Generating iPSCs from MAK-associated RP Fibroblasts

Following informed consent, skin biopsies were collected from three patients with MAK-associated RP and used for fibroblast isolation and iPSC generation as described previously [6 (link)–8 (link)]. Briefly, dermal fibroblast cells were reprogrammed using the CytoTune non-integrating Sendai virus reprogramming kit according to the manufacturers protocol (Invitrogen/Thermo Fisher Scientific, Waltham, MA; CytoTune-iPS Reprogramming Kit; Cat#: A16517) [9 (link)]. Fibroblasts were plated on 6-well tissue culture plates and infected at a multiplicity of infection (MOI) of 5. At 12–16 h following transduction cells were washed and fed with fresh fibroblast cell growth media [(DMEM/F12, 5% heat inactivated FBS (Invitrogen/Thermo Fisher Scientific) and 0.2% primocin (Invivogen, San Diego, CA)]. At 7 days post-infection, cells were passaged onto 6-well LN521-coated cell culture dishes at a density of 30,000 cells/well and fed every day with E8 pluripotency media (Invitrogen/Thermo Fisher Scientific). At 3 weeks post-viral transduction, iPSC colonies were picked, passaged, and clonally expanded on fresh LN521-coated cell culture dishes. During reprogramming and maintenance of pluripotency, cells were cultured at 5% CO₂, 10% O₂, and 37 °C.