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RETINA Publication-derived

Self-Formation of Optic Cups and Storable Stratified Neural Retina from Human ESCs

Source Nakano et al. · <UNKNOWN>

👤 Tokushige Nakano, Satoshi Ando, Nozomu Takata, Masako Kawada, Keiko Muguruma, Kiyotoshi Sekiguchi, Koichi Saito, Shigenobu Yonemura, Mototsugu Eiraku, Yoshiki Sasai ⏱ 126 days 📋 6 phases 🧫 Human ESC (KhES-1)

Abstract

This protocol describes the self-formation of optic cups and stratified neural retina tissues from human embryonic stem cells (hESCs) using a serum-free floating culture of embryoid body-like aggregates with quick re-aggregation (SFEBq). The resulting neural retina develops photoreceptor layers with functional cilia and can be cryopreserved for long-term storage and later differentiation.

Cell source
Human ESC (KhES-1)
Application
Developmental study, disease modeling platform, drug screening

Protocol overview

26 steps across 6 phases

Initial aggregation and early retinal specification Days 0-12
  1. 1 Preparation of hESC aggregates in V-bottomed wells
  2. 2 Addition of differentiation medium and growth factor inhibitors
  3. 3 Addition of Matrigel from day 2
  4. 4 Maintain standard culture conditions for days 0-12
Retinal progenitor enhancement (NR-selective pathway) Days 12-18
  1. 5 Removal of IWR1e and addition of pro-retinal signaling factors
  2. 6 Culture with FBS and SAG through day 18
  3. 7 Optional: verify retinal specification by immunostaining
Optic cup formation (days 15-26) Days 15-26
  1. 8 Addition of Wnt agonist for balanced NR and RPE generation
  2. 9 Transfer aggregates to retina culture medium on day 18
  3. 10 Observation of optic vesicle formation
  4. 11 Optic cup invagination (days 19-26)
  5. 12 Continued culture and pigmentation of RPE
Isolation and long-term culture of neural retina Days 18-126
  1. 13 Isolation of Rx::venus[+] neural retina epithelium on day 18
  2. 14 Culture in suspension with high oxygen conditions
  3. 15 Observation of epithelial eversion and stratification (days 18-30)
  4. 16 Retinal progenitor expansion (days 30-60)
  5. 17 Multilayered stratification and early photoreceptor accumulation (days 60-126)
Acceleration of photoreceptor differentiation by Notch inhibition Days 29-43
  1. 18 Treatment with Notch inhibitor DAPT
  2. 19 Monitoring photoreceptor accumulation
  3. 20 Observation of tissue architectural changes
Cryopreservation of neural retina epithelium Days 18-40 (or up to day 105)
  1. 21 Preparation of NR tissues for cryopreservation
  2. 22 Pretreatment with cryoprotectants
  3. 23 Vitrification freezing
  4. 24 Storage of frozen tissues
  5. 25 Thawing of frozen NR tissues
  6. 26 Post-thaw culture and evaluation

Full SOP

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Attribution

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

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