Methods in molecular biology最新文献

A Simplified and Robust Immunofluorescence Labeling Method for Complex 3D Cell Cultures: Minimizing Manipulation and Maximizing Data in Whole-Mount Analysis of Organoids, Spheroids, and Co-culture Models. 复杂3D细胞培养的一种简化和稳健的免疫荧光标记方法：在类器官，球体和共培养模型的全mount分析中，最小化操作和最大化数据。

Methods in molecular biology Pub Date : 2025-06-05 DOI: 10.1007/7651_2025_634

Gamze Demirel, Olgu E Tok, Ranan G Aktas

引用次数: 0

Stem Cell Niche: iPSC-Based Assembloids for Modeling Human Hematopoiesis. 干细胞生态位：基于ipsc的人类造血模型组装体。

Methods in molecular biology Pub Date : 2025-06-04 DOI: 10.1007/7651_2025_629

Madeline J Caduc, Marcelo A S de Toledo, Steffen Koschmieder, Simón Méndez-Ferrer

{"title":"Stem Cell Niche: iPSC-Based Assembloids for Modeling Human Hematopoiesis.","authors":"Madeline J Caduc, Marcelo A S de Toledo, Steffen Koschmieder, Simón Méndez-Ferrer","doi":"10.1007/7651_2025_629","DOIUrl":"https://doi.org/10.1007/7651_2025_629","url":null,"abstract":"The bone marrow (BM) niche is a highly specialized and dynamic microenvironment that tightly regulates hematopoiesis in both health and disease. In this chapter, we present a protocol for generating patient-specific 3D BM-mimicking assembloids, which offer precise control over cellular composition and genetic background. This in vitro platform enables the dissection of mechanisms underlying hematopoietic regulation and BM niche remodeling. We describe, in detail, the stepwise differentiation of induced pluripotent stem cells (iPSCs) into hematopoietic and endothelial lineages, the isolation of human primary mesenchymal stromal cells (MSCs) from femoral heads, and the assembly of BM-mimicking 3D assembloids. Single-cell RNA sequencing of these assembloids identified key myeloid populations and non-hematopoietic lineages such as endothelial cells and various MSC clusters, all crucial for stem cell fate determination and niche maintenance. Furthermore, assembloids harboring the JAK2V617F driver mutation successfully recapitulated key features of myeloproliferative neoplasms, demonstrating the platform's potential for mechanistic studies in human hematopoiesis. This approach provides a powerful tool to model both physiological and neoplastic BM niches, facilitating preclinical research and drug development while potentially reducing reliance on animal models.","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":"144208948","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":"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.","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

Protocols to Assess the Aging of Human Mesenchymal Stem Cells. 评估人类间充质干细胞衰老的方案。

Methods in molecular biology Pub Date : 2025-06-04 DOI: 10.1007/7651_2025_631

Merdiye Mavis, Nedime Serakinci

引用次数: 0

Paraffin Embedding and Histological Staining of Intestinal Organoids. 肠类器官石蜡包埋及组织学染色。

Methods in molecular biology Pub Date : 2025-06-04 DOI: 10.1007/7651_2025_633

Rachel Edens, Renata Rocha do Nascimento, Kristen A Engevik, Amy C Engevik

{"title":"Paraffin Embedding and Histological Staining of Intestinal Organoids.","authors":"Rachel Edens, Renata Rocha do Nascimento, Kristen A Engevik, Amy C Engevik","doi":"10.1007/7651_2025_633","DOIUrl":"https://doi.org/10.1007/7651_2025_633","url":null,"abstract":"Organoids have emerged as a powerful in vitro model system for biomedical research, providing a physiologically relevant alternative to traditional cell lines. Intestinal organoids recapitulate the complex cellular composition and function of the intestinal epithelium, making them valuable for studying intestinal biology and disease. These three-dimensional (3D) cultures can be differentiated to contain all the major intestinal cell types-including enterocytes, Paneth cells, goblet cells, stem cells, enteroendocrine cells, and tuft cells-allowing for more accurate modeling of intestinal function. However, their 3D structure presents challenges for high-resolution imaging and histological analysis. Common methods for embedding intestinal organoids, such as frozen sectioning or pre-embedding in semi-solid gels, can compromise morphology and sectioning integrity. To address these limitations, we present an optimized paraffin-embedding protocol that provides robust immunofluorescent staining and imaging of intestinal organoids while preserving cellular architecture. This approach provides researchers with an improved tool for analyzing organoid-based models of intestinal function and disease.","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":"144208946","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

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

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

引用次数: 0

Isolation and Culturing of Newborn Rat Nasal Chondrocytes. 新生大鼠鼻腔软骨细胞的分离与培养。

Methods in molecular biology Pub Date : 2025-05-22 DOI: 10.1007/7651_2025_632

Ekaterina I Smirnova, Victoria A Shestakova, Elena V Isaeva, Anastas A Kisel, Elena M Yatsenko, Peter V Shegay, Andrey D Kaprin, Ilya D Klabukov, Denis S Baranovskii

引用次数: 0

Microwells as Minimalistic Niches to Study Heterotypic Interactions of Stromal and Hematopoietic Stem Cells. 微孔作为研究基质干细胞和造血干细胞异型相互作用的极简龛。

Methods in molecular biology Pub Date : 2025-05-22 DOI: 10.1007/7651_2025_628

Adrian Candelas, Thomas Bessy, Benoit Vianay, Manuel Théry, Stephane Brunet

{"title":"Microwells as Minimalistic Niches to Study Heterotypic Interactions of Stromal and Hematopoietic Stem Cells.","authors":"Adrian Candelas, Thomas Bessy, Benoit Vianay, Manuel Théry, Stephane Brunet","doi":"10.1007/7651_2025_628","DOIUrl":"https://doi.org/10.1007/7651_2025_628","url":null,"abstract":"Hematopoietic stem and progenitor cells (HSPCs) can migrate and reside within the bone marrow in distinct microenvironments or niches. The niches organize around specific stromal cells, such as endothelial cells at the capillary or sinusoid walls, and osteoblasts along the bone matrix. Within each niche, a specific combination of external cues, including secreted and diffusible factors, cell-matrix, and cell-cell interactions, controls HSPCs behavior and fate. Deciphering the interplay between HSPCs and stromal cells of the niches is challenging: in vivo, it is hindered by the opacity of the bone matrix; in vitro, classical co-culture models only poorly recapitulate essential features of the physiological niches. The difficulty is moreover amplified by the exceptional migration capacity of HSPCs.In this chapter, we present a method to overcome these limitations by producing arrays of microwells designed to mimic bone marrow niches in a functional manner. These \"microniches\" promote a long-term interaction between the HSPC and a stromal cell of interest. We describe their microfabrication based on a maskless photolithography method allowing the production of arrays of microwells with reproducible volume and geometry, and the iterative improvement of the geometric design of the wells. We describe the loading and culture of stromal cells with HSPCs. We discuss the potentiality of microwells, in basic and applied research, as a platform to investigate molecular mechanisms involved in direct cell-cell interactions and local effects of diffusible factors, for any adherent and non-adherent cells of interest.","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":"144111314","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