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SKIN Publication-derived

Human pluripotent stem cell-derived skin organoids enabled pathophysiological model of Mycobacterium tuberculosis infection

Source Yue et al., 2025 · Academy of Military Medical Sciences, Beijing, China · 10.1038/s41467-025-65848-z

👤 Liang Yue, Yating Liang, Pengfei Zhong, Leiming Fang, Zhimin Yun, Qisheng Su, Chaofan Li, Meida Xiang, Jie Zhang, Jiru Wang, Leixin Ge, Chengjun Wu, Hebing Chen, Xiaochen Qiu, Xiaochen Bo, Yingxia Tan ⏱ 125 days 📋 8 phases 🧫 Human iPSC

Abstract

This protocol describes the generation of hair-bearing skin organoids (SKOs) from human induced pluripotent stem cells (hiPSCs) and their use as an in vitro model for cutaneous tuberculosis (CTB) pathogenesis. Following Mtb infection of mature SKOs, the model recapitulates key pathological features of CTB, including extracellular matrix remodeling, fibrosis, and alterations in cell populations, providing a platform for investigating CTB mechanisms and testing antifibrotic therapeutics.

Cell source
Human iPSC
Application
Disease modeling

Protocol overview

41 steps across 8 phases

hiPSC Culture and Maintenance Ongoing until differentiation
  1. 1 Prepare culture plates with Matrigel coating
  2. 2 Culture hiPSCs in mTeSR1 medium
  3. 3 Passage hiPSCs at ~80% confluency
Skin Organoid Differentiation (Days 0–45) Days -1 to 45
  1. 1 Prepare single-cell suspension and aggregate formation
  2. 2 Day -1: Dilute Y27632
  3. 3 Day 0: Initiate differentiation with E6SFB medium
  4. 4 Day 3: Supplement with LDN and increase bFGF
  5. 5 Day 6: Partial medium exchange
  6. 6 Day 9: Half-medium replacement
  7. 7 Day 12: Transfer to maturation plates
  8. 8 Day 15: Initial half-medium replacement
  9. 9 Days 18–45: Maintenance with periodic medium changes
Skin Organoid Maturation and Stabilization (Days 45–125) Days 45–125
  1. 1 Days 45–125: Continue maturation with adjusted medium changes
  2. 2 Quality assessment at day 125
Mycobacterium tuberculosis Preparation Before infection (ongoing culture)
  1. 1 Culture Mtb H37Rv to mid-exponential phase
  2. 2 Harvest and prepare bacterial suspension
Mycobacterium tuberculosis Infection of SKOs Days 0–15 post-infection (or as needed)
  1. 1 Pre-infection culture preparation
  2. 2 Transfer organoids and prepare for infection
  3. 3 Add Mtb inoculum
  4. 4 Remove extracellular bacteria
  5. 5 Continue culture post-infection
  6. 6 Collect organoids at specified timepoints
Pharmacological Inhibitor Treatment (optional) Concurrent with infection (days 0–9)
  1. 1 Prepare inhibitor stocks
  2. 2 Simultaneous infection and inhibitor treatment
  3. 3 Collect treated organoids
Sample Collection and Tissue Processing At designated timepoints
  1. 1 Fix organoids for histology
  2. 2 Dehydrate and embed in paraffin
  3. 3 Section paraffin blocks
  4. 4 Collect tissue for RNA/protein extraction
  5. 5 Prepare single-cell suspension for scRNA-seq
Downstream Analyses (Histology, qPCR, Immunostaining, Western Blot, ELISA, scRNA-seq) Immediately after sample collection
  1. 1 Hematoxylin & Eosin (H&E) staining
  2. 2 Acid-Fast (Ziehl-Neelsen) staining for Mtb detection
  3. 3 Sirius Red staining for collagen quantification
  4. 4 Masson's Trichrome staining for fibrosis assessment
  5. 5 Immunohistochemistry (IHC) for protein detection
  6. 6 Immunofluorescence (IF) staining
  7. 7 Real-time quantitative PCR (qPCR) for mRNA quantification
  8. 8 Western blotting for protein phosphorylation and expression
  9. 9 ELISA for MMP quantification
  10. 10 Single-cell RNA sequencing (scRNA-seq) library preparation and sequencing
  11. 11 scRNA-seq data processing and analysis

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Yue et al., 2025. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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