Supplementary Materialssupplement. adult somatic cells into any cell type in the body. iPSCs reprogrammed from adult somatic cells have an exciting potential in human disease modeling as well as cell sources for regenerative medicine. For example, iPSCs which are derived from skin or blood cells can be reprogrammed into beta islet cells to treat diabetes, blood cells to produce new blood, or neurons to treat neurological disorders (Ye et al., 2013, Takahashi et al., 2007, Yu et al., 2007, Nakagawa et al., 2008). Several research groups have utilized iPSCs to differentiate into RPE-like cells with striking similarities to native RPE cells (Carr et al., 2009, Z-VAD-FMK inhibition Buchholz et al., 2009, Kokkinaki et al., 2011, Osakada et al., 2009a). RPE cells derived from iPSCs are analogous to human fetal RPE cells with respect to expression of important RPE markers and display RPE functionalities such as formation of tight junctions, protein secretion, phagocytosis and vitamin A metabolism (Chang et al., 2014). hiPSC-RPE cells have met requirements for use Rabbit Polyclonal to SRPK3 in clinical trials and transplantation therapies have been conducted in patients with eye diseases (Schwartz et al., 2012, Mandai et al., 2017). hiPSCs provide access to physiologically relevant samples without the issues associated with paucity of adequate primary human RPE tissues and their limited proliferation potential. In the current study, we isolated peripheral blood mononuclear cells from healthy donors, reprogrammed them to iPSCs followed by differentiation to RPE cells. hiPSC-RPE displayed all features akin to functionally normal RPE cells including morphology, monolayers and tight junction formation, secretory function and ability to carry out phagocytosis. Exposure to physiological stressors such as A2E and H2O2 mimicked unique phenotypes of pathologic or aged RPE cells with inflammation and decrease in cell viability. Our study provides a unique experimental platform not only to understand unique aspects of RPE function but also to dissect the complex cellular and molecular events in degenerative retinal diseases. 2. Materials and methods 2.1. Generation of human iPSCs Blood samples were collected Z-VAD-FMK inhibition from healthy volunteers and peripheral blood mononuclear cells (PBMCs) were isolated using BD Vacutainer Cell Preparation Tubes made up of sodium citrate. PBMCs were expanded and transduced with STEMCCA lentivirus vector using an earlier published protocol (Sommer et al., 2012). iPSC-like colonies were picked and managed up to passage 10 on Matrigel (Corning Bioscience, USA) coated plates. After each passage differentiated cells were discarded and only iPSC-like colonies were propagated. After passage 10, iPSC-like colonies were tested for expression of pluripotency markers quantitative RT-PCR and immunocytochemistry. All procedures were approved by the Institutional Review Boards (IRBs) at the Case Western Reserve University or college, Cleveland Ohio and adhered to the Declaration of Helsinki. All cell culture procedures were approved by Case Western Reserve University or college Institutional Biosafety Committee. All samples were obtained after patients experienced given knowledgeable Z-VAD-FMK inhibition consent. 2.2. Differentiation of human iPSCs to RPE cells Fully characterized iPSC lines at passage 10 were utilized for differentiation. iPSCs were differentiated to functional RPE using a previously reported protocol (Osakada et al., 2009a, Osakada et al., 2009b). Briefly, cells were plated Z-VAD-FMK inhibition on gelatin coated dish with an inhibitor cocktail of CKI7 (Casein Kinase 1 Inhibitor) (Sigma, St. Lois, MO), SB431542 (Sigma) and ROCKi (Stemcell Technologies, Vancouver, Canada) in ReproCELL ReproStem Cell Culture medium (Stemgent Inc., MA) for one day. Culture medium was replaced by RPE differentiation medium with 20% KSR (ThermoFisher Scientific, MA) on day 1 and 3. On day 5, 7 and 9, KSR was reduced to 15% followed by 10% KSR from day 11 to day 18. Inhibitor cocktail was added up to day 18. Day 19 onwards cells were produced in 10% KSR until dark colonies appeared. Around day 30C35, when dark pigmented colonies appeared, cells were managed in RPE maintenance medium. Around 10 days later, cells were detached and allowed to float as aggregates for 5 days to 2 weeks. Dark pigmented aggregates were then plated on CellStar coated plates (ThermoFisher Scientific, MA). After cells expanded, non-RPE cells were scrapped off manually and cells showing RPE morphology and pigmentation were passaged. RPE cells were allowed to mature for 30 days in RPE maintenance medium with bFGF (Stemcell Technologies) and SB431542 before being used for experiments. 2.3. RPE monolayers RPE monolayers were established on 8 well chamber slides (Osakada et al., 2009a, Osakada et al., 2009b, Germany) or 96 well plates coated with CellStart for 30 days. 30,000C50,000 cells were seeded.