Methods in molecular biology最新文献

Tracking of Transplanted Stem Cells in Mouse Models Using Practical Fluorescence-Based Protocols. 使用实用的基于荧光的方案跟踪小鼠模型中的移植干细胞。

Methods in molecular biology Pub Date : 2026-05-08 DOI: 10.1007/7651_2026_704

Riho Kanai, Arvind Hariharan, Janaki Iyer, Younan Liu, Takashi I, Yoshinori Sumita, Simon D Tran

引用次数: 0

A Review on Nanotoxicology in Stem Cell Research: Challenges and Safety Assessments. 纳米毒理学在干细胞研究中的进展：挑战和安全性评估。

Methods in molecular biology Pub Date : 2026-05-08 DOI: 10.1007/7651_2026_705

Gözde Özsezer, Berk Deniz Tosunoğlu, Yavuz Emre Arslan

{"title":"A Review on Nanotoxicology in Stem Cell Research: Challenges and Safety Assessments.","authors":"Gözde Özsezer, Berk Deniz Tosunoğlu, Yavuz Emre Arslan","doi":"10.1007/7651_2026_705","DOIUrl":"https://doi.org/10.1007/7651_2026_705","url":null,"abstract":"This review presents a balanced synthesis of the efficacy-nanotoxicity window of nanomaterials-including carbon-based structures, metallic nanoparticles, and bioceramic composites-within the context of stem cell-assisted tissue engineering. It emphasizes that increases in conventional differentiation markers may not necessarily correspond to functional maturity or long-term safety. Therefore, a gradual shift from standard viability-based assays toward lineage-specific functional assessments is discussed. The nanotoxicological profile of these materials is examined across major tissue types, highlighting how similar physicochemical properties may yield beneficial or adverse outcomes depending on concentration, exposure time, and microenvironmental conditions. In bone tissue, the review considers the point at which enhanced mineralization may be accompanied by oxidative stress and mitochondrial strain. For cartilage, potential concerns related to ion and degradation product accumulation in avascular environments are addressed, including the possible induction of inflammatory signaling and hypertrophic markers such as collagen type X. In neural and cardiac applications, efforts to improve electrical conductivity are evaluated alongside potential electrophysiological alterations, including Ca2+ imbalance and rhythm disturbances at sub-cytotoxic levels. Vascular differentiation is discussed within the context of pro-angiogenic signaling and the risk of endothelial dysfunction. By comparing experimental systems ranging from 2D cultures to induced pluripotent stem cell (iPSC)-derived organoids, the review underscores the model-dependent and tissue-specific nature of nanotoxic responses. Overall, it outlines considerations for integrating physicochemical characterization with functional and metabolic endpoints to support more reliable evaluation of nanobiomaterials in regenerative medicine.","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2026-05-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147840021","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

Isolation of Keratinocytes from Human Hair Follicles and Selection of a Population Enriched in Epithelial Stem and Progenitor Cells by Flow Cytometry. 人毛囊角质形成细胞的分离及上皮干细胞和祖细胞富集群体的流式细胞术筛选。

Methods in molecular biology Pub Date : 2026-05-08 DOI: 10.1007/7651_2026_703

Theebah Sellathurai, Frédéric Auvré, Fabienne Bloas, Stéphane Commo, Gilles Lemaître, Nicolas O Fortunel

{"title":"Isolation of Keratinocytes from Human Hair Follicles and Selection of a Population Enriched in Epithelial Stem and Progenitor Cells by Flow Cytometry.","authors":"Theebah Sellathurai, Frédéric Auvré, Fabienne Bloas, Stéphane Commo, Gilles Lemaître, Nicolas O Fortunel","doi":"10.1007/7651_2026_703","DOIUrl":"https://doi.org/10.1007/7651_2026_703","url":null,"abstract":"The human hair follicle (HF) harbors distinct populations of epithelial stem and progenitor cells that are essential for hair homeostasis and epidermal wound healing. While the follicular niche, the anatomical equivalent of the murine bulge located at the insertion site of the arrector pili muscle, has historically been the primary target for the isolation of hair follicle stem cells (HFSCs), capturing the full epithelial stem and progenitor hierarchy within the outer root sheath (ORS) is critical for a comprehensive understanding of human hair follicle biology. As integrin α6 (CD49f) is a well-established surface marker associated with basal epithelial stem and progenitor states, and long-term proliferative potential of immature keratinocytes, we have exploited this marker to isolate an epithelial cell population corresponding to the keratinocyte basal layer of the ORS. This chapter describes a protocol for the enrichment of stem and progenitor cells from intact human hair follicles, combining manual microdissection of the entire hair follicle, enzymatic dissociation, and flow cytometry sorting based on CD49f expression.Functional validation using in vitro proliferation and clone-forming assays showed that the CD49fhigh fraction exhibits significantly greater proliferative and clonogenic capacity, compared to the ORS total and CD49flow populations. This method preserved cell viability and phenotypic integrity. Consequently, the isolated keratinocyte populations are suitable for a broad range of downstream uses and applications, including transcriptomic analyses such as single-cell RNA sequencing (scRNA-seq), functional assays, and regenerative studies. Altogether, this workflow provides a robust platform for future approaches dedicated to the treatment of alopecia, promotion of hair longevity, and for tissue engineering applications.","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2026-05-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147840055","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

Fabrication and Biological Evaluation of 3D Bioprinted GelMA Scaffolds with Bone Marrow-Derived Mesenchymal Stem Cells for Osteochondral Tissue Engineering. 骨软骨组织工程用骨髓间充质干细胞生物3D打印GelMA支架的制备及生物学评价

Methods in molecular biology Pub Date : 2026-05-08 DOI: 10.1007/7651_2026_707

Reyhan Yanikoglu, Elif Balikci, Ayse Ceren Calikoglu-Koyuncu

{"title":"Fabrication and Biological Evaluation of 3D Bioprinted GelMA Scaffolds with Bone Marrow-Derived Mesenchymal Stem Cells for Osteochondral Tissue Engineering.","authors":"Reyhan Yanikoglu, Elif Balikci, Ayse Ceren Calikoglu-Koyuncu","doi":"10.1007/7651_2026_707","DOIUrl":"https://doi.org/10.1007/7651_2026_707","url":null,"abstract":"Repairing osteochondral defects remains a significant therapeutic challenge due to the complex hierarchical structure of the tissue, while existing treatment strategies are largely palliative rather than curative. For osteochondral tissue production, three-dimensional (3D) bioprinting has become a potent technique to produce biomimetic scaffolds with regulated architecture and cell distribution. This chapter provides a comprehensive and repeatable procedure for the production and biological assessment of 3D bioprinted gelatin methacryloyl (GelMA) scaffolds that incorporate mesenchymal stem cells (MSCs) obtained from bone marrow. The methodology outlines the techniques for photo-crosslinking, extrusion-based bioprinting, MSC expansion and encapsulation, and bioink preparation in order to guarantee cell viability and structural integrity. Comprehensive in vitro evaluation techniques are also described, such as differentiation evaluation related to osteogenic and chondrogenic commitment, cell viability, and proliferation analysis. This methodical approach offers researchers a useful foundation for creating MSC-laden GelMA constructs appropriate for use in osteochondral tissue engineering and additional translational studies.","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2026-05-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147840064","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

Sequential DLP Bioprinting of Dual-Layered Brain Organoid-Like Neural Microtissues Using Nanocomposite Bioinks. 利用纳米复合生物墨水连续DLP生物打印双层脑类器官样神经微组织。

Methods in molecular biology Pub Date : 2026-05-08 DOI: 10.1007/7651_2026_706

Mehmet Bozdag, Zehra Kanli, Oguzhan Gunduz, Sumeyye Cesur

{"title":"Sequential DLP Bioprinting of Dual-Layered Brain Organoid-Like Neural Microtissues Using Nanocomposite Bioinks.","authors":"Mehmet Bozdag, Zehra Kanli, Oguzhan Gunduz, Sumeyye Cesur","doi":"10.1007/7651_2026_706","DOIUrl":"https://doi.org/10.1007/7651_2026_706","url":null,"abstract":"This chapter provides a detailed, step-by-step protocol for the fabrication of architecturally defined brain organoid-like neural microtissues using a sequential digital light processing (DLP) bioprinting strategy. The method relies exclusively on DLP technology to fabricate complex heterogeneous structures through a layer-by-layer vat-switching technique. Central to this protocol is the formulation of a nanocomposite bioink, where reduced graphene oxide (rGO) nanoparticles and bacterial cellulose are mixed with gelatin methacryloyl (GelMA) matrix. This composition provides enhanced electrical conductivity, printability, and mechanical stability, promoting neural network maturation. We describe the complete workflow from induced pluripotent stem cell (iPSC) culture and neural induction, through bioink preparation and DLP printing parameter optimization, to long-term static culture and functional validation. This protocol addresses key limitations of conventional self-assembly methods, offering improved reproducibility, structural control, and physiological relevance for modeling neurodevelopment and disease.","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2026-05-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147839856","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 Induced Pluripotent Stem Cells from Somatic Cells via Solid Lipid Nanoparticle-Mediated Plasmid DNA Delivery. 通过固体脂质纳米颗粒介导的质粒DNA传递从体细胞诱导多能干细胞。

Methods in molecular biology Pub Date : 2026-05-08 DOI: 10.1007/7651_2026_708

Meryem Akkurt Yildirim, Hanife Sevgi Varli, Cem Bülent Üstündağ

{"title":"Generation of Induced Pluripotent Stem Cells from Somatic Cells via Solid Lipid Nanoparticle-Mediated Plasmid DNA Delivery.","authors":"Meryem Akkurt Yildirim, Hanife Sevgi Varli, Cem Bülent Üstündağ","doi":"10.1007/7651_2026_708","DOIUrl":"https://doi.org/10.1007/7651_2026_708","url":null,"abstract":"Induced pluripotent stem cells (iPSCs) have enabled significant advances in regenerative medicine; however, reprogramming methods using viral vectors pose risks to safety and genomic integrity in clinical applications. This protocol details a non-viral, integration-free approach for delivering plasmid DNA (pDNA) into somatic cells via solid lipid nanoparticles (SLNs). SLNs protect pDNA from enzymatic degradation and ensure high transfection efficiency in cells, thereby preventing permanent genetic alterations in the host genome.The protocol provides step-by-step guidance for the preparation of human fibroblasts, the sequential transcription cycles of SLN-pDNA encoding OSKM factors, and the induction and isolation of iPSC colonies. Furthermore, comprehensive characterization methods are described to confirm pluripotency, including monitoring GFP expression levels, quantitative real-time PCR, immunofluorescence staining, flow cytometry, alkaline phosphatase activity assays, and in vitro tri-lineage differentiation.iPSCs obtained while preserving genomic integrity can be safely used in advanced regenerative medicine applications, such as disease modeling, high-throughput drug screening, and personalized cell therapies. This approach enhances safety and efficacy in clinical settings and provides a valuable platform for the development of iPSC-based therapeutic and research applications.","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2026-05-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147840140","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

Live-Cell Imaging of Fish Epidermal Keratocytes. 鱼类表皮角质细胞的活细胞成像。

Methods in molecular biology Pub Date : 2026-05-01 DOI: 10.1007/7651_2026_702

Chika Okimura, Yoshiaki Iwadate

引用次数: 0

Isolation and Culture of Primary Keratinocytes from Human Skin Grafts. 人皮肤移植原代角质形成细胞的分离与培养。

Methods in molecular biology Pub Date : 2026-04-29 DOI: 10.1007/7651_2026_701

Sarit Pal, Xizhao Chen, Gopika Ashokan, Annica R Stull-Lane, Jinyeon Shin, Geoffrey E Hespe, Babak J Mehrara, Raghu P Kataru

{"title":"Isolation and Culture of Primary Keratinocytes from Human Skin Grafts.","authors":"Sarit Pal, Xizhao Chen, Gopika Ashokan, Annica R Stull-Lane, Jinyeon Shin, Geoffrey E Hespe, Babak J Mehrara, Raghu P Kataru","doi":"10.1007/7651_2026_701","DOIUrl":"https://doi.org/10.1007/7651_2026_701","url":null,"abstract":"The skin, the largest organ of the human body, performs essential physiological functions including barrier protection, thermal regulation, and mechano-sensation. Despite its structural consistency, it exhibits marked regional heterogeneity, particularly under pathological conditions. Our recent investigations have revealed that in secondary lymphedema, the affected skin undergoes distinct pathomorphological changes, including hyperkeratosis, spongiosis, and altered keratinocyte polarity within the epidermis. These findings have prompted deeper studies into the molecular and epigenetic mechanisms contributing to the irreversible progression of this disease. This protocol describes a detailed, reproducible method for isolating and culturing primary keratinocytes from both full-thickness and split-thickness skin grafts obtained from lymphedematous regions. The workflow comprises tissue processing, enzymatic dissociation, selective enrichment of keratinocytes, and optimized culture conditions for downstream molecular analyses. Validation of isolated keratinocytes is performed using Western blotting, flow cytometry, and immunofluorescence histology. This approach preserves native phenotypic traits and epigenetic signatures in patient-derived keratinocytes, thereby enhancing the translational relevance of in vitro studies investigating the pathophysiology of secondary lymphedema.","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2026-04-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147775856","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

Modeling the Glioblastoma Microenvironment Using Stem Cell-Based 3D Organoid Platforms. 基于干细胞的三维类器官平台建模胶质母细胞瘤微环境。

Methods in molecular biology Pub Date : 2026-04-25 DOI: 10.1007/7651_2026_699

Varma Bhumika, Bandbe Tanmay, Pal Saptaparna, Mhatre Sejal, Sandbhor Puja

{"title":"Modeling the Glioblastoma Microenvironment Using Stem Cell-Based 3D Organoid Platforms.","authors":"Varma Bhumika, Bandbe Tanmay, Pal Saptaparna, Mhatre Sejal, Sandbhor Puja","doi":"10.1007/7651_2026_699","DOIUrl":"https://doi.org/10.1007/7651_2026_699","url":null,"abstract":"Glioblastoma multiforme (GBM) is sustained by glioma stem cells (GSCs), a self-renewing tumor-initiating population that contributes to cellular heterogeneity, therapeutic resistance, and disease recurrence. Reproducing the complex tumor-microenvironment interaction of GBM in vitro remains a significant challenge. Stem cell-based three-dimensional (3D) organoid systems have emerged as physiologically relevant models that recapitulate tumor architecture, stemness, and dynamic cellular interactions compared with conventional two-dimensional cultures. In this chapter, readers are provided with standardized and reproducible methodological frameworks to generate, maintain, and validate GBM microenvironment-relevant 3D organoid models. By following the step-by-step protocols described, readers will be able to establish GBM organoids from single-cell suspension and microtumor fragments, while preserving tumor-initiating capacity and intratumoral heterogeneity. The chapter further enables readers to integrate neurospheres into cerebral organoid systems to model tumor-neural interactions within a tissue relevant context and to incorporate extracellular matrix (ECM)-informed strategies, including decellularized brain ECM (BdECM), to mimic brain-specific biochemical/mechanical cues. Readers will also be able to perform long-term (up to 60 days) cortical organoid culture with defined quality control (QC) and validation, including evaluation of structural organization, stemness markers expression, differentiation status, and reproducibility across batches. The chapter further provides practical problem-solving guidance to address common technical challenges during organoid establishment and maintenance. Collectively, these protocols offer a resilient, durable, and versatile toolkit to model GBM biology, interrogate tumor-microenvironment interactions, and evaluate therapeutic efficacy using clinically relevant 3D organoid systems.","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2026-04-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147775858","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

Modeling Somitogenesis and Axial Development In Vitro with Axioloids. 用轴状体模拟体外体细胞发生和轴向发育。

Methods in molecular biology Pub Date : 2026-04-23 DOI: 10.1007/7651_2026_697

Keun-Tae Kim, Sofiane Hamidi, Cantas Alev

{"title":"Modeling Somitogenesis and Axial Development In Vitro with Axioloids.","authors":"Keun-Tae Kim, Sofiane Hamidi, Cantas Alev","doi":"10.1007/7651_2026_697","DOIUrl":"https://doi.org/10.1007/7651_2026_697","url":null,"abstract":"Axioloids are three-dimensional (3D) structures derived from pluripotent stem cells (PSCs) that model key aspects of human somitogenesis and early axis development in vitro. These mesoderm-based aggregates recapitulate essential morphogenetic features of the segmentation process, including axial elongation, sequential formation of epithelial somites with proper rostrocaudal patterning, and oscillatory activity of the segmentation clock. They further reproduce the spatiotemporal organization characteristic of early axial development, such as opposing FGF/WNT and retinoic acid signaling gradients and anteroposterior HOX gene expression patterns similar to those observed in vivo. Together, these attributes highlight axioloids as a robust and versatile platform for studying human axial development and congenital disorders of the spine and axial skeleton. In this chapter, we provide a concise, step-by-step protocol for generating axioloids, along with key developmental readouts and troubleshooting strategies to address common problems and issues encountered. This protocol provides a reproducible framework for producing and characterizing axioloids as an in vitro model of human axial development and disease.","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2026-04-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147775905","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