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SKIN Publication-derived

Skin Organoids for Regenerative Medicine (2024)

Full SOP with Academic or a plan

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

Authors: Imaan A. Ahmed, Jane Sun, Min Jie Kong, Kiarash Khosrotehrani, Abbas Shafiee Duration: 120 days 4 phases Cell source: 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

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

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Attribution

This SOP was written by Organthis based on the published method in Ahmed et al., 2024. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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