Trophoblast differentiation, invasion and hormone secretion in a three-dimensional in vitro implantation model with rhesus monkey embryos.

BACKGROUND: The initiation of primate embryo invasion into the endometrium and the formation of the placenta from trophoblasts, fetal mesenchyme, and vascular components are essential for the establishment of a successful pregnancy. The mechanisms which direct morphogenesis of the chorionic villi, and the interactions between trophectoderm-derived trophoblasts and the fetal mesenchyme to direct these processes during placentation are not well understood due to a dearth of systems to examine and manipulate real-time primate implantation. Here we describe an in vitro three-dimensional (3-D) model to study implantation which utilized IVF-generated rhesus monkey embryos cultured in a Matrigel explant system. METHODS: Blastocyst stage embryos were embedded in a 3-D microenvironment of a Matrigel carrier and co-cultured with a feeder layer of cells generating conditioned medium. Throughout the course of embryo co-culture embryo growth and secretions were monitored. Embedded embryos were then sectioned and stained for markers of trophoblast function and differentiation. RESULTS: Signs of implantation were observed including enlargement of the embryo mass, and invasion and proliferation of trophoblast outgrowths. Expression of chorionic gonadotropin defined by immunohistochemical staining, and secretion of chorionic gonadotropin and progesterone coincident with the appearance of trophoblast outgrowths, supported the conclusion that a trophoblast cell lineage formed from implanted embryos. Positive staining for selected markers including Ki67, MHC class I, NeuN, CD31, vonWillebrand Factor and Vimentin, suggest growth and differentiation of the embryo following embedding. CONCLUSIONS: This 3-D in vitro system will facilitate further study of primate embryo biology, with potential to provide a platform for study of genes related to implantation defects and trophoblast differentiation.

Array-based assay detects genome-wide 5-mC and 5-hmC in the brains of humans, non-human primates, and mice.

BACKGROUND: Methylation on the fifth position of cytosine (5-mC) is an essential epigenetic mark that is linked to both normal neurodevelopment and neurological diseases. The recent identification of another modified form of cytosine, 5-hydroxymethylcytosine (5-hmC), in both stem cells and post-mitotic neurons, raises new questions as to the role of this base in mediating epigenetic effects. Genomic studies of these marks using model systems are limited, particularly with array-based tools, because the standard method of detecting DNA methylation cannot distinguish between 5-mC and 5-hmC and most methods have been developed to only survey the human genome. RESULTS: We show that non-human data generated using the optimization of a widely used human DNA methylation array, designed only to detect 5-mC, reproducibly distinguishes tissue types within and between chimpanzee, rhesus, and mouse, with correlations near the human DNA level (R(2) > 0.99). Genome-wide methylation analysis, using this approach, reveals 6,102 differentially methylated loci between rhesus placental and fetal tissues with pathways analysis significantly overrepresented for developmental processes. Restricting the analysis to oncogenes and tumor suppressor genes finds 76 differentially methylated loci, suggesting that rhesus placental tissue carries a cancer epigenetic signature. Similarly, adapting the assay to detect 5-hmC finds highly reproducible 5-hmC levels within human, rhesus, and mouse brain tissue that is species-specific with a hierarchical abundance among the three species (human > rhesus >> mouse). Annotation of 5-hmC with respect to gene structure reveals a significant prevalence in the 3'UTR and an association with chromatin-related ontological terms, suggesting an epigenetic feedback loop mechanism for 5-hmC. CONCLUSIONS: Together, these data show that this array-based methylation assay is generalizable to all mammals for the detection of both 5-mC and 5-hmC, greatly improving the utility of mammalian model systems to study the role of epigenetics in human health, disease, and evolution.

ACP5 (Uteroferrin): phylogeny of an ancient and conserved gene expressed in the endometrium of mammals.

Type 5 acid phosphatase (ACP5; also known as tartrate-resistant acid phosphatase or uteroferrin) is a metalloprotein secreted by the endometrial glandular epithelium of pigs, mares, sheep, and water buffalo. In this paper, we describe the phylogenetic distribution of endometrial expression of ACP5 and demonstrate that endometrial expression arose early in evolution (i.e., before divergence of prototherian and therian mammals ~166 million years ago). To determine expression of ACP5 in the pregnant endometrium, RNA was isolated from rhesus, mouse, rat, dog, sheep, cow, horse, armadillo, opossum, and duck-billed platypus. Results from RT-PCR and RNA-Seq experiments confirmed that ACP5 is expressed in all species examined. ACP5 was also demonstrated immunochemically in endometrium of rhesus, marmoset, sheep, cow, goat, and opossum. Alignment of inferred amino acid sequences shows a high conservation of ACP5 throughout speciation, with species-specific differences most extensive in the N-terminal and C-terminal regions of the protein. Analysis by Selecton indicated that most of the sites in ACP5 are undergoing purifying selection, and no sites undergoing positive selection were found. In conclusion, endometrial expression of ACP5 is a common feature in all orders of mammals and has been subjected to purifying selection. Expression of ACP5 in the uterus predates the divergence of therians and prototherians. ACP5 is an evolutionary conserved gene that likely exerts a common function important for pregnancy in mammals using a wide range of reproductive strategies.

Placental-derived mesenchyme influences chorionic gonadotropin and progesterone secretion of human embryonic stem cell-derived trophoblasts.

Studies of early placental development in humans are difficult because of limitations on experimental material availability from the perimplantation period. We used a coculture system to determine the effects of various effector cell types on trophoblast differentiation. Enhanced green fluorescent protein (EGFP)-expressing H1 human embryonic stem cells were used in co-suspension with human term placental fibroblasts (TPFs) and dermal fibroblasts (CI2F) to form combination embryoid bodies (EBs), with the goal of recapitulating placental morphogenesis through incorporation of placental mesenchymal cells. Overall, the results demonstrated that when using mesenchymal cells for EB preparation from term placentas (TPF), combination EB-derived trophoblasts secrete higher levels of human chorionic gonadotropin (hCG) and progesterone compared to EBs made without effector cells, whereas there was no effect of dermal fibroblasts. This is due to the secretory activity of the EB-derived trophoblasts and not due to the number of differentiated trophoblasts per EB, demonstrating that nontrophoblast cells of the placenta can influence trophoblast endocrine activity.

Characterization of cynomolgus and vervet monkey placental MHC class I expression: diversity of the nonhuman primate AG locus.

Nonhuman primates are important animal models for the study of the maternal immune response to implantation within the decidua. The objective of this study was to define the placental expression of major histocompatibility complex (MHC) class I molecules in the cynomolgus (Macaca fascicularis) and vervet (African green) (Chlorocebus aethiops) monkeys. Early pregnancy (d36-42) cynomolgus and vervet placentas were obtained by fetectomy and prepared for histological evaluation. A pan-MHC class I monoclonal antibody demonstrated MHC class I expression in both vervet and cynomolgus placental trophoblasts, with particularly high expression in the villous syncytium, as previously shown in the rhesus and baboon. Placental cytotrophoblasts were isolated by enzymatic dispersion and gradient centrifugation and cultured, and multicolor flow cytometry was used to phenotype cell populations. Culture of isolated villous cytotrophoblasts demonstrated that MHC class I expression was linked to syncytiotrophoblast differentiation. A monoclonal antibody against Mamu-AG, the nonclassical MHC class I homolog of HLA-G in the rhesus monkey, demonstrated intense immunostaining and cell surface expression in cynomolgus placental trophoblasts; however, staining with vervet placenta and cells was low and inconsistent. Reverse transcriptase polymerase chain reaction was used to clone MHC class I molecules expressed in cynomolgus and vervet placentas. While Mafa-AG messenger RNA (mRNA) was readily detectable in cynomolgus placental RNA and was >99% identical at the amino acid level with Mamu-AG, 7/8 Chae-AG complementary DNAs had an unusual 16 amino acid repeat in the alpha1 domain, and all clones had an unexpected absence of the early stop codon at the 3'-end of the mRNA diagnostic for rhesus, cynomolgus, and baboon AG mRNAs, as well as HLA-G. We conclude that while the vervet monkey has retained the placental expression of a primate-specific nonclassical MHC class I locus, diversity is also revealed in this locus expressed at the maternal-fetal interface, thought to participate in placental regulation of the maternal immune response to embryo implantation and pregnancy.

Characterization of decidual leukocyte populations in cynomolgus and vervet monkeys.

The objective of this study was the phenotypic and functional evaluation of decidual immune cells in the cynomolgus and vervet monkeys. Early pregnancy (days 36-42) deciduas were obtained by fetectomy for histological evaluation and decidual mononuclear leukocyte (MNL) isolation. While peripheral NK (pNK) cells in these species do not express CD56, CD56(+) NK cells were abundant in decidual samples. The majority of decidual NK (dNK) cells (>80%) had high light-scatter characteristics and were CD56(bright)CD16(+) cells with no or very low levels of natural cytotoxicity receptors (NKp46, NKp30) and NKG2A, while a minor population were small CD56(dim)CD16(-) lymphocytes also expressing less NKp46, NKp30 and NKG2A than pNK cells. All dNK cells were found to be perforin(+); however, their cytotoxic potential was low and cynomolgus dNK cells showed strongly reduced cytotoxicity against target cells compared with pNK cells. Macrophages and T cells together comprised approximately 25-30% of decidual MNL. Decidual T cells contained a higher proportion of the minor T cell subtypes (gammadeltaT cells, CD56(+) T cells) compared with peripheral blood. A subset of DC-SIGN(+) macrophages, with a distribution adjacent to areas of placental attachment in contrast to the widespread setting of general CD68(+) cells, was identified in both species. Together, these results demonstrate that the maternal-fetal interface in both cynomolgus and vervet monkeys is very rich in immune cells that have similar phenotypes to those seen in humans, indicating that both species are excellent models to study the contributions of distinct immune cell populations to pregnancy support.

Stable plasmid-based siRNA silencing of gene expression in human embryonic stem cells.

RNA interference (RNAi) using short inhibitory RNAs (siRNAs) has been widely explored for the suppression of cellular mRNA levels to investigate the function of specific genes, including gene function in differentiation and development. The establishment of human embryonic stem cell (hESC) models for differentiation of selected lineages is an area of intense interest and activity. On the basis of our previous work with stable overexpression of enhanced green fluorescent protein (EGFP) in hESC, we used plasmid vector-based siRNA expression to silence EGFP expression in stably-transfected hESC. After hygromycin selection, we derived several cell lines in which EGFP expression was significantly reduced. At the genomic DNA level, there was no difference between the two cell lines and the parental H1EGFP cell line when analyzed with quantitative PCR; however, there were significant differences among the three cell lines at the RNA and protein levels as analyzed with real-time RT-PCR and Western blotting. From these data, we conclude that the decrease in EGFP expression was caused by RNAi, not by genomic DNA loss. Down-regulation of EGFP expression was sustained through multiple passages of both siEGFP cell lines. This simple silencing system will allow novel investigations of target gene function in hESC self-renewal or differentiation, as well as differentiated function in other cell types.

Maintenance of pluripotency in human embryonic stem cells stably over-expressing enhanced green fluorescent protein.

The availability of human embryonic stem (HES) cells with a readily evaluated genetic marker such as green fluorescent protein (GFP) could facilitate a number of experimental opportunities. We constructed a novel plasmid with two elongation factor-1alpha (EF-1alpha) promoters (YPL2) to obtain a vector with mammalian promoters for simultaneous transgene expression in HES cells. An enhanced green fluorescent protein (EGFP) cDNA was inserted under the control of the first EF-1alpha promoter to construct plasmid YPL2-EGFP. The second EF1-alpha promoter was upstream of the neomycin resistance gene. H1 HES cells were transfected with YPL2-EGFP using Fugene 6. Following 100 microg/ml neomycin selection, individual colonies demonstrating stable EGFP expression were observed. After 4 months of passage under neomycin selection, the cells continued to maintain typical HES cell morphology. Undifferentiated cells showed no change in EGFP expression as determined by FACS analysis. Immunostaining demonstrated maintenance of Oct-3/4 expression in undifferentiated H1EGFP cells that was indistinguishable from wild-type HES cells. Addition of 10 ng/ml bone morphogenic protein-4 (BMP-4) to the cells provoked morphological and functional differentiation to trophoblasts, but no loss of EGFP expression. Following injection of EGFP-HES cells into immunodeficient mice, there was robust formation of teratomas that demonstrated a broad range of morphological pluripotency with widespread EGFP expression. EGFP expression was also maintained in differentiating embryoid bodies formed from EGFP-HES cells. This report demonstrates that ES cells carrying EGFP will be useful in diverse areas of embryonic stem cell research.