Current Protocols in Stem Cell Biology最新文献_第6页

Current Protocols in Stem Cell Biology Pub Date : 2019-01-17 DOI: 10.1002/cpsc.70

引用次数: 0

Organotypic Culture of Bone-Like Structures Using Composite Ceramic-Fibrin Scaffolds 复合陶瓷-纤维蛋白支架骨样结构的器官型培养

Current Protocols in Stem Cell Biology Pub Date : 2019-01-14 DOI: 10.1002/cpsc.79

Alexandra Iordachescu, Richard L. Williams, Philippa A. Hulley, Liam M. Grover

引用次数: 7

Isolation and Flow Cytometry Characterization of Extracellular-Vesicle Subpopulations Derived from Human Mesenchymal Stromal Cells 人间充质基质细胞胞外囊泡亚群的分离和流式细胞术鉴定

Current Protocols in Stem Cell Biology Pub Date : 2019-01-09 DOI: 10.1002/cpsc.76

Cansu Gorgun, Daniele Reverberi, Gianluca Rotta, Federico Villa, Rodolfo Quarto, Roberta Tasso

引用次数: 25

Isolation and Enrichment of Spermatogonial Stem Cells From Human Testis Tissues 人睾丸组织精原干细胞的分离与富集

Current Protocols in Stem Cell Biology Pub Date : 2019-01-04 DOI: 10.1002/cpsc.77

Jingtao Guo, Bradley R. Cairns

引用次数: 11

Combinatorial Utilization of Murine Embryonic Stem Cells and In Vivo Models to Study Human Congenital Heart Disease 联合利用小鼠胚胎干细胞和体内模型研究人类先天性心脏病

Current Protocols in Stem Cell Biology Pub Date : 2018-12-12 DOI: 10.1002/cpsc.75

Abeer Zakariyah, Rashida Rajgara, Michael Shelton, Alexandre Blais, Ilona S. Skerjanc, Patrick G. Burgon

{"title":"Combinatorial Utilization of Murine Embryonic Stem Cells and In Vivo Models to Study Human Congenital Heart Disease","authors":"Abeer Zakariyah, Rashida Rajgara, Michael Shelton, Alexandre Blais, Ilona S. Skerjanc, Patrick G. Burgon","doi":"10.1002/cpsc.75","DOIUrl":"10.1002/cpsc.75","url":null,"abstract":"We have established an in vitro model of the human congenital heart defect (CHD)–associated mutation NKX2.5 R141C. We describe the use of the hanging drop method to differentiate Nkx2.5R141C/+ murine embryonic stem cells (mESCs) along with Nkx2.5+/+ control cells. This method allows us to recapitulate the early stages of embryonic heart development in tissue culture. We also use qRT-PCR and immunofluorescence to examine samples at different time points during differentiation to validate our data. The in vivo model is a mouse line with a knock-in of the same mutation. We describe the isolation of RNA from embryonic day 8.5 (E8.5) embryos and E9.5 hearts of wild-type and mutant mice. We found that the in vitro model shows reduced cardiomyogenesis, similar to Nkx2.5R141C/+ embryos at E8.5, indicating a transient loss of cardiomyogenesis at this time point. These results suggest that our in vitro model can be used to study very early changes in heart development that cause CHD. © 2018 by John Wiley & Sons, Inc.","PeriodicalId":53703,"journal":{"name":"Current Protocols in Stem Cell Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2018-12-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpsc.75","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"36782607","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: 1

Differentiation of Human-Induced Pluripotent Stem Cells to Macrophages for Disease Modeling and Functional Genomics 人诱导多能干细胞分化为巨噬细胞用于疾病建模和功能基因组学

Current Protocols in Stem Cell Biology Pub Date : 2018-12-10 DOI: 10.1002/cpsc.74

Jianting Shi, Chenyi Xue, Wen Liu, Hanrui Zhang

{"title":"Differentiation of Human-Induced Pluripotent Stem Cells to Macrophages for Disease Modeling and Functional Genomics","authors":"Jianting Shi, Chenyi Xue, Wen Liu, Hanrui Zhang","doi":"10.1002/cpsc.74","DOIUrl":"10.1002/cpsc.74","url":null,"abstract":"Macrophages play important roles in many diseases. We describe a protocol and the associated resources for the differentiation of human induced pluripotent stem cell-derived macrophages (IPSDM) and their applications in understanding human macrophage physiology and relevant diseases. The protocol uses an embryoid body–based approach with a combination of serum-free condition for hematopoiesis specification, followed by adherent culture with serum and M-CSF for myeloid expansion and macrophage maturation. The protocol produced an almost pure culture of CD45+/CD18+ macrophages yielding up to 2 × 107 cells per 6-well plate of iPSCs within 24 days, demonstrating high efficiency, purity, and scalability. The IPSDM and monocyte-derived macrophages (HMDM) cultured in the same medium were compared at morphological, functional and transcriptomic levels by RNA-sequencing. IPSDM and HMDM showed broadly similar profiles of coding transcriptome, alternative splicing events, and long noncoding RNAs, with advantages and successful applications in disease modeling using patients-derived and CRISPR-edited iPSC lines. © 2018 by John Wiley & Sons, Inc.","PeriodicalId":53703,"journal":{"name":"Current Protocols in Stem Cell Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2018-12-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpsc.74","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"36760619","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: 14

Issue Information TOC 发布信息TOC

Current Protocols in Stem Cell Biology Pub Date : 2018-11-01 DOI: 10.1002/cpsc.69

引用次数: 0

Isolation of Adipose Tissue–Derived Stem Cells: Enzymatic Digestion in Combination with Mechanical Distortion to Increase Adipose Tissue–Derived Stem Cell Yield from Human Aspirated Fat 分离脂肪组织来源的干细胞:酶消化与机械变形相结合以增加从人抽吸脂肪中提取的脂肪组织来源的干细胞产量

Current Protocols in Stem Cell Biology Pub Date : 2018-10-26 DOI: 10.1002/cpsc.68

Toke Alstrup, Marco Eijken, Anja Bille Bohn, Bjarne Møller, Tine Engberg Damsgaard

{"title":"Isolation of Adipose Tissue–Derived Stem Cells: Enzymatic Digestion in Combination with Mechanical Distortion to Increase Adipose Tissue–Derived Stem Cell Yield from Human Aspirated Fat","authors":"Toke Alstrup, Marco Eijken, Anja Bille Bohn, Bjarne Møller, Tine Engberg Damsgaard","doi":"10.1002/cpsc.68","DOIUrl":"10.1002/cpsc.68","url":null,"abstract":"Mesenchymal stem cells (MSCs) are of great interest due to their properties of immune modulation, tissue regeneration, and multipotent differentiation. Future developments of clinical applications, however, require a higher yield of MSCs, lower number of passages of cells in culture, and shorter time from harvest to use. Optimization and standardization of techniques for mesenchymal adipose tissue–derived stem cell isolation offers solutions to current bottlenecks as a larger amount of MSCs can be isolated. These improvements result in shorter expansion time, fewer passages, less donor material needed, and higher MSC yield. This paper describes an MSC isolation method combining enzymatic digestion with mechanic disruption. This protocol is a standardized and easy-to-implement method for reaching significantly higher MSC yields compared to conventional enzymatic isolation protocols. Based on the results presented, we hypothesize that the combined enzymatic and mechanical method increases the surface area of the adipose tissue, facilitating digestion by enzymes. This approach reduces the amount of adipose tissue and in vitro expansion time needed to reach sufficient amounts of MSCs for clinical purposes. Importantly, the method does not require increased amounts of collagenase, nor does it impair the viability or differentiability of the MSCs. Using this protocol increases MSC yield by a factor of three. As a consequence, these results indicate that the physiological concentration of MSCs in adipose tissue is higher than previously assumed. © 2018 by John Wiley & Sons, Inc.","PeriodicalId":53703,"journal":{"name":"Current Protocols in Stem Cell Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2018-10-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpsc.68","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"40545629","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: 24

FACS-Mediated Isolation of Neuronal Cell Populations From Virus-Infected Human Embryonic Stem Cell–Derived Cerebral Organoid Cultures facs介导的从病毒感染的人胚胎干细胞衍生的脑类器官培养中分离神经细胞群的研究

Current Protocols in Stem Cell Biology Pub Date : 2018-10-24 DOI: 10.1002/cpsc.65

Sylvie Janssens, Michael Schotsaert, Lara Manganaro, Marion Dejosez, Viviana Simon, Adolfo García-Sastre, Thomas P. Zwaka

{"title":"FACS-Mediated Isolation of Neuronal Cell Populations From Virus-Infected Human Embryonic Stem Cell–Derived Cerebral Organoid Cultures","authors":"Sylvie Janssens, Michael Schotsaert, Lara Manganaro, Marion Dejosez, Viviana Simon, Adolfo García-Sastre, Thomas P. Zwaka","doi":"10.1002/cpsc.65","DOIUrl":"10.1002/cpsc.65","url":null,"abstract":"Organoids—or pluripotent stem cell–derived in vitro-grown simplified mini organs—have become a tremendously important model to study human organ development and disease. To restrict the noise inherent to the heterogeneous cell mixtures derived from organoid cultures, we developed a new technique of fluorescence-assisted cell sorting (FACS) of virus-infected cerebral organoid cultures. This method still includes the advantage of growing cells in a more natural environment than traditional cell culture, but now renders samples suitable for downstream cell type-specific multi-omics analyses. The protocol starts from stem cell-derived mature brain organoids and includes steps for: preparing the culture for viral infection, production of the viral stocks, FACS sample preparation, and gating and sorting implementation. The protocol has been developed for Zika virus infection, but can be extrapolated to other viruses or fluorescent marker expression as illustrated in an alternate protocol using a single-cycle lentivirus expressing a fluorescent reporter protein. © 2018 by John Wiley & Sons, Inc.","PeriodicalId":53703,"journal":{"name":"Current Protocols in Stem Cell Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2018-10-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpsc.65","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"36660243","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 14

Highly Efficient CRISPR-Cas9-Mediated Genome Editing in Human Pluripotent Stem Cells 高效crispr - cas9介导的人类多能干细胞基因组编辑

Current Protocols in Stem Cell Biology Pub Date : 2018-10-24 DOI: 10.1002/cpsc.64

Jean Ann Maguire, Fabian L. Cardenas-Diaz, Paul Gadue, Deborah L. French

引用次数: 21