Trophoblast Differentiation in Embryoid Bodies Derived from Human Embryonic Stem Cells

Abstract

Trophoblast differentiation and early placental development are essential for the establishment of pregnancy, yet these critical events are not readily investigated in human pregnancy. We used embryoid bodies (EBs) prepared from human embryonic stem (hES) cells as an in vitro model of early human development. The levels of human chorionic gonadotropin (hCG), progesterone, and estradiol-17beta in medium from hES cell-derived EBs grown in suspension culture for 1 wk were higher than unconditioned culture medium or medium from undifferentiated hES cells or spontaneously differentiated hES cell colonies. EBs were explanted into Matrigel (MG) "rafts" and cultured for up to 53 d. During the first 7-10 d of three-dimensional growth in MG, small protrusions appeared on the outer surface of EBs, some of which subsequently extended into multicellular outgrowths. The secretion of hCG, progesterone, and estradiol-17beta began to increase on approximately d 20 of MG culture and remained dramatically elevated over the next 30 d. EBs maintained in suspension culture failed to demonstrate this elevation in hormone secretion. Suspension-cultured and MG-embedded EBs exhibited widespread expression of cytokeratins 7/8, demonstrating extensive epithelial differentiation as well as consistent hCG expression. We propose that hES cell-derived EBs may be a useful model for investigation of human trophoblast differentiation and placental morphogenesis.

Low O 2 tensions and the prevention of differentiation of hES cells

“…4 A and B). A rather similar phenomenon of disorganized differentiation occurs in EBs derived from hES cells (36). This pattern of differentiation is distinct from that occurring when hES colonies are treated with bone morphogenetic protein-4, which causes a synchronized and larger scale differentiation of trophoblast-like cells at the periphery of the colony (37).…”

“…hES cells can readily be induced to form EBs. To explore the effects of hypoxia on this conversion, H1 cells (Ϸ2 ϫ 10 6 cells per well), which had been maintained under hypoxic conditions for eight successive passages (passages [33][34][35][36][37][38][39][40], were compared for their ability to form EBs with an identical number of cells that had been maintained under normoxic conditions for a comparable time period. During the early stages of culture, both groups of cells formed dense, simple EBs (Fig.…”

Activin/Nodal Signaling Switches the Terminal Fate of Human Embryonic Stem Cell-derived Trophoblasts

“…Bisulfite reaction was carried out using the EpiTect bisulfite kit (Qiagen). The ELF5-2b promoter region was isolated using nested PCR as described previously (2). The primers used were as follows: first PCR forward, GGAA-ATGATGGATATTGAATTTGA; first PCR reverse, CAATA-AAAATAAAAACACCTATAACC; 2nd PCR forward, GAGG-TTTTAATATTGGGTTTATAATG; 2nd PCR reverse, ATAAATAACACCTACAAACAAATCC.…”

Embryonic stem cells as models of trophoblast differentiation: progress, opportunities, and limitations

“…Microarray analysis revealed that when compared with undifferentiated cells, many of the most highly up-regulated genes were associated with trophoblast function, particularly transcription factors (chiefly defined in mouse development), and endocrine markers of human trophoblast differentiation, including hCG, and enzymes important for steroid hormone synthesis (Gerami-Naini et al 2004). Interestingly, it has also been reported that the effect of BMP or related ligands on hESCs is very cell line dependent.…”

“…A second approach with H1 and H9 hESCs for trophoblast differentiation from hESCs was identified with subsequent studies (Gerami-Naini et al 2004). Reasoning that mimicking the spherical configuration of the preimplantation embryo with pluripotent hESCs may initiate signals parallel to those acting during preimplantation embryo development, we directed embryoid body (EB) formation by enzymatic release of undifferentiated ESC colonies from culture plates, and maintained them in suspension culture in bacterial grade culture dishes.…”