Hypoxia and loss of GCM1 expression prevents differentiation and contact inhibition in human trophoblast stem cells [RNA-Seq I]

The placenta develops alongside the embryo and nurtures fetal development to term. During the first stages of embryonic development, due to low blood circulation, the blood and ambient oxygen supply is normally very low (~1-2% O2) and gradually increases upon placental invasion. While a hypoxic environment is associated with stem cell self-renewal and proliferation, persistent hypoxia may have severe effects on differentiating cells and could be the underlying cause of placental disorders. We find that human trophoblast stem cells (TSC) thrive in low oxygen, whereas differentiation of syncytiotrophoblast (STB) and extravillous trophoblast (EVT) is negatively affected by hypoxic conditions. We find that the pro-differentiation factor GCM1 (human Glial Cell Missing-1) is downregulated in low oxygen, and there is substantial reduction of GCM1-regulated genes in hypoxic conditions. Knock-out of GCM1 in TSC caused impaired EVT and STB formation and function, reduced expression of genes that respond to differentiation, and resulted in maintenance of self-renewal genes. Treatment with a PI3K inhibitor reported to reduce GCM1 protein levels likewise counteracts spontaneous or directed differentiation. Chromatin immunoprecipitation of GCM1 showed enrichment of GCM1-specific binding near key transcription factors upregulated upon differentiation including the contact inhibition factor CDKN1C. Loss of GCM1 resulted in downregulation of CDKN1C and corresponding loss of contact inhibition, implicating GCM1 in regulation of this critical process. To determine how hypoixa is regulating gene expression changes in trophoblast stem cell, we culture 3 trophoblast stem cell lines in 20%, 5%, 2% oxygen conditions. 3 replicates were collect at different passages.

胎盘与胚胎同步发育，为胎儿发育提供营养支持直至足月。在胚胎发育早期阶段，由于血液循环水平较低，血液及周围环境的氧供应通常极低（约1%~2% O₂），并会随着胎盘侵入母体组织后逐渐升高。低氧环境与干细胞的自我更新及增殖密切相关，但持续低氧则可能对分化中的细胞产生严重不良影响，或为胎盘疾病的潜在致病诱因。本研究发现，人滋养层干细胞（human trophoblast stem cells, TSC）在低氧环境中生长状态良好，而合体滋养层（syncytiotrophoblast, STB）与绒毛外滋养层（extravillous trophoblast, EVT）的分化过程则会受到低氧条件的抑制。我们还发现，促分化因子GCM1（人神经胶质细胞缺失1，human Glial Cell Missing-1）在低氧环境中表达下调，且低氧条件下GCM1所调控的基因表达量显著降低。在TSC中敲除GCM1会导致EVT与STB的形成及功能受损，分化相关基因的表达水平下降，并维持了自我更新相关基因的表达。使用据报道可降低GCM1蛋白水平的PI3K抑制剂进行处理，同样能够抑制滋养层细胞的自发分化与定向分化。针对GCM1的染色质免疫共沉淀（Chromatin Immunoprecipitation）实验显示，在分化过程中上调的关键转录因子（包括接触抑制因子CDKN1C）的邻近区域，存在GCM1特异性结合的富集现象。GCM1缺失会导致CDKN1C表达下调，并相应丧失接触抑制现象，这表明GCM1参与调控这一关键生物学过程。为探究低氧如何调控滋养层干细胞的基因表达变化，我们将3株滋养层干细胞系分别置于氧浓度为20%、5%、2%的培养环境中，并在不同传代代次下收集了3份生物学重复样本。