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SKIN Publication-derived

Generation and Characterization of Hair-Bearing Skin Organoids

Source Lee et al. · Boston Children's Hospital / Harvard Medical School, Boston, USA · 10.1038/s41596-022-00681-y

👤 Lee J, van der Valk WH, Serdy SA, Deakin C, Kim J, Le AP, Koehler KR ⏱ 150 days 📋 5 phases 🧫 Human iPSC / Human ESC

Abstract

Definitive detailed protocol for generating hair-bearing skin organoids from human PSCs over 150 days. Includes comprehensive reagent tables, cell line optimization, troubleshooting, and the novel qCe3D clearing technique for whole-mount immunostaining and 3D imaging.

Cell source
Human iPSC / Human ESC
Application
Skin Biology Research

Protocol overview

21 steps across 5 phases

hPSC Culture and Preparation Pre-differentiation
  1. 1 Thaw frozen hPSCs
  2. 2 Maintain hPSC culture
  3. 3 Passage hPSCs
  4. 4 Freeze hPSCs for long-term storage
Cell Aggregation (Days −2 to −1) Days -2 to -1
  1. 5 Prepare single-cell suspension
  2. 6 Form cell aggregates in 96-well U-bottom plates
  3. 7 Dilute Y-27632 on day −1
Early Differentiation (Days 0–12) Days 0-12
  1. 8 Transition to E6SFB on day 0
  2. 9 Apply LDN and bFGF on day 3
  3. 10 Provide nutrition on day 6
  4. 11 Half medium change on day 9
  5. 12 Transition to floating culture on day 12
Organoid Maturation (Days 15–150) Days 15-150
  1. 13 Half medium change on day 15
  2. 14 Dilute Matrigel on day 18
  3. 15 Perform weekly full and interval half medium changes (days 18–150)
Downstream Assays and Analysis Days 6–150
  1. 16 Dissociate organoids for single-cell RNA-seq
  2. 17 Fix organoids for cryo-embedding
  3. 18 Cryo-embed organoids in OCT
  4. 19 Cryo-section organoids at 12 μm
  5. 20 Immunostain organoid sections
  6. 21 Whole-mount immunostaining with tissue clearing (qCe3D)

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.

This wording is awaiting legal review.

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