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Jie Gong, Mark Fields, Lucian Del Priore; Differentiation of Human Protein Induced Pluripotent Stem Cells (piPS) Towards a Retinal Pigment Epithelium (RPE) Fate. Invest. Ophthalmol. Vis. Sci. 2013;54(15):4053.
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© ARVO (1962-2015); The Authors (2016-present)
Induced pluripotent stem cell (iPS) transplantation is a promising therapeutic approach for the replacement of degenerated neural cells and RPE cells in patients with age-related macular degeneration (AMD), retinitis pigmentosa (RP), and other disorders. The use of piPS cells enables generation of patient-specific pluripotent stem cells that could provide a suitable cell source for transplantation. Herein we demonstrate that protein derived iPS can be induced to differentiate towards an RPE fate.
PiPS from System Biosciences (SBI, Mountain View, CA) were seeded in 6-well low-attachment plates to allow embryoid body (EB) formation for 7 days; cells were then plated on gelatin-coated dishes until pigmented RPE like colonies were visible. RPE were purified by 3-hour exposure to 150units/ml type IV collagenase and manually isolated with a glass pipette. Purified RPE was seeded onto gelatin-coated tissue culture plates and expanded in EGM-2 medium until the desired density was achieved. We performed fluorescence immunocytochemistry on the undifferentiated IPS cells for various pluripotent stem cell markers such as SSEA4, TRA-1-60, OCT4, and TRA-1-81; for differentiated cells using RPE cell markers such as ZO-1, Bestrophin,CRALBP and RPE65. Protein iPS-derived RPE cells and, as a control, human RPE (Lonza Biologics) were then examined phagocytosis function with pHrodo™ Green BioParticles (Life Technologies, Carlsbad, CA).
Undifferentiated piPS cells expressed all pluripotent ES markers SSEA4, TRA-1-60, OCT4, and TRA-1-81, and no RPE markers were detected. During 7 days on low attachment plates, piPS formed EB aggregates. After EB were plated on gelatin coated dishes, pigmented RPE like colonies were visible. Cells were purified (3X) in RPE differentiation medium. Cells expressed hexagonal monolayer RPE phenotype and pigmentation. Differentiated piPS-derived RPE cells expressed RPE markers ZO-1, Bestrophin, CRALBP and RPE65. Phagocytosis function test showed that differentiated piPS-derived RPE also ingested pHrodo™ Green BioParticles.
PiPS generated by use of reprogramming proteins can differentiate towards an RPE fate. RPE derived from piPS exhibited similar physiological properties to those of adult RPE cells. Additional studies are required to analyze functional viability and test cell survival in animal models of retinal degeneration.
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