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RETINA Publication-derived

Generation of a ciliary margin-like stem cell niche from self-organizing human retinal tissue

Source Kuwahara et al., 2015 · RIKEN Center for Developmental Biology, Kobe, Japan · 10.1038/ncomms7286

👤 Atsushi Kuwahara, Chikafumi Ozone, Tokushige Nakano, Koichi Saito, Mototsugu Eiraku, Yoshiki Sasai ⏱ 150 days 📋 7 phases 🧫 Human ESC (hESC; KhES-1 line; Venus-knock-in reporter lines Rx::Venus, Rx::AcGFP, Crx::Venus)

Abstract

This protocol describes a three-dimensional self-organizing culture method for human embryonic stem cells (hESCs) that generates stratified neural retina with an adjacent retinal pigment epithelium, forming a ciliary margin-like growth zone. The method combines selective retinal differentiation using transient BMP4 treatment with an induction-reversal culture approach, producing tissue with functional stem cell niches that expand retinal progenitors peripherally.

Cell source
Human ESC (hESC; KhES-1 line; Venus-knock-in reporter lines Rx::Venus, Rx::AcGFP, Crx::Venus)
Application
Developmental study; Disease modeling; Stem cell niche formation in retinal tissue

Protocol overview

28 steps across 7 phases

hESC maintenance and preparation Ongoing until differentiation
  1. 1 hESC culture on feeder layer
  2. 2 Passage hESCs
Initial aggregation and BMP4 treatment Day 0 to Day 24
  1. 1 Prepare hESC aggregates (SFEBq culture)
  2. 2 Add BMP4 on Day 6
  3. 3 Half-dilute BMP4 every 3 days
  4. 4 Assess neural retina formation by day 24
Long-term NR culture (condition 1) Day 18 to Day 150+
  1. 1 Transfer aggregates to non-adhesive culture dish
  2. 2 Culture in NR-differentiation medium
  3. 3 Monitor NR maturation
Induction-reversal culture for NR–RPE co-generation Day 18 to Day 60+
  1. 1 Transfer aggregates for RPE induction (day 18)
  2. 2 Apply induction medium (RPE-induction phase)
  3. 3 Switch to reversal medium (NR-differentiation phase)
  4. 4 Select two-domain aggregates
  5. 5 Continue long-term culture of two-domain aggregates
Characterization of ciliary margin-like zone Day 50 to Day 150
  1. 1 Assess ciliary margin markers by immunostaining
  2. 2 Verify progenitor dynamics by nucleotide analogue labeling
  3. 3 Confirm presence of de novo NR generation
Retinosphere formation from CM-like tissue Day 60 to Day 85+
  1. 1 Dissect CM-like tissue from two-domain aggregates
  2. 2 Dissociate tissue into single cells
  3. 3 Plate cells for retinosphere formation
  4. 4 Assess primary sphere formation
  5. 5 Generate secondary spheres
  6. 6 Differentiate retinospheres
Optional: SSEA1 cell sorting and retinosphere formation Day 60 to Day 85
  1. 1 Dissect and dissociate CM-like tissue
  2. 2 Immunostain for SSEA1 (FACS or MACS)
  3. 3 Sort SSEA1-positive and SSEA1-negative cells
  4. 4 Plate sorted cells for retinosphere formation
  5. 5 Assess sphere formation and differentiation

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Kuwahara et al., 2015. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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