Methods in molecular biology最新文献_第3页

Protocol Development for CRISPR/Cas9 Knockout of the Anti-inflammatory Protein TNIP1 in HaCaT Keratinocytes.

Methods in molecular biology Pub Date : 2025-03-20 DOI: 10.1007/7651_2025_616

Liam E Carman, Michael L Samulevich, Brian J Aneskievich

{"title":"Protocol Development for CRISPR/Cas9 Knockout of the Anti-inflammatory Protein TNIP1 in HaCaT Keratinocytes.","authors":"Liam E Carman, Michael L Samulevich, Brian J Aneskievich","doi":"10.1007/7651_2025_616","DOIUrl":"https://doi.org/10.1007/7651_2025_616","url":null,"abstract":"Gene editing in cultured cells, including the intent of sequence disruption to achieve a functional knockout of the targeted gene, has been greatly facilitated with the advent of Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9) technology. Primary cell strains and immortalized cell lines from diverse tissue types have been successfully targeted both for basic research examining the effects of loss of the correlating protein and for modeling select loss-of-function disorders. Such accomplishments have extended to cutaneous cells, especially epidermal keratinocytes given their key structural and functional role in barrier formation and surveillance of and response to surface events such as triggering and processing inflammatory responses. Here we describe disruption of the Tumor Necrosis factor-induced protein 3-Interacting Protein 1 (TNIP1) gene in human HaCaT keratinocytes to generate an ongoing loss of expression as a parallel system to transient knockdown we had previously achieved with siRNA transfection. The TNIP1 protein restricts cytoplasmic progression of inflammatory signals. We cover our CRISPR/Cas9 vector choice, enrichment of transfected cells via positive selection for puromycin resistance, their subsequent cloning, and gene disruption and expression analysis. We also emphasize prior keratinocyte-CRISPR/Cas9 literature as a springboard for other investigators and to illustrate the widespread relevance of such editing to the diverse, yet highly consequentially different, genes expressed in keratinocytes.","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-03-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143663738","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 Functional Brain Region-Specific Neural Spheroids for High Throughput Screening.

Methods in molecular biology Pub Date : 2025-02-19 DOI: 10.1007/7651_2024_593

Jiajing Zhang, Angelica Medina, Marc Ferrer, Emily M Lee

{"title":"Generation of Functional Brain Region-Specific Neural Spheroids for High Throughput Screening.","authors":"Jiajing Zhang, Angelica Medina, Marc Ferrer, Emily M Lee","doi":"10.1007/7651_2024_593","DOIUrl":"https://doi.org/10.1007/7651_2024_593","url":null,"abstract":"Therapeutic development and research in the neurodegenerative disease field encounters many challenges such as availability of reproducible and scalable cellular model systems that are biologically, physiologically, and pharmacologically relevant. These cellular models must be informative of cellular mechanisms of diseases and predictive for therapeutics efficacy and toxicity testing during drug discovery and development. Neural spheroids fill the gap of cellular models of the brain that are functional, versatile in neural cell type composition, robust, and scalable for high-throughput screening (HTS). We have previously developed a protocol to aggregate pre-determined ratios of differentiated human-induced pluripotent stem cell (hiPSC)-derived neurons and astrocytes in a scaffold-free environment to form 3D brain-region specific spheroids. By mixing different neuronal types, neural spheroids can be used to simulate the neuronal-type heterogeneity of distinct brain regions in vivo, including the prefrontal cortex (PFC) and ventral tegmental area (VTA). Here, we present a detailed description of a method for generating functional brain region-specific spheroids with HTS-compatible assay readout that monitors changes in neural network activity by measuring calcium oscillations. The versatility of the platform is such that these neural spheroids cellular assays are applicable for a wide range of disease modeling, compound validation, and screening and are limited only by the availability of input cells, including neural subtype, disease cells, and immune cells such as microglia.","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-02-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143441335","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

Transfer of Mitochondria from Healthy Stem Cells to Injured Cells in Stroke with Retinal Impairments.

Methods in molecular biology Pub Date : 2025-02-11 DOI: 10.1007/7651_2024_599

Napasiri Putthanbut, Jea-Young Lee, Cesario V Borlongan

{"title":"Transfer of Mitochondria from Healthy Stem Cells to Injured Cells in Stroke with Retinal Impairments.","authors":"Napasiri Putthanbut, Jea-Young Lee, Cesario V Borlongan","doi":"10.1007/7651_2024_599","DOIUrl":"https://doi.org/10.1007/7651_2024_599","url":null,"abstract":"Stroke is the second leading cause of mortality worldwide, with retinal ischemia as its prominent complication. However, the pathology of retinal ischemia has not been fully elucidated, resulting in a lack of effective treatment. Stem cell therapy has been suggested to be therapeutic in retinal ischemia, with mitochondrial transfer potentially one of the underlying mechanisms. To investigate the mitochondrial function in retinal ischemia and the potential of mitochondrial transfer from mesenchymal stem cells (MSCs), in vivo middle cerebral artery occlusion (MCAO) model and in vitro oxygen-glucose deprivation (OGD) model were utilized in combination. In vivo, rats subjected to MCAO were randomly administered intravenous MSCs or vehicles. Laser doppler was used to measure the blood flow in the brain and the eye, along with immunohistochemical staining for assessing cellular degeneration. In vitro, retinal pigment epithelium (RPE) cells exposed to OGD were cocultured with or without MSCs. Mitochondrial function was measured by mitochondrial respiration, mitochondrial network analysis, mitochondria live cell imaging, and immunocytochemistry. The results demonstrated improved cell survival and restored mitochondrial function following MSC therapy. This chapter details the protocols necessary to produce the in vivo and in vitro models of ischemic stroke along with an assessment of mitochondrial function. Elucidating the mechanisms of mitochondrial transfer will further the knowledge in regenerative medicine and may enable new targets of therapeutics for stroke, especially for retinal ischemia.","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-02-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143391184","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 Retinal Organoids Using Human-Induced Pluripotent Stem Cells.

Methods in molecular biology Pub Date : 2025-02-11 DOI: 10.1007/7651_2024_600

Keith Theodore, Joel Alan Imventarza, Saleha Tahir, Bruna Lopes da Costa, Peter M J Quinn

引用次数: 0

Generation and Characterization of a New Aging Skin Human Dermal Extracellular Matrix Scaffold. 一种新型老化皮肤人真皮细胞外基质支架的制备与表征。

Methods in molecular biology Pub Date : 2025-01-18 DOI: 10.1007/7651_2024_579

Estibaliz Fernández-Carro, Jesús Ciriza

引用次数: 0

A Protocol for Detecting DNA Methylation Changes at CpG Sites of Stemness-Related Genes in Aging Stem Cells. 检测衰老干细胞中干细胞相关基因CpG位点DNA甲基化变化的方案。

Methods in molecular biology Pub Date : 2025-01-17 DOI: 10.1007/7651_2024_590

Mahshid Hodjat, Seyed Mojtaba Daghighi, Mohammad Abdollahi

引用次数: 0

Reproducible, Scale-Up Production of Human Brain Organoids (HBOs) on a Pillar Plate Platform via Spheroid Transfer. 通过球体转移在柱板平台上可重复的，大规模生产人脑类器官（HBOs）。

Methods in molecular biology Pub Date : 2025-01-17 DOI: 10.1007/7651_2024_604

Pranav Joshi, Prabha Acharya, Mona Zolfaghar, Manav Goud Vanga, Sunil Shrestha, Moo-Yeal Lee

{"title":"Reproducible, Scale-Up Production of Human Brain Organoids (HBOs) on a Pillar Plate Platform via Spheroid Transfer.","authors":"Pranav Joshi, Prabha Acharya, Mona Zolfaghar, Manav Goud Vanga, Sunil Shrestha, Moo-Yeal Lee","doi":"10.1007/7651_2024_604","DOIUrl":"https://doi.org/10.1007/7651_2024_604","url":null,"abstract":"Human brain organoids (HBOs) derived from pluripotent stem cells hold great potential for disease modeling and high-throughput compound screening, given their structural and functional resemblance to fetal brain tissues. These organoids can mimic early stages of brain development, offering a valuable in vitro model to study both normal and disordered neurodevelopment. However, current methods of generating HBOs are often low throughput and variable in organoid differentiation and involve lengthy, labor-intensive processes, limiting their broader application in both academic and industrial research. Key challenges include high costs of growth factors, variability in organoid size and function, suboptimal maturation, and manual handling that reduces throughput. Here, we present a standard operating procedure (SOP) for the scalable production of HBOs using a novel pillar plate system that simplifies the spheroid transfer process and allows miniature organoid culture. This method enables the reproducible generation of HBOs without the need for extensive manual intervention, providing a streamlined solution for high-throughput screening (HTS). The resulting assay-ready pillar plate with HBOs is optimized for compound testing, in situ staining, and analysis, offering an efficient platform to advance neurodevelopmental research and therapeutic screening.","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-01-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143008150","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

Biosynthesis of Zinc Oxide Nanoparticles Using Dried Leaves of Camellia sinensis: Methods to Characterize and Assess Their Effects on Mesenchymal Stem Cell Viability. 利用茶树干叶生物合成氧化锌纳米颗粒：表征和评估其对间充质干细胞活力影响的方法

Methods in molecular biology Pub Date : 2025-01-17 DOI: 10.1007/7651_2024_598

Serap Yeşilkır Baydar, Tuba Akgül Çağlar, Fatma Ebru Koç

{"title":"Biosynthesis of Zinc Oxide Nanoparticles Using Dried Leaves of Camellia sinensis: Methods to Characterize and Assess Their Effects on Mesenchymal Stem Cell Viability.","authors":"Serap Yeşilkır Baydar, Tuba Akgül Çağlar, Fatma Ebru Koç","doi":"10.1007/7651_2024_598","DOIUrl":"https://doi.org/10.1007/7651_2024_598","url":null,"abstract":"Stem cell nanotechnology (SCN) is an important scientific field to guide stem cell-based research of nanoparticles. Currently, nanoparticles (NPs) have a rich spectrum regarding the sources from which they are obtained (metallic, polymeric, etc.), the methods of obtaining them (physical, chemical, biological), and their shape, size, electrical charge, etc. properties. It is also essential to expand green synthesis applications for the use of NPs in the field of biomedical sciences. For this purpose, there is a need to produce NPs using biological sources (plant, microorganism, algae, yeast etc.…), characterization and investigation of their effects on biological activities of stem cells. This process involves long and laborious procedures, and there may be differences in methods between individual laboratories.In this protocol, biofabrication and characterization of ZnO NPs using dried leaves of Camellia sinensis is described. This experimental setup includes conventional and novel methods that can be applied to biofabricate and characterize the NPs and to examine the viability, apoptotic, and necrotic effects on human adipose tissue-derived mesenchymal stem cells (ADMSCs) in vitro.","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-01-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143008168","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

Protocol for the Generation and 3D Culture of Fluorescently Labeled Multicellular Spheroids. 荧光标记的多细胞球体的生成和三维培养方案。

Methods in molecular biology Pub Date : 2025-01-17 DOI: 10.1007/7651_2024_592

Emily C Liu, Amy L Ryan, Sinem Koc-Günel

引用次数: 0

RNA Interference Approaches to Study Epidermal Cell Adhesion. 研究表皮细胞粘附的RNA干扰方法。

Methods in molecular biology Pub Date : 2025-01-17 DOI: 10.1007/7651_2024_584

Anamika Dutta, Michele Calder, Lina Dagnino

引用次数: 0