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RETINA Publication-derived

Blood-Derived Human iPS Cells Generate Optic Vesicle–Like Structures with the Capacity to Form Retinal Laminae and Develop Synapses

Source Phillips et al., 2012 · Waisman Center, University of Wisconsin-Madison · 10.1167/iovs.11-9313

👤 M. Joseph Phillips, Kyle A. Wallace, Sarah J. Dickerson, Michael J. Miller, Amelia D. Verhoeven, Jessica M. Martin, Lynda S. Wright, Wei Shen, Elizabeth E. Capowski, E. Ferda Percin, Enio T. Perez, Xiufeng Zhong, Maria V. Canto-Soler, David M. Gamm ⏱ 108 days 📋 7 phases 🧫 Human iPSC (Blood-derived T lymphocytes)

Abstract

This protocol describes the generation of optic vesicle-like structures (OVs) from blood-derived human induced pluripotent stem cells (TiPSCs) and their differentiation into multiple neuroretinal cell types including photoreceptors, ganglion cells, and amacrine cells. The protocol produces stratified retinal laminae capable of expressing synaptic markers critical for intercellular communication.

Cell source
Human iPSC (Blood-derived T lymphocytes)
Application
Retinal cell differentiation and developmental study

Protocol overview

29 steps across 7 phases

T-Cell Activation and Retroviral Transduction Day 0–2
  1. 1 Blood collection and PBMC isolation
  2. 2 Thaw PBMCs and resuspend
  3. 3 T-cell activation with OKT3 and IL-2
  4. 4 Retroviral transduction
  5. 5 Post-transduction incubation
iPSC Colony Expansion Day 2–20
  1. 1 Transfer to MEF feeder layers
  2. 2 Transition to conditioned medium
  3. 3 Identify and isolate iPSC colonies
  4. 4 Hand-pick and propagate iPSC colonies
Embryoid Body Formation and Neural Induction Day 0–16 (relative to differentiation start)
  1. 1 Lift colonies and form embryoid bodies
  2. 2 Replace medium with neural induction medium
  3. 3 Transfer aggregates to laminin-coated plates
  4. 4 Mechanically lift neural clusters
Retinal Differentiation and Optic Vesicle Isolation Day 16–20
  1. 1 Culture in retinal differentiation medium
  2. 2 Identify and isolate optic vesicles
  3. 3 Pool isolated OVs
Retinal Neuron Differentiation and Maturation Day 20–108
  1. 1 Continue culture in RDM
  2. 2 Partial dissociation and plating for differentiation studies
  3. 3 Monitor marker expression over time
Cryosectioning and Laminar Structure Analysis Day 20–105 (as needed)
  1. 1 Fix free-floating OVs
  2. 2 Cryosection OVs
  3. 3 Perform immunocytochemistry on sections
Pluripotency and Genetic Characterization (Quality Control) Performed on established iPSC lines
  1. 1 Genomic DNA PCR for retroviral integration
  2. 2 RT-PCR for transgene silencing and endogenous gene activation
  3. 3 Short tandem repeat (STR) analysis
  4. 4 G-banding karyotype analysis
  5. 5 Teratoma analysis
  6. 6 Flow cytometry for pluripotency markers
  7. 7 Immunocytochemistry for pluripotency markers

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Phillips et al., 2012. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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