Abstract
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 (TSCs) as an in vitro platform for future assessment of primate trophoblast development and function. Macaque TSC lines were generated by isolating first and second trimester placental villous cytotrophoblasts followed by culture in TSC medium to maintain cellular proliferation. TSCs grew as mononuclear colonies, whereas upon induction of syncytiotrophoblast (ST) differentiation multinuclear structures appeared, indicative of syncytium formation. Chorionic gonadotropin secretion was > 4000-fold higher in ST culture media compared to TSC media. The secretion of chorionic gonadotropin by TSC-derived ST reflects a reprogramming of macaque TSCs to an earlier pregnancy phenotype. Characteristic trophoblast hallmarks were defined in TSCs and ST including expression of C19MC miRNAs and the macaque placental nonclassical MHC class I molecule, Mamu-AG. Extravillous trophoblasts (EVTs) were derived that express macaque EVT markers Mamu-AG and CD56, and also secrete high levels of MMP2. Our analyses of macaque TSCs suggests that these cells represent a proliferative, self-renewing population capable of differentiating to STs and EVTs in vitro thereby establishing an experimental model of primate placentation.
Highlights
Nonhuman primates are excellent models for studying human placentation as experimental manipulations in vitro can be translated to in vivo pregnancy
It is postulated that a trophoblast stem cell (TSC) niche resides in the early human first trimester p lacenta[5,6,7,8], where these cells are a proliferative, less differentiated progenitor cell population that gives rise to both villous cytotrophoblasts that fuse to form the syncytia as well as extravillous trophoblast (EVT) progenitor cells at the cell column tips[9]
The present study demonstrates the derivation of macaque TSCs that express key trophoblast features, and are capable of continued proliferation and can be differentiated to STs and putative EVTs
Summary
Nonhuman primates are excellent models for studying human placentation as experimental manipulations in vitro can be translated to in vivo pregnancy. Our analyses of macaque TSCs suggests that these cells represent a proliferative, self-renewing population capable of differentiating to STs and EVTs in vitro thereby establishing an experimental model of primate placentation. TSCs can be derived by culturing extraembryonic ectoderm in the presence of FGF4, heparin and mouse embryonic fibroblasts (or supplementation of TGF-ß1/Activin) and can readily serve as an in vitro experimental platform[19] These methods have not been successfully adapted to human TSC derivation[20], suggestive that FGF4 is dispensable for human TSC renewal. Under differentiation-specific culture media formulations TSCs could be differentiated to either chorionic gonadotropin (CG)-secreting syncytia or HLA-G positive EVTs. The derivation of human trophoblast organoid cultures was recently described by Haider et al.[22] and Turco et al.[23] In both studies, the organoids formed mononuclear trophoblasts on the outer periphery with syncytia and lacunae-like structures within the center. The human TSC media components vary slightly across these studies, collectively they have demonstrated that Wnt activation, EGF signaling, and inhibition of TGF-ß are essential for maintaining proliferation of human trophoblasts in culture
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