Article 文章

Marmoset and human trophoblast stem cells differ in signaling requirements and recapitulate divergent modes of trophoblast invasion
狨猴和人类滋养层干细胞的信号传导需求不同，并概括了滋养层入侵的不同模式

At CS5, the mural trophectoderm (abembryonic trophoblast, distal from the ICM) expanded up to 2 mm to fill the uterine cavity and made loose contact with the opposing uterine wall (Figures S1A and S1B). By CS6, attached mural trophectoderm breached the luminal epithelium and established a secondary implantation site on the uterine wall opposite to the embryo (Figures 1A and 1B). AP2γ+ syncytiotrophoblast appeared at the secondary implantation site (14 μm wide) by CS7 but was thinner compared with the primary implantation site (35 μm wide) (Figures S1C and S1D).^11,34 In most Old World and New World monkeys, including marmosets, the endometrial epithelium is remodeled to form epithelial plaques at the implantation site.^{7,11,34,43,44} This process, which is not observed in Great apes, has been compared with decidualization in human.^10,43 Interestingly, both primary and secondary embryo implantation induced widespread epithelial plaque reaction, transforming SOX17+ endometrial glands into rounded, densely packed clusters of KRT7+, SOX17− epithelial plaque cells (Figures 1A–1C).
在 CS5 时，壁滋养外胚层（胚胎滋养层，远离 ICM）扩张至 2 毫米以填充子宫腔，并与相对的子宫壁形成松散接触（图 S1 A 和 S1B）。到CS6时，附着的壁滋养外胚层突破管腔上皮，并在与胚胎相对的子宫壁上建立了二次植入位点（图1A和1B）。 AP2γ+合体滋养层出现在CS7的二次植入部位（14μm宽），但比初次植入部位（35μm宽）更薄（图S1C和S1D）。 ^{11 34}在大多数旧世界和新世界猴中，包括狨猴，子宫内膜上皮会被重塑，在植入部位形成上皮斑块。 ^{7 11 34 43 44}这一过程在类人猿中没有观察到，但已与人类的蜕膜化进行了比较。^{10 43}有趣的是，初级和次级胚胎植入均诱导了广泛的上皮斑块反应，将 SOX17+ 子宫内膜腺体转化为圆形、密集的 KRT7+、SOX17− 上皮斑细胞簇（图 1 A-1C）。

Outside the primary and secondary implantation sites, the mural trophectoderm remained unattached throughout CS5–7. Luminal epithelium adjacent to unattached trophoblast maintained its columnar morphology (Figure 1B). We refer to this unattached postimplantation trophoblast as luminal trophoblast. In one specimen with a twin pregnancy, we were able to obtain cross-sections at the border between two implanting blastocysts (Figures 1D and 1E). The luminal trophoblast consisted of AP2γ+/KRT7+ epithelial cells, which were clearly distinguishable from PDGFRα+/AP2γ−/KRT7− ExMes lining the luminal trophoblast (Figure 1E).
在初次和二次植入位点之外，在整个 CS5-7 期间，壁滋养外胚层保持不附着。与未附着的滋养层相邻的管腔上皮保持其柱状形态（图1B ）。我们将这种独立的植入后滋养层称为管腔滋养层。在一个双胎妊娠的标本中，我们能够获得两个植入囊胚之间边界的横截面（图 1D和 1E）。管腔滋养层由 AP2γ+/KRT7+ 上皮细胞组成，这些细胞与管腔滋养层内衬的 PDGFRα+/AP2γ−/KRT7− ExMes 明显区分（图 1 E）。

Transcriptome analysis of the pre- to postimplantation transition showed that trophectoderm samples separated from postimplantation trophoblast (Figure 1F). Examination of the postimplantation trophoblast cluster demonstrated no obvious sub-clustering along developmental time (Figure 1F). We identified NOTO, JAM2, and FABP3 as enriched in the trophectoderm, while postimplantation trophoblast samples predominantly upregulated MLLT1, PRR9, CGB3, and CGA (Figures 1G and 1H). Gene Ontology (GO)-term analysis of the pre- to postimplantation transition showed differences in WNT signaling and basement membrane organization (Figure S1E).
植入前到植入后转变的转录组分析表明，滋养外胚层样品与植入后滋养层分离（图1F ）。对植入后滋养层簇的检查表明，随着发育时间的推移，没有明显的亚簇（图 1 F）。我们发现NOTO 、 JAM2和FABP3在滋养外胚层中富集，而植入后滋养层样本主要上调MLLT1 、 PRR9 、 CGB3和CGA （图1G 和 1H）。对植入前到植入后过渡的基因本体 (GO) 术语分析显示 WNT 信号传导和基底膜组织存在差异（图 S1 E）。

To identify postimplantation trophoblast lineages, we annotated trophoblast samples according to their location and transcriptomic signature (see STAR Methods). We analyzed spatially distinct trophoblast lineages: trophectoderm (preimplantation trophoblast of the blastocyst), embryonic trophoblast (postimplantation, attached trophoblast at the primary implantation site), abembryonic trophoblast (postimplantation, attached trophoblast at the secondary implantation site), and luminal trophoblast (postimplantation, unattached trophoblast) (Figures 1I and S1F–S1H). Core trophoblast factors GATA2, GATA3, and KRT7 were expressed throughout all trophoblast lineages (Figure 1I). The marmoset trophectoderm expressed low levels of pluripotency markers POU5F1, SOX2, and NANOG, which were sharply downregulated in all postimplantation trophoblast samples (Figure 1I). Conversely, syncytiotrophoblast-associated genes such as CGA and CGB3 were upregulated upon implantation (Figures 1H and 1I).²³ Human cytotrophoblast-associated transcripts MSX2 and OVOL1 were highest in luminal trophoblast (Figures 1I and S1H). Abembryonic trophoblast samples expressed nearly all postimplantation markers, including MLLT1, NR2F2, CGA, and CGB3, albeit at lower levels than embryonic trophoblast (Figures 1I and S1F). This observation is in line with the delay of trophoblast invasion at the secondary implantation site (Figures 1A and 1B). The interspersed nature of cytotrophoblast and syncytiotrophoblast made it difficult to discriminate between the two lineages. To overcome this challenge, we performed further hierarchical clustering of embryonic trophoblast, which revealed two closely related cell populations (Figure S1I). Differential expression analysis between the two clusters identified them as cytotrophoblast (KRT7+, CDH1+) and syncytiotrophoblast (CGB3+, MLLT1+) (Figure S1J).
为了识别植入后滋养层谱系，我们根据滋养层样本的位置和转录组特征对滋养层样本进行了注释（参见STAR 方法）。我们分析了空间上不同的滋养层谱系：滋养外胚层（囊胚的植入前滋养层）、胚胎滋养层（植入后，在初次植入部位附着的滋养层）、胚胎滋养层（植入后，在二次植入部位附着的滋养层）和管腔滋养层（植入后，未附着的滋养层）（图 1 I 和S1 F–S1H）。核心滋养层因子GATA2 、 GATA3和KRT7在所有滋养层谱系中均有表达（图 1 I）。狨猴滋养外胚层表达低水平的多能性标记物POU5F1 、 SOX2和NANOG ，这些标记物在所有植入后滋养层样本中均急剧下调（图 1 I）。相反，合体滋养层相关基因（例如CGA和CGB3）在植入时上调（图 1H和 1I）。 ²³人细胞滋养层相关转录物MSX2和OVOL1在管腔滋养层中最高（图 1 I 和S1 H）。 胚胎滋养层样本表达几乎所有植入后标记，包括MLLT1 、 NR2F2 、 CGA和CGB3 ，尽管水平低于胚胎滋养层（图 1 I 和S1 F）。这一观察结果与二次植入部位滋养层侵袭的延迟一致（图 1A和 1B）。细胞滋养层和合体滋养层的分散性质使得很难区分这两个谱系。为了克服这一挑战，我们对胚胎滋养层进行了进一步的层次聚类，揭示了两个密切相关的细胞群（图S1 I）。两个簇之间的差异表达分析将其鉴定为细胞滋养层（KRT7+、CDH1+）和合体滋养层（CGB3+、MLLT1+）（图S1 J）。

We conclude that marmoset trophectoderm expresses residual levels of core pluripotency factors that are extinguished upon implantation. Postimplantation trophoblast gives rise to a discontinuous layer of AP2γ (TFAP2C)+/KRT7+ cytotrophoblast and syncytium at the implantation sites, which can be readily demarcated by transcription factors MLLT1, MSX2, and OVOL1, as well as pregnancy hormones CGA and CGB3.
我们得出的结论是，狨猴滋养外胚层表达残留水平的核心多能性因子，这些因子在植入后消失。植入后滋养层在植入位点产生不连续的 AP2γ ( TFAP2C )+/KRT7+ 细胞滋养层和合胞体层，可以通过转录因子MLLT1 、 MSX2和OVOL1以及妊娠激素CGA和CGB3轻松划分。

To further interrogate the developmental identity of marmoset OK cells, we performed full-length single-cell transcriptome profiling and integrated OK cells into our updated marmoset embryo datasets (Figure 1).^34,46 Global dimensionality reduction methods revealed that OK cells clustered closest with postimplantation ExMes cells (Figures 2H and S2C). Global correlation analysis showed that marmoset OK cells had the highest similarity with ExMes at CS6 and expressed known human ExMes markers (Figures S2D and S2E).³¹ Consistent with RT-qPCR data, OK cells expressed low levels of trophoblast markers TFAP2C and KRT7 in comparison to pre- and postimplantation trophoblast lineages in vivo (Figure 2I). Notably, CDX2 is highly expressed in both trophectoderm and ExMes. OK cells upregulated CDX2 and various other ExMes markers, including CDH11, GATA6, and VIM (Figures 2I and 2J). To establish the regional correlation of OK single-cell transcriptomes to the embryo, we performed spatial identity mapping to the embryo (Figure 2K). Naive PSCs correlated to the preimplantation epiblast, as previously reported.³⁴ By contrast, OK cells showed the highest spatial correlation scores with postimplantation ExMes at CS5 and CS6 (Figure 2K). These results are consistent with the recent finding that human naive PSCs can generate both trophoblast and ExMes in OK conditions.³¹ Interestingly, a small number of OK cells clustered closely with postimplantation trophoblast (Figure 2H). To determine if OK conditions promoted a trophoblast phenotype during early differentiation of naive marmoset cells, we monitored KRT7 expression during the first 18 days of OK differentiation (Figures S2F and S2G). We observed that small KRT7+ subpopulations arose by day 12, which were lost in mature OK cultures (day 50+) (Figures S2F and S2G). Thus, human OK TSC medium does not allow direct TSC derivation from naive PSCs in marmoset and promotes ExMes lineage entry instead.
为了进一步探究狨猴 OK 细胞的发育特性，我们进行了全长单细胞转录组分析，并将 OK 细胞整合到我们更新的狨猴胚胎数据集中（图 1 ）。 ^{34 46}全局降维方法显示 OK 细胞与植入后 ExMes 细胞聚类最接近（图 2 H 和S2 C）。全局相关性分析表明，狨猴 OK 细胞与 CS6 的 ExMes 具有最高的相似性，并表达已知的人类 ExMes 标记（图 S2D和 S2E）。 ³¹与 RT-qPCR 数据一致，与体内植入前和植入后滋养层谱系相比，OK 细胞表达低水平的滋养层标记物TFAP2C和KRT7 （图 2 I）。值得注意的是， CDX2在滋养外胚层和 ExMe 中均高度表达。 OK 细胞上调CDX2和各种其他 ExMes 标记物，包括CDH11 、 GATA6和VIM （图 2I和 2J）。为了建立 OK 单细胞转录组与胚胎的区域相关性，我们对胚胎进行了空间同一性映射（图 2 K）。 如先前报道，幼稚 PSC 与植入前外胚层相关。 ³⁴相比之下，OK 细胞在 CS5 和 CS6 处与植入后 ExMe 表现出最高的空间相关性得分（图 2 K）。这些结果与最近的发现一致，即人类初始 PSC 在正常条件下可以产生滋养层和 ExMe。 ³¹有趣的是，少数 OK 细胞与植入后滋养层紧密聚集（图 2 H）。为了确定 OK 条件是否在幼稚狨猴细胞的早期分化过程中促进滋养层表型，我们在 OK 分化的前 18 天监测了 KRT7 的表达（图 S2 F 和 S2G）。我们观察到，在第 12 天时出现了小型 KRT7+ 亚群，这些亚群在成熟的 OK 培养物（第 50 天以上）中消失了（图 S2 F 和 S2G）。因此，人类 OK TSC 培养基不允许从狨猴中的幼稚 PSC 直接衍生 TSC，而是促进 ExMes 谱系进入。

Marmoset OK XAV PD cells could be stably maintained for at least 14 passages and were readily established in a second, independent marmoset PSC line (Figure S3C). In contrast to OK cells, OK XAV PD cells did not express the ExMes marker PDGFRα and upregulated the trophoblast marker AP2γ (TFAP2C) (Figure 3D). Hence, OK XAV PD cells recapitulated the hallmarks of TSCs, exhibiting long-term self-renewing capability and lineage markers expression specific to human TSCs and marmoset cytotrophoblast.
狨猴 OK XAV PD 细胞可以稳定维持至少 14 代，并且很容易在第二个独立的狨猴 PSC 系中建立（图 S3 C）。与 OK 细胞相反，OK XAV PD 细胞不表达 ExMes 标记物 PDGFRα，并上调滋养层标记物 AP2γ ( TFAP2C )（图 3D ）。因此，OK XAV PD 细胞重现了 TSC 的特征，表现出长期自我更新能力和人类 TSC 和狨猴细胞滋养层特有的谱系标记表达。

To assess the developmental authenticity of OK XAV PD cells, we performed single-cell transcriptome profiling. Spatial identity mapping and integrated analysis showed that OK XAV PD cells did not correspond to ExMes but were most similar to postimplantation trophoblast in the marmoset embryo (Figures 3E, 3F, and S3D). Therefore, we denoted OK XAV PD cells as postimplantation TSCs (postTSCs). Notably, ExMes-associated genes, including CDH11, GATA6, and VIM, were extinguished in postTSCs, while trophoblast factors KRT7 and TFAP2C were expressed at the same levels as in the postimplantation trophoblast of the marmoset conceptus (Figure 3G). CDX2 levels were reduced compared with OK cells, ExMes, and preimplantation trophoblast (trophectoderm), but similar to postimplantation trophoblast. Correlation analysis showed that marmoset postTSCs corresponded to postimplantation trophoblast at CS6 (Figure S3E), similar to human TSCs.^23,47
为了评估 OK XAV PD 细胞发育的真实性，我们进行了单细胞转录组分析。空间同一性图谱和整合分析表明，OK XAV PD 细胞与 ExMe 不对应，但与狨猴胚胎中的植入后滋养层最相似（图3E、3F 和S3D ）。因此，我们将 OK XAV PD 细胞称为植入后 TSC（postTSC）。值得注意的是，ExMes相关基因，包括CDH11 、 GATA6和VIM ，在后TSC中消失，而滋养层因子KRT7和TFAP2C的表达水平与狨猴植入后滋养层相同（图3G ）。与 OK 细胞、ExMes 和植入前滋养层（滋养外胚层）相比， CDX2水平降低，但与植入后滋养层相似。相关分析表明，狨猴后 TSC 对应于 CS6 处的植入后滋养层（图 S3 E），与人类 TSC 相似。 ^{23 47}

A defining hallmark of trophoblast cells is their capacity to form syncytiotrophoblast.^3,38,48 Human TSCs are capable of differentiation into a multinucleated syncytium upon cyclic AMP (cAMP) stimulation with forskolin.²⁰ Equally, marmoset postTSCs formed multinucleated cysts in the presence of forskolin (Figures 3H and 3I). Low cellular densities also promoted syncytium formation (Figures 3I and S3F). Moreover, marmoset syncytiotrophoblast genes were enriched in a subset of sequenced postTSCs, suggesting a propensity for syncytiotrophoblast differentiation in postTSCs (Figure S3G). To determine the mechanism by which multinucleated syncytium is formed in vitro, we tracked syncytium formation by live-cell imaging. We generated nuclear GFP and F-actin mCherry-labeled PSCs (nuclear localization sequence (NLS)-GFP LIFEACT) and derived marmoset postTSCs. Time lapse imaging showed that marmoset postTSCs predominantly formed syncytium via endoreduplication (Figures 3J and S3H; Video S1), where mitosis occurred without cytokinesis. On rare occasions, cells would surround the nucleus of another cell, suggesting that cellular fusion may also occur during marmoset syncytium formation (Figure S3I).
滋养层细胞的一个明确标志是它们形成合体滋养层的能力。 ^{3 38 48}人类 TSC 在毛喉素的环 AMP (cAMP) 刺激下能够分化为多核合胞体。 ²⁰同样，狨猴 TSC 后在毛喉素存在下形成多核囊肿（图 3 H 和 3I）。低细胞密度也促进合胞体形成（图 3 I 和S3 F）。此外，狨猴合体滋养层基因在已测序的 postTSC 子集中富集，表明 postTSC 中有合体滋养层分化的倾向（图 S3 G）。为了确定多核合胞体在体外形成的机制，我们通过活细胞成像追踪合胞体的形成。我们生成了核 GFP 和 F-肌动蛋白 mCherry 标记的 PSC（核定位序列 (NLS)-GFP LIFEACT）和衍生的狨猴 postTSC。延时成像显示狨猴后 TSC 主要通过核内复制形成合胞体（图 3 J 和S3 H；视频 S1 ），其中发生有丝分裂而没有胞质分裂。 在极少数情况下，细胞会包围另一个细胞的细胞核，这表明在狨猴合胞体形成过程中也可能发生细胞融合（图S3 I）。

We conclude that marmoset postTSCs closely resemble early postimplantation trophoblast in vivo, are capable of long-term self-renewal, and give rise to syncytiotrophoblast.
我们得出的结论是，狨猴后TSCs与体内早期植入后滋养层非常相似，能够长期自我更新，并产生合体滋养层。

Microvilli are characteristic for marmoset preimplantation trophoblast and thought to play an important role in primate embryo implantation.^6,7 Scanning electron microscopy of PAVS cells revealed extensive microvilli formation, while naive PSCs did not form microvilli (Figure S4B). Examination of cell colony edges showed that microvilli were confined to the apical surface, as observed in implanting primate embryos (Figure S4B).^6,7
微绒毛是狨猴植入前滋养层的特征，被认为在灵长类动物胚胎植入中发挥重要作用。 ^{6 7} PAVS 细胞的扫描电子显微镜显示广泛的微绒毛形成，而初始 PSC 未形成微绒毛（图 S4 B）。对细胞集落边缘的检查表明，微绒毛仅限于顶端表面，正如在植入灵长类动物胚胎时观察到的那样（图 S4 B）。 ^{6 7}

To assess the developmental identity of PAVS-derived TSCs at the transcriptome level, we performed single-cell profiling and mapped PAVS cells to the spatial transcriptomic embryo dataset. Principal-component analysis (PCA) placed PAVS cells in between trophectoderm and early postimplantation trophoblast samples (Figure 4E). Spatial identity mapping revealed greatest correlation scores to trophectoderm and lower correlation to postimplantation trophoblast (Figure 4F). Uniform manifold approximation and projection (UMAP) showed similar results, with some PAVS cells clustering with trophectoderm at CS3 (Figure S4C). Global correlation analysis placed PAVS cells with trophectoderm, in contrast to postTSCs, which more closely correlated with postimplantation trophoblast (Figure S4D). Therefore, we annotated PAVS TSCs as periimplantation trophoblast-like TSCs (periTSCs). periTSCs lacked expression of ExMes-associated transcripts and showed robust trophoblast gene expression (Figure S4E). Importantly, periTSCs upregulated preimplantation-specific mRNAs, including NOTO, MIOX, and FABP3, in contrast to postTSCs and OK cells (Figure 4G). To examine whether periTSCs are able to contribute to trophectoderm, we generated cross-species aggregation chimeras with mouse embryos and marmoset periTSCs (Figure 4H). periTSCs contributed exclusively to the trophectoderm with an overall efficiency of 27% (13.5% and 13.5% for complete and partial trophectoderm contribution, respectively) (Figures 4I, 4J, S4F, and S4G).
为了在转录组水平评估 PAVS 衍生的 TSC 的发育特性，我们进行了单细胞分析并将 PAVS 细胞映射到空间转录组胚胎数据集。主成分分析 (PCA) 将 PAVS 细胞置于滋养外胚层和早期植入后滋养层样本之间（图 4 E）。空间同一性图谱显示与滋养外胚层的相关性最高，与植入后滋养层的相关性较低（图4F ）。均匀流形近似和投影 (UMAP) 显示了类似的结果，一些 PAVS 细胞与滋养外胚层在 CS3 处聚集（图 S4 C）。全局相关性分析将 PAVS 细胞置于滋养外胚层，而后 TSC 与植入后滋养层的相关性更密切（图 S4 D）。因此，我们将 PAVS TSC 注释为植入周围滋养层样 TSC（periTSC）。 periTSC 缺乏 ExMes 相关转录本的表达，并显示出强大的滋养层基因表达（图 S4 E）。重要的是，与 postTSC 和 OK 细胞相比，periTSC 上调了植入前特异性 mRNA，包括NOTO 、 MIOX和FABP3 （图 4 G）。为了检查 periTSC 是否能够促进滋养外胚层，我们用小鼠胚胎和狨猴 periTSC 生成了跨物种聚集嵌合体（图 4 H）。 periTSC 专门对滋养外胚层做出贡献，总体效率为 27%（完全和部分滋养外胚层贡献分别为 13.5% 和 13.5%）（图 4 I、4J、 S4 F 和 S4G）。

Human TSCs exhibit lower global DNA-methylation levels compared with primed PSCs and somatic lineages.²⁰ Little is known about the methylation dynamics in marmoset pre- and postimplantation development. Consequently, we generated single-cell bisulfite sequencing samples of marmoset in vivo postimplantation trophoblast and embryonic disc. Furthermore, we performed bisulfite sequencing on bulk populations of in vitro marmoset naive PSCs, primed PSCs, and periTSCs. Global CpG methylation levels were lowest in naive PSCs and highest in primed PSCs (Figure S4H), consistent with reported differences between naive and primed pluripotent states in human.^49,50 periTSCs exhibited slightly lower DNA methylation levels compared with primed PSCs and characteristic hypomethylation of the ELF5 promoter region, consistent with the dynamics observed in vivo between the postimplantation trophoblast and the embryonic disc (Figures 4K, S4H, and S4I).⁵¹ Interestingly, ELF5 hypomethylation was also observed in naive PSCs, which suggests that some trophoblast factors remain unmethylated in the preimplantation epiblast (Figure S4J). In vivo trophoblast and periTSCs exhibited similar hypomethylation patterns for characteristic trophoblast genes such as KRT7 (Figure S4K). To investigate trophoblast-specific methylation features, we determined differentially methylated genes in naive PSCs versus periTSCs. periTSCs exhibited higher methylation levels of pluripotency factors POU5F1, NANOG, and KLF17 (Figure S4I). SMAD2 and the amnion-specific gene VTCN1 were also more highly methylated in periTSCs (Figure S4I),^52,53,54,55 suggesting trophoblast-specific DNA methylation of epiblast- and amnion-related transcripts.
与引发的 PSC 和体细胞谱系相比，人类 TSC 表现出较低的整体 DNA 甲基化水平。 ²⁰人们对狨猴植入前和植入后发育中的甲基化动态知之甚少。因此，我们生成了狨猴体内植入后滋养层和胚胎盘的单细胞亚硫酸氢盐测序样本。此外，我们对体外狨猴幼稚 PSC、引发的 PSC 和 periTSC 的大量群体进行了亚硫酸氢盐测序。总体 CpG 甲基化水平在初始 PSC 中最低，在引发 PSC 中最高（图 S4 H），与报道的人类初始和引发多能状态之间的差异一致。与引发的 PSC 相比， ^{49 50 个}periTSC 表现出略低的 DNA 甲基化水平和ELF5启动子区域的特征性低甲基化，与体内观察到的植入后滋养层和胚盘之间的动态一致（图 4 K、 S4 H 和 S4I）。 ⁵¹有趣的是，在幼稚 PSC 中也观察到ELF5低甲基化，这表明一些滋养层因子在植入前外胚层中仍未甲基化（图 S4 J）。 体内滋养层和 periTSC 的特征性滋养层基因（例如KRT7 ）表现出相似的低甲基化模式（图 S4 K）。为了研究滋养层特异性甲基化特征，我们确定了幼稚 PSC 与 periTSC 中的差异甲基化基因。 periTSC 表现出较高的多能性因子POU5F1 、 NANOG和KLF17甲基化水平（图 S4 I）。 SMAD2和羊膜特异性基因VTCN1在 periTSC 中的甲基化程度也更高（图 S4 I）， ^{52 53 54 55}表明外胚层和羊膜相关转录物的滋养层特异性 DNA 甲基化。

Human and non-human primate PSCs harbor the potential to differentiate into amnion.^{34,53,54,56,57,58} Marmoset amnion in vivo exhibits a flat squamous epithelial morphology and expresses several trophoblast-associated genes, including TFAP2C and GATA2.³⁴ To determine whether periTSCs or postTSCs expressed molecular features of amnion, we extracted a comprehensive panel of markers that discriminate between amnion and trophoblast in vivo (Figure S4M). Both periTSCs and postTSCs expressed the majority of trophoblast-associated genes and lacked amnion-related transcripts, including VTCN1 and POU5F1 (Figure S4L), consistent with previous global dimensionality reduction methods (Figures 3F, 4E, S3D, and S4C). Immunofluorescence confirmed that periTSCs do not express VTCN1 at a protein level (Figure S4M). The addition of bone morphogenic protein 4 (BMP4) induced VTCN1 expression (Figure S4M), in line with the reported role of BMP signaling for amnion formation.^{34,53,54,56,57}
人类和非人类灵长类动物 PSC 具有分化为羊膜的潜力。 ^{34 53 54 56 57 58}狨猴羊膜在体内表现出平坦的鳞状上皮形态，并表达多种滋养层相关基因，包括TFAP2C和GATA2 。 ³⁴为了确定 periTSC 或 postTSC 是否表达羊膜的分子特征，我们提取了一组可在体内区分羊膜和滋养层的全面标记物（图 S4 M）。 periTSC 和 postTSC 均表达大部分滋养层相关基因，缺乏羊膜相关转录本，包括VTCN1和POU5F1 （图 S4 L），与之前的全局降维方法一致（图 3 F、 4 E、 S3 D 和S4 C） ）。免疫荧光证实 periTSC 在蛋白质水平上不表达 VTCN1（图 S4 M）。 添加骨形态发生蛋白 4 (BMP4) 可诱导 VTCN1 表达（图 S4 M），这与报道的 BMP 信号传导对羊膜形成的作用一致。^{34 53 54 56 57}

We observed that periTSCs, when passaged or cultured at higher densities, formed free-floating vesicles that could be maintained in hanging drops for at least 5 days (Figures 4L and S4O). Immunocytochemistry of periTSC vesicles showed robust expression of the trophectoderm marker GATA3 (Figure 4M). EZRIN and F-actin localized to the outer membrane of periTSC vesicles, which demonstrated that periTSC-derived trophoblast spheroids (Tb-spheroids) exhibit an outward-facing apical domain, similar to the trophectoderm of the blastocyst (Figure 4M). Consistent with this result, Laminin was found on the inner membrane of the POU5F1−/AP2γ+/ZO1+ Tb-spheroids (Figure 4N).
我们观察到，当以较高密度传代或培养时，periTSC 形成自由漂浮的囊泡，可以在悬滴中维持至少 5 天（图 4 L 和S4 O）。 periTSC 囊泡的免疫细胞化学显示滋养外胚层标记物 GATA3 的强劲表达（图 4 M）。 EZRIN和F-肌动蛋白定位于periTSC囊泡的外膜，这表明periTSC衍生的滋养层球体（Tb球体）表现出面向外的顶端结构域，类似于囊胚的滋养外胚层（图4M ）。与此结果一致，在 POU5F1−/AP2γ+/ZO1+ Tb 球体的内膜上发现了层粘连蛋白（图 4 N）。

We conclude that PAVS induces a periimplantation trophectoderm-like TSC state, as determined by transcriptome and methylome profiling, chimeric contribution to the trophectoderm of mouse blastocysts and spontaneous syncytium and trophectoderm-like spheroid formation.
我们得出的结论是，PAVS 诱导植入周围滋养外胚层样 TSC 状态，根据转录组和甲基化组分析、对小鼠囊胚滋养外胚层的嵌合贡献以及自发合胞体和滋养外胚层样球体形成确定。

To test if CDX2 can promote a trophoblast-like phenotype in primed (postimplantation-like) pluripotency, we assessed CDX2 overexpression in self-renewing primed PSC culture conditions (knockout serum replacement [KSR]/basic FGF [bFGF]) (Figures 5C and S5D). CDX2 overexpression in primed cells led to a decrease in OCT4 expression and differentiation (Figure 5D). However, DOX+ cells remained KRT7−, suggesting differentiation into other lineages (Figure 5D). A subset of CDX2 overexpressing PSCs in KSR/bFGF expressed TBXT+, which may indicate acquisition of a primitive streak-like mesodermal cell identity (Figure S5E).^34,36,62 We conclude that CDX2 overrules pluripotency maintenance conditions and promotes trophoblast-like identity in naive, but not primed, marmoset PSCs.
为了测试 CDX2 是否可以促进启动（植入后样）多能性中的滋养层样表型，我们评估了自我更新启动 PSC 培养条件（敲除血清替代物 [KSR]/碱性 FGF [bFGF]）中的CDX2过表达（图 5 C）和S5 D)。致敏细胞中 CDX2 过度表达导致 OCT4 表达和分化减少（图 5D ）。然而，DOX+ 细胞仍然是 KRT7−，表明分化为其他谱系（图 5D ）。 KSR/bFGF 中CDX2过表达 PSC 的子集表达 TBXT+，这可能表明获得了原条状中胚层细胞身份（图 S5 E）。 ^{34 36 62}我们得出的结论是，CDX2 会否决多能性维持条件，并促进幼稚但未启动的狨猴 PSC 中的滋养层样身份。

Analysis of pre- and postimplantation-specific trophoblast signatures (Figure 1) identified the presence of pluripotency factors in marmoset trophectoderm in vivo, including POU5F1 (Figures 5E and S5F). This is in contrast to mouse, where Pou5f1 (OCT4) is rapidly downregulated in the trophectoderm.⁶³ Therefore, we set out to examine the role of POU5F1 in marmoset trophoblast differentiation. To elucidate POU5F1 dynamics during TSC derivation, we performed lineage marker analysis at day 3 during periTSCs differentiation from naive PSCs. SOX2 was downregulated in the majority of cells; however, OCT4 was present in all cells, including early GATA3+ and KRT7+ trophoblast cells (Figures 5F and S5H–S5J). Image quantification showed no anti-correlation (R = 0.1066) between OCT4 and GATA3 expression (Figure S5J). Equally, CDX2 started to become upregulated in a subset of OCT4+ cells (Figure S5G). To test whether POU5F1 upregulation and sustained expression would prevent differentiation into trophoblast-like cells, we generated marmoset doxycycline-inducible POU5F1 overexpression PSCs (Figure 5G). We confirmed tight control over the POU5F1-transgene (Figure 5H) and derived periTSCs in the presence and absence of doxycycline (DOX+/−) (Figures 5I and 5J). In the absence of DOX (DOX−), POU5F1-inducible naive PSCs converted into KRT7+/CDX2+/POU5F1− periTSCs (Figure 5J), similar to wild-type cells (Figure 4C). In the presence of DOX (DOX+) in PAVS for 2 passages, POU5F1-inducible naive PSCs equally flattened out, acquired a trophoblast-like morphology, and could be readily propagated (Figure 5I). Sustained POU5F1 expression did not interfere with the robust expression of trophoblast markers KRT7, GATA3, and CDX2 (Figures 5J–5L). We conclude that POU5F1 does not inhibit trophoblast-specific gene expression in vitro.
对植入前和植入后特异性滋养层特征的分析（图1 ）确定狨猴体内滋养外胚层中存在多能因子，包括POU5F1 （图5E和S5F ）。这与小鼠相反，小鼠的滋养外胚层中的Pou5f1 (OCT4) 快速下调。 ⁶³因此，我们着手研究POU5F1在狨猴滋养层分化中的作用。为了阐明 TSC 衍生过程中的POU5F1动态，我们在 periTSC 与初始 PSC 分化期间的第 3 天进行了谱系标记分析。 SOX2 在大多数细胞中下调；然而，OCT4 存在于所有细胞中，包括早期 GATA3+ 和 KRT7+ 滋养层细胞（图5F 和S5H -S5J）。图像量化显示 OCT4 和 GATA3 表达之间没有反相关性 (R = 0.1066)（图 S5 J）。同样，CDX2 在 OCT4+ 细胞的子集中开始上调（图 S5 G）。为了测试POU5F1上调和持续表达是否会阻止分化为滋养层样细胞，我们生成了狨猴强力霉素诱导的POU5F1过表达 PSC（图 5 G）。 我们确认了在存在和不存在强力霉素（DOX+/-）的情况下对POU5F1转基因（图5H）和衍生的 periTSC 的严格控制（图5I 和 5J）。在缺乏 DOX (DOX−) 的情况下， POU5F1诱导型幼稚 PSC 转化为 KRT7+/CDX2+/POU5F1− periTSC（图 5 J），与野生型细胞类似（图 4 C）。在 PAVS 中存在 DOX (DOX+) 的情况下传代 2 代， POU5F1诱导的幼稚 PSC 同样变平，获得滋养层样形态，并且可以轻松繁殖（图 5 I）。持续的POU5F1表达不会干扰滋养层标记物 KRT7、GATA3 和 CDX2 的稳健表达（图 5 J-5L）。我们得出结论， POU5F1在体外不会抑制滋养层特异性基因表达。

To assess marmoset Tb-spheroid implantation in vitro, we allowed spheroids to attach on a Matrigel bed after release from the microgels (Figure 6D). Marmoset Tb-spheroids maintained regular spherical shapes and expanded lumina (Figures 6E, S6D, and S6E), consistent with the superficial implantation mode of marmoset embryos (Figure 1A), where the blastocyst expands to fill the uterine cavity. By contrast, human Tb-spheroids exhibited an irregular and deflated morphology (Figures S6C–S6E). These findings suggest that marmoset TSCs recapitulate some morphological aspects of superficial attachment (Figure S6F).
为了评估狨猴 Tb 球体的体外植入，我们允许球体从微凝胶释放后附着在基质胶床上（图 6D ）。狨猴 Tb 球体保持规则的球形形状和扩张的管腔（图 6 E、 S6 D 和 S6E），与狨猴胚胎的浅表植入模式一致（图 1 A），囊胚扩张以填充子宫腔。相比之下，人类 Tb 球体表现出不规则且扁平的形态（图 S6 C-S6E）。这些发现表明狨猴 TSC 概括了浅表附着的一些形态学方面（图 S6 F）。

Primate trophectoderm differentiates into primary syncytium upon contact with the uterine epithelium in vivo.¹³ To evaluate the ability of Tb-spheroids to recapitulate primary syncytium formation upon attachment, Tb-spheroids were transferred to adherent culture conditions. Tb-spheroids formed a multinucleated syncytium upon contact with thinly coated Matrigel or collagen IV (Figures 6F, 6G, and S6G). We confirmed that syncytium formation was specific to TSCs by plating marmoset primed PSC-derived spheroids, which did not give rise to multinucleated cells (Figure 6G). Notably, attachment of Tb-spheroids on thicker beds of Matrigel or collagen IV did not form syncytium (Figures 6H and 6I), suggesting that substrate stiffness may play a role in the regulation of primary syncytium formation.
灵长类滋养外胚层在体内与子宫上皮接触后分化为初级合胞体。 ¹³为了评估 Tb 球体在附着后重现初级合胞体形成的能力，将 Tb 球体转移到贴壁培养条件下。 Tb 球体在与薄层基质胶或胶原 IV 接触后形成多核合胞体（图 6F 、6G 和S6G ）。我们通过铺板狨猴引发的 PSC 衍生球体来确认合胞体形成对 TSC 具有特异性，该球体不会产生多核细胞（图 6 G）。值得注意的是，Tb 球体附着在较厚的基质胶或胶原 IV 床上并没有形成合胞体（图 6H和 6I），这表明基质硬度可能在调节初级合胞体形成中发挥作用。

Migratory EVT differentiation is essential for immune, endometrial, and vessel remodeling during placentation. Despite significantly delayed trophoblast invasion in the marmoset compared with human, migratory trophoblast cells have been observed in the marmoset endometrium and uteroplacental arteries in vivo.⁴¹ To examine if marmoset TSCs are able to differentiate into invasive trophoblast, we cultured periTSCs and postTSCs in human EVT conditions²⁰ (Figure 6J). Supplementation of NRG1 and ECM together with TGF-β/NODAL inhibition robustly induced epithelial-mesenchymal transition and expression of the EVT marker MMP2 by day 6, similar to human EVT differentiation (Figures 6K, 6L, and S6I–S6K). The human EVT marker HLA-G showed very limited conservation in the marmoset (Figure S6H).^66,67 To determine the migratory potential of marmoset EVT-like cells, we performed transwell migration assays. Marmoset EVT differentiation increased migratory behavior, similar to EVT differentiation in human (Figure S6L). This result suggests at least partial conservation of EVT specification in human and marmoset.
迁移性 EVT 分化对于胎盘期间的免疫、子宫内膜和血管重塑至关重要。尽管与人类相比，狨猴的滋养层侵袭显着延迟，但在狨猴体内的子宫内膜和子宫胎盘动脉中观察到了迁移的滋养层细胞。 ⁴¹为了检查狨猴 TSC 是否能够分化为侵袭性滋养层，我们在人类 EVT 条件下培养 periTSC 和 postTSC ²⁰ （图 6 J）。补充 NRG1 和 ECM 以及抑制 TGF-β/NODAL 可在第 6 天强烈诱导上皮间质转化和 EVT 标记物 MMP2 的表达，类似于人类 EVT 分化（图 6K 、6L 和S6 I-S6K）。人类 EVT 标记 HLA-G 在狨猴中表现出非常有限的保守性（图 S6 H）。 ^{66 67}为了确定狨猴 EVT 样细胞的迁移潜力，我们进行了 Transwell 迁移测定。狨猴 EVT 分化增加了迁徙行为，类似于人类的 EVT 分化（图 S6 L）。这一结果表明，人类和狨猴中 EVT 规范至少部分保留。

Together, these experiments show that marmoset periTSCs undergo superficial attachment, form primary syncytia, and are capable of EVT differentiation, thus recapitulating important aspects of New World monkey trophoblast development.
总之，这些实验表明，狨猴 periTSC 经历表面附着，形成初级合胞体，并且能够进行 EVT 分化，从而概括了新世界猴滋养层发育的重要方面。

To determine potential drivers of primate EVT and syncytiotrophoblast formation, we generated a single-cell embryo profiling compendium for the trophoblast lineages of human,⁹ cynomolgus monkey,⁶⁹ and marmoset.³⁴ Integrated analysis revealed the formation of two branches, representing syncytiotrophoblast and EVT differentiation (Figures 7E and S7A). Trophectoderm and early cytotrophoblast clustered in close proximity at the root of the syncytiotrophoblast and EVT trajectories in human and cynomolgus. Notably, marmoset samples did not form EVT at the examined stages (CS5–7), demonstrating a species-specific adaptation. This observation is consistent with the reported delayed formation of proliferative trophoblast projections in marmoset compared with human.⁷⁰ Our previous experiments suggested that NRG1 is a conserved agonist of EVT differentiation in both marmoset and human (Figure 6). Notably, NRG1 is secreted by decidualized stromal cells in human.⁷¹ In accordance with delayed EVT formation, marmoset decidualized stromal cells did not express NRG1 at CS5–7 (Figure 7F). To further identify species-specific adaptations in cell signaling, we performed signaling pathway analysis between human and marmoset cytotrophoblasts. GO showed that WNT signaling was significantly enriched in the marmoset trophoblast (Figure 7G), suggesting that endogenous WNT may play a role in preventing premature EVT differentiation. Consistent with this notion, the soluble WNT inhibitor DKK1 was highly expressed in human decidualized endometrial stroma³³ but scarcely detectable in marmoset (Figure 7F). These data highlight a potential role for WNT signaling in human EVT differentiation in vivo.
为了确定灵长类动物 EVT 和合体滋养层形成的潜在驱动因素，我们为人类、 ⁹食蟹猴、 ⁶⁹和狨猴的滋养层谱系生成了单细胞胚胎分析纲要。 ³⁴综合分析揭示了两个分支的形成，代表合体滋养层和 EVT 分化（图 7 E 和S7 A）。在人和食蟹猴中，滋养外胚层和早期细胞滋养层紧密聚集在合体滋养层和 EVT 轨迹的根部。值得注意的是，狨猴样本在检查阶段（CS5-7）并未形成 EVT，这表明了物种特异性的适应。这一观察结果与报道的狨猴与人类相比增殖性滋养层投射的形成延迟一致。 ⁷⁰我们之前的实验表明，NRG1 在狨猴和人类中都是 EVT 分化的保守激动剂（图 6 ）。值得注意的是，NRG1 由人类蜕膜基质细胞分泌。 ⁷¹根据 EVT 形成延迟，狨猴蜕膜基质细胞在 CS5-7 处不表达 NRG1（图 7 F）。为了进一步确定细胞信号传导中的物种特异性适应性，我们对人类和狨猴细胞滋养层之间的信号传导途径进行了分析。 GO显示WNT信号在狨猴滋养层中显着富集（图7G ），这表明内源性WNT可能在防止EVT过早分化中发挥作用。与这一观点一致，可溶性 WNT 抑制剂DKK1在人类蜕膜化子宫内膜基质中高度表达^33，但在狨猴中几乎检测不到（图 7 F）。这些数据强调了 WNT 信号在人类 EVT体内分化中的潜在作用。

WNT activation is a core component of OK medium for TSC self-renewal and is suggested to stabilize the cytotrophoblast state^20,21 (Shannon et al.,⁷²). We tested if WNT activation with the GSK-3β inhibitor CHIR99021 could prevent EVT differentiation in PAVS conditions (Figure 7H). Naive human PSCs were cultured in PAVS and PAVS plus CHIR99021 (PAVS+CHIR). In both media, naive PSCs flattened out and generated epithelial colonies within 3–5 days (Figure 7H). By passage 5, PAVS+CHIR samples sustained a TSC-like epithelial morphology, whereas PAVS TSCs differentiated into EVT-like cells (Figures 7C and 7I). Transcriptional profiling of lineage markers of naive PSCs, PAVS, and PAVS CHIR at passage 5 showed that human cells in PAVS downregulated cytotrophoblast markers, including GATA2, GATA3, and NR2F2, and showed strong upregulation of EVT markers HLA-G and ANXA4 (Figure 7J). However, PAVS+CHIR robustly expressed GATA3, GATA2, TFAP2C, NR2F2, and OVOL1, consistent with TSC identity (Figure 7J). At the protein level, human PAVS cells showed widespread upregulation of EVT-specific HLA-G and CGB at passage 6 (Figures 7K and S7B). By contrast, PAVS+CHIR cultured human cells did not express trophoblast differentiation markers, maintained epithelial morphology, and were positive for AP2γ (TFAP2C) (Figure 7K). These results demonstrate that in human, but not marmoset, TSCs require WNT signaling to block EVT differentiation in PAVS conditions.
WNT 激活是 TSC 自我更新 OK 培养基的核心成分，并被认为可以稳定细胞滋养层状态^{20 21} (Shannon 等人， ⁷² )。我们测试了 GSK-3β 抑制剂 CHIR99021 激活 WNT 是否可以阻止 PAVS 条件下的 EVT 分化（图 7 H）。初始人类 PSC 在 PAVS 和 PAVS 加 CHIR99021 (PAVS+CHIR) 中培养。在两种培养基中，初始 PSC 在 3-5 天内变平并产生上皮集落（图 7 H）。到第 5 代时，PAVS+CHIR 样品维持了 TSC 样上皮形态，而 PAVS TSC 分化为 EVT 样细胞（图7C 和 7I）。第 5 代幼稚 PSC、PAVS 和 PAVS CHIR 谱系标记物的转录谱显示，PAVS 中的人类细胞下调了细胞滋养层标记物，包括GATA2 、 GATA3和NR2F2 ，并且 EVT 标记物HLA-G和ANXA4强烈上调（图 7） J）。然而，PAVS+CHIR 强烈表达GATA3 、 GATA2 、 TFAP2C 、 NR2F2和OVOL1 ，与 TSC 身份一致（图 7 J）。 在蛋白质水平上，人 PAVS 细胞在第 6 代时表现出 EVT 特异性 HLA-G 和 CGB 的广泛上调（图7K 和S7B ）。相比之下，PAVS+CHIR培养的人细胞不表达滋养层分化标记物，维持上皮形态，并且AP2γ（ TFAP2C ）呈阳性（图7K ）。这些结果表明，在人类（而非狨猴）中，TSC 需要 WNT 信号传导来阻断 PAVS 条件下的 EVT 分化。

Profiling of marmoset TSCs derived from marmoset blastocysts or first trimester placenta was not included due to technical limitations. We were not able to perform chimeric integration with marmoset embryos due to embryo scarcity. Matrigel alone is insufficient to faithfully recapitulate the maternal environment. Future studies must incorporate endometrial glands and stromal cells. While we have performed significant validation of in vivo marmoset sample identity, the inability to identify cell borders during tissue lysis makes samples with multiple cell types possible.
由于技术限制，未包括源自狨猴囊胚或妊娠早期胎盘的狨猴 TSC 分析。由于胚胎稀缺，我们无法与狨猴胚胎进行嵌合整合。单独的基质胶不足以忠实地再现母体环境。未来的研究必须纳入子宫内膜腺体和基质细胞。虽然我们对体内狨猴样品的身份进行了重要验证，但在组织裂解过程中无法识别细胞边界，使得具有多种细胞类型的样品成为可能。

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Thorsten Boroviak (teb45@cam.ac.uk).
有关资源和试剂的更多信息和请求应直接发送给主要联系人 Thorsten Boroviak ( teb45@cam.ac.uk ) ，并由其完成）。

Plasmids and marmoset lines generated in this study are available from the lead contact upon request.
本研究中产生的质粒和狨猴系可根据要求从主要联系人处获得。