187 Phenotype switch of human fibroblasts into trophoblastic cells

Abstract

The first differentiation event in mammalian embryos is the formation of the trophectoderm, which is crucial for implantation of the blastocyst and gives rise to specialised populations of trophoblast cells in the definitive placenta. However, our understanding of these early differentiation events is limited, particularly in humans, because of ethical and legal restrictions on the isolation and manipulation of human embryogenesis. Here we describe experiments aimed at converting human fibroblasts into trophoblastic cells, using 5-azacytidine (5-aza-CR) to erase the original phenotype and a cocktail containing bone morphogenic protein 4 (BMP4) and inhibitors of the activin/nodal/ERK signalling pathways, to drive trophoblastic differentiation. The method required 3 main steps: (1) preparation of mouse embryonic fibroblasts (MEF) monolayer, and culture and collection of MEF conditioned medium (CM); (2) culture of human fibroblasts, obtained from a skin biopsy, and epigenetic erasure with 5-aza-CR for 18 h; (3) use of CM, with BMP4 (50 ng mL⁻¹), PD0325901 (1 µM), CHIR99021 (1 µM), and PD173074 (0.1 µM) to differentiate erased human fibroblasts to trophoblast cells. Morphology changes were monitored along the process. In the initial phase of differentiation, a mixture of cell types appeared, including small cells growing in clusters and giant cells possessing very large nuclei. After 2 weeks of culture, cells displayed a distinct trophoblast-like morphology and showed the presence of typical clustering/lacunae monolayer patterning. Cells continued to proliferate and maintained a normal karyotype. BMP4-mediated differentiation was also assessed by quantitative RT-PCR using primers specific for 2 trophoblast markers: keratin 7 (KRT7) and caudal type homeobox 2 (CDX2), that are absent in the original fibroblasts. Their expression appeared by Day 3 of induction and was strong and steady throughout the process, confirming that the acquisition of a trophoblast-like morphology was supported by the activation of trophoblast-specific gene transcription. The results demonstrate the possibility of obtaining trophoblast-like cells through the conversion of human fibroblasts and confirm the involvement of BMP4-together with the inhibition of the activin/nodal/ERK signalling pathway-to activate early trophoblast differentiation in vitro. The challenge for future experiments will be to determine the precise role of BMP signals in human trophoblast definition in vivo, which, to our knowledge, has not been elucidated yet. The method here described is efficient and reproducible and has the advantage of utilising easily accessible cells as a starting population. It can be used for a variety of applications, including drug discovery and stem cell research, as well as to implement studies on the pathogenesis of developmental disorders with trophoblast defects.

This research was supported by Carraresi Foundation. Authors are members of the COST Actions CA16119 and CM1406.