Current Protocols in Stem Cell Biology最新文献_第9页

hPSC-derived Midbrain Dopaminergic Neurons Generated in a Scalable 3-D Biomaterial 在可扩展的三维生物材料中生成hpsc衍生的中脑多巴胺能神经元

Current Protocols in Stem Cell Biology Pub Date : 2018-02-28 DOI: 10.1002/cpsc.47

Maroof M. Adil, David V. Schaffer

引用次数: 4

CRISPR-Cas9-Based Genome Editing of Human Induced Pluripotent Stem Cells 基于crispr - cas9的人诱导多能干细胞基因组编辑

Current Protocols in Stem Cell Biology Pub Date : 2018-02-28 DOI: 10.1002/cpsc.46

Joseph C. Giacalone, Tasneem P. Sharma, Erin R. Burnight, John F. Fingert, Robert F. Mullins, Edwin M. Stone, Budd A. Tucker

{"title":"CRISPR-Cas9-Based Genome Editing of Human Induced Pluripotent Stem Cells","authors":"Joseph C. Giacalone, Tasneem P. Sharma, Erin R. Burnight, John F. Fingert, Robert F. Mullins, Edwin M. Stone, Budd A. Tucker","doi":"10.1002/cpsc.46","DOIUrl":"10.1002/cpsc.46","url":null,"abstract":"Human induced pluripotent stem cells (hiPSCs) are the ideal cell source for autologous cell replacement. However, for patients with Mendelian diseases, genetic correction of the original disease-causing mutation is likely required prior to cellular differentiation and transplantation. The emergence of the CRISPR-Cas9 system has revolutionized the field of genome editing. By introducing inexpensive reagents that are relatively straightforward to design and validate, it is now possible to correct genetic variants or insert desired sequences at any location within the genome. CRISPR-based genome editing of patient-specific iPSCs shows great promise for future autologous cell replacement therapies. One caveat, however, is that hiPSCs are notoriously difficult to transfect, and optimized experimental design considerations are often necessary. This unit describes design strategies and methods for efficient CRISPR-based genome editing of patient- specific iPSCs. Additionally, it details a flexible approach that utilizes positive selection to generate clones with a desired genomic modification, Cre-lox recombination to remove the integrated selection cassette, and negative selection to eliminate residual hiPSCs with intact selection cassettes. © 2018 by John Wiley & Sons, Inc.","PeriodicalId":53703,"journal":{"name":"Current Protocols in Stem Cell Biology","volume":"44 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-02-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpsc.46","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"35889819","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

Direct Conversion of Human Pluripotent Stem Cells to Osteoblasts With a Small Molecule 用小分子直接转化人多能干细胞为成骨细胞

Current Protocols in Stem Cell Biology Pub Date : 2018-02-28 DOI: 10.1002/cpsc.44

Heemin Kang, Yu-Ru V. Shih, Shyni Varghese

引用次数: 12

Controlling the Effective Oxygen Tension Experienced by Cells Using a Dynamic Culture Technique for Hematopoietic Ex Vivo Expansion 利用动态培养技术控制造血体外扩增细胞的有效氧张力

Current Protocols in Stem Cell Biology Pub Date : 2018-02-28 DOI: 10.1002/cpsc.42

Abhilasha Tiwari, Cynthia S. Wong, Lakshmi P. Nekkanti, James A. Deane, Courtney McDonald, Jingang Li, Yen Pham, Amy E. Sutherland, Graham Jenkin, Mark A. Kirkland

{"title":"Controlling the Effective Oxygen Tension Experienced by Cells Using a Dynamic Culture Technique for Hematopoietic Ex Vivo Expansion","authors":"Abhilasha Tiwari, Cynthia S. Wong, Lakshmi P. Nekkanti, James A. Deane, Courtney McDonald, Jingang Li, Yen Pham, Amy E. Sutherland, Graham Jenkin, Mark A. Kirkland","doi":"10.1002/cpsc.42","DOIUrl":"10.1002/cpsc.42","url":null,"abstract":"Clinical hematopoietic stem/progenitor cell (HSPC) transplantation outcomes are strongly correlated with the number of cells infused. Hence, to generate sufficient HSPCs for transplantation, the best culture parameters for expansion are critical. It is generally assumed that the defined oxygen (O2) set for the incubator reflects the pericellular O2 to which cells are being exposed. Studies have shown that low O2 tension maintains an undifferentiated state, but the expansion rate may be constrained because of limited diffusion in a static culture system. A combination of low ambient O2 and dynamic culture conditions has been developed to increase the reconstituting capacity of human HSPCs. In this unit, the protocols for serum-free expansion of HSPCs at 5% and 20% O2 in static and dynamic nutrient flow mode are described. Finally, the impact of O2 tension on HSPC expansion in vitro by flow cytometry and colony forming assays and in vivo through engraftment using a murine model is assessed. © 2018 by John Wiley & Sons, Inc.","PeriodicalId":53703,"journal":{"name":"Current Protocols in Stem Cell Biology","volume":"44 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-02-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpsc.42","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"35890118","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: 2

Isolation of Human Melanoma Stem Cells Using ALDH as a Marker 用ALDH作为标记分离人黑色素瘤干细胞

Current Protocols in Stem Cell Biology Pub Date : 2018-02-16 DOI: 10.1002/9780470151808.sc0308s26

Yuchun Luo, Nicholas Nguyen, Mayumi Fujita

{"title":"Isolation of Human Melanoma Stem Cells Using ALDH as a Marker","authors":"Yuchun Luo, Nicholas Nguyen, Mayumi Fujita","doi":"10.1002/9780470151808.sc0308s26","DOIUrl":"10.1002/9780470151808.sc0308s26","url":null,"abstract":"<div>\u0000 \u0000 This unit describes a protocol for establishing a patient-derived tumor xenograft model (PDTX model) of human melanoma and isolating human melanoma stem cells from human melanoma specimens using aldehyde dehydrogenase (ALDH) as a marker. One major limitation of analyzing a small fraction of cancer stem cells from patient tumor samples is that substantial quantities of fresh tumor tissues are not available. To overcome this challenge, we have established a PDTX model of human melanoma. In this model, human tumor tissues obtained from melanoma patients are dissected into small pieces and subsequently implanted into immunocompromised mice. The PDTX tumors retain fundamental genotypic and phenotypic features of the original tumors and are suitable for further biological analyses. Using the PDTX model, we have analyzed ALDH-labeled human melanoma stem cells. This unit will describe how to establish a PDTX model using human tumor samples. We will also describe how to isolate and analyze ALDH-labeled human melanoma stem cells using this model. Curr. Protoc. Stem Cell Biol. 26:3.8.1-3.8.10. © 2013 by John Wiley & Sons, Inc.</div>","PeriodicalId":53703,"journal":{"name":"Current Protocols in Stem Cell Biology","volume":"26 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-02-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"32101526","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: 22

Array-Based High-Throughput Screening in Mouse Embryonic Stem Cells with shRNAs 基于阵列的shrna小鼠胚胎干细胞高通量筛选

Current Protocols in Stem Cell Biology Pub Date : 2018-02-16 DOI: 10.1002/9780470151808.sc05c03s26

Chia-Hui Wang, Nianhan Ma, Yu-Tsen Lin, Cheng-Chung Wu, Hong-Jin Wu, Ching-Chia Yu, Michael Hsiao, Frank Leigh Lu, Scott C. Schuyler, Jean Lu

{"title":"Array-Based High-Throughput Screening in Mouse Embryonic Stem Cells with shRNAs","authors":"Chia-Hui Wang, Nianhan Ma, Yu-Tsen Lin, Cheng-Chung Wu, Hong-Jin Wu, Ching-Chia Yu, Michael Hsiao, Frank Leigh Lu, Scott C. Schuyler, Jean Lu","doi":"10.1002/9780470151808.sc05c03s26","DOIUrl":"10.1002/9780470151808.sc05c03s26","url":null,"abstract":"<div>\u0000 \u0000 High-throughput short-hairpin RNA (shRNA) lentivirus screening is a powerful tool for identifying multiple functional regulators in embryonic stem cells (ESCs). shRNA libraries can efficiently down-regulate target genes persistently with high efficiency. The concurrent measurement of relative cell number by alamarBlue (AB) assay and undifferentiated ESC markers via an alkaline phosphatase (ALP) activity assay in the same cell culture well provides an efficient and economical way to pinpoint factors crucial for ESC pluripotency and/or expansion. Most of the renewal pathways affect ALP activity. Thus, multiple positive and negative regulators can be identified by this method. In addition, morphological changes and/or the expression levels of specific pluripotency or differentiation markers examined by immunofluorescence can be used as secondary screens for target-gene selection. In summary, we describe an efficient way to identify multiple regulators of ESC renewal using shRNAs. Curr. Protoc. Stem Cell Biol. 26:5C.3.1-5C.3.19. © 2013 by John Wiley & Sons, Inc.</div>","PeriodicalId":53703,"journal":{"name":"Current Protocols in Stem Cell Biology","volume":"26 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-02-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"32101527","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

Transplantation Models to Characterize the Mechanisms of Stem Cell–Induced Islet Regeneration 移植模型表征干细胞诱导的胰岛再生机制

Current Protocols in Stem Cell Biology Pub Date : 2018-02-16 DOI: 10.1002/9780470151808.sc02b04s26

Gillian I. Bell, Ayesh K. Seneviratne, Grace N. Nasri, David A. Hess

{"title":"Transplantation Models to Characterize the Mechanisms of Stem Cell–Induced Islet Regeneration","authors":"Gillian I. Bell, Ayesh K. Seneviratne, Grace N. Nasri, David A. Hess","doi":"10.1002/9780470151808.sc02b04s26","DOIUrl":"10.1002/9780470151808.sc02b04s26","url":null,"abstract":"<div>\u0000 \u0000 This unit describes our current knowledge regarding the isolation human bone marrow–derived progenitor cells for the paracrine stimulation of islet regeneration after transplantation into immunodeficient mouse models of diabetes. By using high aldehyde dehydrogenase (ALDHhi) activity, a conserved function in multiple stem cell lineages, a mixed population of hematopoietic, endothelial, and mesenchymal progenitor cells can be efficiently purified using flow cytometry. We describe in vitro approaches to characterize and expand these distinct cell types. Importantly, these cell types can be transplanted into immunodeficient mice rendered beta-cell deficient by streptozotocin (STZ) treatment, in order monitor functional recovery from hyperglycemia and to characterize endogenous islet regeneration via paracrine mechanisms. Herein, we provide detailed protocols for: (1) isolation and characterization of ALDHhi cells for the establishment of hematopoietic and multipotent-stromal progenitor lineages; (2) intravenous and intrapancreatic transplantation of human stem cell subtypes for the quantification of glycemic recovery in STZ-treated immunodeficient mice; and (3) immunohistochemical characterization of islet recovery via the stimulation of islet neogenic, beta-cell proliferative, and islet revascularization programs. Collectively, these systems can be used to support the pre-clinical development of human progenitor cell–based therapies to treat diabetes via islet regeneration. Curr. Protoc. Stem Cell Biol. 26:2B.4.1-2B.4.35. © 2013 by John Wiley & Sons, Inc.</div>","PeriodicalId":53703,"journal":{"name":"Current Protocols in Stem Cell Biology","volume":"26 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-02-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/9780470151808.sc02b04s26","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"32101524","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: 4

Intraspinal Transplantation of Mouse and Human Neural Precursor Cells 小鼠和人神经前体细胞的椎管内移植

Current Protocols in Stem Cell Biology Pub Date : 2018-02-16 DOI: 10.1002/9780470151808.sc02d16s26

Jason G. Weinger, Lu Chen, Ronald Coleman, Ronika Leang, Warren C. Plaisted, Jeanne F. Loring, Thomas E. Lane

引用次数: 5

Differentiation of Hepatocytes from Pluripotent Stem Cells 多能干细胞与肝细胞的分化

Current Protocols in Stem Cell Biology Pub Date : 2018-02-16 DOI: 10.1002/9780470151808.sc01g04s26

Sunil K. Mallanna, Stephen A. Duncan

引用次数: 96

Single-Cell Functional Analysis of Stem-Cell Derived Cardiomyocytes on Micropatterned Flexible Substrates 微图案柔性基质上干细胞衍生心肌细胞的单细胞功能分析

Current Protocols in Stem Cell Biology Pub Date : 2018-02-13 DOI: 10.1002/cpsc.40

Jan David Kijlstra, Dongjian Hu, Peter van der Meer, Ibrahim J. Domian

{"title":"Single-Cell Functional Analysis of Stem-Cell Derived Cardiomyocytes on Micropatterned Flexible Substrates","authors":"Jan David Kijlstra, Dongjian Hu, Peter van der Meer, Ibrahim J. Domian","doi":"10.1002/cpsc.40","DOIUrl":"10.1002/cpsc.40","url":null,"abstract":"Human pluripotent stem–cell derived cardiomyocytes (hPSC-CMs) hold great promise for applications in human disease modeling, drug discovery, cardiotoxicity screening, and, ultimately, regenerative medicine. The ability to study multiple parameters of hPSC-CM function, such as contractile and electrical activity, calcium cycling, and force generation, is therefore of paramount importance. hPSC-CMs cultured on stiff substrates like glass or polystyrene do not have the ability to shorten during contraction, making them less suitable for the study of hPSC-CM contractile function. Other approaches require highly specialized hardware and are difficult to reproduce. Here we describe a protocol for the preparation of hPSC-CMs on soft substrates that enable shortening, and subsequently the simultaneous quantitative analysis of their contractile and electrical activity, calcium cycling, and force generation at single-cell resolution. This protocol requires only affordable and readily available materials and works with standard imaging hardware. © 2017 by John Wiley & Sons, Inc.","PeriodicalId":53703,"journal":{"name":"Current Protocols in Stem Cell Biology","volume":"43 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-02-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpsc.40","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"35252303","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: 5