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SKIN Publication-derived

iPSC-Derived Epithelial and Mesenchymal (EM) Organoid Differentiation and Transplantation for Localized Scleroderma Therapy

Source Ma et al., 2022 · Peking Union Medical College Hospital, Chinese Academy of Medical Sciences · 10.1002/advs.202106075

👤 Jie Ma, Wei Li, Ruiyuan Cao, Dunqin Gao, Qiyu Zhang, Xiao Li, Biyou Li, Luye Lv, Mansheng Li, Junyi Jiang, Yujie Wang, Jun Li, Zhihong Wu, Yunping Zhu, Wu Zhong, Shuyang Zhang, Ling Leng ⏱ 17 days 📋 7 phases 🧫 Human iPSC (Nuwacell RC01001-B)

Abstract

This protocol describes the generation of human iPSC-derived epithelial and mesenchymal (EM) organoids in a 3D culture system over 16-17 days, followed by transplantation into a bleomycin-induced localized scleroderma mouse model. The EM organoids comprise diverse cell populations including mesenchymal stem cells, epithelial cells, and neural crest-derived cells, which collectively promote recovery of skin fibrosis, enhance epidermal and appendage regeneration, and restore angiogenic capacity in diseased skin tissues.

Cell source
Human iPSC (Nuwacell RC01001-B)
Application
Disease modeling and therapeutic intervention for localized scleroderma

Protocol overview

29 steps across 7 phases

hiPSC Expansion and Preparation Days -7 to 0 (prior to differentiation)
  1. 1 Culture hiPSC Colonies on Matrigel-Coated Plates
  2. 2 Prepare Single-Cell Suspension from hiPSC Colonies
Organoid Aggregate Formation (Days 0-3) Days 0 to 3
  1. 1 Distribute Cells into U-Bottom 96-Well Plates
  2. 2 Incubate Initial Cell Aggregates
  3. 3 Begin EM Organoid Differentiation Medium
  4. 4 Induce Cranial Neural Crest Cell Formation at Day 3
Organoid Maturation and Self-Assembly (Days 12-17) Days 12 to 17
  1. 1 Transfer Aggregates to Low-Attachment Plates
  2. 2 Medium Replacement and Continued Culture
  3. 3 Harvest Organoids at Day 17
Bleomycin-Induced Scleroderma Model Establishment and Organoid Transplantation Weeks 0-8
  1. 1 Prepare Bleomycin Solution
  2. 2 Establish Scleroderma Mouse Model via Daily Bleomycin Injections
  3. 3 Surgical Transplantation of EM Organoids at 4 Weeks
  4. 4 Continue Bleomycin Injections and Monitor Recovery
  5. 5 Perform Microvascular Imaging at Week 8
  6. 6 Harvest Skin Tissues for Analysis
Pathological and Molecular Analysis Post-harvest tissue processing
  1. 1 Hematoxylin and Eosin (H&E) Staining
  2. 2 Immunofluorescence Staining for Organoid and Tissue Characterization
  3. 3 Confocal Microscopy Imaging of Organoids
  4. 4 Masson's Trichrome Staining for Collagen Quantification
Proteomics Analysis Post-tissue harvest
  1. 1 Tissue Homogenization and Sample Preparation
  2. 2 Protein Transfer and Urea Buffer Treatment
  3. 3 Protein Reduction and Alkylation
  4. 4 Buffer Exchange with Uric Acid
  5. 5 Ammonium Bicarbonate Buffer Treatment
  6. 6 Tryptic Digestion
  7. 7 Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) Analysis
Single-Cell RNA Sequencing (scRNA-seq) for Organoid Characterization Day 16 post-differentiation
  1. 1 Organoid Dissociation to Single-Cell Suspension
  2. 2 Cell Viability Assessment and Preparation
  3. 3 Single-Cell 3′ RNA-seq Library Generation

Full SOP

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Attribution

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

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