Maintenance of mouse trophoblast stem cells in KSR-based medium allows conventional 3D culture.

Mouse trophoblast stem cells (TSCs) can differentiate into trophoblast cells, which constitute the placenta. Under conventional culture conditions, in a medium supplemented with 20% fetal bovine serum (FBS), fibroblast growth factor 4 (FGF4), and heparin and in the presence of mouse embryonic fibroblast cells (MEFs) as feeder cells, TSCs maintain their undifferentiated, proliferative status. MEFs can be replaced by a 70% MEF-conditioned medium (MEF-CM) or by TGF-ß/activin A. To find out if KnockOutTM Serum Replacement (KSR) can replace FBS for TSC maintenance, we cultured mouse TSCs in KSR-based, FBS-free medium and investigated their proliferation capacity, stemness, and differentiation potential. The results indicated that fibronectin, vitronectin, or laminin coating was necessary for adhesion of TSCs under KSR-based conditions but not for their survival or proliferation. While the presence of FGF4, heparin, and activin A was not sufficient to support the proliferation of TSCs, the addition of a pan-retinoic acid receptor inverse agonist and a ROCK-inhibitor yielded a proliferation rate comparable to that obtained under the conventional FBS-based conditions. TSCs cultured under the KSR-based conditions had a gene expression and DNA methylation profile characteristic of TSCs and exhibited a differentiation potential. Moreover, under KSR-based conditions, we could obtain a suspension culture of TSCs using extracellular matrix (ECM) coating-free dishes. Thus, we have established here, KSR-based culture conditions for the maintenance of TSCs, which should be useful for future studies.

DNA methylation and its role in the trophoblast cell lineage.

DNA methylation functions as cellular memory beyond generations of cells and is involved in many biological processes. Because of its relatively stable nature compared with the transcriptome, the DNA methylation profile of cells can also be used to evaluate developmental similarity and cellular phenotypes. Recent insights into 5-hydroxymethylcytosine have started to reshape our view of the epigenetic regulation of mammalian development. Both global DNA methylation and hydroxymethylation levels change dynamically during preimplantation embryogenesis. It is known that DNA methylation plays an essential role in embryonic cell fate restriction, whereas its role in trophoblast development requires further research. Two distinct blastocyst-derived stem cell lines, embryonic stem (ES) cells and trophoblast stem (TS) cells, are used to study the epigenetic mechanisms underlying cell lineage maintenance and the regulation of cell differentiation. Such studies will allow us to understand the details of the epigenetic landscape of trophoblast development, which should offer valuable information for managing pregnancy-related diseases in humans.

Trophoblast-specific DNA methylation occurs after the segregation of the trophectoderm and inner cell mass in the mouse periimplantation embryo.

The first cell differentiation in the mammalian development separates the trophoblast and embryonic cell lineages, resulting in the formation of the trophectoderm (TE) and inner cell mass (ICM) in blastocysts. Although a lower level of global DNA methylation in the genome of the TE compared with ICM has been suggested, the dynamics of the DNA methylation profile during TE/ICM differentiation has not been elucidated. To address this issue, first we identified tissue-dependent and differentially methylated regions (T-DMRs) between trophoblast stem (TS) and embryonic stem (ES) cells. Most of these TS-ES T-DMRs were also methylated differentially between trophoblast and embryonic tissues of embryonic day (E) 6.5 mouse embryos. Furthermore, we found that the human genomic regions homologous to mouse TS-ES T-DMRs were methylated differentially between human placental tissues and ES cells. Collectively, we defined them as cell-lineage-based T-DMRs between trophoblast and embryonic cell lineages (T-E T-DMRs). Then, we examined TE and ICM cells isolated from mouse E3.5 blastocysts. Interestingly, all T-DMRs examined, including the Elf5, Pou5f1 and Nanog loci, were in the nearly unmethylated status in both TE and ICM and exhibited no differences. The present results suggest that the establishment of DNA methylation profiles specific to each cell lineage follows the first morphological specification. Together with previous reports on asymmetry of histone modifications between TE and ICM, the results of the current study imply that histone modifications function as landmarks for setting up cell-lineage-specific differential DNA methylation profiles.

Bridging sequence diversity and tissue-specific expression by DNA methylation in genes of the mouse prolactin superfamily.

Much of the DNA in genomes is organized within gene families and hierarchies of gene superfamilies. DNA methylation is the main epigenetic event involved in gene silencing and genome stability. In the present study, we analyzed the DNA methylation status of the prolactin (PRL) superfamily to obtain insight into its tissue-specific expression and the evolution of its sequence diversity. The PRL superfamily in mice consists of two dozen members, which are expressed in a tissue-specific manner. The genes in this family have CpG-less sequences, and they are located within a 1-Mb region as a gene cluster on chromosome 13. We tentatively grouped the family into several gene clusters, depending on location and gene orientation. We found that all the members had tissue-dependent differentially methylated regions (T-DMRs) around the transcription start site. The T-DMRs are hypermethylated in nonexpressing tissues and hypomethylated in expressing cells, supporting the idea that the expression of the PRL superfamily genes is subject to epigenetic regulation. Interestingly, the DNA methylation patterns of T-DMRs are shared within a cluster, while the patterns are different among the clusters. Finally, we reconstituted the nucleotide sequences of T-DMRs by converting TpG to CpG based on the consideration of a possible conversion of 5-methylcytosine to thymine by spontaneous deamination during the evolutionary process. On the phylogenic tree, the reconstituted sequences were well matched with the DNA methylation pattern of T-DMR and orientation. Our study suggests that DNA methylation is involved in tissue-specific expression and sequence diversity during evolution.