p63 Inhibits Extravillous Trophoblast Migration and Maintains Cells in a Cytotrophoblast Stem Cell-Like State

Abstract

Proper differentiation of placental epithelial cells, called trophoblast, is required for implantation. Early during placentation, trophoblast cell columns help anchor the developing embryo in the uterine wall. Although proximally continuous with villous cytotrophoblast (CTB) distally, these cells differentiate into invasive extravillous trophoblast. We previously reported that p63, a p53 family member, is highly expressed in proliferative villous CTB and required for induction of the trophoblast lineage in human pluripotent stem cells. We now further explore its function in human trophoblast by using both primary CTB from the early placenta and established trophoblast cell lines. We show that p63 is expressed in epidermal growth factor receptor-positive CTB and that its expression decreases with differentiation into HLA-G(+) extravillous trophoblast. In trophoblast cell lines, p63 is expressed in JEG3 cells but absent from HTR8 cells. Overexpression of p63 in both cell lines enhances cell proliferation and significantly reduces cell migration; conversely, down-regulation of p63 in JEG3 cells reduces cell proliferation and restores cell migration. Analysis of epithelial-to-mesenchymal transition, cell adhesion, and matrix degradation pathways shows that p63 blocks epithelial-to-mesenchymal transition, promotes a CTB-specific cell adhesion profile, and inhibits expression of matrix metalloproteinases. Taken together, these data show that p63 maintains the proliferative CTB state, at least partially through regulation of epithelial-to-mesenchymal transition, cell adhesion, and matrix degradation pathways.

Human pluripotent stem cells as a model of trophoblast differentiation in both normal development and disease

“…These data indicate that, with only 4 d of low-dose BMP4 treatment in this defined medium, hPSCs uniformly assume a CTB stem-like phenotype. Interestingly, as in isolated primary CTBs but unlike p63 + CTBs in vivo (26), hPSCderived CTBs were not highly proliferative and could not be propagated under these culture conditions (Fig. S2).…”

“…To confirm the utility of hPSCs for modeling trophoblast differentiation, we instead asked whether these cells can recapitulate functional phenotypes of primary trophoblasts during both normal development and disease. We previously have identified p63, a member of the p53 family of nuclear proteins, as a marker specific to proliferative cytotrophoblasts (CTBs) in the human placenta (21,25,26). We now have identified a subpopulation of CTBs in the early human placenta that are double-positive for p63 and CDX2; this CTB subpopulation is greatly reduced in the second trimester and is temporally associated with the loss of bipotential differentiation of CTBs (27).…”

Notch1 controls development of the extravillous trophoblast lineage in the human placenta

“…On the other hand, suppression of cell fusion and downregulation of p63/TEAD4 by N1ICD could negatively affect vCTB self-renewal and thereby trigger CCT formation and EVT differentiation. Along those lines, silencing of p63 in JEG-3 choriocarcinoma cells was shown to increase migration, a characteristic feature of invasive EVTs (28).…”

“…Descriptive analyses in first trimester tissues and trophoblast cell models have been performed to gain insights into the expression patterns and epigenetic profiles of some of these genes (37,40,48,49). However, few of them have been functionally examined by using choriocarcinoma cells as surrogate for human primary trophoblasts (28,50). To extend our present knowledge, we herein analyzed expression of key regulators in the two purified CTB progenitor subtypes and performed functional analyses in these cells.…”

“…S8). In contrast, ΔNp63 and TEAD4, controlling self-renewal of vCTBs and murine trophoblast stem cells (28,29), respectively, were suppressed ( Fig. 5 D and F and SI Appendix, Fig.…”