The procedure to induce early stages of retinal differentiation was based on a previously described protocol with major modifications14 (link),22 (link),29 (link). Briefly, on day 0 (D0) of differentiation, hiPSC were enzymatically detached by dispase treatment, dissociated into small clumps, and cultured in suspension with mTeSR1 medium and 10 µM Blebbistatin (Sigma) to induce aggregate formation. Aggregates were gradually transitioned into neural-induction medium (NIM) containing Dulbecco’s modified eagle medium (DMEM)/F12 (1:1), 1% N2 supplement (Invitrogen), 1x minimum essential media-non essential amino acids (NEAA), 2µg ml−1 heparin (Sigma), by replacing the medium with a 3:1 ratio of mTeSR1/NIM on D1, 1:1 on D2, and 100% NIM on D3. On D7, aggregates (average size of 0.22 ± 0.05 mm) were seeded onto Matrigel (growth-factor-reduced; BD Biosciences) coated dishes containing NIM at an approximate density of 20 aggregates per cm2 (link), and switched to DMEM/F12 (3:1) supplemented with 2% B27 (without vitamin A, Invitrogen), 1x NEAA, and 1% antibiotic-antimycotic (Gibco) on D16. Thereafter, the medium was changed daily.
Protocol detail
Retinal Differentiation Protocol for hiPSCs
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Antibiotic
Blebbistatin
Dishes
Dispase
Eagle
Essential amino acids
Growth factor
Heparin
Hipsc
Matrigel
Neural
Retinal
Vitamin a
Corresponding Organization :
Other organizations : Johns Hopkins Medicine, Johns Hopkins University, Hefei National Center for Physical Sciences at Nanoscale, University of Science and Technology of China, McGovern Institute for Brain Research, Sidney Kimmel Comprehensive Cancer Center, Indiana University – Purdue University Indianapolis, University of Wisconsin–Madison, Smith-Kettlewell Eye Research Institute
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Variable analysis
independent variables
- Blebbistatin (10 µM) treatment
- Transition of cell aggregates from mTeSR1 medium to neural-induction medium (NIM)
- Induction of early stages of retinal differentiation
- Enzymatic detachment of hiPSC using dispase
- Dissociation of hiPSC into small clumps
- Aggregate formation in suspension culture
- Seeding of aggregates onto Matrigel-coated dishes
- Supplementation of media with N2, NEAA, heparin, B27 (without vitamin A), and antibiotic-antimycotic
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