analysis of fibrosis markers

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Kim et al., 2022. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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