Skip to content
← Back to browse
SKIN Publication-derived

Generating Skin-Derived Precursor-Like Cells From Human-Induced Pluripotent Stem Cell-Derived Skin Organoids

Source Ahmed et al., 2024 · Frazer Institute, Faculty of Medicine, The University of Queensland · 10.1111/exd.70017

👤 Imaan A. Ahmed, Jane Sun, Min Jie Kong, Kiarash Khosrotehrani, Abbas Shafiee ⏱ 120 days 📋 4 phases 🧫 Human iPSC

Abstract

This protocol describes the isolation and culture of skin-derived precursor-like cells (SKP-like cells) from human iPSC-derived skin organoids (SKOs). SKP-like cells exhibit characteristic spheroid morphology, express key markers including SOX2, fibronectin, and S100β, and retain self-renewal capacity in defined SKP growth medium, offering a scalable source of progenitor cells for hair regeneration therapies.

Cell source
Human iPSC
Application
Hair follicle regeneration and isolation of progenitor cells for therapeutic applications

Protocol overview

24 steps across 4 phases

Isolation of Skin-Derived Precursor-Like Cells From Skin Organoids Days 75–120 of SKO culture
  1. 1 Transfer and mechanical dissociation of mature SKOs
  2. 2 Transfer and wash dissociated tissue
  3. 3 Enzymatic digestion with Accutase
  4. 4 Stop digestion with SKP basic media
  5. 5 Centrifuge to pellet cells
  6. 6 Resuspend in SKP proliferation media
  7. 7 Seed cells in low-attachment plates
  8. 8 Culture cells in defined SKP medium
  9. 9 Monitor spheroid formation and passage
Isolation of Skin Organoid-Derived Fibroblasts After mature SKO dissociation
  1. 1 Digest mature SKOs to single cell suspension
  2. 2 Resuspend in Rheinwald and Green's medium with ROCK inhibitor
  3. 3 Culture in 2D adherent conditions for five passages
  4. 4 Perform immunostaining for fibronectin confirmation
  5. 5 Transfer to low-attachment culture in SKP proliferation media
  6. 6 Culture for 2 days and assess spheroid formation
Immunofluorescence Analysis and Characterization At P1 Day 3 for SKP-like cells; Day 2 for SKO-FB spheroids; fresh and freeze-thawed samples
  1. 1 Harvest and fix SKP-like cell spheroids
  2. 2 Perform immunofluorescence staining for SKP markers
  3. 3 Acquire immunofluorescence images
  4. 4 Analyze SOX2 expression quantitatively
  5. 5 Assess freeze-thaw retention of SOX2 expression
Statistical Analysis of SOX2 Expression After immunofluorescence analysis of P0, P1, and P2 samples
  1. 1 Collect SOX2 quantification data across passage numbers and hiPSC lines
  2. 2 Perform Mann-Whitney U test for passage number comparison
  3. 3 Perform Kruskal-Wallis ANOVA for hiPSC line comparison
  4. 4 Report statistical results and interpret findings

Full SOP

🔬

Create a free account to access this protocol

Join OrganMatch to unlock step-by-step procedures, reagent concentrations, QC checklists, and downloadable batch record templates.

Create free account

Already registered? Log in

Attribution

This SOP was authored by Organthis based on the published method in Ahmed et al., 2024. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

This wording is awaiting legal review.

Something wrong with this entry? Report an issue with this protocol

Need a commercial licence?
Use this protocol in your therapeutic or diagnostic pipeline.
Request Commercial Licence