{"doi":"10.21769/bioprotoc.4042","title":"Generation of Mouse Pluripotent Stem Cell-derived Trunk-like Structures: An in vitro Model of Post-implantation Embryogenesis","abstract":"Post-implantation mammalian embryogenesis involves profound molecular, cellular, and morphogenetic changes. The study of these highly dynamic processes is complicated by the limited accessibility of in utero development. In recent years, several complementary in vitro systems comprising self-organized assemblies of mouse embryonic stem cells, such as gastruloids, have been reported. We recently demonstrated that the morphogenetic potential of gastruloids can be further unlocked by the addition of a low percentage of Matrigel as an extracellular matrix surrogate. This resulted in the formation of highly organized trunk-like structures (TLSs) with a neural tube that is frequently flanked by bilateral somites. Notably, development at the molecular and morphogenetic levels is highly reminiscent of the natural embryo. To facilitate access to this powerful model, here we provide a detailed step-by-step protocol that should allow any lab with access to standard cell culture techniques to implement the culture system. This will provide the user with a means to investigate early mid-gestational mouse embryogenesis at an unprecedented spatiotemporal resolution., [摘要]植入后胚胎的哺乳动物涉及深刻分子，细胞，和形态结构的变化。这些高度动态的过程的研究由于子宫内发育的可及性有限而变得复杂。近年来，几种互补体外系统，其包括小鼠胚胎干细胞的自我组织组件，例如gastruloids ，已经报道了。我们最近证明，通过添加低百分比的Matrigel作为细胞外基质替代物，可以进一步释放类固醇的形态发生潜能。这导致带有神经管的高度组织化的躯干状结构（TLS）形成，该神经管经常两侧为双侧牙节。值得注意的是，在分子和形态发生水平上的发育高度让人联想到天然胚胎。为了方便访问此强大的模型，在此我们提供详细的分步协议，该协议应允许任何实验室都能使用标准细胞培养技术来实施培养系统。这将为用户提供一种以前所未有的时空分辨率研究早期妊娠中期小鼠胚胎发生的方法。[背景]原肠胚形成和早期器官表示是成功产生功能体计划的关键发育事件。在哺乳动物中，这些过程只在胚胎植入后开始在子宫内，并在几天内，各种各样的形态和功能多样的组织出现。目前，很难在体内研究这些高度动态的变化，并且植入后小鼠胚胎的离体培养费力，昂贵并且需要严格的训练，这对于大多数实验室而言通常是不切实际的。这些障碍导致了大量的努力，以模型植入后和早期妊娠中期人的发展在体外用胚胎干细胞（综述Shahbazi; Shahbazi和Zernicka -戈茨2018 。等，2019;贝利-本森等人。2020年； Veenvliet和Herrmann，2021年）。特别是，植入后的发育可以用自组织的类胃体，小鼠或人类胚胎干细胞（mESC / hESC ）聚集体进行建模（van den Brink等人，2014和2020；Moris等人，2020）。原始的小鼠胃二倍体培养方案可产生具有类似于枕后小鼠胚胎的类似胚胎的表达结构域的细长结构，并且三个体轴的位置正确，但形态发生受限（van den Brink等人，2014； Baillie-Johnson等人，2015;贝卡利等人，2018一和201 8B ;特纳。等人，2017）。最近的努力已经设法通过改变细胞环境来引入胚样形态特征，例如形成类似松节状结构或心管（van den Brink et al。，2020; Rossi et al。，202 1 ）。进一步的进展已经表明，在加入细胞外基质（ECM）的替代物，以gastruloids Ç一个触发器的更胚样与肠管以及体系结构体节侧翼神经管（Veenvliet等人，2020）。我们戏称这些胚胎组织体干状结构（TLSS），因为它们类似于早期妊娠中期的树干的核心部分人胚胎（〜萌芽阶段（E）8.5-9）。重要的是，在TLS诱导的时间范围内（聚集后96-120小时），基因调控程序与发育中的胚胎高度相似。此外，分割时钟，振荡器驱动的节律沉积体节体内，是活性在胚样步伐在TLS（Pourquié ，2003; Veenvliet 。等人，2020）。 TLS模型易于访问，跟踪，操纵和缩放，这使其成为研究碟中植入后和妊娠中期早期哺乳动物发育的强大工具。在这里，我们提供了一个全面的分步过程，以简化树干状结构的生成。我们还描述了如何处理TLS进行下游分析，包括整个安装的免疫荧光染色和（单细胞）RNA测序。","journal":"BIO-PROTOCOL","year":2021,"id":193949,"datarank":0.3596842909197557,"base_score":2.3978952727983707,"endowment":2.3978952727983707,"self_citation_contribution":0.3596842909197557,"citation_network_contribution":0.0,"self_endowment_contribution":0.3596842909197557,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":10,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.95,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2021-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":234447,"name":"Leah Haut","orcid":null,"position":1,"is_corresponding":false},{"id":763066,"name":"Seher Ipek Gassaloglu","orcid":"0000-0002-6307-378X","position":2,"is_corresponding":false},{"id":763773,"name":"Polly Burton","orcid":null,"position":3,"is_corresponding":false},{"id":13682,"name":"Helene Kretzmer","orcid":"0000-0002-0723-4980","position":4,"is_corresponding":false},{"id":232560,"name":"René Buschow","orcid":"0000-0002-9800-2578","position":5,"is_corresponding":false},{"id":769,"name":"Alexander Meissner","orcid":"0000-0001-8646-7469","position":6,"is_corresponding":false},{"id":31011,"name":"Bernhard G. Herrmann","orcid":"0000-0002-2192-8188","position":7,"is_corresponding":false},{"id":232554,"name":"Jesse V. Veenvliet","orcid":"0000-0002-1191-2975","position":8,"is_corresponding":false},{"id":232555,"name":"Adriano Bolondi","orcid":"0000-0002-1096-9435","position":0,"is_corresponding":true}],"reference_count":17,"raw_metadata":{"citation_network_status":"fetched"},"created_at":"2026-07-18T23:49:55.282992Z","pmid":"34250208","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}