Rhesus macaque iPS cells were generated from primary fibroblast cultures transduced with retroviral vectors as previously described.
24 Established iPS colonies were maintained on inactivated mouse embryonic fibroblast (mEF) cells in Dulbecco's modified Eagle's medium/F12 (Invitrogen, Carlsbad, CA, USA) supplemented with 15% fetal bovine serum (HyClone, Erie, UK), 1% nonessential amino acids (Invitrogen), 2 mM L-glutamine (Invitrogen), 0.1 mM β-mercaptoethanol (Sigma-Aldrich, St. Louis, MO, USA), and 1% penicillin streptomycin (Invitrogen). On 80% confluence, iPS colonies were incubated with 1 mg/mL collagenase and gently collected using a cell lifter (Corning Inc., Corning, NY, USA). Intact colonies were plated on low-attachment six-well plates (Corning) for embryoid body formation followed by distribution on Matrigel (BD Biosciences, San Jose, CA, USA) -coated plates. RPE differentiation was directed by exposing cells to knockout serum replacement media supplemented with Noggin (R&D Systems, Minneapolis, MN, USA), SB431542 (Tocris, Bristol, UK), Activin A (R&D Systems), and nicotinamide (Sigma-Aldrich, St. Louis, MO, USA),
25 a protocol similar to previously published differentiation methods for human RPE.
26–30 Pigmented colonies were manually picked, dissociated with trypsin, and plated on Primaria plates (BD Biosciences) in THT medium for further enrichment.
31,32 RPE cells were transduced to express green fluorescent protein (GFP) using AAV2-CMV-eGFP a minimum of 2 weeks before transplantation. In vitro characterization of RPE cells included PCR and immunocytochemistry. Total RNA was extracted (RNeasy Kit; Qiagen, Valencia, CA, USA) from iPS cell–derived RPE, and PCR reactions were performed for RPE65, pigment-epithelium-derived factor (PEDF), cellular retinaldehyde-binding protein (CRALBP), Bestrophin-1 (BEST1), zonula occludens protein 1 (ZO-1), and microphthalmia-associated transcription factor (MITF). Additional cells were plated on Nunc Lab-Tek II chamber slides (Sigma-Aldrich) for immunostaining using antibodies against RPE65, 1:250 (Abcam, Cambridge, UK); ZO-1, 1:100 (Invitrogen); CRALBP, 1:100 (Abcam); premelanosome (PMEL) 17, 1:500 (Abcam); and MITF, 1:100 (Millipore, Billerica, MA, USA).