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Ramesh Kaini, Heuy-Ching Hetty Wang; Xeno-free defined conditions for differentiation of protein-induced pluripotent stem cells towards retinal precursor cells. Invest. Ophthalmol. Vis. Sci. 2016;57(12):1130.
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© ARVO (1962-2015); The Authors (2016-present)
Induced pluripotent stem cells (iPSCs) have been successfully differentiated into different retinal cell types, thus instilling hopes for stem cell-based therapy in various retinal degenerative diseases and trauma. However, iPSCs used in these studies were reprogrammed using retroviral or lentiviral approaches and differentiated using animal derived products that may have potential risks of oncogenesis, zoonotic disease transmission, and immune rejection. Therefore, we sought to differentiate genetic integration free, protein-induced pluripotent stem cells (piPSCs) towards retinal lineage and generate clinical-grade retinal precursor cells in defined xeno-free conditions.
Commercially available piPSCs were maintained in xeno-free TeSR-E8 medium and vitronectinXF coated culture plates for at least 5 passages. piPSCs were differentiated towards retinal lineage by using a modified protocol by Sridhar A et al. Cells were dissociated using ReLeSRTM and then generated as uniform sized EBS of 9000 cells/EB using Aggrewell . EBs were gradually transitioned to neural induction medium (NIM) on day 3 of differentiation and maintained in suspension culture in rotary cell culture system (Synthecon) for 7 days. EBs were plated on vitronectinXF coated plates on day 8. On Day 17, aggregates were dislodged and transferred to rotary cell culture system in retinal differentiation medium (RDM).
piPSCs 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. During retinal differentiation, piPSCs acquired expression of various neural and retinal specification markers including Otx2, Sox2, LHX2, RAX , PAX6, and CHX10 at different time points of differentiation.
In this study, we have confirmed the capacity of piPSCs to differentiate into retinal precursor cells using a modified xeno-free retinal differentiation protocol. Our results suggest that pluripotent stem cells developed using protein-based reprogramming can be a viable option for generating a safe source of patient-specific cells for retinal regenerative therapies.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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