Purchase this article with an account.
Ramesh Kaini, Anthony James Johnson, Teresa A Burke, Dallas Golden, Heuy-Ching Hetty Wang; Xeno-free 3D retinal differentiation of human induced-pluripotent stem cells. Invest. Ophthalmol. Vis. Sci. 2014;55(13):1369.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Advances made in recent years to generate different retinal precursor cells from pluripotent stem cells have instilled hopes for cell replacement therapy in retinal degeneration diseases and retinal trauma. However, stem cells must be derived, maintained and differentiated in xeno-free condition for potential clinical use in human. In this study, we sought to differentiate human induced-pluripotent stem (iPS) cells towards neural retinal lineage and produce clinical-grade retinal progenitor cells.
Commercially available iPS cells, IMR90-4, were maintained in VitronectinXF coated culture plates with xeno-free TeSR-E8 medium. iPS cells were dissociated and quickly reaggregated using Sumilon PrimeSurface96V culture plates in GMEM medium containing 20% KnockOut Serum Replacement XenoFree . To start the xeno-free 3D differentiation, VitronectinXF (10 ug/ml) was added in the retinal differentiation medium from day 2 onwards to day 18. 3D cell aggregates were treated with Wnt inhibitor, IWR-1-endo, for the first 12 days. KnockOut SR Growth Factor cocktail was added on day 12 and Smoothened Agonist (SAG) on day 15. Aggregates were then switched to DMEM/F12-Glutamax medium with N2 supplement on day 18. Expression of different markers of eye field and retinal progenitor cells were studied at different time points.
iPS cells maintained in vitronectinXF coated plates with TeSR-E8 medium retained the expression of pluripotent markers, including OCT4, Nanog, SSEA-3, and TRA-1-60 after five passages. Aggregates of xeno-free 3D differentiation started expressing different neural and eye field markers, including Otx2, Sox2, Rx, LHX2, Six6, PAX6, MITF, and CHX10 at different time points of differentiation. Retinal progenitor cell marker, CHX10 was observed on day 16 onwards.
In this study, we observed that neural retinal lineage cells can be derived from human iPS cells in a 3D culture system using defined, xeno-free components. We are in the process of developing stratified neural retina from iPS cells under defined, xeno-free condition and generating different retinal precursor cells for cell replacement therapy.
This PDF is available to Subscribers Only