Derivation of macaque trophoblast stem cells

Abstract

200 words max) Nonhuman primates are excellent models for studying human placentation as experimental manipulations in vitro can be translated to in vivo pregnancy. Our objective was to develop macaque trophoblast stem cells (TSC) as an in vitro platform for future assessment of primate trophoblast development and function. Macaque TSC lines were generated by isolating first trimester placental villous cytotrophoblasts followed by culture in TSC medium to "reprogram" the cells to a proliferative state. TSCs grew as mononuclear colonies, whereas upon induction of syncytiotrophoblast (ST) differentiation multinuclear structures appeared, indicative of syncytium formation. Chorionic gonadotropin secretion was >4,000-fold higher in ST culture media compared to TSC media. Characteristic trophoblast hallmarks were defined in TSCs and ST including expression of C19MC miRNAs and macaque placental nonclassical MHC class I molecule, Mamu-AG. TSC differentiation to extravillous trophoblasts (EVTs) with or without the ALK-5 inhibitor A83-01 resulted in differing morphologies but similar expression of Mamu-AG and CD56 as assessed by flow cytometry, hence further refinement of relevant EVT markers is needed. Our preliminary characterization of macaque TSCs suggests that these cells represent a proliferative, self-renewing TSC population capable of differentiating to STs in vitro thereby establishing an experimental model of primate placentation.