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通讯作者:

王红梅(1973-)女,内蒙古自治区赤峰市人,博士生导师,主要从事哺乳动物胚胎与胎盘发育方面研究。E-mail:wanghm@ioz.ac.cn

中图分类号:Q112

文献标识码:A

文章编号:2096-8965(2020)01-0035-08

DOI:10.12287/j.issn.2096-8965.20200106

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目录contents

    摘要

    早期胚胎发育关乎生命健康本源。解析人类早期胚胎发育调控机制可从源头提升人口健康,提高人口素质,从而促进经济社会可持续发展。然而,受限于技术和伦理,人类早期胚胎发育调控机制尚未得到全面解析。近年来,随着哺乳动物胚胎体外培养和“类胚胎”等技术的发展,人类进入解开自身早期胚胎发育之谜的最好时代,同时也迎来了新一轮伦理挑战。本文系统地回顾了以啮齿类和灵长类为主的哺乳动物早期胚胎发育领域的研究进展,探讨了人类胚胎研究相关的伦理争议,展望了早期胚胎发育研究的发展方向,希冀为人类认知早期胚胎发育事件和出生缺陷及多种发育源性疾病的发病机制与诊疗提供新思路和新手段。

    Abstract

    The importance of early embryonic development in ensuring human health has long been recognized. Insights into the mechanisms of human early embryonic development can potentially contribute towards improving public health and reversing population aging, and promoting economic and social development. However, our knowledge of regulation networks that underpin human early embryonic development is far from complete due to technological and ethical constrains. Recently, incredible progress in scientific research on in vitro culture of mammalian embryos and embryoid technology represents a great opportunity in disclosing the mystery of human early embryonic development but also presents the toughest ethical challenges. Here we systematically review the major progress of research on early mammalian (rodent and primate) embryonic development, discuss the ethical debates about human embryo research, and lay out the future direction for the study of the early embryonic development regulation for both basic research and clinical application.

  • 前言

  • 人口健康是决定人口质量的关键因素,是国家经济稳定和社会可持续发展的重要保证。当前,出生缺陷等重大疾病严重危害人口健康,我国出生缺陷发生率为5.6%,平均每30 秒钟就会有一名缺陷儿出生[1],为患儿家庭和国家带来沉重经济负担。 这样严峻的现实使得人类不得不从源头着手,寻求从根本上提升人口健康的科学途径。早期胚胎发育关乎生命健康本源,早期发育异常是先天性心脏病、神经管缺陷等多种发育源性出生缺陷疾病的起因,而严重的异常早期胚胎发育则导致反复种植失败和早期妊娠丢失。解析早期胚胎发育调控机制有助于我们深入理解早期胚胎发育过程、早期器官发生、多器官相互作用及其调控网络与疾病发生的关系,为防治出生缺陷及多种发育源性疾病提供理论基础和可行路径。

  • 1 早期胚胎发育研究进展

  • 哺乳动物机体由200多种细胞类型、40余种器官构成[2]。这些细胞类群和器官在早期胚胎发育的不同阶段逐渐形成。

  • 哺乳动物胚胎发育起始于受精卵,受精卵在输卵管向子宫移行过程中经过多次卵裂形成囊胚。囊胚由位于内部的内细胞团(Inner Cell Mass,ICM) 和位于外部的滋养外胚层(Trophectoderm,TE) 组成。内细胞团未来发育成胎儿,滋养外胚层未来发育成胎盘。胎盘是哺乳动物妊娠过程中连接母体和胎儿的临时内分泌器官,在妊娠过程中扮演物质运输、免疫耐受、激素分泌等角色。滋养外胚层与内细胞团接触的部分为极滋养外胚层(polar TE),不与内细胞团接触的部分为壁滋养外胚层(Mural TE)(图1)。囊胚从输卵管迁移入子宫,并逐渐从透明带中孵化,进入着床(植入)准备期。着床的过程分为三个阶段:定位(Apposition)、附着(Adhesion) 和侵入(Invasion)。

  • 图1 小鼠早期胚胎发育过程模式图[20]

  • 内细胞团在胚胎植入过程中分化为上胚层(Epiblast,EPI) 和下胚层(Hypoblast,也称原始内胚层,即Primitive Endoderm,PrE)。胚胎植入子宫之后,由于上胚层细胞多能性退出和细胞极性出现,上胚层中间出现“花环”结构(Rosette)[3]。 随后“花环”结构逐渐扩大形成原羊膜腔。之后上胚层和下胚层经过分化和形态演变,形成对称的杯状结构(啮齿类) 或盘状结构(灵长类)。

  • 随着上、下胚层和滋养外胚层的相互作用和分化,胚胎后部(Posterior) 上胚层的部分细胞会分化为表达Branchury基因的原肠运动细胞(Gastrulating Cells,Gast)[4, 5],标志着原肠运动(Gastrulation) 开启。原肠运动过程伴随胚胎后部上胚层凹陷形成。随着原肠运动进行,一些上胚层细胞仍保留在外侧,形成外胚层(Ectoderm),另一部分靠近原条的上胚层细胞开始增殖,并逐渐侵入下胚层, 从而形成胚胎的定型内胚层(Definitive Endoderm, DE)[6, 7]。在紧贴上胚层脏侧的原始内胚层,即脏内胚层(Visceral Endoderm,VE)中,有一部分细胞从后部向前部(Anterior) 迁移,形成前部脏内胚层(Anterior Visceral Endoderm,AVE),同样参与内胚层分化。还有一些原条细胞会迁移进入内外胚层中间, 形成第三胚层,即中胚层(Mesoderm) 发生的基础[4, 8],形成三胚层的胚胎被称为原肠胚(Gastrula)。

  • 随着原肠胚的发育,不同胚层之间发生更加广泛、复杂而有序的相互作用[9, 10]。外胚层进一步特化为位于中央的神经板(Neural Plate) 和两侧对称的表皮区(Epidermis)。神经板由位于中间的神经外胚层(Neural Ectoderm,NE) 和双侧对称的边缘(Border) 细胞组成。边缘细胞未来将分化为神经嵴(Neural Crest,NC) 细胞。神经板以背侧中间轴为中心,对称性向腹侧凹陷折叠,边缘处逐渐在背侧中央发生融合,最终神经外胚层分化为神经管(Neural Tube,NT)。从神经板开始出现,到神经管闭合期间的胚胎,被称为神经胚(Neurula)。同时神经嵴细胞在神经管的背侧发生广泛迁移,参与多种神经细胞和脏器的分化。因此,神经嵴又被称为“第四胚层”。表皮区未来将主要参与皮肤的构建。此外,脏内胚层和定型内胚层共同参与肠管(Gut Tube,GT)的发育,肠管细胞在未来将参与包括消化道、肺脏、胰腺、肝脏等多种内脏器官的构建。中胚层细胞将参与骨骼、骨骼肌、结缔组织、 循环系统、泌尿系统等的构建。

  • 啮齿类动物胚胎是研究哺乳动物胚胎早期发育常用的实验模型。同时,非人灵长类动物胚胎在结构和发育时间节点上与人类早期胚胎发育最为相近。因此本文分别以小鼠、食蟹猴和人类胚胎为例综述哺乳动物早期胚胎发育研究进展。

  • 1.1 啮齿类动物早期胚胎发育

  • 小鼠受精卵在多次卵裂的同时发生复杂的细胞命运决定,这种细胞命运决定表现在卵裂球中的单细胞转录组、基因甲基化水平、非编码RNA等的差异,与细胞极性、空间位置等密切相关。 已发现的参与细胞命运决定的基因和长链非编码RNA包括 Carm1Prmd14Oct4Sox2Cdx2 和LinGET等[11-16]

  • 小鼠胚胎于E(Embryonic Day)3.5 形成囊胚。 小鼠囊胚于E4.0-E4.5 通过壁滋养外胚层种植到母体子宫,发生着床[17]。上胚层此时聚集成“花环” 结构[3],其壁侧被顶壁内胚层(Parietal Endoderm, ParE) 包裹。顶壁内胚层沿壁滋养外胚层对称性伸展[17]。E5.5阶段,极滋养外胚层分化为胚外外胚层(Extra-embryonic Ectoderm,ExE)。上胚层与胚外外胚层共同形成原羊膜腔结构。同时由原始内胚层发育而来的脏内胚层包裹上胚层向壁滋养外胚层侧进一步延伸,最终形成由上胚层和脏内胚层包裹的中空“杯状”结构[18](Egg Cylinder)(图1)。杯状结构的形成是小鼠胚胎原肠运动的基础[10, 17, 19]

  • 小鼠胚胎于E6.5 进入原肠运动阶段,在胚胎后部形成原条。原肠运动持续至E8.5,胚胎逐渐进入早期器官发育阶段。2019 年,英国剑桥大学的Berthold Göttgens团队首次利用单细胞转录组测序技术揭示了小鼠E6.5-E8.5 胚胎的细胞类群和分化特征,探讨了脏内胚层和原条来源的内胚层融合。 同年,美国Sloan Kettering研究所的Anna-Katerina团队同样利用单细胞转录组测序技术,研究小鼠囊胚至E8.75的细胞谱系发生,确定肠管的发育由上胚层分化的定型内胚层和原始内胚层分化而来的脏内胚层共同参与[20]。由于小鼠原肠运动的发生伴随三维空间的不对称分化,因此来自中国科学院生物化学与细胞生物学研究所的景乃禾团队先后利用空间转录组技术,在三维层面还原小鼠胚胎发育特征和细胞谱系分化轨迹,对小鼠胚胎原肠运动进行多维探讨[7, 21]。总之,目前小鼠E3.5 至E8.75 阶段胚胎的发育事件和细胞谱系发生可借助多组学技术进行连续描述。受限于技术手段以及器官发育的多样性,E8.75-E10.5的早期器官发育至更广泛器官发育阶段的细胞谱系分化研究仍非常有限。E8.5 之后, 胚胎三胚层的细胞同时发生分化和迁移,共同参与多器官的发育。同时器官的发育在空间结构和细胞来源上也存在复杂的调控。例如,E9.5阶段小鼠胚胎的脑部在结构上可划分为端脑、前脑、中脑、后脑和脑桥,在细胞类群上包括神经细胞、小胶质细胞、血管内皮细胞、间质细胞等。这些结构和细胞都是由不同胚层贡献的,但其具体的分化路径、迁移轨迹等基本科学问题尚未被全面诠释。

  • 除研究体内胚胎,科学家还尝试胚胎的体外孕育。早在1982 年,来自美国约翰霍普金斯大学的Hsu团队将小鼠囊胚体外培养至接近E9.5 阶段[22], 证明小鼠胚胎可在无母体支持条件下发育至早期器官形成阶段。近年来,伴随小鼠胚胎体外培养体系的优化,小鼠原肠胚之前的结构演变过程和发育特征被不断揭示[3, 23]。未来随着培养体系进一步优化和体内细胞示踪技术的发展,更多小鼠胚胎发育相关关键事件的发生机制将被阐明。

  • 1.2 灵长类动物早期胚胎发育

  • 以人类胚胎为例,人类受精卵于E5形成囊胚, E6 由极滋养外胚层与母体子宫接触并发生着床。 胚胎着床后,上胚层的中间部位形成原羊膜腔。 E12-E13,上胚层背侧细胞特化成鳞状羊膜细胞, 腹侧细胞特化成柱状上皮细胞[24]。上胚层细胞此时类似“盘状”。在上胚层腹侧,脏内胚层与顶壁内胚层形成初级卵黄囊(Primary Yolk Sac)。初级卵黄囊细胞增殖和重排后形成次级卵黄囊(Secondary Yolk Sac)。脏内胚层形态也类似盘状,所以上胚层和脏内胚层共同构成灵长类胚胎典型的双胚盘结构(Bilaminar Disk)(图2)。

  • 图2 人类早期胚胎发育过程模式图[26]

  • 由于科研伦理限制,科学家无法直接研究人类体内胚胎发育过程,因此体外胚胎培养对于我们了解人类早期胚胎发育至关重要。人类胚胎的体外研究存在必须在E14终止的伦理限制[25]。2016年,英国剑桥大学的Zernicka-Goetz和美国洛克菲勒大学的Brivanlou团队分别报道了将人类胚胎体外培养至E13的研究,并且描述了人类胚胎早期发育过程中不同细胞谱系的典型基因表达特征[26, 27]。2019 年,北京大学汤富酬和乔杰团队再次利用该培养技术,将人类胚胎培养至E14,并结合单细胞多组学技术系统分析了人类植入后胚胎细胞的分化路径和甲基化特征,发现雌性胚胎在植入过程中出现X染色体随机失活,启动X染色体连锁基因的等位基因特异性表达[28]。2020年,来自昆明理工大学的李天晴和季维智团队构建了三维胚胎培养系统,将人类胚胎体外培养至原条原基(Primitive Streak Anlage, PSA) 阶段,并结合单细胞转录组分析,进一步描述了上、下胚层和滋养外胚层的基因调控网络[29]

  • 灵长类动物胚胎的原肠运动等生物学事件发生于E14 之后,由于伦理学限制(14 天准则),我们无法利用人类胚胎研究E14之后的发育事件。非人灵长类动物胚胎在早期胚胎发育阶段与人类胚胎高度相似,是研究人类胚胎发育事件的可靠模型。 2016年,日本京都大学的Saitou团队首次系统报道了食蟹猴胚胎植入后上胚层发育至早期原肠运动过程,并借助单细胞转录组学分析,揭示了非人灵长类动物上胚层发育过程中多能性相关基因的表达变化特征,深入研究了发生原肠运动胚胎的细胞类群(图3)[24]。由于非人灵长类动物资源宝贵,其体内正常发育的胚胎不易获得,因此科学家也尝试将非人灵长类动物胚胎进行体外培养。1995 年,来自澳大利亚墨尔本大学的Watkins团队首次将狨猴囊胚体外培养至E11,并研究了植入后胚胎的形态特征,但受限于当时的技术,胚胎的更多生物学事件无法被进一步揭示[30]。考虑到非人灵长类动物胚胎体外研究不受“14 天准则”的伦理学限制,科学家们希望在体外维持胚胎发育至更后期阶段。2019 年,来自中国科学院动物研究所的王红梅和李磊团队,以及昆明理工大学的谭韬和季维智等团队同时将食蟹猴胚胎体外培养至E20,并在此基础上研究了灵长类动物胚胎原肠运动发育特征[31, 32]。这是国际上首次将非人灵长类动物胚胎培养至原肠运动,并将非人灵长类动物原肠胚结构进行三维重构及在单细胞转录组层面进行深入研究。同时,体外培养至E20的胚胎形成了类神经沟结构[32]。通过优化体外培养体系,延长胚胎体外发育,灵长类动物早期器官发生过程中更加复杂的生物学事件将被逐渐揭示。

  • 图3 食蟹猴早期胚胎发育过程模式图[24]

  • 1.3 哺乳动物类胚胎的研究进展

  • 类胚胎(Embryoid) 是利用干细胞自组装特性建立的简化的胚胎模型,也被称为干细胞组装胚胎模型,主要用来模拟胚胎的部分特征,进而研究胚胎发育过程。类胚胎与在体胚胎相比具有诸多优势,如不受受精过程限制、易于大量获取和操作、 均一性高等。目前,类胚胎已被用于揭示发育过程的关键事件,如胚胎植入、原肠运动和神经胚发育等[33-35]

  • 1981年,英国剑桥大学的Evans和Kaufman以及美国加利福尼亚大学Martin成功建立小鼠胚胎干细胞系(Mouse Embryonic Stem Cells,mESCs)[36, 37], 随后科学家开始利用mESCs的简单聚集构建拟胚体(Embryoid Body,EB),探究细胞谱系分化及其发生机制,但拟胚体的分化与胚胎协同有序的发育模式不同。直到2008 年,美国霍华德·休斯医学研究所Nusse团队发现拟胚体的基因表达呈前后极性分布,后部区域出现中胚层的特化,此时的拟胚体类似于胚胎原条的特征。2014年,英国剑桥大学Arias团队利用悬滴法培养mESCs,使其聚集形成类原肠胚结构(Gastruloid)[38],该结构在震荡后出现前后轴、背腹轴和中间外侧轴[39],并可在三维培养条件下重现体节发生[40]。同年,Zernicka-Goetz团队利用细胞外基质三维培养mESCs自发形成类似于胚胎植入后的“花环”结构[10]。2017 年,ZernickaGoetz团队在单一的mESCs体系中加入小鼠滋养层干细胞(Mouse Trophoblast Stem cells,mTSCs), 产生的类胚胎出现了原始生殖细胞和中胚层细胞[41]。 2018年,他们将培养体系进一步优化,并加入小鼠胚外内胚层干细胞(Extra-embryonic Endoderm Stem Cells,XENs),将类胚胎发育延长至原肠运动阶段[23]。2019年,中国农业大学韩建永团队在震荡体系中利用上述三种干细胞(mESCs、mTSCs和XENs) 构建出植入后类胚胎,该类胚胎被转移入小鼠子宫后可引发子宫发生类似于接受正常胚胎时的蜕膜化[42]。除了构建植入后的类胚胎,2018年,荷兰马斯特里赫特大学Rivron团队先后将mESCs和mTSCs加到微孔中形成包含内外结构的“类囊胚”, 将类囊胚植入小鼠子宫可触发蜕膜化现象[43]。2019 年,美国Salk研究所Belmonte团队利用小鼠扩展多能性干细胞(Mouse Extended Pluripotent Stem Cell, mEPSCs) 成功构建类囊胚结构[44];同年,ZernickaGoetz团队用mEPSCs和mTSCs也构建了类囊胚结构[45]。这两个团队构建的类囊胚发育潜能明显提高, 可在体外发育至植入后阶段,且植入后都能使小鼠子宫出现蜕膜化反应。

  • 人类类胚胎的构建研究目前仍停留在单种胚胎干细胞(Human Embryonic Stem Cells,hESCs) 诱导阶段。构建获得的类胚胎主要模拟植入后胚胎的发育特征。2014 年,Brivanlou团队首先利用平面圆形微模式(Micropattern) 研究干细胞自组装特性,先后模拟了原肠胚和神经胚细胞的分化过程[33, 46]。美国密西根大学傅剑平团队着力开发三维培养体系和微流控芯片培养体系,已成功诱导hESCs体外重现羊膜腔和神经胚形成等事件[35, 47]。Arias团队利用hESCs聚集,结合不同的信号诱导,构建出类原肠胚结构[48]。相信随着培养体系的优化,未来类胚胎会更好地模拟生理状态发育的胚胎,从而更好地服务于临床出生缺陷等多种发育疾病的诊疗。

  • 1.4 早期胚胎发育为预防出生缺陷及发育源性疾病提供理论基础和研究模型

  • 大量研究表明,早期胚胎发育异常是反复种植失败、反复妊娠丢失和出生缺陷等多种不良妊娠结局的主要诱因。因此,基于早期胚胎发育基础研究体系搭建的研究平台和基于其分子调控网络建立的多维组学数据库将为胚胎发育异常病因学研究和诊治提供理论基础,并为基础研究成果向应用类研究转化提供了强有力的科学途径。

  • 目前,对早期胚胎发育的应用类研究多集中于导致胚胎发育异常的环境因素、致病基因、调控网络等发病机制的挖掘和筛选。例如Arias团队利用人类类原肠胚验证了维甲酸破坏胚胎发育过程中的轴向模式而引起先天性畸形的机制[48]。Berthold Göttgens团队利用单细胞转录组测序技术发现Tal1 基因的突变可导致早期中胚层分化的多样性缺陷, 从而导致恶性贫血和胚胎致死[6]。Brivanlou团队利用类神经胚模型证明亨廷顿突变蛋白通过破坏细胞骨架引发神经胚形态变化,从而导致神经管结构无法形成[33]。此外,Pax3[49]Lrp2[50]Wnt2bWnt7b[51] 等基因的突变与神经管闭合也密切相关。 来自美国Sloan Kettering研究所的Anna-Katerina团队发现Apela信号肽的缺失可导致胚胎血液循环构建后低外显性心血管缺失,从而导致胚胎致命的心脏缺陷[52]。虽然目前多数哺乳动物早期胚胎发育的应用类研究还处于发病机制的探索阶段,但随着研究基础的积累,多学科交叉汇聚与多技术跨界融合,未来早期胚胎发育研究将为出生缺陷及多种发育源性疾病的诊疗提供重要靶点,并最终展现其在发育源性疾病防治领域的多重应用价值。

  • 2 人类胚胎及类胚胎研究的伦理挑战

  • 从生命伦理学的发展历程来看,生命科学领域的重大突破时常伴随巨大的生命伦理争议。人类早期胚胎发育研究涉及人类胚胎,必然存在伦理问题。支持或反对人类胚胎研究的主要分歧在于,是否将胚胎视为人或是潜在的人。例如,基于天主教教义,胚胎是神造的,其自受精之时起便已然为人,应当拥有与出生之人同等的伦理地位,因而不得使用或毁坏任何发育阶段的胚胎。而于其他宗教(例如犹太教、伊斯兰教、佛教等) 而言,胚胎的伦理地位是随着发育进程而逐渐提高的。上世纪70 年代末80 年代初,在有关辅助生殖的讨论中, 已有关于是否可用人类胚胎进行研究的相关探讨。 为应对体外受精、胚胎研究中产生的伦理问题, 1979 年美国卫生教育和福利部伦理咨询委员会首次公开倡议“14天准则”,要求胚胎的体外培养时间不得超过E14或原肠运动发生。该准则在一定程度上尊重了胚胎的伦理地位,同时为决策者、管理者提供了一个高效、可操作的政策工具。1984年英国《沃诺克报告》(Report of the Committee of Inquiry into Human Fertilisation and Embryology) 吸纳该准则,并建议设置特别机构,通过立法来监管胚胎相关研究及应用。该准则及相关建议随后被1990 年英国 《人类受精与胚胎学法案》 吸收,并深远地影响着包括中国在内的多国立法政策。“14 天准则”被认为是迄今为止在生殖科学和医学领域最具国际共识的规则之一。实践证明,此类伦理规范不仅没有阻碍科研的发展,反而为胚胎研究能够获得公众信任和支持以及科研监管领域的国际交流合作提供了合理、合法的通路。随着胚胎发育研究的发展,诸如“14 天准则”等相关伦理规范正受到前所未有的新挑战。如前所述,当下研究14 天以后人类胚胎发育已经在技术可行范围之内,因而14 天伦理界限“硬标准”是否仍然适应今天的科学环境已是一个愈发有挑战性的问题。

  • 最近几年,可部分重现围植入期甚至E14之后人类胚胎结构的“类胚胎”研究不断涌现并飞速发展。类胚胎是否也应遵守“14 天准则”逐渐成为热议问题。2020 年,Arias团队构建出与E18-E21 人类原肠胚发育阶段相似的类原肠胚,部分结构超越了14 天准则规定的发育阶段。他们宣称其所获得的类原肠胚结构发育潜能有限,无法发育出大脑,同时也因缺乏胚外组织而无法植入母体子宫, 不能称其为真正意义上的胚胎,为方便理解,他们将其构建的类原肠胚结构比作“飞机模拟器”,模拟器是复杂的飞机模型,能使我们学到驾驶飞机的知识以及模拟飞行中的潜在问题,但它无法载人, 始终不会是真正的飞机,因而没有违反“14 天准则”(http://amapress.gen.cam.ac.uk/? p=2469)。然而,未来更接近在体胚胎的类胚胎研究是否应当落入“14 天准则”限定范围的问题极具争议。就当前而言,科学家普遍认为,使用这种不完整的类胚胎模拟某些重要器官的发育反而可能会降低使用人类在体胚胎的必要性,且现有的类胚胎仍只可重现胚胎发育中的有限部分,因此被认为不应与在体胚胎享有同等的伦理地位[17]。然而,从长远来看,在何处划定界限方可确保类胚胎研究符合伦理仍是一个值得深入探讨的问题。

  • 3 展望

  • 未来研究人员一方面可利用工程学、材料学等领域的技术进一步稳定和优化培养体系,提高体外胚胎的发育率及质量,以获得发育时程更长,且更接近于在体胚胎的体外胚胎,以研究多级胚胎发育事件,另一方面可结合多维组学、生物成像及基因编辑等技术建立人类生理、病理胚胎模型,探索人类早期胚胎发育相关疾病的发病机制,开发新的防治手段。随着科学技术的发展,人类从健康需求出发获知人类早期胚胎发育基本原理的渴望日渐增强。与此同时,诸多伦理问题(如“14 天准则” 是否延长,类胚胎的伦理、法律地位何为等) 亟待解答。针对此类伦理问题的探讨应考量多学科学者的意见,并经过公众广泛讨论。唯有如此,人类胚胎及类胚胎研究才能逐渐走出伦理困境、获取更多的支持与信任,实现自身跨越式发展并最终服务于人类健康。

  • 参考文献

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    • [3] CHRISTODOULOU N,KYPRIANOU C,WEBERLING A,et al.Sequential formation and resolution of multiple rosettes drive embryo remodelling after implantation[J].Nat Cell Biol,2018,20(11):1278-1289.

    • [4] TAM P P L,LOEBEL D A F.Gene function in mouse embryogenesis:get set for gastrulation[J].Nat Rev Genet,2007,8(5):368-381.

    • [5] WILKINSON D G,BHATT S,HERRMANN B G.Expression pattern of the mouse T gene and its role in mesoderm formation[J].Nature,1990,343(6259):657-659.

    • [6] PIJUAN-SALA B,GRIFFITHS J A,GUIBENTIF C,et al.A single-cell molecular map of mouse gastrulation and early organogenesis[J].Nature,2019,566(7745):490-495.

    • [7] PENG G D,SUO S B,CUI G Z,et al.Molecular architecture of lineage allocation and tissue organization in early mouse embryo[J].Nature,2019,572(7770):528-532.

    • [8] ZORN A M,WELLS J M.Vertebrate endoderm development and organ formation[J].Annu Rev Cell Dev Biol,2009,25:221-251.

    • [9] SAUKA-SPENGLER T,BRONNER-FRASER M.A gene regulatory network orchestrates neural crest formation[J].Nat Rev Mol Cell Biol,2008,9(7):557-568.

    • [10] BEDZHOV I,ZERNICKA-GOETZ M.Self-organizing properties of mouse pluripotent cells initiate morphogenesis upon implantation[J].Cell,2014,156(5):1032-1044.

    • [11] GUO S,JIANG X,DUO S,et al.Tracing the origin of the placental trophoblast cells in mouse embryo development[J].Biology of Reproduction,2020,102(3):598-606.

    • [12] MORRIS S A,TEO R T,LI H,et al.Origin and formation of the first two distinct cell types of the inner cell mass in the mouse embryo[J].Proc Natl Acad Sci USA,2010,107(14):6364-6369.

    • [13] WANG J,WANG L,FENG G,et al.Asymmetric expression of lincget biases cell fate in two-cell mouse embryos[J].Cell,2018,175(7):1887-1901.

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    • [15] BEDZHOV I,GRAHAM S J,LEUNG C Y,et al.Developmental plasticity,cell fate specification and morphogenesis in the early mouse embryo[J].Philos Trans R Soc Lond B Biol Sci,2014,369(1657):20130538.

    • [16] LIM H Y G,ALVAREZ Y D,GASNIER M,et al.Keratins are asymmetrically inherited fate determinants in the mammalian embryo[J].Nature,2020,585(7825):404-409.

    • [17] CHRISTODOULOU N,WEBERLING A,STRATHDEE D,et al.Morphogenesis of extra-embryonic tissues directs the remodelling of the mouse embryo at implantation[J].Nat Commun,2019,10(1):3557.

    • [18] SAYKALI B,MATHIAH N,NAHABOO W,et al.Distinct mesoderm migration phenotypes in extra-embryonic and embryonic regions of the early mouse embryo[J].Elife,2019,8:e42434.

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    • [23] SOZEN B,AMADEI G,COX A,et al.Self-assembly of embryonic and two extra-embryonic stem cell types into gastrulating embryo-like structures[J].Nat Cell Biol,2018,20(8):979-989.

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    • [26] SHAHBAZI M N,JEDRUSIK A,VUORISTO S,et al.Self-organization of the human embryo in the absence of maternal tissues[J].Nat Cell Biol,2016,18(6):700-708.

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    • [30] LOPATA A,KOHLMAN D J,BOWES L G,et al.Culture of marnoset blastocysts on matrigel a model of differentiation during the implantation period[J].The Anatomical Record,1995,241:469-486.

    • [31] NIU Y,SUN N,LI C,et al.Dissecting primate early postimplantation development using long-term in vitro embryo culture[J].Science,2019,366(6467):eaaw5754.

    • [32] MA H,ZHAI J,WAN H,et al.In vitro culture of cynomolgus monkey embryos beyond early gastrulation[J].Science,2019,366(6467):eaax7890.

    • [33] HAREMAKI T,METZGER J J,RITO T,et al.Self-organizing neuruloids model developmental aspects of huntington's disease in the ectodermal compartment[J].Nat Biotechnol,2019,37(10):1198-1208.

    • [34] SIMUNOVIC M,METZGER J J,ETOC F,et al.A 3D model of a human epiblast reveals BMP4-driven symmetry breaking[J].Nat Cell Biol,2019,21(7):900-910.

    • [35] ZHENG Y,XUE X,SHAO Y,et al.Controlled modelling of human epiblast and amnion development using stem cells[J].Nature,2019,573(7774):421-425.

    • [36] EVANS M J,KAUFMAN M H.Establishment in culture of pluripotential cells from mouse embryos[J].Nature,1981,292(5819):154-156.

    • [37] MARTIN G R.Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells[J].Proc Natl Acad Sci USA,1981,78(12):7634-7638.

    • [38] VAN DEN BRINK S C,BAILLIE-JOHNSON P,BALAYO T,et al.Symmetry breaking,germ layer specification and axial organisation in aggregates of mouse embryonic stem cells[J].Development,2014,141(22):4231-4242.

    • [39] BECCARI L,MORIS N,GIRGIN M,et al.Multi-axial self-organization properties of mouse embryonic stem cells into gastruloids[J].Nature,2018,562(7726):272-276.

    • [40] VAN DEN BRINK S C,ALEMANY A,VAN BATENBURG V,et al.Single-cell and spatial transcriptomics reveal somitogenesis in gastruloids[J].Nature,2020,582(7812).

    • [41] HARRISON S E,SOZEN B,CHRISTODOULOU N,et al.Assembly of embryonic and extraembryonic stem cells to mimic embryogenesis in vitro[J].Science,2017,356(6334):eaal1810.

    • [42] ZHANG S,CHEN T,CHEN N,et al.Implantation initiation of self-assembled embryo-like structures generated using three types of mouse blastocyst-derived stem cells[J].Nat Commun,2019,10(1):496.

    • [43] RIVRON N C,FRIAS-ALDEGUER J,VRIJ E J,et al.Blastocyst-like structures generated solely from stem cells[J].Nature,2018,557(7703):106-111.

    • [44] Li R H,ZHONG C Q,YU Y.et al.Generation of blastocyst-like structures from mouse embryonic and adult cell cultures[J].Cell,2019,179(3):687-702.

    • [45] SOZEN B,COX A L,JONGHE J D,et al.Self-organization of mouse stem cells into an extended potential blastoid[J].Developmental Cell,2019,51(6):698-712.

    • [46] WARMFLASH A,SORRE B,ETOC F,et al.A method to recapitulate early embryonic spatial patterning in human embryonic stem cells[J].Nature Methods,2014,11(8):847-854.

    • [47] ZHENG Y,XUE X,RESTO-IRIZARRY A M,et al.Dorsal-ventral patterned neural cyst from human pluripotent stem cells in a neurogenic niche[J].Science Advances,2019,5(12):eaax5933.

    • [48] MORIS N,ANLAS K,VAN DEN BRINKS C,et al.An in vitro model of early anteroposterior organization during human development[J].Nature,2020,582(7812):410-415.

    • [49] OHNISHI T,MIURA I,OHBA H,et al.A spontaneous and novel Pax3 mutant mouse that models Waardenburg syndrome and neural tube defects[J].Gene,2017,607:16-22.

    • [50] SABATINO J A,STOKES B A,ZOHN I E.Prevention of neural tube defects in Lrp2 mutant mouse embryos by folic acid supplementation[J].Birth Defects Research,2017,109(1):16-26.

    • [51] BAI B,CHEN S,ZHANG Q,et al.Abnormal epigenetic regulation of the gene expression levels of Wnt2b and Wnt7b:Implications for neural tube defects[J].Molecular Medicine Reports,2016,13(1):99-106.

    • [52] FREYER L,HSU C W,NOWOTSCHIN S,et al.Loss of apela peptide in mice causes low penetrance embryonic lethality and defects in early mesodermal derivatives[J].Cell Reports,2017,20(9):2116-2130.

  • 参考文献

    • [1] 卫生部,中国出生缺陷防治报告(2012)[J].中国药房,2012,23(39):3693.

    • [2] HAN X P,ZHOU Z M,FEI L J,et al.Construction of a human cell landscape at single-cell level[J].Nature,2020,581(7808):303-309.

    • [3] CHRISTODOULOU N,KYPRIANOU C,WEBERLING A,et al.Sequential formation and resolution of multiple rosettes drive embryo remodelling after implantation[J].Nat Cell Biol,2018,20(11):1278-1289.

    • [4] TAM P P L,LOEBEL D A F.Gene function in mouse embryogenesis:get set for gastrulation[J].Nat Rev Genet,2007,8(5):368-381.

    • [5] WILKINSON D G,BHATT S,HERRMANN B G.Expression pattern of the mouse T gene and its role in mesoderm formation[J].Nature,1990,343(6259):657-659.

    • [6] PIJUAN-SALA B,GRIFFITHS J A,GUIBENTIF C,et al.A single-cell molecular map of mouse gastrulation and early organogenesis[J].Nature,2019,566(7745):490-495.

    • [7] PENG G D,SUO S B,CUI G Z,et al.Molecular architecture of lineage allocation and tissue organization in early mouse embryo[J].Nature,2019,572(7770):528-532.

    • [8] ZORN A M,WELLS J M.Vertebrate endoderm development and organ formation[J].Annu Rev Cell Dev Biol,2009,25:221-251.

    • [9] SAUKA-SPENGLER T,BRONNER-FRASER M.A gene regulatory network orchestrates neural crest formation[J].Nat Rev Mol Cell Biol,2008,9(7):557-568.

    • [10] BEDZHOV I,ZERNICKA-GOETZ M.Self-organizing properties of mouse pluripotent cells initiate morphogenesis upon implantation[J].Cell,2014,156(5):1032-1044.

    • [11] GUO S,JIANG X,DUO S,et al.Tracing the origin of the placental trophoblast cells in mouse embryo development[J].Biology of Reproduction,2020,102(3):598-606.

    • [12] MORRIS S A,TEO R T,LI H,et al.Origin and formation of the first two distinct cell types of the inner cell mass in the mouse embryo[J].Proc Natl Acad Sci USA,2010,107(14):6364-6369.

    • [13] WANG J,WANG L,FENG G,et al.Asymmetric expression of lincget biases cell fate in two-cell mouse embryos[J].Cell,2018,175(7):1887-1901.

    • [14] HUPALOWSKA A,JEDRUSIK A,ZHU M,et al.CARM1 and paraspeckles regulate pre-implantation mouse embryo development[J].Cell,2018,175(7):1902-1916.

    • [15] BEDZHOV I,GRAHAM S J,LEUNG C Y,et al.Developmental plasticity,cell fate specification and morphogenesis in the early mouse embryo[J].Philos Trans R Soc Lond B Biol Sci,2014,369(1657):20130538.

    • [16] LIM H Y G,ALVAREZ Y D,GASNIER M,et al.Keratins are asymmetrically inherited fate determinants in the mammalian embryo[J].Nature,2020,585(7825):404-409.

    • [17] CHRISTODOULOU N,WEBERLING A,STRATHDEE D,et al.Morphogenesis of extra-embryonic tissues directs the remodelling of the mouse embryo at implantation[J].Nat Commun,2019,10(1):3557.

    • [18] SAYKALI B,MATHIAH N,NAHABOO W,et al.Distinct mesoderm migration phenotypes in extra-embryonic and embryonic regions of the early mouse embryo[J].Elife,2019,8:e42434.

    • [19] SHAHBAZI M N,SCIALDONE A,SKORUPSKA N,et al.Pluripotent state transitions coordinate morphogenesis in mouse and human embryos[J].Nature,2017,552(7684):239-243:e42434.

    • [20] NOWOTSCHIN S,SETTY M,YING-YI K,et al.The emergent landscape of the mouse gut endoderm at singlecell resolution[J].Nature,2019,569(7756):361-367.

    • [21] CHEN J,SUO S,TAM P P,et al.Spatial transcriptomic analysis of cryosectioned tissue samples with Geo-seq[J].Nat Protoc,2017,12(3):566-580.

    • [22] CHENL T,HSU Y C.Development of mouse embryos in vitro:preimplantation to the limb bud stage[J].Science(New York,N.Y.),1982,218(4567):66-68.

    • [23] SOZEN B,AMADEI G,COX A,et al.Self-assembly of embryonic and two extra-embryonic stem cell types into gastrulating embryo-like structures[J].Nat Cell Biol,2018,20(8):979-989.

    • [24] NAKAMURA T,OKAMOTO I,SASAKI K,et al.A developmental coordinate of pluripotency among mice,monkeys and humans[J].Nature,2016,537(7618):57-62.

    • [25] ROSSANT J,TAM P P L.New insights into early human development:lessons for stem cell derivation and differentiation[J].Cell Stem Cell,2017,20(1):18-28.

    • [26] SHAHBAZI M N,JEDRUSIK A,VUORISTO S,et al.Self-organization of the human embryo in the absence of maternal tissues[J].Nat Cell Biol,2016,18(6):700-708.

    • [27] DEGLINCERTI A,CROFT G F,PIETILA L N,et al.Selforganization of the in vitro attached human embryo[J].Nature,2016,533(7602):251-254.

    • [28] ZHOU F,WANG R,YUAN P,et al.Reconstituting the transcriptome and DNA methylome landscapes of human implantation[J].Nature,2019,572(7771):660-664.

    • [29] XIANG L,YIN Y,ZHENG Y,et al.A developmental landscape of 3D-cultured human pre-gastrulation embryos[J].Nature,2020,577(7791):537-542.

    • [30] LOPATA A,KOHLMAN D J,BOWES L G,et al.Culture of marnoset blastocysts on matrigel a model of differentiation during the implantation period[J].The Anatomical Record,1995,241:469-486.

    • [31] NIU Y,SUN N,LI C,et al.Dissecting primate early postimplantation development using long-term in vitro embryo culture[J].Science,2019,366(6467):eaaw5754.

    • [32] MA H,ZHAI J,WAN H,et al.In vitro culture of cynomolgus monkey embryos beyond early gastrulation[J].Science,2019,366(6467):eaax7890.

    • [33] HAREMAKI T,METZGER J J,RITO T,et al.Self-organizing neuruloids model developmental aspects of huntington's disease in the ectodermal compartment[J].Nat Biotechnol,2019,37(10):1198-1208.

    • [34] SIMUNOVIC M,METZGER J J,ETOC F,et al.A 3D model of a human epiblast reveals BMP4-driven symmetry breaking[J].Nat Cell Biol,2019,21(7):900-910.

    • [35] ZHENG Y,XUE X,SHAO Y,et al.Controlled modelling of human epiblast and amnion development using stem cells[J].Nature,2019,573(7774):421-425.

    • [36] EVANS M J,KAUFMAN M H.Establishment in culture of pluripotential cells from mouse embryos[J].Nature,1981,292(5819):154-156.

    • [37] MARTIN G R.Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells[J].Proc Natl Acad Sci USA,1981,78(12):7634-7638.

    • [38] VAN DEN BRINK S C,BAILLIE-JOHNSON P,BALAYO T,et al.Symmetry breaking,germ layer specification and axial organisation in aggregates of mouse embryonic stem cells[J].Development,2014,141(22):4231-4242.

    • [39] BECCARI L,MORIS N,GIRGIN M,et al.Multi-axial self-organization properties of mouse embryonic stem cells into gastruloids[J].Nature,2018,562(7726):272-276.

    • [40] VAN DEN BRINK S C,ALEMANY A,VAN BATENBURG V,et al.Single-cell and spatial transcriptomics reveal somitogenesis in gastruloids[J].Nature,2020,582(7812).

    • [41] HARRISON S E,SOZEN B,CHRISTODOULOU N,et al.Assembly of embryonic and extraembryonic stem cells to mimic embryogenesis in vitro[J].Science,2017,356(6334):eaal1810.

    • [42] ZHANG S,CHEN T,CHEN N,et al.Implantation initiation of self-assembled embryo-like structures generated using three types of mouse blastocyst-derived stem cells[J].Nat Commun,2019,10(1):496.

    • [43] RIVRON N C,FRIAS-ALDEGUER J,VRIJ E J,et al.Blastocyst-like structures generated solely from stem cells[J].Nature,2018,557(7703):106-111.

    • [44] Li R H,ZHONG C Q,YU Y.et al.Generation of blastocyst-like structures from mouse embryonic and adult cell cultures[J].Cell,2019,179(3):687-702.

    • [45] SOZEN B,COX A L,JONGHE J D,et al.Self-organization of mouse stem cells into an extended potential blastoid[J].Developmental Cell,2019,51(6):698-712.

    • [46] WARMFLASH A,SORRE B,ETOC F,et al.A method to recapitulate early embryonic spatial patterning in human embryonic stem cells[J].Nature Methods,2014,11(8):847-854.

    • [47] ZHENG Y,XUE X,RESTO-IRIZARRY A M,et al.Dorsal-ventral patterned neural cyst from human pluripotent stem cells in a neurogenic niche[J].Science Advances,2019,5(12):eaax5933.

    • [48] MORIS N,ANLAS K,VAN DEN BRINKS C,et al.An in vitro model of early anteroposterior organization during human development[J].Nature,2020,582(7812):410-415.

    • [49] OHNISHI T,MIURA I,OHBA H,et al.A spontaneous and novel Pax3 mutant mouse that models Waardenburg syndrome and neural tube defects[J].Gene,2017,607:16-22.

    • [50] SABATINO J A,STOKES B A,ZOHN I E.Prevention of neural tube defects in Lrp2 mutant mouse embryos by folic acid supplementation[J].Birth Defects Research,2017,109(1):16-26.

    • [51] BAI B,CHEN S,ZHANG Q,et al.Abnormal epigenetic regulation of the gene expression levels of Wnt2b and Wnt7b:Implications for neural tube defects[J].Molecular Medicine Reports,2016,13(1):99-106.

    • [52] FREYER L,HSU C W,NOWOTSCHIN S,et al.Loss of apela peptide in mice causes low penetrance embryonic lethality and defects in early mesodermal derivatives[J].Cell Reports,2017,20(9):2116-2130.

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