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RETINA Publication-derived

Three-Dimensional Retinal Organoids Facilitate the Investigation of Retinal Ganglion Cell Development, Organization and Neurite Outgrowth from Human Pluripotent Stem Cells

Source Fligor et al., 2018 · Department of Biology, Indiana University Purdue University Indianapolis · 10.1038/s41598-018-32871-8

👤 Clarisse M. Fligor, Kirstin B. Langer, Akshayalakshmi Sridhar, Yuan Ren, Priya K. Shields, Michael C. Edler, Sarah K. Ohlemacher, Valentin M. Sluch, Donald J. Zack, Chi Zhang, Daniel M. Suter, Jason S. Meyer ⏱ 80 days 📋 9 phases 🧫 Human ESC (H9), Human iPSC (miPS2), CRISPR-engineered hPSC lines (A81-H7, E4-H7)

Abstract

This protocol describes the differentiation of human pluripotent stem cells into three-dimensional retinal organoids that recapitulate the spatial and temporal development of retinal ganglion cells (RGCs). The organoids enable investigation of RGC layer formation, neurite outgrowth modulation via substrate and growth factor signaling, and characterization of guidance receptor diversity through single-cell transcriptomics.

Cell source
Human ESC (H9), Human iPSC (miPS2), CRISPR-engineered hPSC lines (A81-H7, E4-H7)
Application
Disease modeling, developmental study, cell replacement therapy development

Protocol overview

29 steps across 9 phases

hPSC Maintenance and Expansion Ongoing (passages every 4-5 days)
  1. 1 Culture hPSCs on Matrigel substrate
  2. 2 Passage hPSCs mechanically when at ~70% confluency
Embryoid Body Formation and Neural Induction Days 0-7
  1. 1 Lift hPSCs from Matrigel and form embryoid bodies
  2. 2 Gradually transition EBs to neural induction medium (NIM)
Retinal Specification and Organoid Formation Days 7-16
  1. 1 Plate EBs and switch to adhesion culture
  2. 2 Remove FBS and maintain in NIM
  3. 3 Transition to Retinal Differentiation Medium (RDM)
Organoid Maturation and RGC Layer Development Days 16-70+
  1. 1 Maintain retinal organoids in RDM
  2. 2 Monitor organoid morphology and marker expression
Organoid Dissociation and RGC Neurite Outgrowth Analysis Days 40-80
  1. 1 Identify and harvest organoids at day 40
  2. 2 Plate organoid aggregates on substrate-coated coverslips
  3. 3 Culture aggregates for 24 hours to allow neurite outgrowth
  4. 4 Fix cells and prepare for immunocytochemistry
Single-Cell Dissociation and Extended Culture (Optional) Days 40-80
  1. 1 Dissociate organoids to single cells
  2. 2 Plate dissociated cells on laminin-coated coverslips
  3. 3 Maintain dissociated cells for additional differentiation
Immunocytochemistry and Imaging Same day or after fixation
  1. 1 Prepare samples for immunostaining
  2. 2 Apply primary antibodies
  3. 3 Wash and apply secondary antibodies
  4. 4 Mount and image samples
Growth Cone Imaging and Time-Lapse Analysis Day 40 (after plating aggregates)
  1. 1 Fix samples for growth cone imaging
  2. 2 Block and stain with phalloidin and antibodies
  3. 3 Apply secondary antibodies and mount
  4. 4 Perform time-lapse imaging of live growth cones
Single-Cell qRT-PCR Analysis Day 40 (from dissociation) to same day completion
  1. 1 Dissociate BRN3B:tdTomato organoids to single cells
  2. 2 Sort tdTomato-positive cells by FACS
  3. 3 Load sorted cells into microfluidic array
  4. 4 Lyse cells and generate cDNA
  5. 5 Perform qPCR on Biomark HD

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Fligor et al., 2018. 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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