Title: Rhesus macaque iPSCs for in vitro modeling of genetic frontotemporal dementia
Abstract: The autosomal dominant MAPT R406W mutation has been linked to frontotemporal dementia (FTD). At the Wisconsin National Primate Research Center, we have identified a family of rhesus macaques carrying MAPT R406W. Induced pluripotent stem cells (iPSCs) derived from these macaques present a unique opportunity for in vitro modeling; however, methods for rhesus iPSC (RhiPSC) derivation are not as robust as human iPSC derivation. The goal of this study was to To develop a reproducible method for RhiPSC generation to support in vitro modeling of MAPT R406W FTD. Skin-derived fibroblasts were collected from a MAPT wild type (1.8 years old, male) and a R406W (4.6 years old, male) monkey. Optimization of the fibroblast derivation included identifying a biopsy digestion media (hi-glucose DMEM, FBS, collagenase I, DNase I) and digestion time (overnight) to maximize the number of low passage fibroblasts for reprogramming. RhiPSC derivation with Sendai vs. oriP/EBNA1 vectors identified the latter as the most efficient. Electroporation conditions for plasmid delivery were optimized for each fibroblast line to produce the highest transduction efficiency and cell survival after 48 hours. Two clones per RhiPSC line were selected for expansion and characterization. RhiPSCs were best maintained on mouse embryonic fibroblasts (compared to Matrigel, vitronectin, or laminin-52l) in a commercial media supplemented with 1uM IWR1 and 0.5uM CHIR99021 (compared to E12+IWR1 media). In these conditions, each clone expressed OCT3/4, SOX2, and NANOG, were cytogenetically normal, and formed teratomas containing all three germ lineages following injection to immunocompromised mice. Neuroprogenitors were generated using a monolayer protocol and expressed PAX6 and NESTIN after 21 days of differentiation. Overall, we have established a reliable method for deriving RhiPSCs. The MAPT R406W RhiPSCs will be a critical resource for evaluating the molecular underpinnings of the mutation and tau-related neurodegeneration across primate species.
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