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Isolation, Transplantation, and Long-Term Noninvasive Tracking of DiD-Labeled EpCAM+ Human Fetal Hepatic Progenitors in Mouse Livers. 小鼠肝脏中did标记EpCAM+人胎肝祖细胞的分离、移植和长期无创追踪。
Methods in molecular biology Pub Date : 2025-07-22 DOI: 10.1007/7651_2025_658
Chaturvedula Tripura, Sandeep Kumar Vishwakarma, Srinivas Gunda
{"title":"Isolation, Transplantation, and Long-Term Noninvasive Tracking of DiD-Labeled EpCAM+ Human Fetal Hepatic Progenitors in Mouse Livers.","authors":"Chaturvedula Tripura, Sandeep Kumar Vishwakarma, Srinivas Gunda","doi":"10.1007/7651_2025_658","DOIUrl":"https://doi.org/10.1007/7651_2025_658","url":null,"abstract":"<p><p>Chronic liver disease (CLD) is a progressive condition characterized by the deterioration of liver structure and function, resulting from persistent injury and inflammation. Liver cell therapy has emerged as a promising alternative bridging strategy for patients waiting for the availability of a suitable donor liver for transplantation. Fetal human hepatic progenitor cells (fHPCs) hold great potential as a source for liver regeneration and restoration of liver function in individuals with CLD. A key challenge in liver cell therapy lies in the ability to effectively track transplanted donor cells, monitoring their homing, repopulation, and functional integration into the recipient's liver.This protocol outlines a comprehensive methodology for isolating fHPCs, enrichment of EpCAM positive cells, and labeling them with DiD dye. It also details the procedure for inducing liver fibrosis in SCID mice, transplanting the donor fHPCs, and conducting noninvasive, long-term imaging to track the transplanted cells in recipient SCID mice. Furthermore, we outline a thorough approach to confirm the presence and functional integration of the transplanted cells within the recipient livers.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-07-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144675259","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
A Deep Learning Approach to Assessing Cell Identity in Stem Cell-Based Embryo Models. 基于干细胞的胚胎模型中评估细胞身份的深度学习方法。
Methods in molecular biology Pub Date : 2025-07-22 DOI: 10.1007/7651_2025_654
Nazmus Salehin, Martin Proks, Joshua M Brickman
{"title":"A Deep Learning Approach to Assessing Cell Identity in Stem Cell-Based Embryo Models.","authors":"Nazmus Salehin, Martin Proks, Joshua M Brickman","doi":"10.1007/7651_2025_654","DOIUrl":"https://doi.org/10.1007/7651_2025_654","url":null,"abstract":"<p><p>Since the generation of embryoid bodies from embryonic stem cells (ESCs), three-dimensional differentiation has been used to mimic developmental processes. To what extent do these in vitro cell types reflect the cells generated by the embryo? We used deep learning (DL) to develop an integrated model of early human development leveraging existing single-cell RNA-seq (scRNA-seq) and using scvi-tools to both integrate and classify cell types. This tool can interrogate in vitro cell types and assign them both identity and provide an entropy score for the reliability of this classification. In this protocol we explain how to use state-of-the-art tools and our associated, publicly available DL models for early embryonic development to explore phenotypes and cell types derived in vitro. Our tools represent an important new resource to interrogate stem cell-based embryo models and the fidelity with which they recapitulate development.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-07-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144675257","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
Computational Approaches for Delineating Lysosomes and Related Intracellular Trafficking Vesicles in Confocal and Other Fluorescence Datasets. 共聚焦和其他荧光数据集中描述溶酶体和相关细胞内运输囊泡的计算方法。
Methods in molecular biology Pub Date : 2025-07-22 DOI: 10.1007/7651_2025_657
Charles Ellis, David S Chatelet, J Arjuna Ratnayaka
{"title":"Computational Approaches for Delineating Lysosomes and Related Intracellular Trafficking Vesicles in Confocal and Other Fluorescence Datasets.","authors":"Charles Ellis, David S Chatelet, J Arjuna Ratnayaka","doi":"10.1007/7651_2025_657","DOIUrl":"https://doi.org/10.1007/7651_2025_657","url":null,"abstract":"<p><p>Fluorescence datasets from investigations into intracellular trafficking compartments produce images of variable quality, scales, and complexities. Investigators are therefore confronted with a choice of how to analyze this information. Here, we have used confocal immunofluorescence images of lysosomes from retinal pigment epithelial cells as an exemplar dataset, and employed three freely accessible computational approaches (Fiji, CellProfiler and Icy) to showcase their workings. A step-by-step workflow for each pipeline is described with non-specialist users in mind. These produce results including lysosomal number and shape, but also 3D outputs such as volume. Features of the three methods alongside their advantages and limitations are subsequently summarized. An important consideration, however, is that results generated from the different approaches are not necessarily comparable. Hence, users should adopt only a single method to analyze their dataset which best suit their specific requirements.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-07-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144675258","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
Electrospun Poly(vinyl alcohol)/Silver Nitrate (PVA/AgNO₃) Nanofibers Incorporated with Mesenchymal Stem Cells for Wound Dressing Applications. 电纺丝聚（乙烯醇）/硝酸银（PVA/AgNO₃）纳米纤维与间充质干细胞结合用于伤口敷料应用。
Methods in molecular biology Pub Date : 2025-07-17 DOI: 10.1007/7651_2025_659
Aysegul Tiryaki, Ayse Ceren Calikoglu-Koyuncu
{"title":"Electrospun Poly(vinyl alcohol)/Silver Nitrate (PVA/AgNO₃) Nanofibers Incorporated with Mesenchymal Stem Cells for Wound Dressing Applications.","authors":"Aysegul Tiryaki, Ayse Ceren Calikoglu-Koyuncu","doi":"10.1007/7651_2025_659","DOIUrl":"https://doi.org/10.1007/7651_2025_659","url":null,"abstract":"<p><p>This chapter presents a novel approach for developing antibacterial wound dressings by electrospinning a composite of polyvinyl alcohol (PVA) and silver nitrate (AgNO₃). The three dimensional (3D) wound dressing combines the biocompatibility and favorable mechanical properties of PVA with the antibacterial properties of silver ions. The electrospinning process provides structural integrity and controlled release of silver ions to prevent bacterial infection. Mesenchymal stem cells (MSCs) are used to assess biocompatibility of electrospun 3D PVA/AgNO<sub>3</sub> nanofiber scaffolds for tissue engineering applications.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-07-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144642931","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 the Nonintegrated Human Bilaminar Embryo Model (Bilaminoid) from Naïve Pluripotent Stem Cells. 从Naïve多能干细胞生成非整合人双层胚胎模型（Bilaminoid）。
Methods in molecular biology Pub Date : 2025-07-15 DOI: 10.1007/7651_2025_652
Takumi Okubo, Yasuhiro Takashima
{"title":"Generation of the Nonintegrated Human Bilaminar Embryo Model (Bilaminoid) from Naïve Pluripotent Stem Cells.","authors":"Takumi Okubo, Yasuhiro Takashima","doi":"10.1007/7651_2025_652","DOIUrl":"https://doi.org/10.1007/7651_2025_652","url":null,"abstract":"<p><p>Human embryogenesis has been problematic to study due to technical and ethical issues. Recently, a human embryo model generated from pluripotent stem cells (PSCs) to mimic human embryogenesis, which has attracted attention as an invaluable tool for studying human embryonic development.We have successfully developed a method to efficiently induce the differentiation of naïve human PSCs, which correspond to preimplantation epiblasts, into extraembryonic cells of the blastocyst. Furthermore, by combining these cells, we developed a novel nonintegrated human embryo model called \"bilaminoid\" that reproduces development from preimplantation to peri-gastrulation stages. Here, we describe a detailed protocol for the bilaminoid formation.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-07-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144626695","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
Extended In Vitro Culture of Human Embryos. 人类胚胎体外扩展培养。
Methods in molecular biology Pub Date : 2025-07-15 DOI: 10.1007/7651_2025_641
Sylwia M Czukiewska, Felicitas Azpiroz, Susana M Chuva de Sousa Lopes, Mina Popovic
{"title":"Extended In Vitro Culture of Human Embryos.","authors":"Sylwia M Czukiewska, Felicitas Azpiroz, Susana M Chuva de Sousa Lopes, Mina Popovic","doi":"10.1007/7651_2025_641","DOIUrl":"https://doi.org/10.1007/7651_2025_641","url":null,"abstract":"<p><p>Recent innovations in extended in vitro culture (IVC) systems have revolutionized our understanding of human peri-implantation development. Building on foundational animal studies, refined protocols now support human embryo culture beyond the blastocyst stage, providing unprecedented access to previously elusive developmental events. These systems have yielded critical insights into early morphogenetic processes, lineage specification, and tissue organization, significantly advancing developmental biology. Here, we provide our current protocol for the extended culture of human embryos, followed by immunofluorescence for lineage markers of interest. Unveiling human peri-implantation development also promises to improve reproductive medicine, potentially addressing challenges related to implantation failure, chromosomal instability in embryos, as well as congenital disorders. Insights gained from this research may pave way for novel therapeutic approaches and advancements in medically assisted reproduction.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-07-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144626694","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
Microfluidic Device Manufacturing by Light-Based 3D Printing for Organoid Vascularization. 基于光基3D打印的类器官血管化微流控装置制造。
Methods in molecular biology Pub Date : 2025-07-11 DOI: 10.1007/7651_2025_639
Rochelle Aubry, Idris Salmon, Adrian Ranga
{"title":"Microfluidic Device Manufacturing by Light-Based 3D Printing for Organoid Vascularization.","authors":"Rochelle Aubry, Idris Salmon, Adrian Ranga","doi":"10.1007/7651_2025_639","DOIUrl":"https://doi.org/10.1007/7651_2025_639","url":null,"abstract":"<p><p>3D printing by light-based vat polymerization enables the manufacturing of a variety of microfluidic devices which can be used to study growth, patterning, vascularization, and tissue interactions of stem cell-derived spheroids, organoids, and tissue explants. This technology allows to design and manufacture compartmentalized devices for precise seeding of cells and organoids, combined with the possibility to generate controlled media flow. Here, we detail the steps involved in the fabrication of such microfluidic devices, including the printing and post-processing stages using light-based 3D printing. We also give an example of how such a 3D printed microfluidic device can be used to culture and vascularize cerebral organoids. The use of 3D printing provides a rapid and inexpensive way to generate microfluidic devices without the need for cleanroom facilities and is therefore a technology accessible to every life science research lab. In addition, this high throughput method facilitates organoid studies in a more controlled environment, thereby representing a significant advancement in reproducibility for organoid research.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-07-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144600938","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
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
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