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Lineage Tracing Reveals that the Activation of Endogenous Sox9+ Cells Promotes Kidney Regeneration After Acute Kidney Injury. 谱系追踪揭示内源性Sox9+细胞的激活促进急性肾损伤后肾脏再生。
Methods in molecular biology Pub Date : 2025-07-05 DOI: 10.1007/7651_2025_655
Haozheng Liu, Rui Li, Zongjin Li
{"title":"Lineage Tracing Reveals that the Activation of Endogenous Sox9<sup>+</sup> Cells Promotes Kidney Regeneration After Acute Kidney Injury.","authors":"Haozheng Liu, Rui Li, Zongjin Li","doi":"10.1007/7651_2025_655","DOIUrl":"https://doi.org/10.1007/7651_2025_655","url":null,"abstract":"<p><p>Acute kidney injury (AKI), characterized by a sudden and sustained decline in renal function, is linked to significant morbidity and mortality. The regeneration of the kidney following AKI is a complex process in which the activation of stem and progenitor cells plays a crucial role. Numerous studies have demonstrated that endogenous Sox9<sup>+</sup> cells contribute to this regeneration. Traditionally, the status of kidney regeneration after AKI has been evaluated through histopathological examination and renal function indices, which are limited in providing real-time and dynamic insights. To address these limitations, we propose a novel approach using two-photon live imaging to track lineage-labeled endogenous Sox9<sup>+</sup> cells in AKI mouse models, allowing long-term monitoring and visualization of the kidney regeneration process.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-07-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144560448","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 Hemichannel and Gap Junctional Channel Activities in HaCaT Keratinocyte Cells. HaCaT角化细胞半通道和间隙连接通道活性的评估。
Methods in molecular biology Pub Date : 2025-07-05 DOI: 10.1007/7651_2025_650
Ece Inal, M Azra Yildirim, Gulistan Mese
{"title":"Assessing Hemichannel and Gap Junctional Channel Activities in HaCaT Keratinocyte Cells.","authors":"Ece Inal, M Azra Yildirim, Gulistan Mese","doi":"10.1007/7651_2025_650","DOIUrl":"https://doi.org/10.1007/7651_2025_650","url":null,"abstract":"<p><p>Gap junctions (GJ), formed by connexins (Cx), are mediators of intercellular communication between adjacent cells by allowing the transfer of small molecules. Connexins assemble to form hemichannels, which are then transported to the plasma membrane. There, they can either function independently or dock with adjacent hemichannels to form gap junctions. The epidermis is an avascular tissue and depends on connexin-mediated communication to maintain epidermal cell homeostasis and dysregulation of connexins can lead to human disorders. Keratitis-ichthyosis-deafness (KID) syndrome is caused by mutations in the GJB2 gene, encoding Cx26 protein, and the severity of the phenotypes varies among individuals carrying distinct mutations. The effects of mutations on channel functions can be assessed by various techniques. Here, we describe approaches to assess hemichannel and gap junctional activity in HaCaT cells that constitutively express Cx26 KID syndrome mutations. Ethidium bromide (EtBr) can be used in scrape loading assay to investigate GJ channel activity, and neurobiotin in dye uptake assay can be used to determine hemichannel functionality using fluorescence microscopy.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-07-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144560524","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
Interaction Between Pluripotent Stem Cells and Amniotic Epithelium for the In Vitro Study of Human Embryogenesis. 多能干细胞与羊膜上皮相互作用对人胚胎发生的体外研究。
Methods in molecular biology Pub Date : 2025-07-03 DOI: 10.1007/7651_2025_643
Daniela Ávila-González, Axel Castro-Abrego, Jonathan Salazar-Alonso, Omar Martínez Alarcon, Alejandro Martinez-Juarez, Anayansi Molina Hernández, Nestor Emmanuel Diaz-Martinez, Wendy Portillo, Nestor Fabián Diaz
{"title":"Interaction Between Pluripotent Stem Cells and Amniotic Epithelium for the In Vitro Study of Human Embryogenesis.","authors":"Daniela Ávila-González, Axel Castro-Abrego, Jonathan Salazar-Alonso, Omar Martínez Alarcon, Alejandro Martinez-Juarez, Anayansi Molina Hernández, Nestor Emmanuel Diaz-Martinez, Wendy Portillo, Nestor Fabián Diaz","doi":"10.1007/7651_2025_643","DOIUrl":"https://doi.org/10.1007/7651_2025_643","url":null,"abstract":"<p><p>During the peri-implantation stage in primate embryos, the epiblast coexists with amniotic epithelium, suggesting a pivotal role for this tissue in embryogenesis. This interaction has attracted considerable interest in developmental biology and regenerative medicine due to its potential influence on embryonic patterning and tissue specification. In this protocol, we examined whether co-culturing human amniotic epithelial cells (hAEC) with human embryonic stem cells (hESC) enhances the latter's capacity for differentiation toward human primordial germ cell-like cells (hPGCLC).</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-07-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144540813","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
Generating Human Blastoids Following Transient Naïve Resetting of Primed Pluripotent Stem Cells. 诱导多能干细胞瞬时重置后生成人类囊胚。
Methods in molecular biology Pub Date : 2025-07-03 DOI: 10.1007/7651_2025_649
Maria Carolina Zimara, Carlos A Pinzon-Arteaga, Kun Liu, Toshihiko Ezashi, Seiya Oura, Shijian Lyu, Menaka Sanghvi, Ye Yuan, Jun Wu
{"title":"Generating Human Blastoids Following Transient Naïve Resetting of Primed Pluripotent Stem Cells.","authors":"Maria Carolina Zimara, Carlos A Pinzon-Arteaga, Kun Liu, Toshihiko Ezashi, Seiya Oura, Shijian Lyu, Menaka Sanghvi, Ye Yuan, Jun Wu","doi":"10.1007/7651_2025_649","DOIUrl":"https://doi.org/10.1007/7651_2025_649","url":null,"abstract":"<p><p>Human pluripotent stem cells (hPSCs) exist in at least two distinct states of pluripotency: naïve and primed. While naïve hPSCs possess the unique ability to generate blastocyst-like structures, they are often genetically and epigenetically unstable, which compromises the quality and developmental potential of naïve hPSC-derived blastoids. This protocol presents an optimized human blastoid protocol through a transient resetting method that converts primed hPSCs into a naïve-like state, addressing the stability issues associated with long-term naïve hPSC maintenance. The approach is compatible with both feeder-free and feeder-based culture systems and demonstrates high efficiency in generating human blastoids directly from primed hPSCs. This advancement provides a more robust and reliable strategy for blastoid formation, circumventing the limitations of suboptimal naïve hPSC cultures.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-07-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144540812","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 Blastoids from Human Naïve Pluripotent Stem Cells. 从人Naïve多能干细胞生成囊胚。
Methods in molecular biology Pub Date : 2025-06-04 DOI: 10.1007/7651_2025_642
Hiroo Sasaki, Iori Sasaki, Jennifer Nichols, Ayaka Yanagid
{"title":"Generation of Blastoids from Human Naïve Pluripotent Stem Cells.","authors":"Hiroo Sasaki, Iori Sasaki, Jennifer Nichols, Ayaka Yanagid","doi":"10.1007/7651_2025_642","DOIUrl":"https://doi.org/10.1007/7651_2025_642","url":null,"abstract":"<p><p>Human embryo research is essential for understanding the development of human embryos and their unique features that cannot be investigated in mouse embryos. Natural development of human embryos remains challenging to study both in vivo and in vitro owing to ethical concerns, technical difficulties, and limited availability of embryos for research purposes. Until recently, access to human embryo research, especially the implantation period, was limited. However, optimization of a stem cell-based model known as a blastoid has opened a new era for human embryo research. In contrast with mouse embryonic stem cells, human naïve pluripotent stem cells retain extended cell lineage plasticity. They can differentiate into hypoblast and trophectoderm while retaining characteristic resembling the naïve epiblast in the inner cell mass region. Taking this unique differentiation potential and inherent propagation capacity as pluripotent stem cells, human blastoids are generated by the self-organization of naïve pluripotent stem cells. They resemble human blastocyst structures, consisting of the three founder cell lineages that closely resemble the gene expression profile of human blastocysts. This protocol for generating blastoids solely from naïve human pluripotent stem cells utilizing simple, efficient, and scalable procedures is a robust tool for advancing aspects of human embryo research.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-06-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144208945","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
3D In Vitro Culture of Early Mouse Embryos and Trophoblast Tissue Explants. 小鼠早期胚胎和滋养细胞组织的3D体外培养。
Methods in molecular biology Pub Date : 2025-05-22 DOI: 10.1007/7651_2025_646
Devila Prit, Shashank Jaitly, Niraimathi Govindasamy, Adrian Ranga, Ivan Bedzhov
{"title":"3D In Vitro Culture of Early Mouse Embryos and Trophoblast Tissue Explants.","authors":"Devila Prit, Shashank Jaitly, Niraimathi Govindasamy, Adrian Ranga, Ivan Bedzhov","doi":"10.1007/7651_2025_646","DOIUrl":"https://doi.org/10.1007/7651_2025_646","url":null,"abstract":"<p><p>The cellular dynamics during peri-implantation embryogenesis and the concurrent interactions at the embryo-maternal interface are inherently difficult to study due to intrauterine development in mammals. To model certain aspects of these processes in vitro, we have generated a biomimetic environment resembling the mechanical properties of the murine uterine stroma. Here we describe a step-by-step methodology for 3D culture of mouse embryos and ectoplacental cone explants in synthetic hydrogels that allow ex utero trophoblast invasion.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-05-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144111095","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 Naïve Pluripotent Stem Cell-Derived Blastoids in Thermoformed Microwell Platforms. 人Naïve多能干细胞衍生囊胚在热形成微孔平台的生成。
Methods in molecular biology Pub Date : 2025-05-22 DOI: 10.1007/7651_2025_644
Dorian G Luijkx, Erik J Vrij, Ge Guo, Stefan Giselbrecht
{"title":"Generation of Human Naïve Pluripotent Stem Cell-Derived Blastoids in Thermoformed Microwell Platforms.","authors":"Dorian G Luijkx, Erik J Vrij, Ge Guo, Stefan Giselbrecht","doi":"10.1007/7651_2025_644","DOIUrl":"https://doi.org/10.1007/7651_2025_644","url":null,"abstract":"<p><p>Due to the inaccessibility of early human embryos for large, robust studies, many questions regarding the mechanisms of early embryogenesis remain. To address these questions, multiple research groups have developed human stem cell-based models of the pre-implantation blastocyst stage. These models, known as blastoids, mimic several key processes that natural blastocysts undergo as they prepare to implant into the uterine wall. One of the main advantages of blastoids is their scalability, making them suitable for both screenings and molecular studies. To leverage this advantage, we established a protocol for the parallel formation and culture of blastoids in thermoformed microwell arrays. Thin-walled thermoformed microwell platforms allow for uniform, large-scale generation of blastoids and enable in situ high-resolution imaging through both widefield and confocal microscopy. Here we present a step-by-step protocol for the culture of blastoids in thermoformed microwell platforms.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-05-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144111218","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
Application of CRISPR-Based Epigenome Editing Tools for Engineering Programmable Embryo Models. 基于crispr的表观基因组编辑工具在工程可编程胚胎模型中的应用
Methods in molecular biology Pub Date : 2025-05-22 DOI: 10.1007/7651_2025_637
Gerrald A Lodewijk, Sayaka Kozuki, Carly Guiltinan, Benjamin R Topacio, S Ali Shariati
{"title":"Application of CRISPR-Based Epigenome Editing Tools for Engineering Programmable Embryo Models.","authors":"Gerrald A Lodewijk, Sayaka Kozuki, Carly Guiltinan, Benjamin R Topacio, S Ali Shariati","doi":"10.1007/7651_2025_637","DOIUrl":"https://doi.org/10.1007/7651_2025_637","url":null,"abstract":"<p><p>Stem cell-based embryo models (SEMs) have the potential to transform our understanding of early human embryogenesis. A critical step in engineering SEMs is the generation of the major cell types that compose preimplantation embryos including two primary extraembryonic lineages: (i) trophoblast cells, which are crucial for implantation and the establishment of maternal-fetal exchange, and (ii) hypoblast cells, which contribute to yolk sac formation. In addition, both cell types provide key signaling cues necessary for embryonic development. CRISPR-based epigenome editors are programmable devices that allow for efficient and precise activation (CRISPRa) or repression (CRISPRi) of cell fate-determining factors by modulating endogenous regulatory elements. Here, we present a step-by-step method to implement CRISPRa for controlling cell fate in embryonic stem cells based on our work in generation of CRISPR-programmed mouse embryo models.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-05-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144111193","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
From Single Stem Cells to an In Vitro Model of the Post-implantation Human Embryo: A Step-by-Step Guide. 从单个干细胞到植入后人类胚胎的体外模型：一步一步的指南。
Methods in molecular biology Pub Date : 2025-05-22 DOI: 10.1007/7651_2025_647
Seher Ipek Gassaloglu, Monique Pedroza, Berna Sozen
{"title":"From Single Stem Cells to an In Vitro Model of the Post-implantation Human Embryo: A Step-by-Step Guide.","authors":"Seher Ipek Gassaloglu, Monique Pedroza, Berna Sozen","doi":"10.1007/7651_2025_647","DOIUrl":"https://doi.org/10.1007/7651_2025_647","url":null,"abstract":"<p><p>Recent advances in three-dimensional (3D) modeling of post-implantation human embryos using human pluripotent stem cells (hPSCs) have revolutionized our ability to investigate this crucial yet enigmatic stage of development. Here we detail the generation of the human extra-embryoid (hEE), a 3D stem cell-based embryo model that uniquely captures key spatiotemporal events of peri-gastrulation development through the formation and co-development of post-implantation embryonic and extra-embryonic lineages, with high efficiency and robustness across genetic backgrounds. This chapter provides a detailed protocol for generating hEEs in vitro, including guidance on hPSC maintenance, expected cell morphology, troubleshooting strategies, and key culture techniques.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-05-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144111261","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
Linking Single-Cell Dynamics to Cell Fate in Differentiating hPSCs. 分化造血干细胞中单细胞动力学与细胞命运的联系。
Methods in molecular biology Pub Date : 2025-05-22 DOI: 10.1007/7651_2025_638
Seth Teague, Zhiyuan Yu, Idse Heemskerk
{"title":"Linking Single-Cell Dynamics to Cell Fate in Differentiating hPSCs.","authors":"Seth Teague, Zhiyuan Yu, Idse Heemskerk","doi":"10.1007/7651_2025_638","DOIUrl":"https://doi.org/10.1007/7651_2025_638","url":null,"abstract":"<p><p>Protocols for human pluripotent stem cell differentiation commonly yield a heterogeneous mix of cell types. To understand the source of heterogeneity at the single-cell level, it may be necessary to link final cell state to the cell's history and initial state, for example to determine gene expression or morphogen signaling over the course of differentiation. Here we present methods to quantify and track single cells in time-lapse fluorescence microscopy during stem cell differentiation and link single-cell dynamics to the final resulting state in the same cells.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-05-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144111270","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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