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Alan D Marmorstein, Benjamin Gillies, Lori Bachman, Joshua Upton, Samuel Cross, Adiv A Johnson, Lihua Y Marmorstein; Maintenance of phenotype following Expansion of iPS cell derived RPE cultures through multiple passages.. Invest. Ophthalmol. Vis. Sci. 2016;57(12):1142.
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© 2017 Association for Research in Vision and Ophthalmology.
The use of induced pluripotent stem (iPS) cells in research on the retinal pigment epithelium (RPE) is limited by the ability to grow sufficient numbers of iPSC-derived RPE cells. Methods for the differentiation of iPSC-derived RPE require expensive media supplements, frequent feeding, extended periods of growth, and yet often yield small numbers of cells. This study sought to define conditions for the growth of large numbers of iPS-derived RPE cells using inexpensive media.
The iPS cell line 006-BIOTR-0001 was obtained from the Mayo Clinic BioTrust and cultured in mTeSR1™ media on Geltrex™ coated plates. Differentiation of iPSCs to RPE was performed on Matrigel™ coated plates following a protocol using defined media. Differentiation was assessed using morphologic, biochemical, and functional criteria. Cells were then subject to additional passages during which they were switched to DMEM containing 1% FBS at 0, 7, 14, or 28 days after plating. Cells were passaged using one of 4 different enzymatic preparations.
iPSC-derived RPE formed a pigmented monolayer of hexagonal cells 60-80 days after initiation of differentiation. Differentiated cells formed monolayers of cuboidal epithelia with apical microvilli, melanin granules, and junctional complexes. The RPE marker protein Best1 was expressed and localized to the basolateral plasma membrane. Western blot demonstrated that cells expressed RPE markers Best1, CRALBP, PEDF, and RPE65. 80 days into the differentiation protocol we passaged cells at a 1:3 ratio using Accumax, Tryp LE, trypsin/EDTA, or dispase. Phenotype was best preserved using Accumax or Tryp LE. Cells were then transferred from DMEM containing B27 supplement to DMEM containing 1% FBS at 0, 7, 14, or 28 days, followed by passaging at 30 or 60 days using Accumax (1x). Following passage feeding was reduced from daily to 3x/week. Phenotype was preserved through at least 3 additional passages in cells following transfer to DMEM/1% FBS and was most effective after cells were preconditioned to DMEM/FBS for at least 16 days prior to passage.
Using these data we have developed a protocol that permits a single 6 cm plate of iPSCs to yield up to 81 x 6 cm plates of iPSC-derived RPE cells in as little as 170 days with the majority of those plates maintained in DMEM/FBS and fed three times per week.
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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