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SKIN Publication-derived

Hair-Bearing Human Skin Generated Entirely from Pluripotent Stem

Source Lee et al. · Indiana University School of Medicine / Boston Children's Hospital, Boston, USA · 10.1038/s41586-020-2352-3

👤 Lee J, Rabbani CC, Gao H, Steinhart MR, Woodruff BM, Pflum ZE, Kim A, Heller S, Liu Y, Shipchandler TZ, Koehler KR ⏱ 150 days 📋 7 phases 🧫 Human iPSC / Human ESC

Abstract

Landmark protocol for generating complex hair-bearing skin entirely from human pluripotent stem cells using stepwise TGFβ and FGF modulation. Over 4–5 months, organoids develop stratified epidermis, pigmented hair follicles, sebaceous glands, and sensory neurons recapitulating fetal skin architecture.

Cell source
Human iPSC / Human ESC
Application
Regenerative Medicine & Skin Grafting

Protocol overview

22 steps across 7 phases

hPSC Culture and Maintenance Day -30 to Day 0
  1. 1 Coat culture plates with Vitronectin
  2. 2 Culture hPSCs in Essential 8 Flex medium
  3. 3 Passage hPSCs at ~80% confluency
Cell Aggregate Formation and Pre-Differentiation Day -2 to Day 0
  1. 1 Dissociate hPSCs to single cells
  2. 2 Prepare high Y-concentration cell suspension
  3. 3 Seed cells into U-bottom 96-well plates
  4. 4 Incubate aggregates at 37°C
  5. 5 Add fresh medium to dilute Y (day –1)
Initial Differentiation and Epithelial Induction (Day 0–3) Day 0 to Day 3
  1. 1 Transfer aggregates to differentiation medium (day 0)
  2. 2 Induce CNCC formation (day 3)
Expansion and Maturation in Suspension (Day 6–12) Day 6 to Day 12
  1. 1 Add fresh medium (day 6)
  2. 2 Perform half-medium changes (days 8 and 10)
  3. 3 Transfer aggregates to 24-well plates with organoid maturation medium (day 12)
  4. 4 Place plates on orbital shaker
Organoid Maturation and Differentiation (Day 15–45) Day 15 to Day 45
  1. 1 Perform half-medium change (day 15)
  2. 2 Perform half-medium changes every 3 days (days 18–45)
Late Maturation and Hair Follicle Development (Day 45–70) Day 45 to Day 70
  1. 1 Perform half-medium changes every other day (days 45–150)
  2. 2 Perform full-medium changes once weekly (starting day 45)
  3. 3 Monitor for hair follicle emergence (days 70 ± 5)
Continued Maturation and Hair Follicle Growth (Day 70–150) Day 70 to Day 150
  1. 1 Increase medium volume if necessary (day 80 onwards)
  2. 2 Maintain half-medium changes every other day and full-medium changes once weekly
  3. 3 Harvest organoids for analysis or grafting (day 100–150)

Full SOP

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Attribution

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

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