Transcriptomic signatures of villous cytotrophoblast and syncytiotrophoblast in term human placenta.

During pregnancy, the placenta ensures multiple functions, which are directly involved in the initiation, fetal growth and outcome of gestation. The placental tissue involved in maternal-fetal exchanges and in synthesis of pregnancy hormones is the mononucleated villous cytotrophoblast (VCT) which aggregates and fuses to form and renew the syncytiotrophoblast (ST). Knowledge of the gene expression pattern specific to this endocrine and exchanges tissue of human placenta is of major importance to understand functions of this heterogeneous and complex tissue. Therefore, we undertook a global analysis of the gene expression profiles of primary cultured-VCT (n=6) and in vitro-differentiated-ST (n=5) in comparison with whole term placental tissue from which mononucleated VCT were isolated. A total of 880 differentially expressed genes (DEG) were observed between VCT/ST compared to whole placenta, and a total of 37 and 137 genes were significantly up and down-regulated, respectively, in VCT compared to ST. The 37 VCT-genes were involved in cellular processes (assembly, organization, and maintenance), whereas the 137 ST-genes were associated with lipid metabolism and cell morphology. In silico, all networks were linked to 3 transcriptional regulators (PPARγ, RARα and NR2F1) which are known to be essential for trophoblast differentiation. Furthermore, a subset of DEG were validated by RT-qPCR or by immunohistochemistry. To conclude, recognition of these pathways is fundamental to increase our understanding of the molecular basis of human trophoblast differentiation. The present study provides for the first time a gene expression signature of the VCT and ST compared to their originated term human placental tissue. One microgram of total RNA from each sample preparation was amplified using the MessageAmp RNA kit (Ambion), and 3 µg of amplified RNA (aRNA) was Cy-dye labeled using the CyScribe first-strand cDNA labeling kit (Amersham Biosciences). To compare the gene expression pattern obtained from each cultured cells (VCT or ST) with the one obtained from corresponding total placental extracts, amplified RNA from cultured cells was labeled with Cy5, while the aRNA from placental tissue was labeled with Cy3. A total of six (VCT) or five (ST) individual cDNA microarrays were performed in this condition.

妊娠期间，胎盘发挥多项功能，直接参与妊娠启动、胎儿生长及妊娠结局的调控。参与母胎物质交换与妊娠激素合成的胎盘组织成分为单核绒毛细胞滋养层细胞（mononucleated villous cytotrophoblast, VCT），该细胞可聚集并融合，以形成并更新合体滋养层细胞（syncytiotrophoblast, ST）。解析人类胎盘这一兼具内分泌与物质交换功能的组织的特异性基因表达谱，对于理解这一异质性复杂组织的功能具有重要意义。为此，本研究针对原代培养的VCT（n=6）与体外分化获得的ST（n=5）的基因表达谱开展了全局分析，并以分离得到VCT的足月胎盘整体组织作为对照。与足月胎盘整体组织相比，VCT与ST中共筛选得到880个差异表达基因（differentially expressed genes, DEG）；且相较于ST，VCT中分别有37个基因显著上调、137个基因显著下调。这37个VCT特异性基因参与细胞过程（包括组装、组织及维持），而137个ST特异性基因则与脂质代谢及细胞形态相关。生物信息学分析显示，所有差异基因的调控网络均与3种转录调控因子（PPARγ、RARα及NR2F1）相关，上述因子已被证实对滋养层细胞分化至关重要。此外，本研究通过逆转录实时定量聚合酶链式反应（RT-qPCR）与免疫组织化学技术对部分差异表达基因进行了验证。综上，明确上述通路机制，对于加深我们对人类滋养层细胞分化的分子基础的理解具有关键作用。本研究首次报道了相较于其来源的足月人类胎盘组织，VCT与ST的基因表达特征谱。每份样本制备物提取的1μg总RNA采用MessageAmp RNA试剂盒（Ambion）进行扩增，随后取3μg扩增后的RNA（amplified RNA, aRNA）使用CyScribe第一链cDNA标记试剂盒（Amersham Biosciences）进行Cy染料标记。为比较培养细胞（VCT或ST）与对应胎盘总提取物的基因表达模式，培养细胞的扩增RNA采用Cy5标记，而胎盘组织来源的aRNA则采用Cy3标记。本研究共针对VCT组（6例）与ST组（5例）分别开展了独立的cDNA微阵列实验。