VGLL1 regulates extraembryonic lineage specification in human [RNA-seq II]

Placenta abnormality is one of the key reasons for early pregnancy loss but the regulatory mechanisms underlying placenta formation are largely unknown. Here, we applied a human naïve pluripotent stem cell (PSC) derived TE model to explore the driven regulatory machinery. We demonstrate that VGLL1 (vestigiallikefamilymember1) is crucial for TE self-renewal and TE-specific gene expression, thus suppressing VGLL1 leads to severely impaired cell proliferation and skewed TE induction. VGLL1 exerts its function through interacting with TEAD4 (TEA domain transcription factor 4) and colocalize at target gene promoters and enhancers. Further investigation uncovers the enrichment of H3K27ac active histone marks at genomic regions occupied by VGLL1-TEAD4 complex, indicates a close association between them. Overall, our data reveals the critical function model of VGLL1-TEAD4 in human TE derivation, highlighting a potential interspecies difference between human and mouse TE induction. Human naïve PSCs were dissociated into single cells with a 1:1 mixture of 0.5 mM EDTA and TrypLE Express and plated at a density of 100,000 cell/well of 6-well plate on Geltrex in TE induction medium (1:1 mix of Neurobasal medium (Gibco) and DMEM/F12 (HyClone) supplemented with N2 (Gibco) and B27 (Gibco), penicillin-streptomycin (HyClone), Glutamax (Gibco), β-mercaptoethanol (Sigma), 1 µM PD0325901 (Axon) and 1 µM A83-01 (Selleck)) for five days. TELCs at day 5 were used for downstream analysis throughout this study. For generating expandable TSCs, TELCs at day 5 were dissociated into single cells with TrypLE and plated on Geltrex-coated six-well plates at a 1:4-1:8 split ratio in TSC medium (DMEM/F12 supplemented with N2 and B27, penicillin-streptomycin, Glutamax, β-mercaptoethanol, 1.5 µg/ml L-ascorbic acid, 50 ng/ml EGF (PeproTech), 0.5 µM A83-01 (Selleck), 1 µM SB431542 (Selleck), 2 µM CHIR99021 (Axon), 0.8 mM VPA (Vetec) and 5 µM Y-27632 (Axon)) supplemented with 10 µM Y-27632.

胎盘异常是早期妊娠丢失的关键诱因之一，但胎盘形成的核心调控机制仍未完全明确。本研究利用人类初始态多能干细胞（naïve pluripotent stem cell, PSC）构建的滋养外胚层（trophectoderm, TE）模型，探究其背后的驱动调控机制。研究证实，VGLL1（Vestigial-like family member 1）对滋养外胚层的自我更新及特异性基因表达至关重要；抑制VGLL1会导致细胞增殖严重受损，并使滋养外胚层诱导过程出现偏斜。VGLL1通过与TEAD4（TEA domain transcription factor 4）相互结合发挥功能，并在靶基因的启动子与增强子区域共定位。进一步研究发现，VGLL1-TEAD4复合物结合的基因组区域富集有组蛋白H3赖氨酸27乙酰化（H3K27ac）活性修饰标记，提示二者之间存在紧密关联。综上，本研究的数据揭示了VGLL1-TEAD4复合物在人类滋养外胚层诱导生成中的关键功能模型，并凸显了人类与小鼠滋养外胚层诱导过程中潜在的种间差异。

本研究中，人类初始态多能干细胞先用0.5mM EDTA与TrypLE Express按1:1体积比混合的溶液解离为单细胞悬液，随后以每孔10万个细胞的密度接种于包被有Geltrex的6孔板中，采用滋养外胚层诱导培养基培养5天。该诱导培养基为Neurobasal培养基（Gibco）与DMEM/F12培养基（HyClone）按1:1体积比混合而成，并添加N2添加剂（Gibco）、B27添加剂（Gibco）、青霉素-链霉素（HyClone）、Glutamax添加剂（Gibco）、β-巯基乙醇（Sigma）、1μM PD0325901（Axon）以及1μM A83-01（Selleck）。本研究全程均采用第5天的滋养外胚层样细胞（TE-like cells, TELCs）进行后续实验分析。

为构建可扩增的滋养层干细胞（trophoblast stem cells, TSCs），将第5天的滋养外胚层样细胞用TrypLE解离为单细胞悬液，以1:4至1:8的传代比例接种于包被有Geltrex的6孔板中，采用添加了以下成分的DMEM/F12培养基作为滋养层干细胞培养基：N2添加剂、B27添加剂、青霉素-链霉素、Glutamax添加剂、β-巯基乙醇、1.5μg/ml L-抗坏血酸、50ng/ml EGF（PeproTech）、0.5μM A83-01（Selleck）、1μM SB431542（Selleck）、2μM CHIR99021（Axon）、0.8mM VPA（Vetec）以及5μM Y-27632（Axon），且额外添加10μM Y-27632（Axon）。