Transcriptomic signature of trophoblast differentiation in a human embryonic stem cell model.

Identification of genes involved in trophoblast differentiation is of great interest in understanding cellular and molecular mechanisms involved in placental development and is relevant clinically to fetal development, fertility, and maternal health. Herein, we investigated differentiation of human embryonic stem cells (hESCs) down the trophoblast lineage by culture with bone morphogenetic protein 4 (BMP4) over a 10-day period. Within 2 days, the stemness markers POU5F1 and NANOG were markedly down-regulated, followed temporally by up-regulation of the CDX2, KRT7, HLA-G, ID2, CGA, and CGB trophoblast markers. To understand, on a global scale, changes in the transcriptome during the differentiation of hESCs down the trophoblast lineage, a large-scale microarray analysis was performed. Through whole-genome analysis, more than 3800 genes displayed statistically significant and 2-fold or greater changes in expression during the time course. Of those genes that showed the largest increases, many were involved in processes associated with trophoblast biology; however, novel genes were also identified. Some of them are hypothesized to be associated mainly with extracellular matrix remodeling (e.g., NID2) and cell migration and invasion (e.g., RAB25). Using Ingenuity pathways analysis software to identify signaling pathways involved in trophoblast differentiation or function, we discovered that many genes are involved in WNT/beta-catenin, ERK/MAPK, NFKB, and calcium signaling pathways, suggesting potential roles for these families in trophoblast development. This work provides an in vitro functional genomic model with which to identify genes involved in trophoblast development.

Developmental competence of immature and failed/abnormally fertilized human oocytes in nuclear transfer.

Somatic cell nuclear transfer holds great promise for basic studies of reprogramming human somatic cells and for the potential development of novel cell-based therapeutics. The aim of this study was to examine experimental aspects of human nuclear transfer via use of an abundant source of oocytes, those that are routinely discarded from assisted reproduction clinics. The results suggest and reinforce several findings based on the analysis of multiple parameters: first, it was observed that supplementation of commercial culture media with hormones promoted embryo development after parthenogenetic activation. Second, the use of the chemical activation reagent puromycin resulted in significant differences in cleavage rates in oocytes that were failed/abnormally fertilized after intracytoplasmic sperm injection relative to those from IVF (P < 0.05). Third, cycloheximide promoted cleavage rates >/=40% in both groups of oocytes; moreover, two blastocysts were produced following cycloheximide treatment. Finally, the use of a subset of oocytes for nuclear transfer resulted in cleaved embryos that expressed green fluorescent protein from a transgene in donor nuclei from human embryonic stem cells. In light of these results, it is suggested that the discarded oocytes can be used to investigate new human nuclear transfer protocols for embryonic stem cell derivation.