Our experimental approach demonstrates for the first time, that although most genes present in the two main cell types of the placenta (cytotrophoblasts and fibroblasts) exhibit similar promoter methylation patterns, some specific genes show differential promoter methylation. Such differences may in part explain the underlying biologic basis of cell-specific differentiation within the developing human placenta.
We used serial enzymatic digestion followed by Ficoll gradient centrifugation and magnetic bead separation, to maximize the separation of the two main proliferating cell types within placental villi in the second trimester, namely villous cytotrophoblasts and mesenchymal fibroblasts. Most investigators use some, but not all, of these steps to obtain villous cytotrophoblast cells for trophoblast differentiation studies.13
To the best of our knowledge, there are no published reports on the separation of large quantities of fibroblasts from placenta. These cells are more difficult to separate from the extracellular matrix, but they exhibit significant growth potential in culture. Therefore only small amounts are necessary to generate large colonies. We did not culture fibroblasts because cell culture conditions could potentially affect epigenetic marks and become a source of bias.24
Our immunocytochemical (cytokeratin-7/vimentin antibodies) assessment of the remaining samples showed effective purification of villous cytotrophoblasts, but only enrichment of the fibroblast fraction, despite the addition of a double immunomagnetic bead separation step. Despite the protocol limitations, our exploratory data do demonstrate epigenetic differences between these two cell types. Although the data do not permit precise quantification of the methylation differences between cytotrophoblasts and fibroblasts, they do support the hypothesis of cell-type specific gene regulation by differential DNA methylation.
Genes that demonstrate cell-type specific differences in methylation in our study included hCG genes and tumor suppressor genes. Since hCG is produced mainly in cytotrophoblasts,25
the lower promoter methylation levels of several genes coding for the hCG β subunit is consistent with epigenetically regulated cell specific expression.
Moreover, several aspects of early placental development, especially in extra-villous trophoblast cells, rely on rapid cell proliferation and invasion that is analogous to tumor behavior.22–24
Promoter regions of tumor suppressor genes have been found to be methylated in association with reduced expression in tumors.26,27
Some of these same promoter regions have also been reported to be methylated in placenta.19–21,28
On the other hand, most normal tissues exhibit low methylation in these genomic regions, suggesting some molecular methylation signatures of tumor suppressor genes are shared by tumors and placenta. If this epigenetic signature is driven by the placental cell type with invasive behavior, it would be expected that methylation of tumor suppressor genes in cytotrophoblasts should be higher than in fibroblasts. In our list of selected probes with higher methylation in cytotrophoblasts, there was enrichment for CpG sites mapping to tumor suppressor gene promoter regions. Our data therefore suggest that repression, by promoter methylation, of tumor suppressor genes in cytotrophoblasts, facilitates extensive proliferation of this cell type within immature intermediate villi. Subsequent demethylation of these genes is one candidate pathway by which the third trimester placenta could reduce the proliferative potential of villous cytotrophoblasts near term.
There are several positive outcomes from our research. We have improved on previous efforts to separate populations of uncultured fibroblast cells from placenta. Further, we have generated a list of genomic regions for which there are clear cell type-specific DNA methylation differences. This list is likely to represent only a subset of such differences. Other differentially methylated probes may have been masked by the suboptimal fibroblast purification. New microarrays with better probe coverage of the genome will certainly extend this list.
In summary, we analyzed the DNA methylation patterns of cytotrophoblast and fibroblast enriched placental cell fractions for global and specific differences. Although the specific cell types do not demonstrate genome wide differentiation in DNA methylation, we identified several genomic regions for which the methylation pattern is significantly different, likely representing true biological differences between cytotrophoblasts and fibroblasts. The identification of such differences underscores the importance of epigenetic mechanisms in normal human placental development and provides a useful resource for the interpretation of placental DNA methylation data. Importantly, these data support the use of placental villi for epigenetic studies targeting cytotrophoblast cells. Further, they provide a framework from which to propose hypotheses regarding critical epigenetic modifications that drive normal placental development as well as important epigenetic alterations that could cause placental related diseases such as preeclampsia and intrauterine growth restriction. In severe placental insufficiency syndromes, complicated by severe early-onset preeclampsia or intrauterine growth restriction, histologic evidence of defective differentiation of the extravillous trophoblast or of the chorionic villi (comprising stroma, endothelium and a covering of villous trophoblast) has been found.29
Defective epigenetic regulation affecting the differentiation of these different cell types may be a unifying mechanism that could illuminate currently unresolved questions about placental development.30
The normal function of these tissues likely depends not only on the correct epigenetic profiles but also the normal proportion of the different cell types carrying each profile. Variations in the cell-specific profiles or the proportions of specific cell types among samples of the same tissue could result in abnormal development and/or function of this tissue. We undertook a study of cell specific methylation profiles in the placenta with the expectation that these data could provide a framework for understanding epigenetic regulation and cell-type specificity in the normal placentas. Such data would be expected to generate hypothesis-driven research into the epigenetic basis of normal placental development across gestation as well as the role of epigenetic alteration in diseases of the placenta and the fetus.