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

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. Overall design: ChIP-seq was performed on day3 differentiated EVT to determine GCM1 transcriptional targets. 2 biological replicates were generated for this experiment.

胎盘与胚胎同步发育，为胎儿发育至足月提供滋养支持。在胚胎发育早期，由于血液循环尚未充分建立，血液及环境氧供应通常极低（约1%~2% O₂），并随胎盘侵袭过程逐步升高。低氧环境与干细胞自我更新及增殖过程密切相关，但持续性低氧可对分化中的细胞产生严重不良影响，或为胎盘疾病的潜在诱因。
本研究发现，人类滋养层干细胞（TSC）在低氧环境中生长状态良好，而合体滋养层细胞（STB）与绒毛外滋养层细胞（EVT）的分化则会受到低氧条件的抑制。我们观察到，促分化因子GCM1（人类神经胶质细胞缺失因子1，human Glial Cell Missing-1）在低氧环境中表达下调，且低氧条件下GCM1调控的靶基因数量显著减少。在TSC中敲除GCM1会导致EVT与STB的形成及功能受损，降低分化相关基因的表达水平，并维持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抑制剂处理细胞，同样能够抑制自发或定向分化过程。对GCM1进行染色质免疫共沉淀（Chromatin immunoprecipitation）分析显示，GCM1特异性结合位点富集于分化过程中上调的关键转录因子区域，其中包括接触抑制因子CDKN1C。GCM1缺失会导致CDKN1C表达下调，并相应丧失接触抑制，表明GCM1参与调控这一关键生物学过程。
整体实验设计：对分化至第3天的EVT进行染色质免疫共沉淀测序（ChIP-seq），以鉴定GCM1的转录靶标。本实验共设置2个生物学重复。