Isolation and characterisation of a novel trophoblast side-population from first trimester placentae

Abstract

The placenta is responsible for all nutrient and gas exchange between mother and baby during pregnancy. The differentiation of specialised placental epithelial cells called trophoblasts is essential for placental function, but we understand little about how these populations arise. Mouse trophoblast stem cells have allowed us to understand many of the factors that regulate murine trophoblast lineage development, but the human placenta is anatomically very different from the mouse, and it is imperative to isolate a human trophoblast stem cell to understand human placental development. Here we have developed a novel methodology to isolate a Hoechst side-population of trophoblasts from early gestation placentae and compared their transcriptome to differentiated trophoblast populations (cytotrophoblasts and extravillous trophoblasts) using microarray technology. Side-population trophoblasts clustered as a transcriptomically distinct population but were more closely related to cytotrophoblasts than extravillous trophoblasts. Side-population trophoblasts up-regulated a number of genes characteristic of trophectoderm and murine trophoblast stem cells in comparison to cytotrophoblasts or extravillous trophoblasts and could be distinguished from both of these more mature populations by a unique set of 22 up-regulated genes, which were enriched for morphogenesis and organ development and the regulation of growth functions. Cells expressing two of these genes (LAMA2 and COL6A3) were distributed throughout the cytotrophoblast layer at the trophoblast/ mesenchymal interface. Comparisons to previously published trophoblast progenitor populations suggest that the side-population trophoblasts isolated in this work are a novel human trophoblast population. Future work will determine whether these cells exhibit functional progenitor/stem cell attributes. Reproduction (2015) 150 449-462

Development of the human placenta

“…While a number of trophoblast cell lines exist, a major problem with using these has been the lack of consensus about how to define the human trophoblast in vitro, with a wide range of features being used in each case (King et al, 2000a). This is the issue for the various cell lines that have been derived from first-trimester or term placentas, so their identity is still not clear (Feng et al, 2005;Genbacev et al, 2011Genbacev et al, , 2016Graham et al, 1993;James et al, 2007James et al, , 2015Perez-Garcia et al, 2018;Straszewski-Chavez et al, 2009;Takao et al, 2011;Zdravkovic et al, 2015). Care must be taken, as contaminating maternal epithelial and fetal mesenchymal cells can outgrow trophoblast isolated from the placenta (Heazlewood et al, 2014;Turco et al, 2018).…”

Human trophoblast stem cells: Real or not real?

“…Based on their gene expression profile, the side population cells were more closely related to CTB than EVT, and their proportion did not change significantly between 6 and 12 weeks of gestation [35]. Interestingly, extracellular matrix (ECM)-related genes were significantly enriched within these side population cells [35], indicating that the ECM likely plays a crucial role for establishment and maintenance of this potential stem cell niche. Additional studies are necessary to determine if these side population cells are truly multipotent TSC.…”

Human trophoblasts are primarily distinguished from somatic cells by differences in the pattern rather than the degree of global CpG methylation

“…In order to examine the global DNA methylation levels of individual trophoblast populations and compare them to that of other cell types, three distinct primary trophoblast populations – Hoechst side-population trophoblasts [a candidate trophoblast-stem cell population (James et al, 2015)], villous cytotrophoblasts and extravillous trophoblasts – were isolated from first trimester placentae (Fig. 2) and subjected to RRBS.…”

“…Hoechst-low side-population trophoblasts ( cells/sample, n =4), Hoechst-high ß4-integrin positive cytotrophoblasts ( cells/sample, n =4) and HLA-G positive extravillous trophoblasts ( cells/sample, n =4) were isolated from the same four first trimester human placentae using fluorescence activated cell sorting as previously described (James et al, 2015). In brief, first trimester villous tissue underwent two enzymatic diests in 10 ml of phosphate buffered saline (PBS) containing 0.25% trypsin (Gibco) and 200 μg/ml DNAse I (Sigma-Aldrich).…”