Abstract
The first differentiation event in mammalian embryos is the formation of the trophectoderm, which is the progenitor of the outer epithelial components of the placenta, and which supports the fetus during the intrauterine life. However, the epigenetic and paracrine controls at work in trophectoderm differentiation are still to be fully elucidated and the creation of dedicated in vitro models is desirable to increase our understanding. Here we propose a novel approach based on the epigenetic conversion of adult dermal fibroblasts into trophoblast-like cells. The method combines the use of epigenetic erasing with an ad hoc differentiation protocol. Dermal fibroblasts are erased with 5-azacytidine (5-aza-CR) that confers cells a transient high plasticity state. They are then readdressed toward the trophoblast (TR) phenotype, using MEF conditioned medium, supplemented with bone morphogenetic protein 4 (BMP4) and inhibitors of the Activin/Nodal and FGF2 signaling pathways in low O2 conditions. The method here described allows the generation of TR-like cells from easily accessible material, such as dermal fibroblasts, that are very simply propagated in vitro. Furthermore, the strategy proposed is free of genetic modifications that make cells prone to instability and transformation. The TR model obtained may also find useful application in order to better characterize embryo implantation mechanisms and developmental disorders based on TR defects.
Highlights
Reproduction in eutherian mammals strictly depends on the placenta that is required for both maintenance of pregnancy and fetal development
Fibroblasts obtained from skin biopsies grew out of the original explants within 6 days of culture (Figure 1A, left panel) and formed a monolayer (Figure 1A, right panel)
These features closely resemble those previously identified in embryonic stem cells (ESCs) [43, 44] and induced pluripotent cells [45], indicating the acquisition of morphological characteristics distinctive of a high plasticity phenotype
Summary
Reproduction in eutherian mammals strictly depends on the placenta that is required for both maintenance of pregnancy and fetal development. The placenta is a transient extraembryonic organ that nourishes and supports the fetus during intrauterine life. Trophoblast (TR) cells represent the major placental cell type and are highly specialized in each of the above mentioned functions [2, 3]. These cells originate from a simple epithelial sheet that surrounds the blastocoel and the inner cell mass (ICM) at the blastocyst stage, namely the embryonic trophectoderm (TE). Comparative studies on placental morphology revealed that TR tissue can range from a single layer of cells to a complex multilayered organization, based on the different types of placentation [4]
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