Studies on the Function of Stromal Cell for Embryonic Stem Cell Growth and Decidual Differentiation

Abstract

Stromal cells are connective cells that provide structure and play supportive roles for functional cells residing within specific tissues. A long history of research has uncovered key biological roles for many of the growth factors and cytokines secreted by stromal cells. In the present study, we attempted to develop a technique for using bone marrow stromal cells (BMSCs) as feeder cells to improve the culture efficiency of embryonic stem cells (ESCs). B6CBAF1 blastocysts or ESCs stored after their establishment were seeded onto a feeder layer of either SCA-1+/CD45-/CD11b-BMSCs or mouse embryonic fibroblasts (MEFs). Feeder cell activity that promoted ESC establishment and maintenance from blastocysts did not differ significantly when using either BMSC or MEF feeders. However, after culturing of inner cell mass cells of blastocysts, the highest efficiency of colony formation was observed with the BMSC line, which secreted the largest quantities of leukemia inhibitory factor (LIF). Exogenous LIF was essential for ESC establishment with BMSC feeders, but not for ESC maintenance. Neither changes in stem cell-specific gene expression nor increases in stem cell aneuploidy was detected after the use of BMSC feeders. We therefore conclude that BMSCs can be utilized as ESC feeders, a method that improves culture efficiency. Stromal cells can also differentiate into specific types of functional somatic cells. In this study, we focused on understanding the differentiation process of endometrial stromal cells into decidual cells. Decidualization is a crucial cellular change required for successful embryo implantation and the maintenance of pregnancy. During this process, endometrial stromal cells differentiate into decidual cells in response to a number of ovarian steroid hormones in early pregnancy. The extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) are known to regulate cell proliferation and apoptosis in multiple cell types, including uterine endometrial cells. Aberrant activation of ERK1/2 has recently been implicated in the pathological processes of endometriosis and endometrial cancer. However, the exact function of ERK1/2 signaling during implantation and decidualization is poorly understood yet. In order to determine the role and regulation of ERK1/2 signaling during the implantation and decidualization processes, we examined the expression profiles of ERK1/2 signaling molecules in the mouse uterus during early pregnancy using immunohistochemistry and qPCR techniques. Interestingly, levels of phospho-ERK1/2 were highest within decidual cells located at the implantation sites, while expression levels of ERK1/2 target genes were also significantly increased at implantation sites compared to both inter-implantation sites and during the pre-implantation period. To determine the role of ERK1/2 signaling in human endometrial decidualization, we examined levels of phospho-ERK1/2 in cultured human endometrial stromal cells during in vitro decidualization. Levels of phospho-ERK1/2 were markedly increased during in vitro decidualization. Treatment with the ERK1/2 inhibitor U0126 significantly decreased the expression of the known decidualization marker genes, IGFBP1 and PRL as well as inhibited the induction of known ERK1/2 target genes

FOS, MSK1, STAT1, and STAT3. Additionally, phosphorylation of CCAAT/enhancer binding protein β (C/EBPβ), a protein previously shown to be critical for decidualization, was significantly reduced in this model. These results suggest that ERK1/2 signaling is required for successful decidualization in mice as well as human endometrial stromal cells and implicates C/EBPβ as a downstream target of ERK1/2.