mTOR activity paces human blastocyst stage developmental progression

doi:10.1016/j.cell.2024.08.048

Cell

Available online 26 September 2024
2024 年 9 月 26 日上线

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正在出版，更正的证明

生物学TOPSCI升级版生物学1区SCI基础版生物1区IF 45.5 如果45.5SWJTU A++ 西南交通大学A++

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Highlights 亮点

•
Human pluripotent cells are capable of entering a reversible dormant state
人类多能细胞能够进入可逆的休眠状态
•
Human blastoids under mTOR inhibition show a diapause-like response
mTOR 抑制下的人类胚细胞表现出滞育样反应
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Dormant human blastoids show altered developmental progression and attachment
休眠的人类胚细胞显示出改变的发育进程和附着
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Species-specific metabolic profiles of mouse and human cells can be seen in dormancy
在休眠状态下可以看到小鼠和人类细胞的物种特异性代谢特征

Summary 概括

Many mammals can temporally uncouple conception from parturition by pacing down their development around the blastocyst stage. In mice, this dormant state is achieved by decreasing the activity of the growth-regulating mTOR signaling pathway. It is unknown whether this ability is conserved in mammals in general and in humans in particular. Here, we show that decreasing the activity of the mTOR signaling pathway induces human pluripotent stem cells (hPSCs) and blastoids to enter a dormant state with limited proliferation, developmental progression, and capacity to attach to endometrial cells. These in vitro assays show that, similar to other species, the ability to enter dormancy is active in human cells around the blastocyst stage and is reversible at both functional and molecular levels. The pacing of human blastocyst development has potential implications for reproductive therapies.
许多哺乳动物可以通过在胚泡阶段放慢发育速度来暂时将受孕与分娩分开。在小鼠中，这种休眠状态是通过降低生长调节 mTOR 信号通路的活性来实现的。目前尚不清楚这种能力在哺乳动物中，特别是在人类中是否保守。在这里，我们发现，降低 mTOR 信号通路的活性会诱导人多能干细胞 (hPSC) 和胚泡进入休眠状态，其增殖、发育进程和附着子宫内膜细胞的能力受到限制。这些体外测定表明，与其他物种类似，人类细胞在囊胚期左右进入休眠的能力是活跃的，并且在功能和分子水平上都是可逆的。人类囊胚发育的节奏对生殖治疗具有潜在影响。

Graphical abstract 图文摘要

Keywords 关键词

diapause

dormancy

mTOR

human

blastoid

pluripotent stem cells

development

滞育休眠mTOR人母细胞多能干细胞发育

Introduction 介绍

Embryonic development comprises a series of cell commitment and differentiation events famously depicted by the Waddington model. According to this model, a cell serially restricts its developmental choices by following a continuous developmental path (“rolling downhill”). Complementing this widely accepted model of continuous development, many mammalian species evolved a means to temporarily delay development by “restraining the ball’s downhill roll.” This phenomenon of dormancy, termed embryonic diapause, often preserves the embryo around the blastocyst stage by delaying its developmental progression and implantation into the uterine wall.¹^,²
胚胎发育包括一系列由沃丁顿模型描述的细胞定型和分化事件。根据该模型，细胞通过遵循连续的发育路径（“滚下坡”）来连续限制其发育选择。作为对这种被广泛接受的持续发育模型的补充，许多哺乳动物物种进化出了一种通过“限制球的下坡滚动”来暂时延迟发育的方法。这种休眠现象被称为胚胎滞育，通常通过延迟胚胎的发育进程和植入子宫壁来保留囊胚期左右的胚胎。 ¹ ²

Diapause is triggered by a variety of factors, including changes in photoperiod (exposure to daylight), lactation, or starvation in a species-specific manner,³^,⁴^,⁵ and is mediated by hormonal changes.¹^,³^,⁴^,⁶ During diapause, the dormant embryo shows minimal anabolic activity and diminished proliferation⁴^,⁷ yet remains responsive to signals that can kick-start its reactivation and resume development.⁸^,⁹^,¹⁰ To successfully enter, maintain, and subsequently exit dormancy, embryos rewire transcriptional, epigenetic, and metabolic programs in a tissue- and stage-specific manner.¹¹^,¹²^,¹³^,¹⁴^,¹⁵^,¹⁶^,¹⁷^,¹⁸^,¹⁹ For example, in the diapaused mouse epiblast (EPI), pluripotency characteristics of the blastocyst stage are retained, indicating that this normally transient primordial state can be stabilized and maintained in vivo for extended periods.²⁰
滞育由多种因素触发，包括光周期（暴露在日光下）的变化、哺乳或物种特异性的饥饿³ ⁴ ^5，并由激素变化介导。 ¹ ³ ⁴ ⁶在滞育期间，休眠胚胎表现出最低的合成代谢活性和增殖减弱⁴ ⁷但仍然对可以启动其重新激活和恢复发育的信号做出反应。 ⁸ ⁹ ¹⁰为了成功进入、维持并随后退出休眠，胚胎以组织和阶段特定的方式重新连接转录、表观遗传和代谢程序。¹¹ ¹² ¹³ ¹⁴ ¹⁵ ¹⁶ ¹⁷ ¹⁸ ¹⁹例如，在滞育小鼠外胚层 (EPI) 中，囊胚阶段的多能性特征得以保留，表明这种通常短暂的原始状态可以在体内长期稳定和维持。²⁰

To date, over 130 mammalian species have been shown to employ a spectrum of diapause states as part of their reproductive cycle. In addition, interspecies uterine transfer experiments suggest that the capacity to enter a dormant state may be retained in the blastocysts of more species without being necessarily exploited for diapause.²¹^,²² Indeed, sheep blastocysts that do not naturally diapause can enter this dormant state upon transfer into mouse uteri induced for diapause.²¹ Given that different species employ either conserved or divergent regulatory networks, signaling pathways, and morphological organization to support blastocyst development,²³^,²⁴^,²⁵^,²⁶^,²⁷^,²⁸^,²⁹ whether more mammals harbor this intrinsic capacity for dormancy is unclear.
迄今为止，超过 130 种哺乳动物已被证明采用一系列滞育状态作为其生殖周期的一部分。此外，种间子宫移植实验表明，进入休眠状态的能力可能保留在更多物种的囊胚中，而不必用于滞育。 ²¹ ²²事实上，非自然滞育的绵羊囊胚在转移到诱导滞育的小鼠子宫中后可以进入这种休眠状态。 ²¹鉴于不同物种采用保守或不同的调控网络、信号通路和形态组织来支持囊胚发育， ²³ ²⁴ ²⁵ ²⁶ ²⁷ ²⁸ ²⁹是否有更多的哺乳动物具有这种内在的休眠能力尚不清楚。

In humans, the possibility of diapause has been anecdotally raised.³⁰^,³¹ Since diapause reveals itself most reliably as a delay in implantation, investigating the possibility of human embryo diapause would require precise measurements of the implantation event in large cohorts, which is measurable using pregnancy tests. Such studies to date have not found evidence for abnormally delayed implantation under physiological conditions.
据称，人类滞育的可能性有所增加。 ³⁰ ³¹由于滞育最可靠地表现为着床延迟，因此研究人类胚胎滞育的可能性需要对大群体的着床事件进行精确测量，这可以使用妊娠测试来测量。迄今为止，此类研究尚未发现生理条件下植入异常延迟的证据。

Few attempts at triggering diapause in human embryos have delivered promising first insights, yet hallmarks of diapause such as maintenance of cellular identities and embryo morphology, reversibility of dormancy, and prevention of implantation were not sufficiently fulfilled.¹⁴^,³² Importantly, the reversibility of dormancy needs to be documented functionally and/or molecularly to make a substantiated case of a potential for human cells to undergo dormancy. Technical hurdles and justified ethical principles preclude experimental investigations of human embryogenesis in vivo.³³ However, surplus in vitro fertilized embryos, pluripotent stem cells (PSCs) derived from early embryos, cells reprogrammed to generate induced PSCs (iPSCs), and stem cell-based embryo models can be used to test the potential of human progenitor cells to time their development and implantation.
在人类胚胎中触发滞育的尝试很少提供有希望的初步见解，但滞育的标志，例如细胞特性和胚胎形态的维持、休眠的可逆性和植入的预防尚未得到充分实现。 ¹⁴ ³²重要的是，休眠的可逆性需要在功能和/或分子水平上进行记录，以证实人类细胞有可能经历休眠。技术障碍和合理的伦理原则阻碍了对体内人类胚胎发生的实验研究。 ³³然而，剩余的体外受精胚胎、源自早期胚胎的多能干细胞 (PSC)、经过重新编程以产生诱导性 PSC (iPSC) 的细胞以及基于干细胞的胚胎模型可用于测试人类祖细胞的潜力。他们的发展和植入。

We have previously discovered the mTOR pathway as a major regulator of embryonic diapause in mice.³⁴ Inhibition of mTOR (mTORi) alone induces a diapause-like dormant state in mouse blastocysts in vitro, limiting blastocyst growth and developmental progression for several weeks. In addition, mouse PSCs treated with mTORi can be propagated in a dormant state that resembles the diapaused EPI.²⁰^,³⁴ Notably, this dormant state is reversible, and reactivated embryos and PSCs can give rise to live, fertile mice and high-grade chimeras, respectively.³⁴ Downstream of mTORi, dormant cells display reduced global anabolic activity along with altered metabolic and transcriptional networks reminiscent of in vivo diapause embryos.¹⁵^,³⁵
我们之前发现 mTOR 通路是小鼠胚胎滞育的主要调节因子。 ³⁴单独抑制 mTOR (mTORi) 可在体外诱导小鼠囊胚进入滞育样休眠状态，从而限制囊胚生长和发育进程长达数周。此外，用 mTORi 处理的小鼠 PSC 可以在类似于滞育 EPI 的休眠状态下繁殖。 ²⁰ ³⁴值得注意的是，这种休眠状态是可逆的，重新激活的胚胎和 PSC 可以分别产生活的、可育的小鼠和高级嵌合体。 ³⁴ mTORi 下游的休眠细胞表现出整体合成代谢活性降低以及代谢和转录网络改变，让人想起体内滞育胚胎。 ¹⁵ ³⁵

Here, we reveal a conserved dormancy response in a human stem cell-based model of the blastocyst (blastoids) as well as blastocyst-stage PSCs that are triggered by mTORi. We show that embryonic and extraembryonic cells maintain their cellular identity, undergo restricted proliferation and developmental progression in a functionally reversible way. As observed during mouse diapause, we also show a tissue- and stage-specific response and a lack of potency to attach to hormonally stimulated human endometrial cells in vitro. These results pinpoint a conserved role for mTOR to regulate the growth and developmental progression of human blastocyst-stage-like cells, which raises the possibility of modulating the timing of early human development by extending the time window of developmental competence at the pre-implantation stage.
在这里，我们揭示了基于人类干细胞的囊胚（胚泡）模型以及由 mTORi 触发的囊胚期 PSC 的保守休眠反应。我们发现胚胎和胚胎外细胞保持其细胞身份，以功能可逆的方式经历受限的增殖和发育进程。正如在小鼠滞育期间观察到的，我们还表现出组织和阶段特异性反应，并且缺乏在体外附着于激素刺激的人类子宫内膜细胞的效力。这些结果明确了 mTOR 在调节人类囊胚期样细胞的生长和发育进程中的保守作用，这提高了通过延长植入前阶段发育能力的时间窗口来调节人类早期发育时间的可能性。

Results 结果

Conserved pattern of mTOR activation via IGF1 sensing in mouse and human embryos
小鼠和人类胚胎中通过 IGF1 感应激活 mTOR 的保守模式

The mTOR protein kinase is a major controller of the dormancy vs. proliferation decision in the mouse blastocyst, as the inhibition of its catalytic activity allows maintenance of the mouse blastocyst for weeks in vitro with little developmental progression in a state similar to diapause.¹⁵^,³⁴ To probe eventual similarities between mouse and human blastocysts, we first compared the pattern of mTOR pathway activity (Figure 1). In late blastocysts of both species, the phosphorylation of ribosomal protein S6 (S6), a downstream target of mTOR, shows a similar pattern, with the highest signal in the polar trophectoderm (pTE) and lower signals in the mural TE and the inner cell mass (ICM; Figures 1A and 1B). In mouse blastocysts, other mTOR targets p4EBP1, pAKT, and nascent translation show patterns similar to pS6, with consistently higher activity in the pTE (Figure S1A). The pTE was previously shown to be less amenable to repression during mouse diapause, which may be due to its high mTOR activity.⁷ The conserved pattern of pS6 in mouse and human embryos suggests that the mTOR pathway may contribute similarly to blastocyst development in both species.³⁶
mTOR 蛋白激酶是小鼠囊胚休眠与增殖决定的主要控制者，因为抑制其催化活性可以使小鼠囊胚在体外维持数周，而在类似于滞育的状态下几乎没有发育进展。 ¹⁵ ³⁴为了探究小鼠和人类囊胚之间最终的相似性，我们首先比较了 mTOR 通路活性的模式（图 1 ）。在两个物种的晚期囊胚中，mTOR 下游靶标核糖体蛋白 S6 (S6) 的磷酸化显示出相似的模式，在极滋养外胚层 (pTE) 中信号最高，在壁 TE 和内细胞中信号较低质量（ICM；图 1 A 和 1B）。在小鼠囊胚中，其他 mTOR 靶点 p4EBP1、pAKT 和新生翻译显示出与 pS6 类似的模式，并且 pTE 中的活性始终较高（图 S1 A）。之前的研究表明，pTE 在小鼠滞育期间不太容易受到抑制，这可能是由于其高 mTOR 活性所致。 ⁷ pS6 在小鼠和人类胚胎中的保守模式表明，mTOR 通路可能对这两个物种的囊胚发育有类似的贡献。 ³⁶

The mTOR pathway regulates cellular growth and proliferation in response to nutrients and certain growth factors in the cellular environment. The insulin growth factor (IGF) is an upstream main regulator of the mTOR pathway and is involved in diapause regulation in several species.³⁷^,³⁸ IGF1 is expressed from the maternal uterine tissue in an estrogen-dependent manner for paracrine regulation of the blastocyst.³⁹^,⁴⁰ In mice, reduced estrogen levels block embryo implantation and induce diapause.⁴¹ To test whether mTOR pathway activity is similarly sensed and adjusted in human and mouse blastocysts, we subjected these to IGF1 (in the human either from day 2 or day 5 onwards, based on embryo availability; Figures 1C–1E). Two doses of IGF1 were used: 1.7 and 17 nM, the latter being the estimated concentration in the human reproductive tract.⁴²^,⁴³ Low IGF1 supplementation increased human blastocyst formation rate (58% vs. 27% in control, readout: blastocoel cavity; Figures 1C and S1B), and the number of ICM cells per blastocyst in both species by tendency (Figures 1D and 1E), consistent with previous findings.⁴³ High IGF1 concentration did not improve blastocyst formation but increased ICM cell numbers similar to 1.7 nM IGF1, an observation that correlated with a plateau of pS6 (Figure S1C). We concluded that, in both species, the mTOR pathway is active, actionable by IGF1, and exploited differently by the TE and ICM tissues.
mTOR 通路响应细胞环境中的营养物质和某些生长因子来调节细胞生长和增殖。胰岛素生长因子 (IGF) 是 mTOR 通路的上游主要调节因子，参与多个物种的滞育调节。 ³⁷ ³⁸ IGF1 以雌激素依赖性方式从母体子宫组织表达，用于囊胚的旁分泌调节。 ³⁹ ⁴⁰在小鼠中，雌激素水平降低会阻碍胚胎着床并诱导滞育。 ⁴¹为了测试 mTOR 通路活性在人类和小鼠囊胚中是否有类似的感知和调整，我们将这些囊胚置于 IGF1 中（在人类中，从第 2 天或第 5 天开始，根据胚胎的可用性；图 1 C-1E）。使用两种剂量的 IGF1：1.7 和 17 nM，后者是人类生殖道中的估计浓度。 ⁴² ⁴³低 IGF1 补充剂可提高人类囊胚形成率（58% vs. 27%为对照，读数：囊胚腔；图 1 C 和S1 B），以及两个物种中每个囊胚的 ICM 细胞数量（按趋势）（图 1 D 和 1E），与之前的发现一致。 ⁴³高 IGF1 浓度不会改善囊胚形成，但会增加 ICM 细胞数量，类似于 1.7 nM IGF1，这一观察结果与 pS6 的平台期相关（图 S1 C）。我们的结论是，在这两个物种中，mTOR 通路都是活跃的，可被 IGF1 激活，并被 TE 和 ICM 组织以不同的方式利用。

mTOR activity regulates TE development and attachment
mTOR 活性调节 TE 的发育和附着

The dormant states of diapause are characterized by slowed-down proliferation and prevention of blastocyst implantation in utero. Following on the gain-of-function experiment in human blastocysts that suggested a role for mTOR in ICM and TE proliferation (Figure 1D), we tested the impact of inhibiting mTOR pathway activity (Figure 2). To achieve this with large enough sample numbers that allow statistical analyses, we used blastoids, the stem cell-based embryo model of blastocysts. Blastoids are generated from naive human PSCs (hPSCs) cultured in PXGL conditions and morphologically and transcriptionally represent the day 5–7 human blastocyst.³⁶^,⁴⁴ The structures were treated with the catalytic mTOR inhibitor RapaLink-1 starting at day 2 of blastoid formation until days 4/5, a time window during which the TE analog proliferates.³⁶ RapaLink-1 is a rapamycin-INK128 conjugate that blocks both the allosteric and catalytic sites on mTOR, thereby effectively reducing target phosphorylation (Figure S2A; condition denoted as mTORi). Under both control and mTORi conditions, blastoids formed with high efficiency (86% vs. 83% in control vs. mTORi) and generated the analogs of the three blastocyst lineages (Figures 2A and S2B). However, mTORi-treated structures formed smaller blastoids comprising fewer TE-like cells (Figures 2B, 2C, and S2B). We concluded that mTOR activity regulates TE development as previously reported in dormant mouse blastocysts¹⁵ ³⁴ (Figures 1D and 1E). Then, we tested whether mTOR activity regulates the capacity of the TE to attach to the endometrium. In addition to reducing proliferation, mTORi treatment significantly decreased the expression of CCR7 (day 4; Figure 2D) and NR2F2 (day 5; Figures 2E and S2C), two molecules marking the differentiation of the pTE and contributing to the implantation of the human blastocyst.⁴⁵^,⁴⁶^,⁴⁷ To test the functionality of pTE, we transferred blastoids on layers of endometrial cells derived from primary endometrial organoids (Figure 2F). As we reported previously,³⁶ blastoids attached to endometrial cells via the polar TE, particularly when these cells were hormonally stimulated (Figure 2F; 6% without vs. 23% with hormonal stimulation). By contrast, mTORi-treated blastoids showed a significantly reduced capacity to attach to hormonally stimulated endometrial cells (Figure 2F; 5%). These data suggest that mTOR signaling activity contributes to human TE development, including TE proliferation, pTE differentiation, and attachment capacity to endometrial cells.
滞育休眠状态的特点是增殖减慢并阻止胚泡在子宫内着床。在人类囊胚中进行的功能获得实验表明 mTOR 在 ICM 和 TE 增殖中发挥作用（图 1D ）后，我们测试了抑制 mTOR 通路活性的影响（图 2 ）。为了通过足够大的样本数量来实现这一目标，以便进行统计分析，我们使用了胚泡，即基于干细胞的囊胚模型。囊胚是由在 PXGL 条件下培养的原始人类 PSC (hPSC) 产生的，在形态和转录上代表第 5-7 天的人类囊胚。 ³⁶ ⁴⁴从胚泡形成的第 2 天开始，直到第 4/5 天（TE 类似物增殖的时间窗口），用催化 mTOR 抑制剂 RapaLink-1 处理这些结构。 ³⁶ RapaLink-1 是一种雷帕霉素-INK128 缀合物，可阻断 mTOR 上的变构和催化位点，从而有效减少靶标磷酸化（图 S2 A；条件表示为 mTORi）。在对照和 mTORi 条件下，胚泡形成效率很高（对照为 86%，mTORi 为 83%），并生成了三个囊胚谱系的类似物（图 2 A 和S2 B）。然而，mTORi 处理的结构形成了更小的胚泡，包含更少的 TE 样细胞（图 2B 、2C 和S2B ）。我们得出的结论是，mTOR 活性调节 TE 发育，正如之前在休眠小鼠囊胚¹⁵ ³⁴中报道的那样（图1D 和 1E）。然后，我们测试了 mTOR 活性是否调节 TE 附着子宫内膜的能力。除了减少增殖之外，mTORi 治疗还显着降低了 CCR7（第 4 天；图 2D ）和 NR2F2（第 5 天；图2E 和S2C ）的表达，这两种分子标志着 pTE 的分化并有助于植入人类囊胚。 ⁴⁵ ⁴⁶ ⁴⁷为了测试 pTE 的功能，我们将胚泡转移到源自原代子宫内膜类器官的子宫内膜细胞层上（图 2 F）。正如我们之前报道的， ^{36 个}胚泡通过极性 TE 附着在子宫内膜细胞上，特别是当这些细胞受到激素刺激时（图2F；无激素刺激时为 6%，无激素刺激时为 6%）。 23% 受荷尔蒙刺激）。相比之下，mTORi 处理的胚泡与激素刺激的子宫内膜细胞的附着能力显着降低（图 2 F；5%）。这些数据表明 mTOR 信号传导活性有助于人类 TE 发育，包括 TE 增殖、pTE 分化和子宫内膜细胞的附着能力。

mTORi activity prolongs the blastocyst-like stage in human blastoids
mTORi 活性延长人胚泡的囊胚样阶段

Mouse diapause slows down the developmental progression of the blastocyst by maintaining its morphology and limiting the proliferation and differentiation of its tissues. We next tested whether the developmental progression of fully formed blastoids could be delayed by mTORi, potentially by limiting the proliferation and differentiation of their tissues. For this, blastoids were generated without any mTORi treatment and were only subject to mTORi after full expansion (day 4; see Figure S2D for effectivity). mTORi-treated blastoids retained the blastocoel morphology (an observation reflecting maintained TE epithelial integrity) and a visible ICM analog for up to 8 days, with ∼40% of mTORi blastoids remaining intact on day 5 vs. 1% without treatment (Figures 3A–3D and S2E). Under mTORi, the TE analog maintained low-level proliferation, resulting in further expansion of the blastocoel, whereas the ICM analog did not proliferate (Figures 3B and S2E–S2G). Such an expansion of the blastocoel and different dynamics of tissue proliferation of TE and ICM phenocopies previously reported features of in vivo-diapaused mouse blastocysts⁷^,⁴⁸ (Figure 3C). mTORi-treated human blastoids expressed markers of pluripotent and extraembryonic lineages similar to untreated blastoids, suggesting that these cells maintain lineage commitment (Figures 3E and S2G). To investigate whether the blastocyst-like stage preserved under mTORi is globally similar to in vivo mouse diapause, we compared the proteome profiles of mTORi-treated human blastoids (day 3; Table S1) and mouse blastocysts¹⁵ (day 5) with those of mouse in vivo-diapaused blastocysts.⁴⁹ For this, we surveyed the expression of 179 proteins that are significantly upregulated in in vivo diapause (Figure 3F). We reasoned that upregulated proteins constitute a more specific expression signature compared with downregulated ones that can reflect a default repressive state resulting from loss of activity. mTORi-treated human blastoids and mouse blastocysts showed a significant enrichment for this in vivo mouse diapause signature, with 41 proteins following the in vivo diapause expression pattern in both mouse and human and 27 and 23 additional proteins specific to the mouse or human, respectively (Figures 3F and 3G; normalized enrichment score: 1.33 for human and 1.36 for mouse; see core enriched proteins in Table S2). By contrast, control blastocysts/blastoids negatively correlated with the in vivo mouse diapause state. We concluded that mTORi treatment of human blastoids limits TE proliferation, prominently decreases ICM proliferation, and elicits a response that molecularly resembles aspects of mouse in vivo diapause.
小鼠滞育通过维持囊胚的形态并限制其组织的增殖和分化来减慢囊胚的发育进程。接下来，我们测试了 mTORi 是否可以通过限制其组织的增殖和分化来延迟完全形成的胚泡的发育进程。为此，在没有任何 mTORi 处理的情况下生成胚泡，并且仅在完全扩增后才接受 mTORi（第 4 天；参见图 S2 D 的有效性）。 mTORi 处理的母细胞在长达 8 天的时间内保留了囊胚腔形态（反映了维持 TE 上皮完整性的观察结果）和可见的 ICM 类似物，其中约 40% 的 mTORi 母细胞在第 5 天保持完整，而未经处理的 mTORi 母细胞为 1%（图 3 A） –3D 和S2 E)。在 mTORi 下，TE 类似物保持低水平增殖，导致囊胚腔进一步扩张，而 ICM 类似物没有增殖（图 3B和S2 E-S2G）。这种囊胚腔的扩张以及 TE 和 ICM 表型的组织增殖的不同动力学先前报道了体内滞育小鼠囊胚的特征⁷ ⁴⁸ （图 3 C）。 mTORi 处理的人胚泡表达与未处理的胚泡类似的多能和胚胎外谱系标记，表明这些细胞维持谱系定向（图 3E和S2G ）。为了研究 mTORi 下保存的囊胚样阶段是否与小鼠体内滞育总体相似，我们将 mTORi 处理的人囊胚（第 3 天；表 S1 ）和小鼠囊胚¹⁵ （第 5 天）的蛋白质组谱与小鼠的蛋白质组谱进行了比较体内滞育的囊胚。 ⁴⁹为此，我们调查了 179 种在体内滞育期间显着上调的蛋白质的表达（图 3 F）。我们推断，与下调的蛋白质相比，上调的蛋白质构成了更特异的表达特征，可以反映由于活性丧失而导致的默认抑制状态。 mTORi 处理的人胚泡和小鼠囊胚显示出小鼠体内滞育特征的显着富集，其中 41 种蛋白质遵循小鼠和人类体内滞育表达模式，另外分别有 27 种和 23 种小鼠或人类特有的蛋白质。图3F和3G；标准化富集分数：人类为1.33，小鼠为1.36；参见表S2中的核心富集蛋白质。相比之下，对照囊胚/胚泡与体内小鼠滞育状态呈负相关。我们得出的结论是，mTORi 处理人胚细胞限制了 TE 增殖，显着降低 ICM 增殖，并引发分子上类似于小鼠体内滞育的反应。

Inhibition of translation alone fails to fully recapitulate the effect of mTORi
单独抑制翻译并不能完全重现 mTORi 的作用

mTOR controls cellular growth largely via ribosomal translation. To test the extent to which reduced translation contributes to the mTORi-induced state, we directly inhibited translation by culturing the blastoids in cycloheximide (CHX). At a concentration commonly used to inhibit translation in vitro (100 ng/μL), CHX treatment did not maintain blastoid morphology (Figure S2E). At a 10-fold higher CHX dose (1,000 ng/μL), blastoid morphology was maintained; however, the ICM was compromised and lost cells over time, in contrast to mTORi (Figures S2E–S2H). These observations recapitulate the outcome of CHX treatment in mouse blastocysts³⁴ and show that mTORi leads to tissue-specific changes that go beyond reduced translation. To get insights into these changes, we profiled and compared the proteomes of CHX- and mTORi-treated blastoids (day 3). Principal component and Pearson correlation analyses showed that the CHX and mTORi proteomes are more similar to each other than to the control blastoids (Figures S3A and S3B). Still, the mTORi- and CHX-induced states were clearly distinguishable (PC2: 34.8%), with the CHX signature primarily related to cytoplasmic translation, whereas the mTORi signature containing adhesion-, signaling-, and metabolism-related proteins, including FOXO1, a regulator of mouse diapause¹⁵ (Figure S3A; gene ontology terms related to top 200 differentially enriched proteins are shown in Figure S3C). To further compare mTORi and CHX responses, we defined the Protein Set: mTORi, which comprises 233 proteins that are significantly differentially expressed in mTORi vs. control blastoids (Figure S3D; Tables S1, S2, and S3). Protein expression changes due to CHX treatment were overall positively correlated with Protein Set: mTORi (normalized enrichment score: 1.3); however, only 41 of these were significantly differentially expressed in CHX-treated blastoids (Tables S2 and S3). The remaining 192 proteins that are only significantly differentially expressed in mTORi-treated blastoids include metabolic (e.g., fatty acid-related ACOX1), cell adhesion (e.g., adherens junctions CDH1 and lamina-related ITGA6, LAMA1/B1), and signaling factors (e.g., HIPPO-related YAP1, TEAD3). These data show that the mTORi-induced state encompasses translation but is not limited to it, and induction of a dormant state may require altered adhesion, developmental signaling, and metabolic activities.
mTOR 主要通过核糖体翻译控制细胞生长。为了测试翻译减少对 mTORi 诱导状态的影响程度，我们通过在放线菌酮 (CHX) 中培养胚细胞来直接抑制翻译。在通常用于抑制体外翻译的浓度 (100 ng/μL) 下，CHX 处理不能维持胚细胞形态（图 S2 E）。在高 10 倍的 CHX 剂量 (1,000 ng/μL) 下，胚细胞形态得以维持；然而，与 mTORi 相比，随着时间的推移，ICM 受到损害并丢失细胞（图 S2 E-S2H）。这些观察结果概括了小鼠囊胚³⁴中 CHX 治疗的结果，并表明 mTORi 导致的组织特异性变化超出了翻译减少的范围。为了深入了解这些变化，我们对 CHX 和 mTORi 处理的胚泡（第 3 天）的蛋白质组进行了分析和比较。主成分和 Pearson 相关分析表明，CHX 和 mTORi 蛋白质组彼此比对照胚泡更相似（图 S3 A 和 S3B）。尽管如此，mTORi 和 CHX 诱导的状态是明显可区分的 (PC2: 34.8%），CHX 特征主要与细胞质翻译相关，而 mTORi 特征包含粘附、信号传导和代谢相关蛋白，包括 FOXO1，小鼠滞育的调节因子¹⁵ （图 S3 A；与前 200 个差异富集的蛋白质如图 S3 C) 所示。为了进一步比较 mTORi 和 CHX 反应，我们定义了蛋白质组： mTORi ，其中包含 233 种蛋白质，这些蛋白质在 mTORi 与对照母细胞中表达显着差异（图 S3 D；表 S1 、 S2和S3 ）。 CHX 处理引起的蛋白质表达变化总体上与蛋白质组：mTORi呈正相关（归一化富集评分：1.3）；然而，其中只有 41 个在 CHX 处理的胚泡中显着差异表达（表 S2和S3 ）。其余 192 个蛋白质仅在 mTORi 处理的胚泡中显着差异表达，包括代谢（例如，脂肪酸相关的 ACOX1）、细胞粘附（例如，粘附连接 CDH1 和层板相关的 ITGA6、LAMA1/B1）和信号因子（例如，HIPPO 相关的 YAP1、TEAD3)。这些数据表明，mTORi 诱导的状态包括但不限于翻译，并且休眠状态的诱导可能需要改变粘附、发育信号传导和代谢活动。

mTOR-inhibited blastoids maintain a transcriptome reflecting the blastocyst stage
mTOR 抑制的胚泡维持反映囊胚阶段的转录组

Our results so far indicate a tissue-specific response to mTORi in the TE and ICM. To further investigate this response, we performed single-cell RNA sequencing (scRNA-seq) on control blastoids (day 4 of formation) and blastoids treated with mTORi (treated for 3 days after day 4 of formation, ∼1,000 blastoids per condition; Figure S4A). Cells formed three clusters, each containing intermingled control and mTORi-treated cells (Figures 4A and S4B). Tissue-specific markers showed that these three clusters represented the three blastocyst lineages (Figures 4A and S4C; GATA3 for TE, NANOG for EPI, and PDGFRA for hypoblast [HYPO], along with a larger gene set in Figure S4D). In order to accurately assess the developmental stage of these cells, we projected this scRNA-seq data on two reference maps of human embryogenesis: one that we curated by combining publicly available datasets (Figures 4B, S5A, and S5B; see STAR Methods) and another that was established by an independent consortium⁴⁴ (Figures S5C and S5D). This benchmarking showed that control blastoids comprised more than 96% of cells whose transcriptomes overlapped with those of the human blastocyst at E5–6, as we have previously reported³⁶ (Figures 4C, left, 4D, and S5B). Most cells of mTORi-treated blastoids matched to the E5–6 blastocyst stage (55%; Figure 4C, right) with a significant fraction of the TE analog progressing to the E7–8 blastocyst stage (35% of total, 67.5 % of TE; Figures 4C, right, and S5B). This observation correlates with the sustained proliferation of the TE in mTORi-treated human blastoids as well as in vivo diapaused and mTORi-treated mouse blastocysts (Figure 3B). KRT8, a previously identified marker for mouse TE differentiation,⁵⁰ is upregulated in the TE analog of mTORi-treated human blastoids (Figure S5E). In comparison to the TE, a smaller proportion of EPI analogs continued to develop under mTOR inhibition (∼10% of total, 21% of EPI). These cells mostly reflected the post-implantation E9–10 EPI and amnion (Figures 6D and S5B). Of note, the EPI of diapaused mouse blastocysts often progresses into a post-implantation rosette structure after three days.¹⁸ Genes reflecting the slight developmental progression of mTORi-treated blastoids included HAND1 (early TE marker, downregulated), KLF4 (naive EPI marker, downregulated), NR2F2 (TE maturation marker, upregulated), and DNMT3B (core EPI marker, upregulated; Figure 4E). We concluded that cells from mTORi-treated human blastoids largely retain transcriptomes reflecting the blastocyst stages (>90% at E5–8) that the TE is more prone to proliferation compared with EPI but a subset of the EPI is more prone to differentiation beyond the blastocyst stages.
迄今为止，我们的结果表明 TE 和 ICM 中对 mTORi 存在组织特异性反应。为了进一步研究这种反应，我们对对照胚泡（形成第 4 天）和用 mTORi 处理的胚泡（形成第 4 天后处理 3 天，每种条件约 1,000 个胚泡）进行了单细胞 RNA 测序 (scRNA-seq)；图S4 A）。细胞形成三个簇，每个簇包含混合的对照细胞和 mTORi 处理的细胞（图 4 A 和S4 B）。组织特异性标记显示这三个簇代表三个囊胚谱系（图4A 和S4C ；GATA3 代表 TE，NANOG 代表 EPI，PDGFRA 代表下胚层 [HYPO]，以及图 S4D中更大的基因集）。为了准确评估这些细胞的发育阶段，我们将这些 scRNA-seq 数据投影到人类胚胎发生的两个参考图上：一个是我们通过结合公开可用的数据集来策划的（图 4B 、 S5A和 S5B；参见STAR 方法））和另一个由独立财团⁴⁴建立的（图S5C和S5D）。该基准测试表明，对照胚泡包含超过 96% 的细胞，其转录组与 E5-6 处的人类囊胚的转录组重叠，正如我们之前报道的^{36 个}（图 4 C、左、4D 和S5 B）。 mTORi 处理的囊胚期的大多数细胞与 E5-6 囊胚期相匹配（55%；图 4 C，右），其中很大一部分 TE 类似物进展至 E7-8 囊胚期（占总数的 35%，占囊胚期的 67.5%）。 TE；图 4 C，右，和S5 B)。这一观察结果与 mTORi 处理的人胚泡以及体内滞育和 mTORi 处理的小鼠囊胚中 TE 的持续增殖相关（图 3 B）。 KRT8 是先前确定的小鼠 TE 分化标记物⁵⁰ ，在 mTORi 处理的人胚细胞的 TE 类似物中上调（图 S5 E）。与 TE 相比，在 mTOR 抑制下继续发育的 EPI 类似物比例较小（约占总数的 10%，EPI 的 21%）。这些细胞主要反映植入后 E9-10 EPI 和羊膜（图 6 D 和S5 B）。值得注意的是，滞育小鼠囊胚的 EPI 通常在三天后发展成植入后玫瑰花结结构。^{18 个}反映 mTORi 处理的胚泡轻微发育进展的基因包括 HAND1（早期 TE 标记，下调）、KLF4（初始 EPI 标记，下调）、NR2F2（TE 成熟标记，上调）和 DNMT3B（核心 EPI 标记，上调；图4E ）。我们得出的结论是，来自经 mTORi 处理的人胚泡的细胞很大程度上保留了反映囊胚阶段的转录组（E5-8 时>90%），与 EPI 相比，TE 更容易增殖，但 EPI 的一个子集更容易分化为囊胚阶段以外的细胞。囊胚阶段。

Human PSCs resembling the blastocyst stage adopt a reversible dormant state
类似于囊胚阶段的人类 PSC 采取可逆的休眠状态

Diapause mostly preserves mammalian embryos around the blastocyst stage. Likewise, mTORi treatment of cleavage-stage mouse embryos does not stop developmental progression through the cleavage divisions and only acts around the blastocyst stage.³⁴ Our results corroborate these findings in human blastoids (Figures 2 and 3). To further investigate the stage dependency of the mTORi response on pluripotent cells, we used hPSCs that reflect different EPI stages ranging from the blastocyst to post-implantation.⁵¹^,⁵²^,⁵³^,⁵⁴^,⁵⁵^,⁵⁶^,⁵⁷^,⁵⁸^,⁵⁹^,⁶⁰^,⁶¹ mTORi treatment of naive hPSCs, which are cultured in PXGL conditions and reflect the blastocyst-stage EPI, slowed their proliferation while maintaining the compact colony morphology of this non-differentiated state (Figure 5A). These cells could be maintained under mTORi for at least 18 days (maximum tested period) and iteratively released and retreated with mTORi without compromising colony morphology (Figures 5A and S6A). Therefore, the effects of mTORi treatment were reversible. mTORi effectively suppressed mTOR downstream targets without compromising genome integrity and markers of pluripotency (Figures S6B–S6E) with a temporary increase in apoptosis during the first days of the treatment (Figure 5B). Of note, mTORi reduced cellular proliferation but did not block cell cycle progression, with similar outcomes in mouse PSCs (Figures 5C and 5D). These cells progressed through the cell cycle slowly, as evidenced by lower-level integration of the nucleotide analog EdU during replication compared with untreated cells (Figure 5D, right). Three different catalytic mTOR inhibitors reduced proliferation to similar levels, underlining the reproducibility of this effect (Figure 5C). Cells cultured in RSeT medium that reflect an intermediate stage between naive and primed pluripotency⁵¹^,⁵²^,⁵⁵^,⁶²^,⁶³ adopted a lowly proliferative state under mTORi, albeit at much lower efficiency compared with PXGL culture due to high levels of cell death throughout the treatment (Figures 5A and 5B). Notably, the surviving RSeT cells had a minor percentage going through S phase (0.55% in RSeT vs. 2.5% in PXGL; Figure S6E) and successfully reverted to normal proliferation upon withdrawal of mTORi (Figure 5A). In sharp contrast, primed PSCs cultured in mTeSR medium, that reflect the day 10–14 EPI, died within 4 days of mTORi treatment (200 nM RapaLink-1; Figure 5A). We concluded that mTOR activity regulates hPSC proliferation in a stage-specific manner during the gradual progression from naive to primed pluripotency. At the molecular level, the proteins and pathways, the expression of which was altered under mTORi treatment, largely reversed this trend upon release from mTORi in both PXGL and RSeT conditions (Figures 5E–5J; Tables S1, S4, and S5). These changes mostly correspond to protein synthesis, cell division, and cellular respiration (down in mTORi) and lipid metabolic processes and cellular transport (up in mTORi; Figures 5G and 5H; full list in Table S6). Taken together, these data show that hPSCs capturing the blastocyst stage are able to adapt to mTORi by entering a reversible dormant state, which further supports the stage-specific response to mTORi seen in human blastoids and mouse blastocysts.
滞育主要保留囊胚期左右的哺乳动物胚胎。同样，mTORi 对卵裂期小鼠胚胎的治疗不会阻止卵裂期的发育进程，仅在囊胚期左右起作用。 ³⁴我们的结果证实了人类胚细胞中的这些发现（图 2和3 ）。为了进一步研究 mTORi 反应对多能细胞的阶段依赖性，我们使用了反映从囊胚到植入后不同 EPI 阶段的 hPSC。 ⁵¹ ⁵² ⁵³ ⁵⁴ ⁵⁵ ⁵⁶ ⁵⁷ ⁵⁸ ⁵⁹ ⁶⁰ ⁶¹对在 PXGL 条件下培养并反映囊胚期 EPI 的初始 hPSC 进行 mTORi 处理，可减缓其增殖，同时保持这种非分化状态的紧凑集落形态（图 5 A））。这些细胞可以在 mTORi 下维持至少 18 天（最长测试时间），并用 mTORi 反复释放和再处理，而不会影响集落形态（图 5 A 和S6 A）。因此，mTORi 治疗的效果是可逆的。 mTORi 有效抑制 mTOR 下游靶标，而不损害基因组完整性和多能性标记（图 S6 B-S6E），并且在治疗的第一天细胞凋亡暂时增加（图 5 B）。值得注意的是，mTORi 减少了细胞增殖，但没有阻止细胞周期进展，在小鼠 PSC 中具有类似的结果（图5C 和 5D）。与未处理的细胞相比，这些细胞在细胞周期中进展缓慢，复制过程中核苷酸类似物 EdU 的整合水平较低就证明了这一点（图 5D ，右）。三种不同的催化 mTOR 抑制剂将增殖降低到相似的水平，强调了这种效应的可重复性（图 5 C）。在 RSeT 培养基中培养的细胞反映了初始多能性和引发多能性之间的中间阶段⁵¹ ⁵² ⁵⁵ ⁶² ⁶³在 mTORi 下采用低增殖状态，尽管由于整个处理过程中细胞死亡水平较高，因此与 PXGL 培养相比效率低得多（图 5 A 和 5B）。值得注意的是，存活的 RSeT 细胞有一小部分进入 S 期（RSeT 中为 0.55%，PXGL 中为 2.5%；图 S6 E），并在撤除 mTORi 后成功恢复正常增殖（图 5 A）。与此形成鲜明对比的是，在 mTeSR 培养基中培养的引发 PSC（反映 EPI 第 10-14 天）在 mTORi 处理（200 nM RapaLink-1；图 5 A）后 4 天内死亡。我们得出的结论是，在从初始多能性逐渐发展到引发多能性的过程中，mTOR 活性以阶段特异性方式调节 hPSC 增殖。在分子水平上，在 mTORi 处理下表达发生改变的蛋白质和途径在 PXGL 和 RSeT 条件下从 mTORi 释放后很大程度上逆转了这一趋势（图 5 E-5J；表 S1 、 S4和S5 ）。这些变化主要对应于蛋白质合成、细胞分裂和细胞呼吸（mTORi 中下降）以及脂质代谢过程和细胞运输（mTORi 中上升；图5G 和 5H；表 S6中的完整列表）。总而言之，这些数据表明，捕获囊胚阶段的 hPSC 能够通过进入可逆的休眠状态来适应 mTORi，这进一步支持了在人类囊胚和小鼠囊胚中观察到的对 mTORi 的阶段特异性反应。

Reactivated blastoids developmentally progress and permit stem cell line derivation
重新激活的胚泡发育进展并允许干细胞系衍生

In utero, diapaused mouse embryos remain acutely responsive to reactivation cues and can exit dormancy to resume development. We therefore investigated whether the mTORi state of blastoids is reversible at the functional and molecular levels (Figure 6). To this end, we first tested the capacity of mTORi-treated (3 days) then reactivated (via inhibitor withdrawal, “reactivated” from here on) to progress into a post-implantation-like stage. For this, reactivated blastoids were deposited on matrigel-coated plates for further growth in CMRL-1066 medium supplemented with 10% fetal bovine serum (FBS; Figure 6A). Under these conditions, untreated and reactivated blastoids similarly proliferated and gave rise to early derivatives of the EPI (SOX2⁺) and trophoblast (GATA3⁺), including further differentiating TE derivatives that express CGB (Figure 6A) and secrete human chorionic gonadotropin (hCG) within 2–4 days (Figure 6B).
在子宫内，滞育的小鼠胚胎仍然对重新激活信号做出敏锐的反应，并且可以退出休眠状态以恢复发育。因此，我们研究了母细胞的 mTORi 状态在功能和分子水平上是否可逆（图 6 ）。为此，我们首先测试了 mTORi 处理（3 天）然后重新激活（通过抑制剂撤回，从这里开始“重新激活”）进入植入后样阶段的能力。为此，将重新激活的胚泡沉积在基质胶包被的板上，以便在补充有 10% 胎牛血清（FBS；图 6 A）的 CMRL-1066 培养基中进一步生长。在这些条件下，未经处理和重新激活的母细胞同样增殖并产生 EPI (SOX2 ⁺ ) 和滋养层 (GATA3 ⁺ ) 的早期衍生物，包括进一步分化表达 CGB 的 TE 衍生物（图 6 A）和分泌人绒毛膜促性腺激素 (hCG) ）在 2-4 天内（图 6 B）。

To analyze whether reactivated blastoids generate early post-implantation cell types, we performed scRNA-seq (mTORi-treated for 3 days, then cultured for 4 days in post-implantation culture; 150–200 blastoids were pooled in each condition; Figures 6 and S4A). Cells clustered into three distinct groups expressing the markers of the three lineages (Figures 6C–6E; GATA3 for trophoblast, PDGFRA for extraembryonic endoderm, and NANOG for EPI). Control and reactivated cells were intermingled in all 5 subclusters and showed similar gene signatures. Cell type annotations using human embryo datasets revealed the high degree of similarity between differentiating reactivated cells and control cells with a representation of the three lineages (Figure 6F). Similar to the observation we made previously, a few genes were indicative of enhanced developmental progression in reactivated cells (Figure 6E; e.g., synctiotrophoblast [STB] markers CPM and GADD45G and endoderm marker SOX17). These results show that mTORi does not compromise the differentiation competence of blastoid cells.
为了分析重新激活的母细胞是否产生早期植入后细胞类型，我们进行了 scRNA-seq（mTORi 处理 3 天，然后在植入后培养物中培养 4 天；每种条件下汇集 150-200 个母细胞；图 6和S4 A）。细胞聚集成三个不同的组，表达三个谱系的标记物（图6C-6E；GATA3代表滋养层，PDGFRA代表胚外内胚层，NANOG代表EPI）。对照细胞和重新激活的细胞混合在所有 5 个亚簇中，并显示出相似的基因特征。使用人类胚胎数据集的细胞类型注释揭示了区分重新激活的细胞和具有三个谱系代表的对照细胞之间的高度相似性（图6F ）。与我们之前观察到的类似，一些基因表明重新激活的细胞发育进展增强（图6E ；例如，合体滋养层[STB]标记CPM和GADD45G以及内胚层标记SOX17）。这些结果表明 mTORi 不会损害胚细胞的分化能力。

Next, we tested whether reactivated blastoids can attach to hormonally stimulated endometrial cells. Upon reactivation after 2 days of mTORi, 12% of blastoids could attach to these cells, as compared with 29% of control blastoids (Figure S7A). The remaining reactivated blastoids collapsed due to TE fragility. Of note, 95% of both control day 4 blastoids and reactivated blastoids attached to Ishikawa transformed endometrial cells that have a low specificity relative to the species and to the hormonal stimulation as compared with endometrial organoid cells (Figure S7B). We concluded that the mTORi-treated TE analog retained or reacquired the capacity to attach to endometrial cells, albeit less efficiently than control TE. We then tested whether reactivated blastoids are also permissive to the derivation of stem cells. Indeed, we could derive stem cells of all three blastocyst lineages from reactivated blastoids with efficiencies similar to control blastoids (Figures S7C–S7E). These results suggest that mTORi-treated blastoids retained progenitor cells capable of the de novo establishment self-renewing cell lines.
接下来，我们测试了重新激活的胚泡是否可以附着在激素刺激的子宫内膜细胞上。 mTORi 2 天后重新激活时，12% 的母细胞可以附着在这些细胞上，而对照母细胞的这一比例为 29%（图 S7 A）。剩余的重新激活的胚泡由于 TE 脆弱性而崩溃。值得注意的是，95% 的对照第 4 天母细胞和重新激活的母细胞都附着在 Ishikawa 转化的子宫内膜细胞上，与子宫内膜类器官细胞相比，这些细胞相对于物种和激素刺激具有较低的特异性（图 S7 B）。我们得出结论，mTORi 处理的 TE 类似物保留或重新获得了附着子宫内膜细胞的能力，尽管效率低于对照 TE。然后我们测试了重新激活的胚泡是否也允许干细胞的衍生。事实上，我们可以从重新激活的胚泡中衍生出所有三个胚泡谱系的干细胞，其效率与对照胚泡相似（图 S7 C-S7E）。这些结果表明，mTORi 处理的胚泡保留了能够从头建立自我更新细胞系的祖细胞。

Overall, we concluded that the human mTORi state is characterized by the maintenance of blastoid morphology, slower proliferation, and limited differentiation of the EPI and TE analogs, a significant enrichment for proteins that characterize mouse diapause in vivo, and a reduced capacity of the polar TE to attach to hormonally stimulated endometrial cells. We observed that these effects are specific to the blastocyst stage and are reversible both functionally and molecularly, thereby enabling development to resume. These results pinpoint the conservation of tissue- and stage-specific activities and functions of mTOR activities that are consistent with the hallmarks of a dormant, diapause-like state.
总体而言，我们得出的结论是，人类 mTORi 状态的特征是维持胚细胞形态、较慢的增殖以及 EPI 和 TE 类似物的有限分化、小鼠体内滞育特征的蛋白质显着富集以及极性能力降低。 TE 附着在激素刺激的子宫内膜细胞上。我们观察到这些效应是囊胚阶段特有的，并且在功能和分子上都是可逆的，从而使发育得以恢复。这些结果精确指出了 mTOR 活性的组织和阶段特异性活动和功能的保守性，这些活动与休眠、类滞育状态的特征一致。

Diverse routes converge into a shared dormancy response in mouse and human cells
小鼠和人类细胞中的不同途径汇聚成共同的休眠反应

Numerous mammals use diapause as part of their reproductive cycle. Whether cells from different species transition into dormancy via similar routes or whether there are species-specific requirements for this transition is not known, although key regulators such as mTOR, Myc, and FOXO transcription factors appear to play a conserved role.¹⁰^,¹¹^,¹⁵^,²⁰^,³⁴^,⁶⁴ To identify common and distinct cellular pathways used in the mouse and human in vitro dormancy systems, we analyzed the proteomes of PSCs and blastocysts/blastoids (Figure 7). Pathway expression profiles of dormant cells showed an overall positive correlation in both species, with a particularly high agreement between pathways upregulated in dormant mouse blastocysts and human blastoids (Figure 7A). Such “common up” pathways include cellular trafficking/endocytosis and adherens junctions previously shown to be altered in in vivo-diapaused mouse embryos¹⁵ (Figure 7B). Transcription-, translation-, and export-related cellular anabolic pathways were commonly downregulated, highlighting the need to preserve energy during dormancy (Figure 7B). Signaling and metabolic pathways appeared to be differentially used in human and mouse cells, with, e.g., fatty acid degradation and the related factors FOXO and PPAR upregulated in the mouse and galactose metabolism and JAK-STAT signaling factors particularly increasing in hPSCs (Figure 7B). These results support the model that the transition into dormancy is an actively regulated multi-step process that goes beyond reducing cellular energy expenditure.
许多哺乳动物将滞育作为其生殖周期的一部分。尽管 mTOR、Myc 和 FOXO 转录因子等关键调控因子似乎发挥着保守的作用，但不同物种的细胞是否通过相似的途径进入休眠状态，或者这种转变是否存在物种特异性要求，尚不清楚。 ¹⁰ ¹¹ ¹⁵ ²⁰ ³⁴ ⁶⁴为了确定小鼠和人类体外休眠系统中使用的常见和不同的细胞途径，我们分析了 PSC 和囊胚/胚泡的蛋白质组（图 7 ）。休眠细胞的通路表达谱在两个物种中均显示出总体正相关性，休眠小鼠囊胚和人胚泡中上调的通路之间具有特别高的一致性（图7A ）。这种“共同向上”的途径包括细胞运输/内吞作用和粘附连接，先前显示它们在体内滞育小鼠胚胎中发生改变¹⁵ （图7B ）。转录、翻译和输出相关的细胞合成代谢途径通常被下调，凸显了在休眠期间保存能量的必要性（图 7 B）。信号和代谢途径似乎在人和小鼠细胞中的使用有所不同，例如，脂肪酸降解和相关因子 FOXO 和 PPAR 在小鼠中上调，而半乳糖代谢和 JAK-STAT 信号因子在 hPSC 中尤其增加（图 7 B））。这些结果支持这样的模型：向休眠的转变是一个主动调节的多步骤过程，其不仅仅是减少细胞能量消耗。

Mouse cells can be placed into dormancy via multiple methods, such as hormonal regulation or starvation of the mother, mTOR inhibition, and microRNA (miRNA) overexpression.⁵^,¹⁴^,¹⁷^,³⁴^,⁶⁵^,⁶⁶ To devise alternative entry routes into dormancy in human cells, we tested the effects of nutrient depletion (i.e., reducing anabolism), galactose metabolism (to potentially support the energy needs of dormant cells, as predicted by the proteome analysis), and inhibition of IGF receptor (to eliminate the growth signals interpreted by the ICM as shown in Figure 1). Inhibition of IGFR (denoted “IGFRi”) and nutrient depletion (denoted "reduced") alone reduced the percentage of hPSCs in mitosis from ∼17% in normal PXGL culture to ∼8% and 4%, respectively (Figures 7C and 7D). Galactose supplementation alone did not alter cellular proliferation (Figures 7D and 7E), suggesting that it is a supporting feature and not a driver of dormancy. Culture of hPSCs in reduced media with IGFRi and galactose yielded reduced proliferation and stable colonies that resumed proliferation once returned to standard media (Figures 7C–7E). We conclude that human PSCs can be transitioned into dormancy via alternate routes, and the dormant state of different species may be enhanced by adopting distinct culture conditions according to species-specific metabolic needs (Figure 7F).
小鼠细胞可以通过多种方法进入休眠状态，例如激素调节或母体饥饿、mTOR 抑制和 microRNA (miRNA) 过表达。 ⁵ ¹⁴ ¹⁷ ³⁴ ⁶⁵ ⁶⁶为了设计人类细胞进入休眠状态的替代途径，我们测试了营养消耗（即减少合成代谢）、半乳糖代谢（以潜在支持休眠细胞的能量需求，如蛋白质组分析所预测的那样）的影响）和抑制 IGF 受体（以消除 ICM 解释的生长信号，如图 1所示）。单独抑制 IGFR（表示为“IGFRi”）和营养消耗（表示为“减少”）可将有丝分裂中 hPSC 的百分比从正常 PXGL 培养物中的约 17% 分别降低至约 8% 和 4%（图7C 和 7D）。单独补充半乳糖不会改变细胞增殖（图 7D和 7E），表明它是一种支持功能，而不是休眠的驱动因素。在含有 IGFRi 和半乳糖的还原培养基中培养 hPSC 会产生增殖减少和稳定的集落，一旦返回标准培养基，集落就会恢复增殖（图7C-7E）。我们的结论是，人类 PSC 可以通过其他途径进入休眠状态，并且不同物种的休眠状态可以通过根据物种特定的代谢需求采用不同的培养条件来增强（图 7 F）。

Discussion 讨论

Many mammals use diapause as a strategy to maintain fertilized embryos in utero for extended time periods. Embryos of most species enter diapause at the blastocyst stage. In diapause, embryos switch to a dormant state that is characterized by a low energetic state that (1) limits proliferation, (2) maintains developmental competence, and (3) remains acutely responsive to reactivation cues. Notably, although embryos morphologically do not advance beyond the blastocyst stage, tissues within the blastocyst maintain residual proliferation either initially (e.g., in mouse) or continually (e.g., in roe deer). Additionally, the EPI of a subset of embryos progresses to generate rosettes typically attributed to early post-implantation embryos.¹⁸ As such, diapause appears to restrain the developmental progression of the embryo rather than stopping it. Here, we show that human PSCs and blastoids can enter a diapause-like dormant state in response to mTOR inhibition that lasts a maximum of 8 days in culture. mTORi-treated human blastoids have limited proliferation, developmental progression, and capacity to attach to hormonally stimulated endometrial cells. mTORi-treated blastoids can give rise to stem cell derivatives of the three blastocyst lineages and can continue to differentiate in post-implantation culture conditions. These observations correlate with the ability of many species to initiate diapause, specifically at the blastocyst stage.
许多哺乳动物使用滞育作为在子宫内长时间维持受精胚胎的策略。大多数物种的胚胎在囊胚阶段进入滞育。在滞育期间，胚胎切换到休眠状态，其特征是低能量状态，（1）限制增殖，（2）维持发育能力，（3）保持对重新激活线索的敏锐反应。值得注意的是，尽管胚胎在形态上不会前进超过囊胚阶段，但囊胚内的组织最初（例如，在小鼠中）或持续地（例如，在狍中）维持残余增殖。此外，胚胎子集的 EPI 会产生通常归因于早期植入后胚胎的玫瑰花结。 ¹⁸因此，滞育似乎会抑制而不是阻止胚胎的发育进程。在这里，我们证明人类 PSC 和胚泡可以响应 mTOR 抑制进入类似滞育的休眠状态，这种状态在培养物中最多持续 8 天。 mTORi 处理的人胚细胞的增殖、发育进程和附着于激素刺激的子宫内膜细胞的能力有限。 mTORi 处理的胚泡可以产生三个囊胚谱系的干细胞衍生物，并且可以在植入后培养条件下继续分化。这些观察结果与许多物种启动滞育的能力相关，特别是在囊胚阶段。

Human embryos are notoriously variable in their capacity to progress through early development, with an estimated 1/3 of pregnancies failing in the first trimester due to genetic and non-genetic abnormalities.⁶⁷^,⁶⁸ In this context, our results have several important implications for technological advance as well as fundamental understanding of human early embryogenesis: (1) stimulation of mTOR/phosphatidylinositol 3-kinase (PI3K) pathway activity through supplementation with IGF1 may promote cell proliferation and boost blastocyst formation rates, supporting earlier findings⁶⁹^,⁷⁰^,⁷¹; (2) inhibition of the mTOR pathway at the blastocyst stage might provide a valuable extended time window for characterization and scoring of embryos or synchronization to the mother’s hormonal cycle during an assisted reproductive technology procedure; and (3) dormant hPSCs and blastoids can be used to understand factors supporting the prolonged maintenance of pluripotency (similar to the role of LIF in mouse pluripotency⁴⁸).
众所周知，人类胚胎在早期发育过程中的进展能力存在很大差异，估计有 1/3 的妊娠因遗传和非遗传异常而在妊娠早期失败。 ⁶⁷ ⁶⁸在这种背景下，我们的结果对技术进步以及对人类早期胚胎发生的基本理解具有几个重要意义：(1) 通过补充 IGF1 刺激 mTOR/磷脂酰肌醇 3-激酶 (PI3K) 通路活性可能会促进细胞增殖和提高囊胚形成率，支持早期发现⁶⁹ ⁷⁰ ⁷¹ ； (2) 在囊胚阶段抑制 mTOR 通路可能会为辅助生殖技术过程中胚胎的表征和评分或与母亲激素周期的同步提供宝贵的延长时间窗口； (3) 休眠 hPSC 和胚泡可用于了解支持多能性长期维持的因素（类似于 LIF 在小鼠多能性中的作用⁴⁸ ）。

Dormant hPSCs and blastoids retain the ability to reactivate upon withdrawal of mTORi. However, the starting population of blastocyst- and peri-implantation-like hPSCs show heterogeneity in terms of the capacity to enter and maintain dormancy. The underlying reasons for this heterogeneity remain to be explored. We speculate that the different states that cells reside in due to, e.g., their cell-cycle phase or transcriptional status at the time of treatment could be contributing factors.⁷² We note that human PSCs have higher levels of apoptosis compared with mouse embryonic stem cells (ESCs) and require periodic use of a ROCK inhibitor for stabilization, particularly at the time of passaging. Thus, maintaining hPSCs under mTORi is more challenging as compared with mouse ESCs. Among the conditions tested in this manuscript, PXGL media offers a more stable platform for further investigations of dormancy regulation in pluripotent human cells. Yet, it is advisable to compare different pluripotent states as well as use blastoids for functional testing of dormancy regulators.
休眠的 hPSC 和母细胞保留在 mTORi 撤除后重新激活的能力。然而，囊胚样和植入周围样 hPSC 的起始群体在进入和维持休眠的能力方面表现出异质性。这种异质性的根本原因仍有待探索。我们推测，由于细胞周期阶段或治疗时的转录状态等原因，细胞所处的不同状态可能是影响因素。 ⁷²我们注意到，与小鼠胚胎干细胞 (ESC) 相比，人类 PSC 的细胞凋亡水平更高，需要定期使用 ROCK 抑制剂来稳定，特别是在传代时。因此，与小鼠 ESC 相比，在 mTORi 下维持 hPSC 更具挑战性。在本手稿测试的条件中，PXGL 介质为进一步研究多能人类细胞的休眠调节提供了更稳定的平台。然而，建议比较不同的多能状态以及使用胚泡进行休眠调节剂的功能测试。

In recent years, regulated translation emerged as an important new player connecting stem cell activity to the microenvironment.⁷³^,⁷⁴^,⁷⁵ Downregulation of translation is one of the main components of the dormancy response observed here. However, in the mouse, neither embryos nor cells can be put into dormancy by inhibition of translation alone.³⁴ The same is true for human blastoids, which show deterioration of EPI upon prolonged inhibition of translation. These results suggest that the mTOR pathway activity is quantitatively adjusted in a tissue-specific manner and that its effect extends beyond translation. mTORi-based rewiring of metabolism appears to be a necessary component for the maintenance of the dormant state.¹⁵ Upstream of mTOR, selective depletion of nutrients or growth factors such as IGF1 could trigger mTOR inhibition and dormancy in humans. As the expression of IGF1 is estrogen-dependent and estrogen deprivation is the trigger for the non-receptivity of the uterus, which induces diapause in the mouse, IGF1 depletion may be a major upstream regulator of diapause. Therefore, human blastocysts, although they may not undergo diapause, may have an ability to modulate mTOR activity to pace the growth and development of the ICM and TE prior to implantation. This property of mTOR activity to pace early development may be a legacy of the evolutionary process that is conserved in human embryos and, although not necessarily exploited for diapause, may contribute to the timing of human blastocyst development and implantation.
近年来，调控翻译成为连接干细胞活动与微环境的重要新参与者。 ⁷³ ⁷⁴ ⁷⁵翻译下调是此处观察到的休眠反应的主要组成部分之一。然而，在小鼠中，胚胎和细胞都不能仅通过抑制翻译来进入休眠状态。 ³⁴人类胚细胞也是如此，在长期抑制翻译后，其 EPI 会恶化。这些结果表明 mTOR 通路活性以组织特异性方式进行定量调整，并且其影响超出了翻译范围。基于 mTORi 的代谢重新布线似乎是维持休眠状态的必要组成部分。 ¹⁵ mTOR 上游的营养物质或生长因子（例如 IGF1）的选择性消耗可能会引发人类 mTOR 抑制和休眠。由于 IGF1 的表达依赖于雌激素，而雌激素剥夺会引发子宫不接受子宫，从而导致小鼠滞育，因此 IGF1 耗竭可能是滞育的主要上游调节因子。因此，人类囊胚虽然可能不会经历滞育，但可能具有调节 mTOR 活性的能力，以在植入前调节 ICM 和 TE 的生长和发育。 mTOR 活性的这种加快早期发育速度的特性可能是人类胚胎中保留的进化过程的遗产，尽管不一定用于滞育，但可能有助于人类囊胚发育和植入的时间。

Limitations of the study 研究的局限性

The results, on which our conclusion of the preserved human dormancy response is based, were generated using human PSCs and blastoids. Human blastoids closely represent the human blastocyst at the morphological and transcriptional levels. However, further experimentation is needed to directly assess the human blastocysts’ capacity to enter and withstand a dormant state that fulfills hallmarks of diapause. A large number of embryos is required to arrive at statistically relevant conclusions due to the inherent variability in the timing of human blastocyst development. The functional reversibility of the response is critical in this context.
我们使用人类 PSC 和胚泡得出的结果是我们对保存的人类休眠反应的结论所依据的。人类囊胚在形态和转录水平上密切代表人类囊胚。然而，还需要进一步的实验来直接评估人类囊胚进入和承受符合滞育特征的休眠状态的能力。由于人类囊胚发育时间的固有变异性，需要大量胚胎才能得出统计相关的结论。在这种情况下，反应的功能可逆性至关重要。

In mouse and in human systems, mTOR is likely only one of the components of dormancy regulation. Even though mTORi is sufficient to trigger dormancy, dormancy culture systems may be improved by further molecular and functional analysis as we recently demonstrated for mouse embryos.¹⁵^,¹⁶^,¹⁷ Under current conditions, the residual proliferation and development of the TE renders the structure fragile and prone to collapse, limiting the functionality upon reactivation. Further optimization of culture conditions may overcome these limitations.
在小鼠和人类系统中，mTOR 可能只是休眠调节的组成部分之一。尽管 mTORi 足以触发休眠，但正如我们最近在小鼠胚胎中所证明的那样，可以通过进一步的分子和功能分析来改善休眠培养系统。 ¹⁵ ¹⁶ ¹⁷在当前条件下，TE 的残余增殖和发展导致结构脆弱且容易崩溃，限制了重新激活后的功能。进一步优化培养条件可能会克服这些限制。

Resource availability 资源可用性

Lead contact 铅接触

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Aydan Bulut-Karslioğlu (aydan.karslioglu@molgen.mpg.de).
有关资源和试剂的更多信息和请求应直接联系首席联系人 Aydan Bulut-Karslioğlu ( aydan.karslioglu@molgen.mpg.de ）。

Materials availability 材料可用性

This study did not generate new, unique reagents.
这项研究并没有产生新的、独特的试剂。

Data and code availability
数据和代码可用性

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Single-cell RNA-seq and proteomics data have been deposited at GEO and to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository,⁷⁶ respectively, and are publicly available as of the date of publication. This paper also analyzes existing, publicly available data. All accession numbers are listed in the key resources table. Microscopy data reported in this paper will be shared by the lead contact upon request.
单细胞 RNA 测序和蛋白质组学数据已存放在 GEO 和 ProteomeXchange 联盟 ( http://proteomecentral.protomexchange.org) ）通过 PRIDE 合作伙伴存储库分别⁷⁶ ，并且自发布之日起可公开获取。本文还分析了现有的公开数据。所有入藏号都列在关键资源表中。本文报告的显微镜数据将根据要求由主要联系人共享。
•
This paper does not report original code.
本文不报告原始代码。
•
Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.
重新分析本文报告的数据所需的任何其他信息可根据要求向主要联系人提供。

Acknowledgments 致谢

We thank members of the Rivron and Bulut-Karslioğlu Labs, Ludovic Vallier, Michelle Percharde, and Helene Kretzmer for critical feedback and MPIMG scientific facilities for excellent service. We additionally thank VBC Biooptics, Proteomics, and Next Generation Sequencing facilities for their outstanding service and Giovani Sestini for his assistance in image analysis. For the purpose of open access, the author has applied a CC BY public copyright license. This project was supported by the German Academic Exchange Service (DAAD) PhD Fellowship (91730547 to D.P.I.), the Swiss National Science Foundation Early Postdoc Mobility fellowship (P2EZP3_195682 to V.A.v.d.W.), the Wellcome HDBI initiative (UK Human Developmental Biology Initiative 360G-Wellcome-215116_Z_18_Z to T.R. and K.K.N.), the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (ERC-Co grant agreement no. 101002317, “BLASTOID: a discovery platform for early human embryogenesis” to N.R.), Marie Skłodowska-Curie grant agreement no. 101026451 to H.H.K., the Austrian Science Fund (FWF) through a Lise Meitner Programme (project no. M3131-B to H.H.K.), the Max Planck Society (E.G.S. and A.B.-K.), and the Sofja Kovalevskaja Award (Humboldt Foundation) to A.B.-K. Work in the laboratory of K.K.N. was supported by the Francis Crick Institute, which receives its core funding from Cancer Research UK (FC001120), the UK Medical Research Council (FC001120), and the Welcome Trust (FC001120).
我们感谢 Rivron 和 Bulut-Karslioğlu 实验室的成员 Ludovic Vallier、Michelle Percharde 和 Helene Kretzmer 提供的重要反馈，感谢 MPIMG 科学设施提供的优质服务。我们还感谢 VBC Biooptics、蛋白质组学和下一代测序设施提供的出色服务，并感谢 Giovani Sestini 在图像分析方面提供的帮助。出于开放获取的目的，作者已申请CC BY公共版权许可。该项目得到了德国学术交流服务（DAAD）博士奖学金（ 91730547至DPI）、瑞士国家科学基金会早期博士后流动奖学金（ P2EZP3_195682至VAvdW）、 Wellcome HDBI计划（英国人类发育生物学计划360G-Wellcome- 215116_Z_18_Z至 TR 和 KKN），欧洲研究理事会(ERC) 根据欧盟地平线 2020 研究和创新计划（ERC-Co 资助协议编号101002317 ，“ BLASTOID：早期人类胚胎发生的发现平台”至 NR）， Marie斯克沃多夫斯卡-居里赠款协议101026451 HHK、奥地利科学基金(FWF) 通过 Lise Meitner 计划（项目编号M3131-B至 HHK）、马克斯·普朗克学会（EGS 和 AB-K.）以及Sofja Kovalevskaja 奖（洪堡基金会） AB-K。在KKN实验室工作该项目得到了弗朗西斯·克里克研究所 (Francis Crick Institute)的支持，该研究所的核心资金来自英国癌症研究中心 ( FC001120 )、英国医学研究委员会( FC001120 ) 和Welcome Trust ( FC001120 )。

Author contributions 作者贡献

Conceptualization, D.P.I., H.H.K., V.A.v.d.W., N.R., and A.B.-K.; investigation, D.P.I., H.H.K., V.A.v.d.W., H.K., T.R., C.S.S., S.E.W., A.M., and I.D.; formal analysis, V.A.v.d.W., S.J.P., and M.N.; visualization, D.P.I., H.H.K., V.A.v.d.W., T.R., and C.S.S.; writing – original draft, D.P.I., H.H.K., V.A.v.d.W., N.R., and A.B.-K.; writing – review & editing, D.P.I., H.H.K., V.A.v.d.W., S.J.P., N.R., and A.B.-K.; resources, E.G.S., K.E., P.S., and L.C.; funding acquisition, D.P.I., H.H.K., V.A.v.d.W., K.K.N., N.R., and A.B.-K.; supervision, K.K.N., N.R., and A.B.-K.
概念化、DPI、HHK、VAvdW、NR 和 AB-K。；调查、DPI、HHK、VAvdW、HK、TR、CSS、SEW、AM 和 ID；形式分析、VAvdW、SJP 和 MN；可视化、DPI、HHK、VAvdW、TR 和 CSS；写作——原稿、DPI、HHK、VAvdW、NR 和 AB-K。写作 – 审阅和编辑、DPI、HHK、VAvdW、SJP、NR 和 AB-K。资源、EGS、KE、PS 和 LC；资金收购、DPI、HHK、VAvdW、KKN、NR 和 AB-K。；监督、KKN、NR 和 AB-K。

Declaration of interests 利益申报

The Institute for Molecular Biotechnology, Austrian Academy of Sciences has filed patent application EP21151455.9 describing the protocols for human blastoid formation, and H.H.K., H.K., and N.R. are the inventors on this patent.
奥地利科学院分子生物技术研究所已提交专利申请EP21151455.9，描述了人类母细胞形成的方案，HHK、HK和NR是该专利的发明人。

STAR★Methods STAR★方法

Key resources table 关键资源表

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
Mouse anti-Oct3/4 (C-10) monoclonal antibody	Santa Cruz Biotechnology	Cat# sc-5279, RRID:AB_628051
Rabbit anti-GATA-3 polyclonal antibody	Santa Cruz Biotechnology	Cat# sc-9009, RRID:AB_640893
Rat anti-Gata-4 monoclonal antibody	Thermo Fisher Scientific	Cat# 14-9980-82, RRID:AB_763541
Rat anti-SOX2 monoclonal antibody	Thermo Fisher Scientific	Cat# 14-9811-82, RRID:AB_11219471
Mouse anti-hCG beta monoclonal antibody	Abcam	Cat# ab9582, RRID:AB_296507
Human AP-2 gamma Antibody	R and D Systems	Cat# AF5059, RRID:AB_2255891
Rabbit anti-Phospho-S6 Ribosomal Protein (Ser235/236) (D57.2.2E) monoclonal antibody	Cell Signaling Technology	Cat# 4858, RRID:AB_916156
Goat anti-human Sox17 polyclonal antibody	R and D Systems	Cat# AF1924, RRID:AB_355060
Rabbit anti-NR2F2 monoclonal antibody	Abcam	Cat# ab211776, RRID:AB_2893028
Mouse anti-CDX-2 monoclonal antibody	BioGenex	Cat# MU392-UC, RRID:AB_2335627
Human TROP-2 antibody, Alexa Fluor® 488-conjugated	R and D Systems	Cat# FAB650G, RRID:AB_3101996
PDGF Receptor α (D13C6) monoclonal antibody, Alexa Fluor® 647-Conjugated	Cell Signaling Technology	Cat# 5876, RRID:AB_2797623
CD197 (CCR7) monoclonal antibody (3D12), PE-Conjugated	Thermo Fisher Scientific	Cat# 12-1979-42, RRID:AB_10670625
Rat anti-Cytokeratin 8 antibody	DSHB	Cat# TROMA-I, RRID:AB_531826
Rabbit anti-phospho AKT (Ser473) (D9E) XP monoclonal antibody	Cell Signaling Technology	Cat# 4060, RRID:AB_2315049
Purified mouse anti-Ki-67	BD Pharmingen/BD Biosciences	Cat#556003; RRID:AB_393778
Rabbit anti-Histone H3 (phospho S10) polyclonal antibody	Abcam	Cat#ab5176; RRID:AB_304763
Rabbit anti-Nanog recombinant monoclonal antibody [EPR2027(2)]	Abcam	Cat# ab109250; RRID:AB_10863442
Alexa Fluor® 488 anti-H2A.X Phospho (Ser139) Antibody	Biolegend	Cat#613405; RRID:AB_528914
Phospho-4E-BP1 (Thr37/46) (236B4) Rabbit mAb	Cell Signaling Technology	Cat# 2855; RRID:AB_2292749
Goat anti-NANOG polyclonal antibody	R and D Systems	Cat# AF1997, RRID:AB_355097
Goat anti-Gata-3 polyclonal antibody	R and D Systems	Cat# AF2605, RRID:AB_2108571
Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 568	Thermo Fisher Scientific	Cat# A10042, RRID: AB_2534017
Donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 488	Thermo Fisher Scientific	Cat# 21202, RRID: AB_141607
Donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 647	Thermo Fisher Scientific	Cat# A-31571, RRID:AB_162542
Donkey anti-Goat IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 647	Thermo Fisher Scientific	Cat# A-21447, RRID:AB_2535864
Donkey anti-Rat IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 488	Thermo Fisher Scientific	Cat# A-21208, RRID:AB_2535794
Chemicals, peptides, and recombinant proteins
Recombinant Human IGF-I/IGF-1 Protein, CF	R and D Systems	Cat#291-G1
DMEM/F-12	Media lab, in-house, Vienna Biocenter	N/A
Neurobasal medium	Media lab, in-house, Vienna Biocenter	N/A
DMEM high glucose	Media lab, in-house, Vienna Biocenter	N/A
GlutaMAX	Gibco	Cat#35050038
Sodium pyruvate	Gibco	Cat#11360070
HEPES	Media lab, in-house, Vienna Biocenter	N/A
β-Mercaptoethanol	Gibco	Cat#31350010
Penicillin/streptomycin	Sigma	Cat#P0781
MEM non-essential amino acid (NEAA)	Gibco	Cat#11140035
N-2 supplement	Gibco	Cat#17502048
B-27 supplement	Gibco	Cat#17504044
BSA (35%)	Sigma	Cat#A7979
Fetal bovine serum (FBS)	Sigma	Cat#F7524
Fetal bovine serum (FBS)	Gibco	Cat#16140
Phosphate buffered saline (PBS)	Media lab, in-house, Vienna Biocenter	N/A
Gelatin solution	Sigma	Cat#G1393
Accutase	BioLegend	Cat#423201
TrypLE Express Enzyme (1×), no phenol red	Gibco	Cat#12604039
Mirdametinib (PD0325901)	MedChemExpress	Cat#HY-10254
XAV-939	MedChemExpress	Cat#HY-15147
Gö 6983	MedChemExpress	Cat#HY-13689
Human leukemia inhibitory factor	In-house, Vienna Biocenter	N/A
A 83-01	MedChemExpress	Cat#HY-10432
1-Oleoyl Lysophosphatidic Acid	BioGems	Cat#2256236
Y-27632 dihydrochloride	MedChemExpress	Cat#HY-10583
CMRL Medium, no glutamine	Gibco	Cat#21530027
Progesterone	Sigma	Cat#P8783
β-Estradiol	Sigma	Cat#E2758
Corning® Matrigel® Growth Factor Reduced (GFR)	Corning	Cat#356231
Pierce™ 16% Formaldehyde (w/v), Methanol-free	Thermo Fisher Scientific	Cat#28906
Insulin-Transferrin-Selenium-Ethanolamine (ITS -X) (100X)	Gibco	Cat#51500056
EGF Protein	MedChemExpress	Cat#HY-P7109
Valproic acid sodium salt	Sigma	Cat#P4543
Laduviglusib (CHIR-99021)	MedChemExpress	Cat#HY-10182
L-Ascorbic acid	Sigma	Cat#A4403
SB-431542	MedChemExpress	Cat#HY-10431
Geltrex™ LDEV-Free, hESC-Qualified, Reduced Growth Factor Basement Membrane Matrix	Gibco	Cat#A1413301
Insulin-Transferrin-Selenium	In-house, Vienna Biocenter	N/A
N-acetyl-L-cysteine	Sigma	Cat#A9165
Nicotinamide	Sigma	Cat#N0636
Recombinant FGF2-G3 protein	Qkine	Cat#Qk053
Recombinant human HGF (NK1) protein	Qkine	Cat#Qk013
Recombinant FGF-10 protein	Qkine	Cat#Qk003
SB 202190	MedChemExpress	Cat#HY-10295
R-Spondin1-conditioned medium	In-house, Vienna Biocenter	N/A
Noggin-conditioned medium	In-house, Vienna Biocenter	N/A
Triton X-100	Sigma	Cat#X100
Tween 20	Sigma	Cat#P1379
TrypLE™ Select Enzyme (10X), no phenol red	Gibco	Cat#A1217702
μ-Slide 18 Well Flat dish	Ibidi	Cat#81826
SYTOX AADvanced	Thermo Fisher Scientific	Cat#S10274
Annexin V Ready Flow Conjugates for Apoptosis Detection	Thermo Fisher Scientific	Cat#R37174
Fx-Cycle violet	Thermo Fisher Scientific	Cat#F10347
INK128	MedChemExpress/Biozol	Cat#MCE-HY-13328
mTeSR	StemCell Technologies	Cat#85850
RSeT	StemCell Technologies	Cat#05975
RapaLink-1	Apexbio/Biozol	Cat#APE-A8764
Torin1	Abcam	Cat#ab218606
Galactose	Sigma	Cat#G5388
Linsitinib(IGF-1R)	MedChemExpress/Biozol	Cat#HY-10191
Vectashield with DAPI	Vector Labs	Cat#H-2000
Critical commercial assays
meditrol hCG Schwangerschaftstest	Medichem	Cat#159083
Click-iT EdU Alexa Fluor 488 Flow Cytometry Assay kit	Thermo Fisher Scientific	Cat#C10425
iST 96x	PreOmics	Cat#P.O.00027
EasySep™ Dead Cell Removal (Annexin V) Kit	Stem Cell Technologies	Cat#17899
NC-Slide A8™	ChemoMetec	Cat#942-0003
Solution 18, AO DAPI Staining Reagent	ChemoMetec	Cat#910-3018
Chromium Next GEM Single Cell 3ʹ LT Kit v3.1	10x Genomics	Cat#PN-1000325
Chromium Next GEM Chip L Single Cell Kit	10x Genomics	Cat#PN-1000321
Chromium Next GEM Single Cell 3’ GEM, Library & Gel Bead Kit	10x Genomics	Cat#PN-1000128
Chromium Next GEM Chip G Single Cell Kit	10x Genomics	Cat#PN-1000127
Deposited data
scRNA-seq	This paper	GEO: GSE267302
scRNA-seq	https://doi.org/10.1016/j.cell.2016.03.023 ²⁵	EBI: E-MTAB-3929
scRNA-seq	https://doi.org/10.1038/s41586-019-1500-0 ⁷⁷	GEO: GSE109555
scRNA-seq	https://doi.org/10.1016/j.stem.2021.04.031 ⁷⁸	GEO: GSE171820
scRNA-seq	https://doi.org/10.1016/j.stem.2021.04.027 ⁴⁷	EBI: PRJEB30442
scRNA-seq	https://doi.org/10.1038/s41586-019-1875-y ⁷⁹	GEO: GSE136447
scRNA-seq	https://doi.org/10.1038/nsmb.2660 ²⁶	GEO: GSE36552
scRNA-seq	https://doi.org/10.1038/s41586-021-04158-y ⁸⁰	EBI: E-MTAB-9388
Proteomics data	This paper	PRIDE: PXD036258, PXD029513, PXD052209. Dataset PXD036258 contains more time-point data than presented in the figures.
Experimental models: Cell lines
WAe009-A, H9 female human ESC	WiCell Research Institute (WA)	WAe009-A (RRID:CVCL_9773)
Ishikawa cell line	Sigma-Aldrich	99040201-1VL
ES-E14 male mouse ESC	Sarah Kinkley Lab, Max Planck Institute for Molecular Genetics	RRID:CVCL_C320
Zip13k2 female human iPSCs	Franz-Joseph Müller Lab, for Molecular Genetics	RRID:CVCL_UF44
Endometrial organoids	Hossein Baharvand Lab, Royan Institute for Stem Cell Biology and Technology	N/A
Experimental models: Organisms/strains
Mouse: wild-type: CD1	In-house, Max Planck Institute for Molecular Genetics	N/A
Mouse: wild-type: C57Bl/6xCBA	In-house, Max Planck Institute for Molecular Genetics	N/A
Software and algorithms
FIJI	Schindelin et al.⁸¹	https://imagej.net/software/fiji/
FlowJo V10	FlowJo, LLC	https://www.flowjo.com/
Adobe Creative Suite	Adobe	https://www.adobe.com/creativecloud.html#
Rstudio	Rstudio	https://rstudio.com
Prism 8	Graphpad Software Inc.	https://www.graphpad.com
Cellranger	10X Genomics	https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/installation
Spectronaut 18.5	Biognosys	https://biognosys.com/resources/spectronaut-18/
MS2Go	In-house, Vienna Biocenter	N/A
MaxQuant software	Cox and Mann⁸²	https://www.maxquant.org
The Differential Enrichment analysis of Proteomics data (DEP) package	Zhang et al.⁸³	https://doi.org/10.18129/B9.bioc.DEP
ggplot2	Wickham et al.⁸⁴	https://ggplot2.tidyverse.org/
clusterProfiler R package	Yu et al.⁸⁵ and Wu et al.⁸⁶	https://git.bioconductor.org/packages/clusterProfiler
Gene Set Enrichment Analysis (GSEA)	Subramanian et al.⁸⁷	https://doi.org/10.1073/pnas.050658010 https://www.gsea-msigdb.org/gsea/index.jsp
Bitplane IMARIS 9.7.0 software	Oxford Instruments	https://imaris.oxinst.com/support/imaris-release-notes/9-7-0
FACSDIVA Software v8.0.1	BD Biosciences	https://www.bdbiosciences.com/en-us/products/software/instrument-software/bd-facsdiva-software
A Comprehensive Human Embryogenesis Reference Tool using Single-Cell RNA-Sequencing Data	Zhao et al.⁴⁴	https://doi.org/10.1101/2021.05.07.442980
DiaNN (v1.8)	Demichev et al.⁸⁸	https://doi.org/10.1038/s41592-019-0638-x https://github.com/vdemichev/DiaNN
batchelor v1.18.1	Haghverdi et al.⁸⁹	https://doi.org/10.18129/B9.bioc.batchelor
Seurat v5	Hao et al.⁹⁰	https://github.com/satijalab/seurat
SeuratWrappers v0.3.5	Satija Lab, New York Genome Center	https://github.com/satijalab/seurat-wrappers
scran package v1.30.2	Lun et al.⁹¹	https://bioconductor.org/packages/release/bioc/html/scran.html
ggscatter	Alboukadel Kassambara	https://doi.org/10.32614/CRAN.package.ggpubr
Zen Black software v2.3	Zeiss	https://www.zeiss.com/microscopy/en/products/software/zeiss-zen.html
Zen Blue software v2.3	Zeiss	https://www.zeiss.com/microscopy/en/products/software/zeiss-zen.html
CellProfiler software v4.2.1	Carpenter et al.⁹²	https://cellprofiler.org/
Prism v9	GraphPad	https://www.graphpad.com
Other
Evos M7000	Invitrogen	AMF7000
timsTOF SCP mass spectrometer	Bruker	https://www.bruker.com/en/products-and-solutions/mass-spectrometry/timstof/timstof-scp.html

Experimental model and study participant details
实验模型和研究参与者详细信息

Ethics statement 道德声明

Human embryos were donated to the research project by informed consent under UK Human Fertilization and Embryo Authority (HFEA) License number R0162. Approval was also obtained from the Health Research Authority’s Cambridge Central Research Ethics Committee, IRAS project ID 272218 (Cambridge Central reference number 19/EE/0297). The approval process entailed independent peer review along with approval from both the HFEA Executive Licensing Panel and the Executive Committees. Our research is compliant with the HFEA Code of Practice and has undergone independent HFEA inspections since the license was granted. Patient consent was obtained from Bourn Hall Clinic. Informed consent was obtained from all couples that donated surplus embryos following IVF treatment. Before giving consent, donors were provided with all of the necessary information about the research project, an opportunity to receive counselling, and details of the conditions that apply within the license and the HFEA Code of Practice. Specifically, patients signed a consent form authorizing the use of their embryos for research including stem cell derivation and for the results of these studies to be published in scientific journals. No financial inducements were offered for donation. Patient information sheets and the consent documents provided to patients are publicly available (https://www.crick.ac.uk/research/a-z-researchers/researchers-k-o/kathy-niakan/hfea-licence/). Embryos were cryopreserved, donated, and transferred to the Francis Crick Institute where they were thawed and used in the research project. Data were collected independently from the blastoid and PSC experiments to investigate the effects of IGF.
根据英国人类受精和胚胎管理局 (HFEA) 许可证号 R0162，经知情同意，将人类胚胎捐赠给该研究项目。还获得了卫生研究局剑桥中央研究伦理委员会的批准，IRAS 项目 ID 272218（剑桥中央参考号 19/EE/0297）。批准过程需要独立的同行评审以及 HFEA 执行许可小组和执行委员会的批准。我们的研究符合 HFEA 实践守则，并且自获得许可证以来已经接受了独立的 HFEA 检查。从 Bourn Hall Clinic 获得了患者的同意。所有在体外受精治疗后捐赠剩余胚胎的夫妇都获得了知情同意。在给予同意之前，向捐助者提供了有关研究项目的所有必要信息、接受咨询的机会以及许可证和 HFEA 业务守则中适用的条件的详细信息。具体来说，患者签署了一份同意书，授权使用其胚胎进行研究，包括干细胞衍生，并将这些研究的结果发表在科学期刊上。没有为捐赠提供经济诱因。向患者提供的患者信息表和同意文件是公开的（ https://www.crick.ac.uk/research/az-researchers/researchers-ko/kathy-niakan/hfea-licence/ ）。胚胎被冷冻保存、捐赠并转移到弗朗西斯·克里克研究所，在那里它们被解冻并用于研究项目。独立于胚泡和 PSC 实验收集数据，以研究 IGF 的作用。

Human embryo culture 人类胚胎培养

Vitrified embryos frozen in straws were thawed by quickly transferring the contents of the straw from liquid nitrogen directly into thaw solution (Irvine Scientific Vitrification Thaw Kit) and thawed per manufacturer’s instructions. Embryos frozen in cryopets were thawed for 3 seconds in a 37°C water bath before transferring into thaw solution (Irvine Scientific Vitrification Thaw Kit). Embryos frozen in glass ampoules were thawed completely in a 37°C water bath after the top of the vial was removed under liquid nitrogen. The contents were emptied onto a petri dish and the embryo transferred through a gradient of sucrose solutions (Quinn’s Advantage Thaw Kit, Origio) per manufacturer’s instructions. Embryos were routinely cultured in Global Media supplemented with 5 mg/mL LifeGlobal Human Protein Supplement (both LifeGlobal) pre-equilibrated overnight in an incubator at 37°C and 5% CO₂. These conditions were supplemented with IGF1 (291-G1/ CF, R&D) at a final concentration of 1.7 or 17 nM. The influence of sex on the results has not been studied due to limited resources. The applicability of the results to both sexes may be limited.
通过将吸管中的内容物从液氮中直接快速转移到解冻溶液（Irvine Scientific Vitrification Thaw Kit）中来解冻冷冻在吸管中的玻璃化胚胎，并按照制造商的说明进行解冻。将冷冻在低温容器中的胚胎在 37°C 水浴中解冻 3 秒，然后转移到解冻溶液（Irvine Scientific 玻璃化解冻套件）中。在液氮下除去小瓶顶部后，将玻璃安瓿中冷冻的胚胎在 37°C 水浴中完全解冻。将内容物倒空到培养皿中，并按照制造商的说明通过蔗糖溶液梯度（Quinn's Advantage Thaw Kit，Origio）转移胚胎。胚胎常规在补充有 5 mg/mL LifeGlobal 人类蛋白质补充剂（均为 LifeGlobal）的 Global Media 中培养，并在 37°C 和 5% CO ₂的培养箱中预平衡过夜。这些条件补充了终浓度为 1.7 或 17 nM 的 IGF1（291-G1/CF，R&D）。由于资源有限，尚未研究性别对结果的影响。结果对两性的适用性可能有限。

Human ESC and blastoid experiments
人类ESC和胚泡实验

As required by the German Stem Cell Act, all experiments involving hESCs and/or hESC derived blastoids were approved by Robert Koch Institute, Berlin, and the Commission for Science Ethics of the Austrian Academy of Sciences. The Wicell female line H9 (RRID:CVCL_9773) was used under the agreement 20-WO-341 for a research program entitled ‘Modeling early human development: Establishing a stem cell based 3D in vitro model of human blastocyst (blastoids)’. This work did not exceed a developmental stage normally associated with 14 consecutive days in culture after fertilization even though this is not forbidden by the ISSCR Guidelines as far as embryo models are concerned. All experiments complied with all relevant guidelines and regulations, including the 2021 ISSCR guidelines that forbid the transfer of human blastoids into any uterus.⁹³
根据德国干细胞法案的要求，所有涉及 hESC 和/或 hESC 衍生胚泡的实验均得到柏林罗伯特科赫研究所和奥地利科学院科学伦理委员会的批准。 Wicell 雌性系 H9 (RRID:CVCL_9773) 根据协议 20-WO-341 用于题为“模拟早期人类发育：建立基于干细胞的人类胚泡（胚泡）3D体外模型”的研究项目。这项工作没有超过通常与受精后连续培养 14 天相关的发育阶段，尽管就胚胎模型而言 ISSCR 指南并未禁止这样做。所有实验均遵守所有相关指南和法规，包括 2021 年 ISSCR 指南，该指南禁止将人类胚细胞转移到任何子宫中。 ⁹³

Mouse experiments 小鼠实验

All animal experiments were performed according to local animal welfare laws and approved by local authorities Landesamt für Gesund- heit und Soziales (license numbers ZH120, G0284/18, and G021/19) and UK Home Office (license number 70/8560) within the conditions of the Animal (Scientific Procedures) Act 1986. 8-12 week-old F1 (C57Bl/6xCBA) or CD1 females were used. Eight-week-old or older (C57Bl/6xCBA) F1 or CD1 male mice were used. Mice were housed in individually ventilated cages (Techniplast) on bedding (S-SELECT-09322) on a 12 h dark/light cycle and fed ad libitum (Ssniff, V1124-300). The influence of sex on the results has not been studied due to limited resources. The applicability of the results to both sexes may be limited.
所有动物实验均根据当地动物福利法进行，并获得地方当局 Landesamt für Gesundheit und Soziales（许可证号 ZH120、G0284/18 和 G021/19）和英国内政部（许可证号 70/8560）的批准。 1986 年动物（科学程序）法的条件。使用 8-12 周龄的 F1 (C57Bl/6xCBA) 或 CD1 雌性。使用八周龄或以上（C57Bl/6xCBA）F1 或 CD1 雄性小鼠。将小鼠饲养在单独通风的笼子（Techniplast）中，并在床上用品（S-SELECT-09322）上进行 12 小时的暗/光循环，并随意进食（Ssniff，V1124-300）。由于资源有限，尚未研究性别对结果的影响。结果对两性的适用性可能有限。

Stem cell lines and culture conditions
干细胞系和培养条件

Cell lines have not been authenticated. Cells periodically tested negative for mycoplasma. The influence of sex on the results has not been studied due to limited resources. The applicability of the results to both sexes may be limited.
细胞系尚未经过验证。细胞定期检测支原体呈阴性。由于资源有限，尚未研究性别对结果的影响。结果对两性的适用性可能有限。

Primed human PSC cell culture
引发的人 PSC 细胞培养

Wild-type Zip13k2 female hiPSCs (RRID:CVCL_UF44) were cultured without feeders on Matrigel-coated plates (Corning, 354277) in mTeSR media supplemented with 10 μM ROCK inhibitor (ROCKi) (Tocris, 1254) on the day of seeding. ROCKi was withdrawn the day after thawing, the media was changed daily, and cells were passaged every four days. At each passage, cells were dissociated as clumps using EDTA and re-plated with 10 μM ROCKi in a split ratio of 1:5. Cells were maintained at 37°C in 20% O₂ and 5% CO₂ incubator.
野生型 Zip13k2 雌性 hiPSC (RRID:CVCL_UF44) 在接种当天在补充有 10 μM ROCK 抑制剂 (ROCKi) (Tocris, 1254) 的 mTeSR 培养基中的 Matrigel 包被板 (Corning, 354277) 上无饲养层培养。解冻后第二天撤回ROCKi，每天更换培养基，并且每四天传代细胞一次。每次传代时，使用 EDTA 将细胞解离为团块，并以 1:5 的分流比重新铺板，加入 10 μM ROCKi。将细胞维持在37°C、20% O ₂和5% CO ₂培养箱中。

Naive-primed intermediate (RSeT)
天然引发的中间体 (RSeT)

To obtain naive-like cells, primed hiPSCs were plated at medium density (1:3-1:4 of a 75% confluent 10 cm plate) on Matrigel-coated dishes (Corning, 354277) and grown in mTeSR media for 24 hours at 37°C in 20% O₂, 5% CO₂. After 24 hours, mTeSR media was replaced with RSeT^TM Feeder-Free Medium (Stem Cell Technologies, 05975), and the cells were cultured at 37°C in 5% O₂, 5% CO₂. The cells were passaged at least 3 times to completely reprogram to a naive-like state. At each passage, cells were dissociated using TrypLE (Thermo Fisher Scientific, 12604-021) and 10⁶ cells were seeded on a 10 cm culture dish with 5 μM ROCKi. ROCKi was withdrawn the day after thawing.
为了获得幼稚样细胞，将引发的 hiPSC 以中等密度（1:3-1:4，75% 汇合的 10 cm 板）铺在基质胶包被的培养皿（Corning，354277）上，并在 mTeSR 培养基中于37°C，20% O ₂ 、5% CO ₂中。 24小时后，将mTeSR培养基更换为RSeT ^TM无饲养培养基(Stem Cell Technologies, 05975)，并将细胞在37℃、5% O ₂ 、5% CO ₂中培养。细胞至少传代3次才能完全重编程至幼稚状态。在每次传代时，使用TrypLE (Thermo Fisher Scientific, 12604-021)解离细胞，并将10 ^{6 个}细胞接种到含有5 μM ROCKi的10 cm培养皿上。 ROCKi 在解冻后第二天被撤回。

Naive human PSC culture (PXGL)
原始人类 PSC 培养 (PXGL)

H9 hESCs were cultured on gelatin-coated plates including a feeder layer of mitomycin-treated mouse embryonic fibroblasts (MEFs) in PXGL medium. PXGL medium is prepared using N2B27 basal medium supplemented with PD0325901(1 μM, MedChemExpress, HY-10254), XAV-939 (2 μM, MedChemExpress, HY-15147), Gö 6983 (2 μM, MedChemExpress, HY-13689) and human leukemia inhibitory factor (hLIF, 10 ng ml−1, in-house made). N2B27 basal medium contained DMEM/F12 (50%, GIBCO, 11320-074), neurobasal medium (50%, GIBCO, 21103-049), N-2 supplement (Thermo Fisher Scientific, 17502048), B-27 supplement (Thermo Fisher Scientific, 17504044), GultaMAX supplement (Thermo Fisher Scientific, 35050-038), non-essential amino acid, 2-mercaptoethanol (100 μM, Thermo Fisher Scientific, 31350010), and bovine serum albumin solution (0.45%, Sigma-Aldrich, A7979-50ML). Cells were routinely cultured in hypoxic chambers (5% CO₂, 5% O₂) and passaged as single cells every three to four days. All cell lines routinely tested negative for mycoplasma.
H9 hESC 培养在明胶包被的板上，包括 PXGL 培养基中经丝裂霉素处理的小鼠胚胎成纤维细胞 (MEF) 的饲养层。 PXGL 培养基使用补充有 PD0325901(1 μM, MedChemExpress, HY-10254)、XAV-939 (2 μM, MedChemExpress, HY-15147)、Gö 6983 (2 μM, MedChemExpress, HY-13689) 和人的 N2B27 基础培养基制备。白血病抑制因子（hLIF，10 ng ml−1，内部制造）。 N2B27 基础培养基含有 DMEM/F12 (50%, GIBCO, 11320-074)、神经基底培养基 (50%, GIBCO, 21103-049)、N-2 补充剂 (Thermo Fisher Scientific, 17502048)、B-27 补充剂 (Thermo Fisher Scientific, 17504044)、GultaMAX 补充剂 (Thermo Fisher Scientific, 35050-038)、非必需氨基酸、2-巯基乙醇 (100 μM, Thermo Fisher Scientific, 31350010) 和牛血清白蛋白溶液 (0.45%, Sigma-Aldrich, A7979-50ML）。细胞常规培养在低氧室（5% CO ₂ 、5% O ₂ ）中，并每三到四天作为单细胞传代。所有细胞系的支原体常规检测均为阴性。

Mouse ESC culture 小鼠ESC培养

ES-E14 male ESCs (RRID:CVCL_C320) were plated on 0.1% gelatin-coated dishes and grown in DMEM high glucose with Glutamax media (Thermo Fisher Scientific, 31966047) supplemented with 15% FBS (Thermo Fisher Scientific, 2206648RP), 1x NEAA (Thermo Fisher Scientific, 11140-035), 1x β-mercaptoethanol (Thermo Fisher Scientific, 21985023), 1x Penicillin/streptomycin (Life Technologies, 15140148) and 1000U/mL LIF and grown at 37°C in 20% O₂ and 5% CO₂ incubator. At each passage, cells were dissociated using TrypLE (Thermo Fisher Scientific, 12604-021) with daily media change.
将 ES-E14 雄性 ESC (RRID:CVCL_C320) 铺在 0.1% 明胶包被的培养皿上，并在含有补充有 15% FBS (Thermo Fisher Scientific, 2206648RP)、1x NEAA 的 Glutamax 培养基 (Thermo Fisher Scientific, 31966047) 的 DMEM 高葡萄糖中生长(Thermo Fisher Scientific, 11140-035)、1x β-巯基乙醇 (Thermo Fisher Scientific, 21985023)、1x 青霉素/链霉素 (Life Technologies, 15140148) 和 1000U/mL LIF，在 37°C、20% O ₂和 5 中生长％CO ₂培养箱。每次传代时，使用 TrypLE (Thermo Fisher Scientific, 12604-021) 分离细胞，并每日更换培养基。

Method details 方法详情

mTORi treatments mTORi 治疗

Primed 已涂底漆

Cells were treated with the catalytic mTOR inhibitor INK128 (MedChemExpress/ Biozol, MCE-HY-13328) at 200 nM final concentration and 10 μM ROCKi in mTeSR media for 1 day. The following day, ROCKi was withdrawn and cells were cultured in media containing INK128 for six days with daily media change.
在 mTeSR 培养基中用催化 mTOR 抑制剂 INK128 (MedChemExpress/Biozol, MCE-HY-13328) 以 200 nM 终浓度和 10 μM ROCKi 处理细胞 1 天。第二天，撤回 ROCKi，将细胞在含有 INK128 的培养基中培养六天，每天更换培养基。

Primed-naive intermediate
未处理过的中间体

10⁶ cells were plated and grown in RSeT medium without feeders. The media was changed every other day. On the fourth day of culture (approximate colony diameter of 100 μm), media containing 200 nM INK128 and 5 μM ROCKi was added for one day, then replaced with media containing only 200 nM of mTORi. Media was changed every day.
将10 ^{6 个}细胞铺板并在没有饲养层的RSeT培养基中生长。媒体每隔一天更换一次。培养第四天（菌落直径约为100μm），添加含有200nM INK128和5μM ROCKi的培养基一天，然后更换为仅含有200nM mTORi的培养基。媒体每天都会更换。

Naive 幼稚的

4x10⁵ cells were counted and plated onto confluent MEFs in PXGL media with 10 mM ROCKi, Matrigel and 200 nM INK128 for one day. The next day the media was replenished with PXGL and 200 nM INK128. To validate INK128 effect, RapaLink-1 (Biozol, APE-A87764) was used at 200 nM and Torin1 (Abcam, ab218606) was used at 200-1000 nM. To induce dormancy without mTORi, cells were cultured in reduced PXGL media (½ of the original concentration of N2 and B27) supplemented with 20 mM galactose (Sigma, G5388) and 5μM IGFRi (MedChemExpress, HY-10191).
对 4x10 ^{5 个}细胞进行计数并铺板到含有 10 mM ROCKi、Matrigel 和 200 nM INK128 的 PXGL 培养基中的汇合 MEF 上一天。第二天，用 PXGL 和 200 nM INK128 补充培养基。为了验证 INK128 的效果，RapaLink-1（Biozol，APE-A87764）的使用浓度为 200 nM，Torin1（Abcam，ab218606）的使用浓度为 200-1000 nM。为了在没有 mTORi 的情况下诱导休眠，将细胞培养在补充有 20 mM 半乳糖（Sigma，G5388）和 5μM IGFRi（MedChemExpress，HY-10191）的还原 PXGL 培养基（N2 和 B27 原始浓度的 1/2）中。

Mouse ESCs 小鼠ESC

For pausing of mouse ESCs, cells were treated with INK128 at 200 nM final concentration and the cells were cultured for six days. Media was replenished as necessary.
为了暂停小鼠 ESC，用终浓度为 200 nM 的 INK128 处理细胞，并将细胞培养六天。根据需要补充介质。

Mouse blastocysts 小鼠囊胚

For embryo collection, F1 (C57Bl/6xCBA) females were superovulated to obtain fertilized oocytes. Superovulated female mice were set up for mating with eight-week-old or older (C57Bl/6xCBA) F1 male mice. E0.5 embryos were collected from swollen ampulas in FHM medium (Millipore, MR-024-D), treated with hyaluronidase (Sigma-Aldrich, H4272) to remove cumulus cells, and embryos were cultured in drops of pre-equilibrated KSOM medium overlaid with mineral oil (Origio; ART-4008-5P) at 37.5°C in 5% CO₂.
对于胚胎收集，F1 (C57Bl/6xCBA) 雌性超排以获得受精卵母细胞。超数排卵的雌性小鼠被设置为与八周龄或更大的 (C57Bl/6xCBA) F1 雄性小鼠交配。 E0.5胚胎从肿胀的壶腹中收集到FHM培养基（Millipore，MR-024-D）中，用透明质酸酶（Sigma-Aldrich，H4272）处理以去除卵丘细胞，并将胚胎在覆盖的预平衡的KSOM培养基滴中培养在 37.5°C、5% CO ₂中使用矿物油（Origio；ART-4008-5P）。

In vivo diapause
体内滞育

In vivo diapause was induced through ovariectomy after natural mating of CD1 mice as described before.¹⁵^,³⁵^,⁶⁵ Afterwards, the females were injected every other day with 3 mg medroxyprogesterone 17-acetate subcutaneously. Diapaused blastocysts were flushed from uteri in M2 media after four days of diapause at EDG7.5 (equivalent day of gestation).
如前所述，CD1 小鼠自然交配后通过卵巢切除诱导体内滞育。 ¹⁵ ³⁵ ⁶⁵此后，雌性每隔一天皮下注射3 mg 17-醋酸甲羟孕酮。在 EDG7.5（相当于妊娠日）滞育四天后，将滞育的囊胚从子宫中的 M2 培养基中冲洗掉。

Blastoid generation 母细胞生成

Naive hPSCs were cultured under humidified conditions at 37°C in an incubator with 5% O₂ and 5% CO₂. Naive hPSCs were cultured on mitotically inactivated MEFs in PXGL medium. The medium was changed daily, and cells were passaged 4 days before blastoid formation. Blastoids were formed as previously published with minor modifications.³⁶^,⁹⁴^,⁹⁵ In brief, for extended pre-implantation culture, naive hPSCs were seeded into 300 or 400-micrometer hydrogel microwells in N2B27 with Y-27632. The next day, the medium was changed to PALLY medium, and half of the medium was replaced with fresh PALLY daily. After 4 days, blastoids were treated with 100, 200, and 300 nM RapaLink-1, and 100 and 1000 ng/ml cycloheximide in N2B27 medium. The medium remained unchanged thereafter. N2B27 basal medium contained DMEM/F12 (50%, in house made), neurobasal medium (50%, in-house made), 1X N-2 supplement (Thermo Fisher Scientific, 17502048), 1X B-27 supplement (Thermo Fisher Scientific, 17504044), 1X GultaMAX supplement (Thermo Fisher Scientific, 35050-038), 1X non-essential amino acid, 1 mM sodium pyruvate (Sigma), 2-mercaptoethanol (100 μM, Thermo Fisher Scientific, 31350010) and HEPES (10 mM, in house). Phase-contrast images were acquired using the Thermo Fisher Scientific EVOS cell imaging system. The number of blastoids in the microwells was manually counted for each well daily. Since some blastoids continue to grow and pop out from the microwells, quantification was performed only for visible blastoids in the microwells.
将初始 hPSC 在 37°C、5% O ₂和 5% CO ₂的培养箱中在潮湿条件下培养。将初始 hPSC 在 PXGL 培养基中的有丝分裂失活 MEF 上培养。每天更换培养基，并在胚泡形成前 4 天传代细胞。母细胞的形成与之前发布的一样，稍作修改。 ³⁶ ⁹⁴ ⁹⁵简而言之，为了延长植入前培养，将初始 hPSC 接种到含有 Y-27632 的 N2B27 中的 300 或 400 微米水凝胶微孔中。第二天换成PALLY培养基，每天更换一半新鲜PALLY培养基。 4 天后，用 N2B27 培养基中的 100、200 和 300 nM RapaLink-1 以及 100 和 1000 ng/ml 放线菌酮处理胚泡。此后介质保持不变。 N2B27 基础培养基含有 DMEM/F12（50%，自制）、neurobasal 培养基（50%，自制）、1X N-2 补充剂（Thermo Fisher Scientific，17502048）、1X B-27 补充剂（Thermo Fisher Scientific） , 17504044), 1X GultaMAX 补充剂 (Thermo Fisher Scientific, 35050-038), 1X 非必需氨基酸, 1 mM 丙酮酸钠 (Sigma), 2-巯基乙醇 (100 μM, Thermo Fisher Scientific, 31350010) 和 HEPES (10 mM ，在房子里）。使用 Thermo Fisher Scientific EVOS 细胞成像系统获取相差图像。每天对每个孔的微孔中的胚泡数量进行手动计数。由于一些胚泡继续生长并从微孔中弹出，因此仅对微孔中可见的胚泡进行定量。

Blastoid reactivation after mTORi treatment in postimplantation culture conditions
在植入后培养条件下 mTORi 处理后胚泡重新激活

The control and mTORi-treated blastoids were reactivated by culturing them under an extended culture condition previously described for human blastoids.³⁶ Blastoids were selected using a mouth pipette, washed with CMRL1066 medium, and transferred into wells of a 96-well plate coated with Matrigel containing pre-equilibrated media. Initially, the culture medium consisted of CMRL-1066⁹⁶ supplemented with 10% (v/v) FBS, 1 mM L-glutamine (Gibco), 1× N2 supplement, 1× B27 supplement, 1 mM sodium pyruvate (Sigma), 1 μM progesterone and 10 nM estrogen. Half of the medium was changed daily for the next three days, with the new medium supplemented with 5% Matrigel. Cultures were either fixed for staining after 2 or 4 days of culture with 4% PFA or dissociated for scRNA-seq after 4 days.
对照和 mTORi 处理的胚细胞通过在先前针对人类胚细胞描述的扩展培养条件下培养而被重新激活。使用口移液管选择^36个胚泡，用CMRL1066培养基洗涤，并转移至涂有含有预平衡培养基的Matrigel的96孔板的孔中。最初，培养基由 CMRL-1066 ⁹⁶组成，补充有 10% (v/v) FBS、1 mM L-谷氨酰胺 (Gibco)、1× N2 补充剂、1× B27 补充剂、1 mM 丙酮酸钠 (Sigma)、1 μM 黄体酮和 10 nM 雌激素。接下来的三天每天更换一半培养基，新培养基中补充有 5% 基质胶。培养 2 或 4 天后用 4% PFA 固定培养物进行染色，或在 4 天后解离用于 scRNA-seq。

Derivation of stem cell lines from blastoids
从胚泡中衍生干细胞系

The previously described protocols for deriving and culturing naive human pluripotent stem (PS) cells,⁵³ primitive endoderm/extraembryonic mesoderm (PrE/ExEM) like cells⁹⁷ and trophoblast stem cells (TSCs)⁹⁸ with minor modification were used. In brief, individual human blastoids were transferred onto MEFs and cultured in PXGL medium (for naive PS cells), NCL medium (for PrE/ExEM), and human TSC medium (for TSCs). Within two to seven days, the outgrowth became visible. The outgrowths were dissociated using TrypLE Express and passaged onto newly prepared MEF plates or Geltrex-coated plates. Individual colonies were dissociated and cultured in their respective media.
使用先前描述的用于衍生和培养幼稚人类多能干 (PS) 细胞、 ⁵³原始内胚层/胚外中胚层 (PrE/ExEM) 样细胞⁹⁷和滋养层干细胞 (TSC) ⁹⁸的方案，并进行了较小的修改。简而言之，将单个人胚泡转移到 MEF 上并在 PXGL 培养基（用于初始 PS 细胞）、NCL 培养基（用于 PrE/ExEM）和人 TSC 培养基（用于 TSC）中培养。两到七天内，生长物就变得可见。使用 TrypLE Express 解离产物并传代到新制备的 MEF 平板或 Geltrex 包被的平板上。将各个菌落解离并在各自的培养基中培养。

Attachment to endometrial cells
附着于子宫内膜细胞

OFEL 欧菲尔

For assays involving endometrial epithelial cells derived from organoids, open-faced endometrial layers (OFELs) were prepared as previously outlined.⁹⁴ Control or mTORi-treated blastoids were washed twice with CMRL medium and then transferred onto OFELs, where they were maintained at 37°C in a 20% O₂, 5% CO₂ for 48 hours in CMRL medium supplemented with 10 μM Y-27632, as described earlier. After this period, attachment was assessed by gently flushing around the blastoids with a mouth pipette, and attachment efficiency was determined based on the number of attached blastoids relative to the total number transferred.
对于涉及源自类器官的子宫内膜上皮细胞的测定，如前所述制备开放面子宫内膜层（OFEL）。 ⁹⁴对照或 mTORi 处理的胚芽用 CMRL 培养基洗涤两次，然后转移到 OFEL 上，在补充有 10 μM Y- 的 CMRL 培养基中于 37°C、20% O ₂ 、5% CO ₂中维持 48 小时。 27632，如前所述。此后，通过用口移液管轻轻冲洗胚泡周围来评估附着，并根据附着的胚泡数量相对于转移总数确定附着效率。

Ishikawa cell line 石川细胞系

For assays involving the Ishikawa cell line (a receptive endometrial cell line, 99040201-1VL), cells were cultured in DMEM/F-12 medium supplemented with 10% FBS. Two days prior to the assay, cells were seeded into a 96-well plate at a density of 7x10⁴ cells per well in DMEM/F-12 medium supplemented with 10% FBS, 1 μM progesterone and 10 nM estrogen. Control or mTORi-treated blastoids were then transferred onto the cell surface of a fully confluent cell layer in CMRL medium, as described earlier. The plate was subsequently placed in an incubator for 24 hours before attachment was assessed, following the procedure described for OFELs.
对于涉及 Ishikawa 细胞系（接受性子宫内膜细胞系，99040201-1VL）的测定，细胞在补充有 10% FBS 的 DMEM/F-12 培养基中培养。测定前两天，将细胞以每孔 7x10 ^{4 个}细胞的密度接种到 96 孔板的 DMEM/F-12 培养基中，培养基补充有 10% FBS、1 μM 孕酮和 10 nM 雌激素。然后将对照或 mTORi 处理的胚芽转移到 CMRL 培养基中完全汇合的细胞层的细胞表面上，如前所述。随后将板放入培养箱中 24 小时，然后按照 OFEL 描述的程序评估附着情况。

Immunofluorescence (IF) 免疫荧光 (IF)

Cells 细胞

Cells were cultured on glass coverslips and were fixed in 4% PFA for 10 min at room temperature, washed once in PBS, then permeabilized with 0.2% Triton X-100 in PBS for 5 min on ice. After washing once in PBS-T (PBS with 0.2% Tween-20), cells were blocked with blocking buffer (PBS-T, 2% BSA and 5% goat serum (Jackson Immunoresearch/Dianova, 017-000-121) for 1 hour at room temperature. Cells were then stained with primary antibodies against pS6 (CST Cat no: 4858) 1:200, pAKT (CST, 4060T) 1:200, KI67 (BD Pharmingen, 556003) 1:400, H3 phosphoS10 (Abcam, ab5176), 1:1000, OCT4 (Santa Cruz, sc5279) 1:50, NANOG (Abcam, ab109250) 1:200, gH2A.X (Biolegend, 613405) 1:400 overnight at 4°C. The cells were washed thrice with wash buffer (PBS-T, 2% BSA) for 10 min. Anti-rabbit (Thermo Fisher Scientific, A10042) or anti-mouse (Thermo Fisher Scientific, 21202) secondary antibody conjugated with Alexa Fluor was added to cells at a dilution of 1:700 in blocking buffer and incubated for 1 hour at room temperature, followed by 3 washes with wash buffer for 10 min. The coverslips were then mounted with Vectashield with DAPI (Vector labs, H-2000) and sealed with nail polish. Imaging was done using a Zeiss LSM880 Airy microscope using Airy scan mode and image processing was done using Zen black and Zen blue software (version 2.3). Image quantification was done using CellProfiler (version 4.2.1).⁹² Nuclei or cells were denoted as primary objects, intensities of proteins of interest were measured against nuclear or cell area. Data were plotted using GraphPad Prism (version 9).
将细胞培养在玻璃盖玻片上，并在室温下在 4% PFA 中固定 10 分钟，在 PBS 中洗涤一次，然后用 0.2% Triton X-100 的 PBS 溶液在冰上透化 5 分钟。在 PBS-T（含 0.2% Tween-20 的 PBS）中洗涤一次后，用封闭缓冲液（PBS-T、2% BSA 和 5% 山羊血清（Jackson Nutrition/Dianova, 017-000-121）封闭细胞 1 次）然后用针对 pS6 (CST Cat no: 4858) 1:200、pAKT (CST, 4060T) 1:200、KI67 (BD Pharmingen, 556003) 1:400、H3 磷酸化 S10 (Abcam) 的一抗对细胞进行染色。 , ab5176), 1:1000, OCT4 (Santa Cruz, sc5279) 1:50, NANOG (Abcam, ab109250) 1:200, gH2A.X (Biolegend, 613405) 1:400 在 4°C 下洗涤过夜。用洗涤缓冲液（PBS-T，2% BSA）洗涤三次，每次 10 分钟。将与 Alexa Fluor 缀合的抗兔（Thermo Fisher Scientific，A10042）或抗小鼠（Thermo Fisher Scientific，21202）二抗添加到细胞中。在封闭缓冲液中按 1:700 稀释并在室温下孵育 1 小时，然后用洗涤缓冲液洗涤 3 次，持续 10 分钟，然后用带有 DAPI 的 Vectashield（Vector labs，H-2000）封片并用指甲油密封。使用采用 Airy 扫描模式的 Zeiss LSM880 Airy 显微镜进行成像，并使用 Zen black 和 Zen blue 软件（版本 2.3）进行图像处理。使用 CellProfiler（版本 4.2.1）进行图像量化。 ⁹²细胞核或细胞被表示为主要对象，根据细胞核或细胞区域测量感兴趣蛋白质的强度。使用 GraphPad Prism（版本 9）绘制数据。

Embryos 胚胎

Human embryos were fixed in 4% paraformaldehyde in PBS for 1h at 4°C, and mouse embryos 10 min at room temperature. Embryos were washed once in PBS, then permeabilized with 1× PBS with 0.5% Triton X-100 and then blocked in blocking solution (10% FBS in 1× PBS with 0.1% Triton X-100) for 1-2 h at room temperature on a rotating shaker. Embryos were then incubated with primary antibodies diluted in blocking solution overnight at 4 °C on rotating shaker. The following day, embryos were washed once in 1× PBS with 0.1% Triton X-100 at room temperature on a rotating shaker, and then incubated with secondary antibodies diluted in blocking solution for 1 h at room temperature on a rotating shaker in the dark. Embryos were washed in 1× PBS with 0.1% Triton X-100 and counterstained with DAPI. The following antibodies and dilutions were used: anti-pS6 (Cell Signaling, 4858) 1:250, anti-OCT4 (Santa Cruz, 5279) 1:50, anti-NANOG (R&D AF1997) 1:200, anti-CDX2 (BioGenex, MU392-UC) 1:100, anti-pAKT (CST 4060T) 1:100, anti-4EBP1 (CST 2855) 1:100, and anti-GATA3 (R&D AF2605) 1:200. All secondary antibodies were Alexa Fluor (Life Technologies), raised in donkey and used 1:200 to 1:1000. For imaging, embryos were placed on a μ-Slide 18 Well Flat dish (Ibidi, 81826) in PBS and imaged on a Leica Sp8 confocal with a Leica HCX PL APO 63x / 1.3 GLYC CORR CS objective. The z-stack step is 3.5-5 mm. Imaging of mouse embryos was done using a Zeiss LSM880 Airy microscope using Airy scan mode.
人类胚胎在 4% 多聚甲醛的 PBS 溶液中于 4°C 固定 1 小时，小鼠胚胎于室温固定 10 分钟。胚胎在 PBS 中洗涤一次，然后用含有 0.5% Triton X-100 的 1× PBS 透化，然后在封闭液（含有 0.1% Triton X-100 的 1× PBS 中的 10% FBS）中室温封闭 1-2 小时在旋转摇床上。然后将胚胎与在封闭溶液中稀释的一抗在旋转摇床上于 4°C 下孵育过夜。第二天，在旋转摇床上用含 0.1% Triton X-100 的 1× PBS 在室温下将胚胎洗涤一次，然后在旋转摇床上与用封闭液稀释的二抗在室温下在黑暗中孵育 1 小时。胚胎在含有 0.1% Triton X-100 的 1× PBS 中洗涤，并用 DAPI 复染。使用以下抗体和稀释度：抗 pS6 (Cell Signaling, 4858) 1:250、抗 OCT4 (Santa Cruz, 5279) 1:50、抗 NANOG (R&D AF1997) 1:200、抗 CDX2 (BioGenex) 、MU392-UC) 1:100、抗 pAKT (CST 4060T) 1:100、抗 4EBP1 (CST 2855) 1:100 和抗 GATA3 (R&D AF2605) 1:200。所有二抗均为 Alexa Fluor (Life Technologies)，在驴中饲养，使用比例为 1:200 至 1:1000。为了成像，胚胎被放置在 PBS 中的 μ-Slide 18 孔平皿（Ibidi，81826）上，并在 Leica Sp8 共焦和 Leica HCX PL APO 63x / 1.3 GLYC CORR CS 物镜上成像。 z 堆栈步长为 3.5-5 毫米。使用采用艾里扫描模式的蔡司LSM880艾里显微镜对小鼠胚胎进行成像。

Blastoids and postimplantation culture
胚泡和植入后培养

Blastoids were collected using a mouth pipette and transferred to U-bottomed 96-well plates (Merck, BR701330). Once the structures had settled, the medium was washed twice with PBS and fixed with 4% PFA for 30 min at room temperature, followed by three 10-min washes with PBS. Postimplantation culture blastoids were fixed with 4% PFA for 30 min at room temperature, followed by three 10-min washes with PBS. PBS containing 2% BSA and 0.3% Triton X-100 was used for blocking/permeabilization for 3 hours at room temperature. The primary antibody was incubated at 4°C in blocking/permeabilization solution with gentle shaking and washed at least three times with PBS containing 0.1% Triton X-100 for 10 min. The following antibodies and dilutions were used:
使用口移液器收集胚泡并转移至 U 形底 96 孔板（Merck，BR701330）。结构沉降后，用 PBS 洗涤介质两次，并用 4% PFA 在室温下固定 30 分钟，然后用 PBS 洗涤 3 次，每次 10 分钟。植入后培养胚泡在室温下用 4% PFA 固定 30 分钟，然后用 PBS 洗涤 3 次，每次 10 分钟。使用含有2％BSA和0.3％Triton X-100的PBS在室温下封闭/透化3小时。将一抗在封闭/透化溶液中于 4°C 下孵育，轻轻摇动，并用含有 0.1% Triton X-100 的 PBS 洗涤至少 3 次，每次 10 分钟。使用以下抗体和稀释液：

OCT3/4 (C-10) (Santa Cruz Biotechnology, sc-5279) 1:200, GATA3 (Santa Cruz Biotechnology, sc-9009) 1:200, GATA4 (Invitrogen, 14-9980-82) 1:400, SOX2 (Invitrogen, 14-9811-82) 1:400, hCG beta (Abcam, ab9582) 1:100, hAP-2ɣ (TFAP2c, R&D Systems, AF5059) 1:300, phospho-S6 (Ser235/236) (Cell Signaling, 4858) 1:250, SOX17, (R&D Systems, AF1924-SP) 1:100, TROMA-1 (KRT-8) (DSHB) 1:1000, NR2F2 (Abcam, ab211776) 1:100, CDX2 (BioGenex, MU392A-5UC) 1:300.
OCT3/4 (C-10)（圣克鲁斯生物技术，sc-5279）1:200，GATA3（圣克鲁斯生物技术，sc-9009）1:200，GATA4（Invitrogen，14-9980-82）1:400，SOX2 （Invitrogen，14-9811-82）1：400，hCG beta（Abcam，ab9582）1：100，hAP-2ɣ（TFAP2c，R＆D Systems，AF5059）1：300，磷酸-S6（Ser235/236）（细胞信号传导） , 4858) 1:250, SOX17, (R&D Systems, AF1924-SP) 1:100, TROMA-1 (KRT-8) (DSHB) 1:1000, NR2F2 (Abcam, ab211776) 1:100, CDX2 (BioGenex, MU392A-5UC）1：300。

The secondary antibody was diluted in PBS containing 0.1% Triton X-100 and then incubated at room temperature in the dark for 1 hour. Subsequently, the blastoids were washed three times with PBS containing 0.1% Triton X-100 for 10 minutes each and prepared for imaging. For imaging purposes, the blastoids were positioned in glass-bottom plates. Confocal immunofluorescence images of blastoids were captured using an Olympus IX83 microscope equipped with a Yokogawa W1 spinning disk (Software: CellSense 2.3; camera: Hamamatsu Orca Flash 4.0). The acquired confocal images were analyzed, and display images were generated using either FIJI 1.53k or Bitplane IMARIS 9.7.0 software. Cell counting was performed using Bitplane IMARIS software, where parameters for cell size and fluorescence intensity were set for voxels, and overall cell count data were obtained for each image utilizing the IMARIS spot function.
二抗在含有0.1% Triton X-100的PBS中稀释，然后在室温下避光孵育1小时。随后，用含有0.1％Triton X-100的PBS洗涤胚泡3次，每次10分钟，并准备成像。为了成像目的，将胚泡放置在玻璃底板中。使用配备横河W1旋转盘的Olympus IX83显微镜（软件：CellSense 2.3；相机：Hamamatsu Orca Flash 4.0）捕获胚泡的共焦免疫荧光图像。对采集的共焦图像进行分析，并使用 FIJI 1.53k 或 Bitplane IMARIS 9.7.0 软件生成显示图像。使用 Bitplane IMARIS 软件进行细胞计数，其中为体素设置细胞大小和荧光强度参数，并利用 IMARIS 点功能获得每个图像的总体细胞计数数据。

Apoptosis assay 细胞凋亡检测

Adherent and floating cells were collected for the apoptosis assay. Cells were dissociated using TrypLE, washed in cold PBS, and resuspended in Annexin binding buffer (10 mM HEPES, 140 mM NaCl and 2.5 mM CaCl2, pH 7.4). Cell density was adjusted to 400,000 cells in 500 ml Annexin binding buffer. Staining for Annexin V was done according to manufacturer’s instructions (Thermo Fisher Scientific, R37174) along with dead cell stain using SYTOX AADvanced (Thermo Fisher Scientific, S10274) for 15 min at room temperature. A FACS AriaFusion flow cell cytometer was used to analyze cell staining. Data were analyzed using FlowJo (version 10) and plotted using GraphPad Prism (version 9).
收集贴壁和漂浮细胞用于细胞凋亡测定。使用 TrypLE 解离细胞，在冷 PBS 中洗涤，并重悬于膜联蛋白结合缓冲液（10 mM HEPES、140 mM NaCl 和 2.5 mM CaCl2，pH 7.4）中。在 500 ml 膜联蛋白结合缓冲液中将细胞密度调整为 400,000 个细胞。根据制造商的说明（Thermo Fisher Scientific，R37174）进行膜联蛋白 V 染色，并使用 SYTOX AADvanced（Thermo Fisher Scientific，S10274）在室温下进行死细胞染色 15 分钟。使用 FACS AriaFusion 流式细胞仪分析细胞染色。使用 FlowJo（版本 10）对数据进行分析，并使用 GraphPad Prism（版本 9）进行绘图。

Cell cycle assay 细胞周期测定

The Click-iT EdU Alexa Fluor 488 Flow Cytometry Assay kit (Thermo Fisher Scientific, C10425) was used. Cells were incubated at 37°C for 2 hours with 10 μM EdU in 5% O₂, 5% CO₂. Cells were then harvested, washed once with 3 ml of 1% BSA in PBS, centrifuged at 300 g for 5 min and the supernatant was removed. Cells were fixed in 100 ml Click-iT fixative for 15 minutes at room temperature, washed with 3 ml of 1% BSA in PBS, and resuspended in 100 ml 1x Click-iT saponin-based permeabilization and wash reagent and incubated for 15 minutes at room temperature. To this, 500 ml Click-iT reaction cocktail was added and incubated for 30 minutes in the dark at room temperature. After incubation, cells were washed, resuspended in 200 ul of the Saponin wash buffer. Fx-Cycle violet (Thermo Fisher Scientific, F10347) was added to 1 mg/ml to measure DNA content and incubated for 1 hour at room temperature in the dark. FACS AriaFusion cell cytometer was used to acquire data using BD Software v8.0.1. Data were analyzed using FlowJo (version 10) and plotted using GraphPad Prism (version 9).
使用 Click-iT EdU Alexa Fluor 488 流式细胞术测定试剂盒（Thermo Fisher Scientific，C10425）。将细胞与 10 μM EdU 在 5% O ₂ 、5% CO ₂中于 37°C 孵育 2 小时。然后收获细胞，用3ml 1%BSA的PBS溶液洗涤一次，300g离心5分钟并除去上清液。将细胞在 100 ml Click-iT 固定剂中室温固定 15 分钟，用 3 ml 1% BSA 的 PBS 溶液洗涤，然后重悬于 100 ml 1x Click-iT 基于皂苷的透化和洗涤试剂中，并在 30 ℃下孵育 15 分钟。室温。向其中添加500ml Click-iT反应混合物并在黑暗中在室温下孵育30分钟。温育后，洗涤细胞，重悬于200ul皂苷洗涤缓冲液中。添加 Fx-Cycle violet（Thermo Fisher Scientific，F10347）至 1 mg/ml 以测量 DNA 含量，并在室温下避光孵育 1 小时。 FACS AriaFusion 细胞仪用于使用 BD 软件 v8.0.1 采集数据。使用 FlowJo（版本 10）对数据进行分析，并使用 GraphPad Prism（版本 9）进行绘图。

Global proteomics 全球蛋白质组学

Low-input proteomics of stem cells
干细胞的低输入蛋白质组学

5000 cells per sample were lysed in a denaturing buffer, reduced and alkylated, and sequentially digested by Lys-C and trypsin. Peptides originating from about 1000 cells were loaded onto Evotips Pure (Evosep, Odense, Denmark) according to the manufacturer's protocol. Peptide separation was carried out by nanoflow reverse phase liquid chromatography (Evosep One, Evosep), using the Endurance column (15 cm x 150 μm ID, with Reprosil-Pur C18 1.9 μm beads EV1106, Evosep) with the 30 samples a day method (30SPD). The LC system was online coupled to a timsTOF SCP mass spectrometer (Bruker Daltonics, Bremen, Germany) applying the data-independent acquisition (DIA) with parallel accumulation serial fragmentation (PASEF) method.⁹⁹ MS data were processed with DiaNN (v1.8) and searched against in silico-predicted mouse or human spectra.⁸⁸
每个样品 5000 个细胞在变性缓冲液中裂解、还原和烷基化，然后依次用 Lys-C 和胰蛋白酶消化。根据制造商的方案，将源自约 1000 个细胞的肽加载到 Evotips Pure（Evosep，Odense，丹麦）上。通过纳流反相液相色谱（Evosep One，Evosep）进行肽分离，使用 Endurance 柱（15 cm x 150 μm ID，配有 Reprosil-Pur C18 1.9 μm 微珠 EV1106，Evosep），采用每天 30 个样品的方法（ 30SPD）。 LC 系统在线耦合到 timsTOF SCP 质谱仪（Bruker Daltonics，不来梅，德国），应用数据独立采集（DIA）和并行累积串行碎片（PASEF）方法。使用 DiaNN (v1.8) 处理^{99 个}MS 数据，并在计算机预测的小鼠或人类光谱中进行搜索。 ⁸⁸

Proteomics analysis of blastoids
胚泡的蛋白质组学分析

Control, mTORi-treated and CHX-treated blastoids were collected, washed, pelleted, and frozen under each experimental condition. Samples were prepared and analyzed using LC–MS/MS. Briefly, cell pellets were digested using the iST sample preparation kit (PreOmics) following the manufacturer’s instructions. Protein concentration was determined with the Pierce BCA Protein Assay Kit, and each sample was digested with an equal amount of protein (50 μg). Peptide samples underwent LC–MS/MS analysis using an UltiMate 3000 nano HPLC RSLC system coupled to a timsTOF HT mass spectrometer. Peptides were injected onto a pre-column (PepMap C18) with 2% ACN/water containing 0.1% TFA, then separated on a 25 cm Aurora ULTIMATE series HPLC column. Data-independent acquisition parallel accumulation—serial fragmentation (DIA-PASEF) mode was employed for measurement. For proteomics data analysis, Spectronaut 18.5 (Biognosys)¹⁰⁰ was used. The search was conducted against the Homo sapiens UniProt database. Quantification followed Biognosys BGS Factory Default settings, with protein inference using IDPicker. Spectronaut results were exported and converted to Microsoft Excel files using in-house software MS2Go.
在每个实验条件下收集、洗涤、沉淀和冷冻对照、mTORi处理和CHX处理的胚泡。使用 LC-MS/MS 制备和分析样品。简而言之，使用 iST 样品制备试剂盒 (PreOmics) 按照制造商的说明消化细胞沉淀。使用 Pierce BCA 蛋白质测定试剂盒测定蛋白质浓度，并用等量的蛋白质 (50 μg) 消化每个样品。使用 UltiMate 3000 nano HPLC RSLC 系统与 timsTOF HT 质谱仪联用，对肽样品进行 LC-MS/MS 分析。将肽注射到含有 0.1% TFA 的 2% ACN/水的预柱 (PepMap C18) 上，然后在 25 cm Aurora ULTIMATE 系列 HPLC 柱上分离。采用数据独立采集并行累积-串行碎片（DIA-PASEF）模式进行测量。对于蛋白质组学数据分析，使用 Spectronaut 18.5 (Biognosys) ¹⁰⁰ 。该搜索是针对智人 UniProt 数据库进行的。定量遵循 Biognosys BGS 出厂默认设置，并使用 IDPicker 进行蛋白质推断。使用内部软件 MS2Go 将 Spectronaut 结果导出并转换为 Microsoft Excel 文件。

Classical bulk proteomics of stem cells
干细胞的经典本体蛋白质组学

Proteomics sample preparation was done according to a published protocol with minor modifications.¹⁰¹ In brief, 5×10⁶ cells in biological duplicates were lysed under denaturing conditions in 500 μl of a buffer containing 3 M guanidinium chloride (Gdm-Cl), 10 mM tris(2-carboxyethyl)phosphine, 40 mM chloroacetamide, and 100 mM Tris-HCl pH 8.5. Lysates were denatured at 95°C for 10 min shaking at 1000 rpm in a thermal shaker and sonicated in a water bath for 10 min. 100 μl lysate was diluted with a dilution buffer containing 10% acetonitrile and 25 mM Tris-HCl, pH 8.0, to reach a 1 M GdmCl concentration. Then, proteins were digested with LysC (Roche, Basel, Switzerland; enzyme to protein ratio 1:50, MS-grade) shaking at 700 rpm at 37°C for 2 hours. The digestion mixture was diluted again with the same dilution buffer to reach 0.5 M GdmCl, followed by tryptic digestion (Roche, enzyme to protein ratio 1:50, MSgrade) and incubation at 37°C overnight in a thermal shaker at 700 rpm. Peptide desalting was performed according to the manufacturer’s instructions (Pierce C18 Tips, Thermo Fisher Scientific). Desalted peptides were reconstituted in 0.1% formic acid in water and further separated into four fractions by strong cation exchange chromatography (SCX, 3M Purification, Meriden, CT). Eluates were first dried in a SpeedVac, then dissolved in 5% acetonitrile and 2% formic acid in water, briefly vortexed, and sonicated in a water bath for 30 seconds prior to injection to nano-LC-MS/MS.
蛋白质组学样品制备是根据已发布的方案进行的，并进行了少量修改。 ¹⁰¹简而言之，在变性条件下，在 500 μl 含有 3 M 氯化胍 (Gdm-Cl)、10 mM 三(2-羧乙基)膦^、 40 mM 氯乙酰胺和 100 mM Tris-HCl pH 8.5。将裂解物在热摇床上以 1000 rpm 的速度摇动 95°C 变性 10 分钟，并在水浴中超声处理 10 分钟。使用含有 10% 乙腈和 25 mM Tris-HCl（pH 8.0）的稀释缓冲液稀释 100 μl 裂解液，以达到 1 M GdmCl 浓度。然后，用 LysC（Roche，巴塞尔，瑞士；酶与蛋白质比例 1:50，MS 级）消化蛋白质，在 37°C 下以 700 rpm 振荡 2 小时。将消化混合物再次用相同的稀释缓冲液稀释至 0.5 M GdmCl，然后进行胰蛋白酶消化（Roche，酶与蛋白质比例 1:50，MSgrade）并在热摇床中以 700 rpm 的速度在 37°C 下孵育过夜。根据制造商的说明（Pierce C18 Tips，Thermo Fisher Scientific）进行肽脱盐。将脱盐的肽在 0.1% 甲酸水溶液中重构，并通过强阳离子交换色谱（SCX、3M Purification、Meriden、CT）进一步分离成四个级分。首先将洗脱液在 SpeedVac 中干燥，然后溶解在 5% 乙腈和 2% 甲酸水溶液中，短暂涡旋，并在水浴中超声处理 30 秒，然后注入 Nano-LC-MS/MS。

LC-MS/MS was carried out by nanoflow reverse phase liquid chromatography (Dionex Ultimate 3000, Thermo Fisher Scientific) coupled online to a Q-Exactive HF Orbitrap mass spectrometer (Thermo Fisher Scientific), as reported previously.¹⁰² Briefly, the LC separation was performed using a PicoFrit analytical column (75 μm ID × 50 cm long, 15 μm Tip ID; New Objectives, Woburn, MA) in-house packed with 3-μm C18 resin (Reprosil-AQ Pur, Dr. Maisch, Ammerbuch, Germany).
如先前报道，LC-MS/MS 通过纳流反相液相色谱（Dionex Ultimate 3000，Thermo Fisher Scientific）在线耦合到 Q-Exactive HF Orbitrap 质谱仪（Thermo Fisher Scientific）进行。 ¹⁰²简而言之，使用内部填充 3 μm C18 树脂（Reprosil-AQ Pur， Maisch 博士，德国阿默布赫）。

Raw MS data were processed with MaxQuant software (v1.6.10.43) and searched against the mouse proteome database UniProtKB with 55,153 entries, released in August 2019. The MaxQuant processed output files can be found in Table S1, showing peptide and protein identification, accession numbers, % sequence coverage of the protein, q-values, and label-free quantification (LFQ) intensities.
原始 MS 数据使用 MaxQuant 软件 (v1.6.10.43) 进行处理，并根据 2019 年 8 月发布的小鼠蛋白质组数据库 UniProtKB 进行搜索，该数据库包含 55,153 个条目。MaxQuant 处理后的输出文件可在表 S1中找到，显示肽和蛋白质鉴定，登录号、蛋白质序列覆盖率百分比、q 值和无标记定量 (LFQ) 强度。

Differential expression analysis
差异表达分析

The Differential Enrichment analysis of Proteomics data (DEP) package v1.16.0 was used in R for proteomics data preparation and the statistical analysis.⁸³ For human iPSCs, the label-free quantification (LFQ) values were filtered. Only proteins quantified in both replicates of at least one condition were kept. Number of proteins kept after filtering: 4470 in PXGL, 4615 in RSeT, 5872 in primed conditions. For mouse ESCs, only proteins quantified in at least 2 out of 3 replicates of at least one condition were kept. A total of 4,783 proteins were kept after filtering. Both human and mouse proteomics data was background-corrected and normalized by variance stabilizing transformation. Missing values were imputed using random draws from a Gaussian distribution centered around a minimal value. Proteins with a p.adj < 0.05 and |log₂FC| > 1 were considered differentially expressed. The differential protein expression analysis results of the human blastoids can be found in Table S3 and the expression analysis results of the human PSCs and mouse ESCs can be found in Table S4.
R 中使用蛋白质组数据差异富集分析 (DEP) 软件包 v1.16.0 进行蛋白质组数据准备和统计分析。 ⁸³对于人类 iPSC，无标记定量 (LFQ) 值已被过滤。仅保留在至少一种条件的两次重复中定量的蛋白质。过滤后保留的蛋白质数量：PXGL 中为 4470，RSeT 中为 4615，引物条件下为 5872。对于小鼠 ESC，仅保留在至少一种条件下 3 次重复中至少 2 次定量的蛋白质。过滤后共保留4,783个蛋白质。人类和小鼠蛋白质组数据均通过方差稳定变换进行背景校正和标准化。使用从以最小值为中心的高斯分布中随机抽取来估算缺失值。 p.adj < 0.05 且 |log ₂ FC| 的蛋白质> 1 被认为是差异表达。人胚泡的差异蛋白表达分析结果见表S3 ，人PSC和小鼠ESC的表达分析结果见表S4 。

Scatter plots 散点图

The global change in proteome profile was displayed by the mean LFQ values in normal vs mTORi-treated cells using ggplot2.⁸⁴
使用 ggplot2 通过正常细胞与 mTORi 处理细胞的平均 LFQ 值显示蛋白质组谱的整体变化。 ⁸⁴

Gene Ontology analysis 基因本体分析

clusterProfiler⁸⁵ R package was applied on the differentially expressed proteins (DEPs) with a p.adj < 0.05 and |log₂FC| of >1.⁸⁶ The Benjamini-Hochberg correction was used to correct for multiple comparisons and a pvalueCutoff of 0.05 and qvalueCutoff of 0.1 were used. Enriched biological processes were displayed with a cnetplot and dotplot. Selected biological processes were displayed with ggplot2. A full overview of the enriched biological processes is provided in Table S5.
clusterProfiler ⁸⁵ R 软件包应用于差异表达蛋白 (DEP)，p.adj < 0.05 且 |log ₂ FC| >1。 ⁸⁶使用 Benjamini-Hochberg 校正来校正多重比较，并使用 0.05 的 pvalueCutoff 和 0.1 的 qvalueCutoff。使用 cnetplot 和 dotplot 显示丰富的生物过程。使用 ggplot2 显示选定的生物过程。表 S5提供了富集生物过程的完整概述。

K-means clustering K-均值聚类

Mean-centered expression data of DEP was used for k-means clustering in R with the package ‘stats’ (version 4.1.0). 4 clusters were chosen as optimal based on visual inspection of data. PXGL days were collected on d10 of mTORi and d12 of release.
DEP 的均值中心表达数据用于 R 中的 k 均值聚类，其中包含“stats”包（版本 4.1.0）。根据数据的目视检查，选择 4 个簇作为最佳簇。 PXGL 天数在 mTORi 的第 10 天和释放的第 12 天收集。

Global proteomics: human-mouse comparison
全球蛋白质组学：人鼠比较

A total of 2861 proteins are expressed in both human (PXGL) and mouse data. The log₂FC of mTORi vs control of all overlapping proteins was plotted with ggscatter and the Spearman’s Rho correlation coefficient was calculated.
人类 (PXGL) 和小鼠数据中总共表达了 2861 个蛋白质。使用 ggscatter 绘制 mTORi 相对于所有重叠蛋白的对照的 log ₂ FC，并计算 Spearman's Rho 相关系数。

KEGG¹⁰³ pathways containing at least 10 genes symbols were included in the pairwise pathway expression analysis. A total of 146 pathways were shared between mouse and human data. The pathway expression value was defined as the mean log₂FC of proteins between mTORi and control mouse ESCs and human naïve iPSCs, or between different culture conditions for human PSCs. Pathway log2FC for human and mouse are provided in Table S6. The mean log2FC for each pathway for human and mouse data was plotted with ggscatter and the Spearman’s Rho correlation coefficient was calculated.
成对通路表达分析中包括至少包含 10 个基因符号的 KEGG ^{103 条}通路。小鼠和人类数据之间共有 146 条通路。通路表达值定义为 mTORi 和对照小鼠 ESC 与人初始 iPSC 之间，或人 PSC 不同培养条件之间蛋白质的平均 log ₂ FC。表S6中提供了人和小鼠的途径log2FC。使用 ggscatter 绘制人类和小鼠数据的每个路径的平均 log2FC，并计算 Spearman 的 Rho 相关系数。

GSEA Analysis GSEA分析

Gene Set Enrichment Analysis (GSEA)⁸⁷ was used to identify the differences between two biological states, i.e., dormant vs normal and CHX vs normal, in terms of enrichment with a specific predefined gene set. LFQ values were used to compute (normalized) enrichment scores. The term ‘Protein Set: Diapause’ was defined based on all genes (n=179) significantly upregulated in in vivo diapause embryos as previously published.⁴⁹ The term ‘Protein Set:RapaLink’ was defined as all significantly upregulated genes (n=233 genes) identified by comparing the proteome profile of mTORi-treated vs normal human blastoids.
基因集富集分析 (GSEA) ⁸⁷用于识别两种生物状态之间的差异，即休眠与正常状态以及 CHX 与正常状态在特定预定义基因集的富集方面的差异。 LFQ 值用于计算（标准化）富集分数。术语“蛋白质组：滞育”是根据先前发表的体内滞育胚胎中显着上调的所有基因（n=179）来定义的。 ⁴⁹术语“蛋白质集：RapaLink”定义为通过比较 mTORi 处理与正常人胚泡的蛋白质组谱而鉴定的所有显着上调的基因（n = 233 个基因）。

Blastoid dissociation for flow-cytometry and single-cell analysis
用于流式细胞术和单细胞分析的母细胞分离

Control or mTORi-treated blastoids were manually collected using a mouth pipette and polled into 1.5 ml Eppendorf tubes. The tubes were then centrifuged at 400 x g for 4 minutes at room temperature, and the supernatant was aspirated. 500 μl of dissociation mix (TrypLE Select Enzyme (10X) and Accutase® Cell Detachment Solution at a 1:9 ratio) was added to the tubes. The tubes were then incubated at 37°C for 5 minutes, followed by mechanical dissociation through pipetting, and an additional 10-minute incubation period. Next, 1 ml of FACS buffer was added into the tubes, followed by centrifugation at 400g for 4 minutes at room temperature. The supernatants were carefully removed, and the cell pellets were subsequently washed twice. The cells were then resuspended in a FACS buffer and filtered through a strainer. For scRNA-seq, live cell enrichment was performed according to the manufacturer's instructions using the EasySep™ Dead Cell Removal (Annexin V) kit (Stem Cell Technologies, 17899). Post-implantation samples followed a similar preparation process, albeit with two 15-minute periods of dissociation.
使用口移液管手动收集对照或 mTORi 处理的胚泡并分入 1.5 ml Eppendorf 管中。然后将管在室温下以400×g离心4分钟，并吸出上清液。将 500 μl 解离混合物（TrypLE Select Enzyme (10X) 和 Accutase® Cell Detachment Solution，比例为 1:9）添加到试管中。然后将管在 37°C 下孵育 5 分钟，然后通过移液进行机械解离，并再孵育 10 分钟。接下来，将1ml FACS缓冲液添加到管中，然后在室温下以400g离心4分钟。小心地除去上清液，随后将细胞沉淀洗涤两次。然后将细胞重悬于FACS缓冲液中并通过滤网过滤。对于 scRNA-seq，根据制造商的说明，使用 EasySep™ 死细胞去除 (Annexin V) 试剂盒（Stem Cell Technologies, 17899）进行活细胞富集。植入后样品遵循类似的制备过程，尽管有两个 15 分钟的解离期。

Flow cytometry 流式细胞仪

The structures were collected and then dissociated into single cells as described earlier. These single cells were subsequently prepared for immunofluorescent staining, with washing steps carried out in 1.5 ml Eppendorf tubes. Flow cytometry analysis was conducted using unstained controls on a BD FACS Aria III flow cytometer, with data interpretation performed using FlowJo software. The following antibodies and dilutions were used: TROP-2 Alexa Fluor 488-conjugated (R&D, FAB650G) 1:200, PDGF Receptor α Alexa Fluor 647-conjugated (Cell signaling, 5876) 1:100, CD197 (CCR7) PE-conjugated (eBioscience, 12-1979-42) 1:100.
如前所述，收集结构，然后解离成单细胞。随后将这些单细胞制备用于免疫荧光染色，并在 1.5 ml Eppendorf 管中进行洗涤步骤。使用未染色的对照在 BD FACS Aria III 流式细胞仪上进行流式细胞术分析，并使用 FlowJo 软件进行数据解释。使用以下抗体和稀释度：TROP-2 Alexa Fluor 488 缀合（R&D，FAB650G）1:200，PDGF 受体 α Alexa Fluor 647 缀合（细胞信号传导，5876）1:100，CD197 (CCR7) PE 缀合（电子生物科学，12-1979-42）1：100。

Single-cell RNA-seq library preparation and sequencing of blastoids
单细胞 RNA-seq 文库制备和母细胞测序

The structures dissociated into single cells as described earlier. Live cells were enriched using EasySep™ Dead Cell Removal (Annexin V) Kit removal kit (Stem cell technologies). Cells counting was performed using nucleocounter NC-250 (ChemoMetec). Desired number of cells were used to generate Gel Beads in Emulsions (GEMs) using 10X Genomics chromium X at VBCF-NGS, Vienna. For 96 HR samples, 1000 cells were targeted using Chromium Next GEM Single Cell 3ʹ LT Kit v3.1 (PN-1000325) on chip L (PN-1000321) and for post-implantation samples 10000 cells were targeted using Chromium Next GEM Single Cell 3’ GEM, Library & Gel Bead Kit v3.1 (PN-1000128) on chip G (PN-1000127) according to manufacturer’s protocols (CG000399 and CG000315, respectively (10X Genomics, Pleasanton, CA)). cDNA were amplified for 11 cycles. Libraries were purified using Ampure XP bead purification protocol and were analyzed using a fragment analyzer and qPCR for distribution and quantity. Libraries were pooled together and sequenced on NovaSeq instrument series flowcell (Illumina, San Diego, CA) using paired-end read mode for 300 cycles.
如前所述，这些结构解离成单个细胞。使用 EasySep™ Dead Cell Removal (Annexin V) Kit 去除试剂盒（干细胞技术）富集活细胞。使用核计数器NC-250 (ChemoMetec)进行细胞计数。使用 VBCF-NGS（维也纳）的 10X Genomics chromium X，将所需数量的细胞用于在乳液中生成凝胶珠 (GEM)。对于 96 个 HR 样本，使用芯片 L (PN-1000321) 上的 Chromium Next GEM Single Cell 3ʹ LT Kit v3.1 (PN-1000325) 靶向 1000 个细胞；对于植入后样本，使用 Chromium Next GEM Single Cell 靶向 10000 个细胞根据制造商的方案（分别为 CG000399 和 CG000315（10X Genomics，Pleasanton，CA）），在芯片 G (PN-1000127) 上使用 3' GEM、Library & Gel Bead Kit v3.1 (PN-1000128)。 cDNA扩增11个循环。使用 Ampure XP 珠纯化方案纯化文库，并使用片段分析仪和 qPCR 分析分布和数量。将文库汇集在一起，并在 NovaSeq 仪器系列流通池（Illumina，圣地亚哥，加利福尼亚州）上使用双端读取模式进行 300 个循环的测序。

Single-cell RNA-seq data analysis and projection on the human embryo reference map
单细胞RNA-seq数据分析及在人类胚胎参考图上的投影

scRNA-seq reads were processed with Cell Ranger count v7.2.0 using the prebuilt 10X GRCh38 reference (refdata-gex-GRCh38-2020-A) and default settings. Further analyses were performed in R v4.3.2 with Seurat v5.0.1. The Cell Ranger filtered feature-barcode matrices were used, doublets detected by scDblFinder v1.16.0 were removed, and only cells meeting quality thresholds were retained; all preimplantation samples: more than 3000 detected genes and less than 7.5% of mitochondrial and less than 50% of ribosomal RNA reads, all postimplantation samples: more than 2000 detected genes and less than 15% of mitochondrial and less than 50% of ribosomal RNA reads. Count data were log-normalized and scaled (regressing out the difference between the G2M and S phase signature scores). Dimensionality reduction was performed on the top 2000 most variable genes, and canonical correlation analysis (CCA) was used for scaling and alignment of the datasets, followed by projection onto two-dimensional space using Uniform Manifold Approximation and Projection (UMAP) on the top 15 CCA dimensions. An integrated human embryonic reference map and Seurat reference-based mapping were used to map and predict cell type identity for each sample using the FindTransferAnchors and MapQuery functions.
scRNA-seq 读取使用预构建的 10X GRCh38 参考 (refdata-gex-GRCh38-2020-A) 和默认设置，通过 Cell Ranger count v7.2.0 进行处理。在 R v4.3.2 和 Seurat v5.0.1 中进行了进一步分析。使用Cell Ranger过滤的特征条形码矩阵，去除scDblFinder v1.16.0检测到的双峰，仅保留符合质量阈值的细胞；所有植入前样本：超过 3000 个检测到的基因以及少于 7.5% 的线粒体和少于 50% 的核糖体 RNA 读数，所有植入后样本：超过 2000 个检测到的基因以及少于 15% 的线粒体和少于 50% 的核糖体 RNA读。计数数据进行对数归一化和缩放（回归 G2M 和 S 期特征分数之间的差异）。对前 2000 个变化最大的基因进行降维，并使用典型相关分析 (CCA) 对数据集进行缩放和对齐，然后使用统一流形逼近和投影 (UMAP) 将前 15 个基因投影到二维空间CCA 尺寸。使用 FindTransferAnchors 和 MapQuery 函数，使用集成的人类胚胎参考图和基于 Seurat 参考的映射来映射和预测每个样本的细胞类型身份。

The integrated reference map comprised E-MTAB-3929,²⁵ GSE109555,⁷⁷ GSE171820,⁷⁸ PRJEB30442,⁴⁷ GSE136447,⁷⁹ GSE36552,²⁶ and E-MTAB-9388.⁸⁰ Raw scRNA-seq data were reprocessed as previously described,³⁶ then filtered and annotated using the cell annotation from https://petropoulos-lanner-labs.clintec.ki.se/app/ShinyEmbryoProjP,⁴⁴ except for GSE109555, where we used 1,000 randomly subsampled high-quality single cells and the provided annotation. Cells classified as Zygote, 2-4 cell, 8 cell, Ambiguous, HEP, Erythroblasts, Unknown, and PGC were excluded.
^集成参考图包括 E-MTAB-3929、25 ^{GSE109555、77} ^{GSE171820、78} ^{PRJEB30442、47} ^{GSE136447、79} ^{GSE36552、26}和 E-MTAB-9388。 ^{80 个}原始 scRNA-seq 数据按照之前的描述进行重新处理， ^{36 个数据}然后使用来自https://petropoulos-lanner-labs.clintec.ki.se/app/ShinyEmbryoProjP的细胞注释进行过滤和注释， ^44，除了 GSE109555，我们使用了 1,000 个随机二次采样的高质量单细胞和提供的注释。排除分类为受精卵、2-4 细胞、8 细胞、模糊、HEP、成红细胞、未知和 PGC 的细胞。

Genes detected in at least five cells in any dataset were retained. Log-normalization was performed using computeSumFactors in the scran package v1.30.2 and per-batch scaling normalization using multiBatchNorm in batchelor v1.18.1. Datasets were aligned using the fastMNN approach via SeuratWrappers v0.3.5 using the log-normalized batch-adjusted expression values. MNN low-dimensional coordinates were then used for clustering and visualization by UMAP. The projection of cells onto the online human embryonic reference atlas was performed using the website's information, without employing neighborhood calculations.
保留在任何数据集中至少五个细胞中检测到的基因。使用 scran 包 v1.30.2 中的computeSumFactors 执行对数归一化，并使用batcelor v1.18.1 中的multiBatchNorm 执行每批次缩放归一化。使用 fastMNN 方法通过 SeuratWrappers v0.3.5 使用对数归一化批量调整表达值对数据集进行对齐。然后，UMAP 使用 MNN 低维坐标进行聚类和可视化。使用网站信息将细胞投影到在线人类胚胎参考图谱上，而不使用邻域计算。

Quantification and statistical analysis
量化和统计分析

ANOVA, Kolmogorov-Smirnov test, t-test, or Sperman’s correlation were used as appropriate. Data were evaluated manually to determine whether they met assumptions of the statistical approaches. Statistical assays, sample sizes, definition of center, dispersion and precision measures have been described in figure legends. Samples have not been randomized. Data have not been excluded.
酌情使用方差分析、Kolmogorov-Smirnov 检验、t 检验或 Sperman 相关性。手动评估数据以确定它们是否符合统计方法的假设。统计分析、样本大小、中心定义、分散度和精度测量已在图例中进行了描述。样本尚未随机化。数据并未被排除。

Supplemental information 补充信息

What’s this? 这是什么？

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Table S1. Label-free global proteomics of control, mTORi-treated, and mTORi-treated-released human PSCs (2 biological replicates/condition) cultured in PXGL and RSeT and blastoids (3 biological replicates/condition), related to Figures 3, 5, and 7.

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Table S2. GSEA and PCA of human blastoid and mouse blastocyst proteomics data, related to Figure 3.

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Table S3. DEP output for control, CHX-treated, and mTORi-treated human blastoids (3 biological replicates/condition), related to Figure 3.

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Table S4. DEP output for control, mTORi-treated, and mTORi-treated-released PSCs (2 biological replicates/condition), related to Figure 5.

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Table S5. List of Gene Ontology terms associated with DE proteins in PSCs, related to Figure 5.

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Table S6. List of KEGG pathway expression values (mean log2FC) for human and mouse proteomics comparing mTORi-treated vs. control cells or embryos/blastoids and lists of common and species-specific pathways, related to Figure 5.

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Cell

Article 文章mTOR activity paces human blastocyst stage developmental progressionmTOR 活性调节人类囊胚阶段的发育进程

Highlights 亮点

Summary 概括

Graphical abstract 图文摘要

Keywords 关键词

Introduction 介绍

Results 结果

Conserved pattern of mTOR activation via IGF1 sensing in mouse and human embryos小鼠和人类胚胎中通过 IGF1 感应激活 mTOR 的保守模式

mTOR activity regulates TE development and attachmentmTOR 活性调节 TE 的发育和附着

mTORi activity prolongs the blastocyst-like stage in human blastoidsmTORi 活性延长人胚泡的囊胚样阶段

Inhibition of translation alone fails to fully recapitulate the effect of mTORi单独抑制翻译并不能完全重现 mTORi 的作用

mTOR-inhibited blastoids maintain a transcriptome reflecting the blastocyst stagemTOR 抑制的胚泡维持反映囊胚阶段的转录组

Human PSCs resembling the blastocyst stage adopt a reversible dormant state类似于囊胚阶段的人类 PSC 采取可逆的休眠状态

Reactivated blastoids developmentally progress and permit stem cell line derivation重新激活的胚泡发育进展并允许干细胞系衍生

Diverse routes converge into a shared dormancy response in mouse and human cells小鼠和人类细胞中的不同途径汇聚成共同的休眠反应