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Generation of retinal pigmented epithelium from iPSCs derived from the conjunctiva of donors with and without age related macular degeneration.

PloS one (2017-03-11)
Zhouhui Geng, Patrick J Walsh, Vincent Truong, Caitlin Hill, Mara Ebeling, Rebecca J Kapphahn, Sandra R Montezuma, Ching Yuan, Heidi Roehrich, Deborah A Ferrington, James R Dutton
ABSTRACT

Fidelity in pluripotent stem cell differentiation protocols is necessary for the therapeutic and commercial use of cells derived from embryonic and induced pluripotent stem cells. Recent advances in stem cell technology, especially the widespread availability of a range of chemically defined media, substrates and differentiation components, now allow the design and implementation of fully defined derivation and differentiation protocols intended for replication across multiple research and manufacturing locations. In this report we present an application of these criteria to the generation of retinal pigmented epithelium from iPSCs derived from the conjunctiva of donors with and without age related macular degeneration. Primary conjunctival cells from human donors aged 70-85 years were reprogrammed to derive multiple iPSC lines that were differentiated into functional RPE using a rapid and defined differentiation protocol. The combination of defined iPSC derivation and culture with a defined RPE differentiation protocol, reproducibly generated functional RPE from each donor without requiring protocol adjustments for each individual. This successful validation of a standardized, iPSC derivation and RPE differentiation process demonstrates a practical approach for applications requiring the cost-effective generation of RPE from multiple individuals such as drug testing, population studies or for therapies requiring patient-specific RPE derivations. In addition, conjunctival cells are identified as a practical source of somatic cells for deriving iPSCs from elderly individuals.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Triton X-100, laboratory grade
Sigma-Aldrich
Accumax solution, sterile-filtered, suitable for cell culture
Sigma-Aldrich
Anti-TRA-1-81 Antibody, clone TRA-1-81, clone TRA-1-81, Chemicon®, from mouse
Sigma-Aldrich
Anti-Oct-4 Antibody, clone 10H11.2, clone 10H11.2, Chemicon®, from mouse