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Protocol for the Generation of Human EPS-Blastoids Using a Three-Dimensional Two-Step Induction System. 使用三维两步诱导系统生成人体 EPS--类固醇的方案。
Methods in molecular biology Pub Date : 2024-01-01 DOI: 10.1007/7651_2022_471
Yu-Xin Luo, Yang Yu
{"title":"Protocol for the Generation of Human EPS-Blastoids Using a Three-Dimensional Two-Step Induction System.","authors":"Yu-Xin Luo, Yang Yu","doi":"10.1007/7651_2022_471","DOIUrl":"10.1007/7651_2022_471","url":null,"abstract":"<p><p>Stem cell-derived embryos in vitro allow the exploration of the very early stages of human embryogenesis in vitro and are thus promising for widespread applications in developmental biology, related developmental disease modeling, and drug discovery. Several cell resources have been utilized, with different efficiencies and methods for generating human blastoids, a structure similar to natural blastocysts. Human EPS cells were reported to contribute to the embryonic and extraembryonic lineages and therefore can be a practical and efficient cell resource for constructing human blastoids. Here, we developed a three-dimensional, two-step induction system for generating human blastoids using human EPS cells. According to morphological and transcriptomic analysis, EPS-blastoids recapitulate the key developmental processes and cell lineages of human blastocysts. Moreover, in vitro extended culture for 8 and 10 days of EPS-blastoids can result in postimplantation embryonic structures. In this chapter, we describe a protocol that covers the generation, maintenance, and developmental phenocopying of human EPS blastoids.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":" ","pages":"27-41"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9231785","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Use of Epigenetic Cues and Mechanical Stimuli to Generate Blastocyst-Like Structures from Mammalian Skin Dermal Fibroblasts. 利用表观遗传线索和机械刺激从哺乳动物皮肤真皮成纤维细胞中生成类似囊胚的结构
Methods in molecular biology Pub Date : 2024-01-01 DOI: 10.1007/7651_2023_486
Sharon Arcuri, Georgia Pennarossa, Sergio Ledda, Fulvio Gandolfi, Tiziana A L Brevini
{"title":"Use of Epigenetic Cues and Mechanical Stimuli to Generate Blastocyst-Like Structures from Mammalian Skin Dermal Fibroblasts.","authors":"Sharon Arcuri, Georgia Pennarossa, Sergio Ledda, Fulvio Gandolfi, Tiziana A L Brevini","doi":"10.1007/7651_2023_486","DOIUrl":"10.1007/7651_2023_486","url":null,"abstract":"<p><p>Mammalian embryogenesis is characterized by complex interactions between embryonic and extra-embryonic tissues that coordinate morphogenesis, coupling bio-mechanical and bio-chemical cues, to regulate gene expression and influence cell fate. Deciphering such mechanisms is essential to understand early embryogenesis, as well as to harness differentiation disorders. Currently, several early developmental events remain unclear, mainly due to ethical and technical limitations related to the use of natural embryos.Here, we describe a three-step approach to generate 3D spherical structures, arbitrarily defined \"epiBlastoids,\" whose phenotype is remarkably similar to natural embryos. In the first step, adult dermal fibroblasts are converted into trophoblast-like cells, combining the use of 5-azacytidine, to erase the original cell phenotype, with an ad hoc induction protocol, to drive erased cells into the trophoblast lineage. In the second step, once again epigenetic erasing is applied, in combination with mechanosensing-related cues, to generate inner cell mass (ICM)-like spheroids. More specifically, erased cells are encapsulated in micro-bioreactors to promote 3D cell rearrangement and boost pluripotency. In the third step, chemically induced trophoblast-like cells and ICM-like spheroids are co-cultured in the same micro-bioreactors. The newly generated embryoids are then transferred to microwells, to encourage further differentiation and favor epiBlastoid formation. The procedure here described is a novel strategy for in vitro generation of 3D spherical structures, phenotypically similar to natural embryos. The use of easily accessible dermal fibroblasts and the lack of retroviral gene transfection make this protocol a promising strategy to study early embryogenesis as well as embryo disorders.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":" ","pages":"161-173"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9473324","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Guinea Pig Preimplantation Embryos: Generation, Collection, and Immunofluorescence. 豚鼠植入前胚胎：生成、收集和免疫荧光。
Methods in molecular biology Pub Date : 2024-01-01 DOI: 10.1007/7651_2023_488
Jesica Canizo, Savana Biondic, Katherine Vandal Lenghan, Sophie Petropoulos
{"title":"Guinea Pig Preimplantation Embryos: Generation, Collection, and Immunofluorescence.","authors":"Jesica Canizo, Savana Biondic, Katherine Vandal Lenghan, Sophie Petropoulos","doi":"10.1007/7651_2023_488","DOIUrl":"10.1007/7651_2023_488","url":null,"abstract":"<p><p>Studying various animal models is important for comparative biology and to better understand evolutionary development. Furthermore, when aiming to translate findings to human development, it is crucial to select an appropriate animal model that closely resembles the specific aspect of development under study. The guinea pig is highlighted as a useful platform for reproductive studies due to similarities in in utero development and general physiology with the human. This chapter outlines the methods required for guinea pig mating and collection of embryos for in vitro culture and molecular characterization. Specifically, this chapter provides detailed guidance on monitoring the estrus cycle to determine the mating time, performing a vaginal flush and smear to confirm successful mating, performing euthanasia of the guinea pig, and flushing in vivo embryos. Once collected, the embryos can be utilized for numerous downstream applications. Here we will cover embryo culturing and processing embryos for immunofluorescence.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":" ","pages":"275-292"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9587491","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Manual Dissociation of Mammalian Preimplantation Embryos for Single-Cell Genomics. 手动分离哺乳动物植入前胚胎以进行单细胞基因组学研究。
Methods in molecular biology Pub Date : 2024-01-01 DOI: 10.1007/7651_2023_494
Katherine Vandal, Savana Biondic, Jesica Canizo, Sophie Petropoulos
{"title":"Manual Dissociation of Mammalian Preimplantation Embryos for Single-Cell Genomics.","authors":"Katherine Vandal, Savana Biondic, Jesica Canizo, Sophie Petropoulos","doi":"10.1007/7651_2023_494","DOIUrl":"10.1007/7651_2023_494","url":null,"abstract":"<p><p>Single-cell genomics allow the characterization and quantification of molecular heterogeneity from a wide variety of tissues. Here, we describe the manual dissociation and collection of single cells, a method adapted for the characterization of precious small tissues like preimplantation embryos. We also describe the acquisition of mouse embryos by flushing of the oviducts. The cells can then be used in multiple sequencing protocols, for example, Smart-seq2, Smart-seq3, smallseq, and scBSseq.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":" ","pages":"293-305"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9759218","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Dissection of Daytime and Nighttime Thermoresponsive Hypocotyl Elongation in Arabidopsis. 拟南芥白天和夜间热伸长下胚轴伸长的剖析
Methods in molecular biology Pub Date : 2024-01-01 DOI: 10.1007/978-1-0716-3814-9_2
De Fan, Meng Chen
{"title":"Dissection of Daytime and Nighttime Thermoresponsive Hypocotyl Elongation in Arabidopsis.","authors":"De Fan, Meng Chen","doi":"10.1007/978-1-0716-3814-9_2","DOIUrl":"10.1007/978-1-0716-3814-9_2","url":null,"abstract":"<p><p>Hypocotyl elongation in Arabidopsis is widely utilized as a readout for phytochrome B (phyB) signaling and thermomorphogenesis. Hypocotyl elongation is gated by the circadian clock and, therefore, it occurs at distinct times depending on day length or seasonal cues. In short-day conditions, hypocotyl elongation occurs mainly at the end of nighttime when phyB reverts to the inactive form. In contrast, in long-day conditions, hypocotyl elongation occurs during the daytime when phyB is in the photoactivated form. Warm temperatures can induce hypocotyl growth in both long-day and short-day conditions. However, the corresponding daytime and nighttime temperature responses reflect distinct underpinning mechanisms. Here, we describe assays for dissecting the mechanisms between daytime and nighttime thermoresponsive hypocotyl elongation.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2795 ","pages":"17-23"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140860755","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Mathematical Modeling of Photo- and Thermomorphogenesis in Plants. 植物光热蜕变的数学建模
Methods in molecular biology Pub Date : 2024-01-01 DOI: 10.1007/978-1-0716-3814-9_23
Gabriel Rodriguez-Maroto, Pablo Catalán, Cristina Nieto, Salomé Prat, Saúl Ares
{"title":"Mathematical Modeling of Photo- and Thermomorphogenesis in Plants.","authors":"Gabriel Rodriguez-Maroto, Pablo Catalán, Cristina Nieto, Salomé Prat, Saúl Ares","doi":"10.1007/978-1-0716-3814-9_23","DOIUrl":"10.1007/978-1-0716-3814-9_23","url":null,"abstract":"<p><p>Increased day lengths and warm conditions inversely affect plant growth by directly modulating nuclear phyB, ELF3, and COP1 levels. Quantitative measures of the hypocotyl length have been key to gaining a deeper understanding of this complex regulatory network, while similar quantitative data are the foundation for many studies in plant biology. Here, we explore the application of mathematical modeling, specifically ordinary differential equations (ODEs), to understand plant responses to these environmental cues. We provide a comprehensive guide to constructing, simulating, and fitting these models to data, using the law of mass action to study the evolution of molecular species. The fundamental principles of these models are introduced, highlighting their utility in deciphering complex plant physiological interactions and testing hypotheses. This brief introduction will not allow experimentalists without a mathematical background to run their own simulations overnight, but it will help them grasp modeling principles and communicate with more theory-inclined colleagues.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2795 ","pages":"247-261"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140851889","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Assessing Temperature Responses in Roots. 评估根对温度的反应
Methods in molecular biology Pub Date : 2024-01-01 DOI: 10.1007/978-1-0716-3814-9_4
Sanghwa Lee, Wolfgang Busch
{"title":"Assessing Temperature Responses in Roots.","authors":"Sanghwa Lee, Wolfgang Busch","doi":"10.1007/978-1-0716-3814-9_4","DOIUrl":"10.1007/978-1-0716-3814-9_4","url":null,"abstract":"<p><p>Due to global warming, it is important to understand how plants respond to high ambient temperature. Plant growth responses to high ambient temperature are termed thermomophogenesis and have been explored for more than a decade. However, this was mostly focused on the above-ground part of plants, the shoot. In this chapter, we describe a simple method to assess root growth phenotype to high ambient temperatures. In principle, this protocol can be applied for any other treatments to test overall seedling growth.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2795 ","pages":"37-42"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140864155","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
In Silico Typing and Identification Confirmation of Isolates. 菌株的硅学分型和鉴定确认。
Methods in molecular biology Pub Date : 2024-01-01 DOI: 10.1007/978-1-0716-3898-9_2
Matheus de O Costa, Nahuel Fittipaldi
{"title":"In Silico Typing and Identification Confirmation of Isolates.","authors":"Matheus de O Costa, Nahuel Fittipaldi","doi":"10.1007/978-1-0716-3898-9_2","DOIUrl":"10.1007/978-1-0716-3898-9_2","url":null,"abstract":"<p><p>Streptococcus suis is an important zoonotic pathogen causing severe infections in pigs and humans. Serotyping of S. suis strains is crucial for epidemiological surveillance, outbreak investigations, and understanding the pathogenesis of this bacterium. Here, we describe a step-by-step approach that enhances a previously developed pipeline by utilizing a computational script for efficient and accurate typing of S. suis strains. The pipeline is implemented in Perl programming language and leverages the Short Read Sequence Typing for Bacterial Pathogens (SRST2) tool. It integrates various bioinformatics techniques and utilizes multiple databases, including a serotype database, cpsH confirmation database, multi-locus sequence typing (MLST) database, recN species-specific gene database, and virulence gene database. These databases contain comprehensive information on S. suis serotypes, genetic markers, and virulence factors. The script can utilize paired-end or single-end fastq files as input and first confirms the species by sequence read data aligning to the recN gene, ensuring the accurate identification of S. suis strains. The pipeline next performs MLST typing and virulence factor identification using SRST2 while in a parallel processes it performs in silico serotyping of the strains. The pipeline offers a streamlined and semiautomated approach to serotyping S. suis strains, facilitating large-scale studies and reducing the manual effort required for data analysis.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2815 ","pages":"15-21"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141331396","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Generation of Human-Induced Pluripotent Stem Cells from Peripheral Blood Mononuclear Cells of C9ORF72-Associated Amyotrophic Lateral Sclerosis Patients. 从 C9ORF72 相关肌萎缩侧索硬化症患者的外周血单核细胞中生成人类诱导多能干细胞。
Methods in molecular biology Pub Date : 2024-01-01 DOI: 10.1007/978-1-0716-3995-5_12
Kalina Andrysiak, Jacek Stępniewski, Magdalena Spaczyńska-Boczar, Katarzyna Łapicka-Bodzioch, Agnieszka Słowik, Józef Dulak
{"title":"Generation of Human-Induced Pluripotent Stem Cells from Peripheral Blood Mononuclear Cells of C9ORF72-Associated Amyotrophic Lateral Sclerosis Patients.","authors":"Kalina Andrysiak, Jacek Stępniewski, Magdalena Spaczyńska-Boczar, Katarzyna Łapicka-Bodzioch, Agnieszka Słowik, Józef Dulak","doi":"10.1007/978-1-0716-3995-5_12","DOIUrl":"10.1007/978-1-0716-3995-5_12","url":null,"abstract":"<p><p>Disease modeling of neuromuscular disorders, such as amyotrophic lateral sclerosis (ALS), is hindered by limited accessibility of affected cells. This problem can be overcome by generation of human induced pluripotent stem cells (hiPSC), which can be then differentiated into required cells. Here, we describe the detailed protocol of hiPSC establishment from peripheral blood mononuclear cells (PBMC) of two ALS patients with detected expansion of G4C2 (GGGGCC) repeats in the first intron of C9ORF72 gene, known to be linked with the most common form of familial ALS.Successful PBMC reprogramming with non-integrating Sendai vectors was confirmed by expression of pluripotency markers: OCT4, NANOG, SSEA4, and TRA-1-60 in obtained hiPSC and their ability to differentiate into cells of three germ layers.The generated ALS-patient-specific hiPSC create a possibility for deciphering molecular basis of this devastating neuromuscular disease.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2835 ","pages":"135-146"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141893790","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Comprehensive Micro-SPE-Based Bottom-Up Proteomic Workflow for Sensitive Analysis of Limited Samples. 基于 Micro-SPE 的自下而上蛋白质组综合工作流程，可对有限样本进行灵敏分析。
Methods in molecular biology Pub Date : 2024-01-01 DOI: 10.1007/978-1-0716-3934-4_3
Alan J Zimmerman, Michal Greguš, Alexander R Ivanov
{"title":"Comprehensive Micro-SPE-Based Bottom-Up Proteomic Workflow for Sensitive Analysis of Limited Samples.","authors":"Alan J Zimmerman, Michal Greguš, Alexander R Ivanov","doi":"10.1007/978-1-0716-3934-4_3","DOIUrl":"10.1007/978-1-0716-3934-4_3","url":null,"abstract":"<p><p>Clinical and biological samples are often scarce and precious (e.g., rare cell isolates, microneedle tissue biopsies, small-volume liquid biopsies, and even single cells or organelles). Typical large-scale proteomic methods, where significantly higher protein amounts are analyzed, are not directly transferable to the analysis of limited samples due to their incompatibility with pg-, ng-, and low-μg-level protein sample amounts. Here, we report the on-microsolid-phase extraction tip (OmSET)-based sample preparation workflow for sensitive analysis of limited biological samples to address this challenge. The developed platform was successfully tested for the analysis of 100-10,000 typical mammalian cells and is scalable to allow for lower and larger protein amounts and more samples to be analyzed (i.e., higher throughput of analysis).</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2817 ","pages":"19-31"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141437069","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
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