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RETINA Publication-derived

Photoreceptor Outer Segment-like Structures in Long-Term 3D Retinas from Human Pluripotent Stem Cells

Source Wahlin et al., 2017 · Wilmer Eye Institute, Johns Hopkins University · 10.1038/s41598-017-00774-9

👤 Karl J. Wahlin, Julien A. Maruotti, Srinivasa R. Sripathi, John Ball, Juan M. Angueyra, Catherine Kim, Rhonda Grebe, Wei Li, Bryan W. Jones, Donald J. Zack ⏱ 300 days 📋 3 phases 🧫 Human iPSC (IMR90.4, EP1), Human ESC (H7)

Abstract

This protocol describes an in vitro method to differentiate human pluripotent stem cells into 3D retinal structures with photoreceptor outer segment-like features, synaptic ribbons, and neurotransmitter expression. The advanced maturation supports modeling of retinal degenerative diseases and screening of neuroprotective compounds.

Cell source
Human iPSC (IMR90.4, EP1), Human ESC (H7)
Application
Disease modeling and developmental study of retinal photoreceptors

Protocol overview

14 steps across 3 phases

Stem Cell Maintenance and Preparation D-10 to D0
  1. 1 Culture pluripotent stem cells
  2. 2 Passage cells with blebbistatin
Serum-Free Embryoid Body Formation and Optic Vesicle Induction (D0–D18) D0 to D18
  1. 1 Seed cells for aggregate formation
  2. 2 Maintain in hypoxia for first day
  3. 3 Transition to normoxia and begin neural induction
  4. 4 Optimize medium exchanges and add pathway inhibitors
  5. 5 Transfer to suspension and enhance retinal induction
  6. 6 Mechanically isolate optic vesicles
Long-Term Retinal Cup Development and Maturation (D18–D300) D18 to D300
  1. 1 Culture isolated optic vesicles in suspension
  2. 2 Perform periodic grooming and maintenance
  3. 3 Apply all-trans retinoic acid (ATRA) treatment
  4. 4 Add Gamma-secretase inhibitor (DAPT)
  5. 5 Monitor photoreceptor outer segment development
  6. 6 Continue culture to final maturation stage

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Wahlin et al., 2017. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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