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Ramesh Kaini, Whitney Greene, Heuy-Ching Hetty Wang; Secretome of induced pluripotent stem cells-derived optic vesicles.. Invest. Ophthalmol. Vis. Sci. 2017;58(8):4568.
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© ARVO (1962-2015); The Authors (2016-present)
The paracrine factors secreted in the conditioned medium (CdM) of pluripotent stem cells have shown a promising prospect as a therapeutic agent in regenerative medicine. We hypothesize that pluripotent stem cells differentiated towards retinal lineage secrete neuroprotective and neuroregenerative factors. In this study, we sought to differentiate human induced-pluripotent stem (iPS) cells towards neural retinal lineage and study the factors secreted in the CdM.
3D1 iPS cells were differentiated toward retinal lineage by a modified protocol. Embryoid bodies (EBs) of 9000 cells were generated using Aggrewells. EBs were transitioned to neural induction medium on day 3 of differentiation and maintained in a suspension culture system. On Day 17, neuro-rosettes were manually separated and transferred to suspension culture. Fifty rosettes were cultured in each well of 6 well ultralow attachment plate with 2 ml of DMEM/F12 (3:1) medium supplemented with 2% B27. Media was harvested every 72 hours and replaced with fresh 2 ml medium. Harvested CdM were centrifuged at 5k rpm for 5 minutes and stored at -800F.Differentiation of iPS cells towards retinal lineage was confirmed by immunofluorescence analysis of neural (OTX 2, SOX1), eye field (LHX2, SIX6), and retinal precursor marker (CHX10, PAX6). Multiplex luminex Immunoassays were performed for expression of several neuroprotective factors.
EBs derived from 3D1 cells expressed neuronal markers (OTX2, SOX1), eye field markers (SIX6, LHX2) and retinal precursor markers (co-expression of CHX10 and PAX6) at different time points which confirmed to previous studies on step-wise differentiation towards optic vesicle formation. We observed secretion of several neuroprotective factors including Osteopontin, Hepatocyte growth factor (HGF), Stromal cell-derived factor 1 (SDF-1), Insulin-like growth factor 1 (IGF-1), and Mesencephalic astrocyte-derived neurotrophic factor (MANF).
In this study, we observed that iPS-derived optic vesicles secrete several growth factors that are previously described to protect neurons.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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