Current Protocols in Stem Cell Biology最新文献_第4页

Live Cell Monitoring and Enrichment of Stem Cell-Derived β Cells Using Intracellular Zinc Content as a Population Marker 利用细胞内锌含量作为群体标记对干细胞衍生β细胞的活细胞监测和富集

Current Protocols in Stem Cell Biology Pub Date : 2019-10-28 DOI: 10.1002/cpsc.99

Jeffrey C. Davis, Aharon Helman, José Rivera-Feliciano, Christine M. Langston, Elise N. Engquist, Douglas A. Melton

引用次数: 11

Generation of Functional CX26–Gap-Junction-Plaque-Forming Cells with Spontaneous Ca2+ Transients via a Gap Junction Characteristic of Developing Cochlea 通过发育中的耳蜗间隙连接特性产生具有自发Ca2+瞬态的功能性cx26 -间隙连接斑块形成细胞

Current Protocols in Stem Cell Biology Pub Date : 2019-10-28 DOI: 10.1002/cpsc.100

Ichiro Fukunaga, Ayumi Fujimoto, Kaori Hatakeyama, Nagomi Kurebayashi, Katsuhisa Ikeda, Kazusaku Kamiya

{"title":"Generation of Functional CX26–Gap-Junction-Plaque-Forming Cells with Spontaneous Ca2+ Transients via a Gap Junction Characteristic of Developing Cochlea","authors":"Ichiro Fukunaga, Ayumi Fujimoto, Kaori Hatakeyama, Nagomi Kurebayashi, Katsuhisa Ikeda, Kazusaku Kamiya","doi":"10.1002/cpsc.100","DOIUrl":"10.1002/cpsc.100","url":null,"abstract":"Mutation of the gene GJB2, encoding connexin 26 (CX26; also known as gap junction beta 2), is the most frequent cause of hereditary deafness worldwide. CX26 is expressed in cochlear nonsensory cells, such as cochlear supporting cells, and forms gap junction plaques (GJPs) at cell-cell borders. Cochlear CX26-GJP-forming cells (Cx26GJCs) are thought to be an important therapeutic target for treatment of hereditary deafness. Nevertheless, the generation of Cx26GJCs—such as cochlear supporting cells—from embryonic stem/induced pluripotent stem (ES/iPS) cells has not been reported to date. Here, we detail a novel strategy for differentiating iPS cells into functional Cx26GJCs such as are found in cochlea. Several assays to characterize the phenotype of iPS-derived Cx26GJCs are described, including qRT-PCR, immunohistological analysis, morphological analysis, a scrape-loading and dye transfer assay, and calcium imaging. This in vitro model has applications in the establishment of inner-ear cell therapies and in drug screening to target GJB2-related hearing loss. © 2019 by John Wiley & Sons, Inc.Basic Protocol: Induction of mouse stem cells to create CX26-GJP-forming cellsSupport Protocol 1: Maintenance and passage of mouse induced pluripotent stem cellsSupport Protocol 2: Screening for high GJB2 and GJB6 expression in SFEBq culture using quantitative real-time PCRSupport Protocol 3: Characterization of cells at different stages of differentiation by immunostainingSupport Protocol 4: Ultrastructural analyses of cells at different stages of CX26-GJP-forming cell inductionSupport Protocol 5: Functional analyses of stem cell–derived CX26-GJP-forming cells","PeriodicalId":53703,"journal":{"name":"Current Protocols in Stem Cell Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2019-10-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpsc.100","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"86106404","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

Top-Down Inhibition (TDi) and Baseline Activation (BLa): Controlling Signal Transduction When Endogenous Cytokines are Ruining Your Differentiation 自顶向下抑制(TDi)和基线激活(BLa):当内源性细胞因子破坏分化时控制信号转导

Current Protocols in Stem Cell Biology Pub Date : 2019-09-05 DOI: 10.1002/cpsc.98

James Hackland

{"title":"Top-Down Inhibition (TDi) and Baseline Activation (BLa): Controlling Signal Transduction When Endogenous Cytokines are Ruining Your Differentiation","authors":"James Hackland","doi":"10.1002/cpsc.98","DOIUrl":"10.1002/cpsc.98","url":null,"abstract":"In the 20 years since the first human pluripotent stem cell (hPSC) lines were established, there have been a plethora of protocols developed that allow us to generate a wide range of human cell types in vitro. Efforts to achieve a greater degree of specificity and efficiency in generating desired cell types have resulted in increasingly complex approaches. The magnitude and timing of signals has become key, and the concept of a “fully defined” system is a forever sought-after goal with shifting goalposts. This overview discusses two related approaches that can be used to deliver a tightly regulated, intermediate-strength signal, and which can also manage the impact of endogenous signaling variation and enable a switch away from bovine serum albumin–containing medium to a better-defined system without suffering a subsequent loss of robustness or efficiency. The approaches, referred to as top-down inhibition and baseline activation, were developed to deliver intermediate levels of BMP and WNT signaling during neural crest induction from hPSC, but could be applied to a variety of other signals and differentiation systems. © 2019 by John Wiley & Sons, Inc.","PeriodicalId":53703,"journal":{"name":"Current Protocols in Stem Cell Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2019-09-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpsc.98","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"78159781","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

Spatiotemporal Control of Morphogen Delivery to Pattern Stem Cell Differentiation in Three-Dimensional Hydrogels 形态发生素在三维水凝胶中诱导干细胞分化的时空控制。

Current Protocols in Stem Cell Biology Pub Date : 2019-09-05 DOI: 10.1002/cpsc.97

Brian J. O'Grady, Daniel A. Balikov, Ethan S. Lippmann, Leon M. Bellan

{"title":"Spatiotemporal Control of Morphogen Delivery to Pattern Stem Cell Differentiation in Three-Dimensional Hydrogels","authors":"Brian J. O'Grady, Daniel A. Balikov, Ethan S. Lippmann, Leon M. Bellan","doi":"10.1002/cpsc.97","DOIUrl":"10.1002/cpsc.97","url":null,"abstract":"Morphogens are biological molecules that alter cellular identity and behavior across both space and time. During embryonic development, morphogen spatial localization can be confined to small volumes in a single tissue or permeate throughout an entire organism, and the temporal effects of morphogens can range from fractions of a second to several days. In most cases, morphogens are presented as a gradient to adjacent cells within tissues to pattern cell fate. As such, to appropriately model development and build representative multicellular architectures in vitro, it is vital to recapitulate these gradients during stem cell differentiation. However, the ability to control morphogen presentation within in vitro systems remains challenging. Here, we describe an innovative platform using channels patterned within thick, three-dimensional hydrogels that deliver multiple morphogens to embedded cells, thereby demonstrating exquisite control over both spatial and temporal variations in morphogen presentation. This generalizable approach should have broad utility for researchers interested in patterning in vitro tissue structures. © 2019 by John Wiley & Sons, Inc.","PeriodicalId":53703,"journal":{"name":"Current Protocols in Stem Cell Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2019-09-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpsc.97","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"49684943","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

Issue Information TOC 发布信息TOC

Current Protocols in Stem Cell Biology Pub Date : 2019-09-03 DOI: 10.1002/cpsc.72

引用次数: 0

Overview of Methods to Differentiate Sympathetic Neurons from Human Pluripotent Stem Cells 多能干细胞分化交感神经元的方法综述

Current Protocols in Stem Cell Biology Pub Date : 2019-08-02 DOI: 10.1002/cpsc.92

Hsueh Fu Wu, Nadja Zeltner

引用次数: 0

Reliable Protocols for Flow Cytometry Analysis of Intracellular Proteins in Pluripotent Stem Cell Derivatives: A Fit-For-Purpose Approach 流式细胞术分析多能干细胞衍生物中细胞内蛋白的可靠方案:一种适合目的的方法

Current Protocols in Stem Cell Biology Pub Date : 2019-08-02 DOI: 10.1002/cpsc.94

Linda Berg Luecke, Matthew Waas, Rebekah L. Gundry

{"title":"Reliable Protocols for Flow Cytometry Analysis of Intracellular Proteins in Pluripotent Stem Cell Derivatives: A Fit-For-Purpose Approach","authors":"Linda Berg Luecke, Matthew Waas, Rebekah L. Gundry","doi":"10.1002/cpsc.94","DOIUrl":"10.1002/cpsc.94","url":null,"abstract":"Human pluripotent stem cell (hPSC) derivatives are valuable for a variety of research applications and have the potential to revolutionize approaches to personalized medicine. However, differentiation efficiency varies among cell lines and protocols. Therefore, methods to reliably determine cell type identity in cultures of hPSC derivatives in a manner that is consistent among laboratories are needed. While flow cytometry is apt for routine assessment of population heterogeneity, standardized protocols are not available for most cell types. This article describes a workflow for establishing a fit-for-purpose protocol for flow cytometric analysis of hPSC derivatives. Based on the application of this workflow, a standard operating procedure (SOP) was developed for the analysis of cardiac troponin in hPSC-derived cardiomyocytes (hPSC-CM). Throughout the article, important concepts related to antibody validation and gating strategies are presented to enable users to properly validate any antibody of interest and develop a rigorous SOP for their experimental needs. © 2019 by John Wiley & Sons, Inc.","PeriodicalId":53703,"journal":{"name":"Current Protocols in Stem Cell Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2019-08-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpsc.94","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"78320197","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: 6

An Improved Two-Step Protocol for Trophoblast Differentiation of Human Pluripotent Stem Cells 改进的人多能干细胞滋养层分化两步法

Current Protocols in Stem Cell Biology Pub Date : 2019-08-02 DOI: 10.1002/cpsc.96

Mariko Horii, Tony Bui, Ojeni Touma, Hee Young Cho, Mana M. Parast

{"title":"An Improved Two-Step Protocol for Trophoblast Differentiation of Human Pluripotent Stem Cells","authors":"Mariko Horii, Tony Bui, Ojeni Touma, Hee Young Cho, Mana M. Parast","doi":"10.1002/cpsc.96","DOIUrl":"10.1002/cpsc.96","url":null,"abstract":"We previously established a two-step protocol for differentiation of human pluripotent stem cells (hPSCs) into trophoblasts, using a StemPro-based minimal medium (EMIM) with bone morphogenetic protein-4 (BMP4). This protocol was suboptimal, resulting in induction of mixed mesoderm and trophoblast markers. Furthermore, adapting hPSCs to StemPro has proven difficult, and prolonged culture in this medium has been shown to promote genomic instability. Therefore, we moved on to the use of new media, including E8, and most recently, StemFlex, for rapid adaptation from feeder to non-feeder conditions. In the new protocol, we have incorporated the WNT inhibitor IWP2 into the first step, resulting in uniform differentiation of hPSCs into cytotrophoblast (CTB)-like cells, without induction of the mesoderm lineage. We also show that, at the end of the second step, there are distinct populations of terminally differentiated multinucleated human chorionic gonadotropin (hCG)-producing syncytiotrophoblast (STB) and HLAG+ extravillous trophoblast (EVT)-like cells. © 2019 by John Wiley & Sons, Inc.","PeriodicalId":53703,"journal":{"name":"Current Protocols in Stem Cell Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2019-08-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpsc.96","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"81803636","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: 29

Differentiation of Retinal Organoids from Human Pluripotent Stem Cells 视网膜类器官从人多能干细胞分化

Current Protocols in Stem Cell Biology Pub Date : 2019-07-31 DOI: 10.1002/cpsc.95

Valeria Chichagova, Birthe Dorgau, Majed Felemban, Maria Georgiou, Lyle Armstrong, Majlinda Lako

引用次数: 35

A High-Throughput Screening Method to Identify Compounds Displaying Human Vascular Embryonic Toxicity 一种高通量筛选方法鉴定显示人类血管胚胎毒性的化合物

Current Protocols in Stem Cell Biology Pub Date : 2019-07-26 DOI: 10.1002/cpsc.93

Susana Rosa, Patrícia Pitrez, Hugo Fernandes, Lino Ferreira

引用次数: 1