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Current Protocols in Stem Cell Biology最新文献

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The Pluripotency Continuum and Interspecies Chimeras 多能性连续体与种间嵌合体
Current Protocols in Stem Cell Biology Pub Date : 2019-06-11 DOI: 10.1002/cpsc.87
Alejandro De Los Angeles
{"title":"The Pluripotency Continuum and Interspecies Chimeras","authors":"Alejandro De Los Angeles","doi":"10.1002/cpsc.87","DOIUrl":"10.1002/cpsc.87","url":null,"abstract":"<p>Pluripotency refers to the capacity of single cells to form derivatives of the three germ layers—ectoderm, mesoderm, and endoderm. Pluripotency can be captured in vitro as a spectrum of pluripotent stem cell states stabilized in specialized laboratory conditions. The recent discovery that pluripotent stem cells can colonize the embryos of distantly related animal organisms could, with further refinement, enable the generation of chimeric embryos composed of cells of human and animal origin. If achievable, the production of human-animal chimeras will open up new opportunities for regenerative medicine, facilitating human disease modeling and human organ generation inside large animals. However, the generation of human-animal interspecies chimeras is anticipated to require human chimera-competent pluripotent stem cells. Thus, it remains imperative to examine the pluripotency continuum more closely in light of advances that will facilitate the production of human-animal chimeras. This piece will review the current understanding of the pluripotency continuum and interspecies chimeras. © 2019 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":53703,"journal":{"name":"Current Protocols in Stem Cell Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2019-06-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpsc.87","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37316991","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
Issue Information TOC 发布信息TOC
Current Protocols in Stem Cell Biology Pub Date : 2019-05-10 DOI: 10.1002/cpsc.71
{"title":"Issue Information TOC","authors":"","doi":"10.1002/cpsc.71","DOIUrl":"https://doi.org/10.1002/cpsc.71","url":null,"abstract":"<p><b>Cover</b>: In Deshpande et al. (https://doi.org/10.1002/cpsc.80), the image shows lineage differentiation from adult murine neural stem cells.\u0000\u0000 <figure>\u0000 <div><picture>\u0000 <source></source></picture><p></p>\u0000 </div>\u0000 </figure></p>","PeriodicalId":53703,"journal":{"name":"Current Protocols in Stem Cell Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2019-05-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpsc.71","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"92290414","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: 0
Differentiating Human Induced Pluripotent Stem Cells (iPSCs) Into Lung Epithelial Cells 人诱导多能干细胞分化为肺上皮细胞
Current Protocols in Stem Cell Biology Pub Date : 2019-04-18 DOI: 10.1002/cpsc.86
Harshini Surendran, Mohanapriya Rajamoorthy, Rajarshi Pal
{"title":"Differentiating Human Induced Pluripotent Stem Cells (iPSCs) Into Lung Epithelial Cells","authors":"Harshini Surendran,&nbsp;Mohanapriya Rajamoorthy,&nbsp;Rajarshi Pal","doi":"10.1002/cpsc.86","DOIUrl":"10.1002/cpsc.86","url":null,"abstract":"<p>Human induced pluripotent stem cells (hiPSCs) not only offer great opportunities for the study of human development but also have tremendous potential for future clinical cell-based therapies. The protocol outlined here is used to differentiate hiPSCs into lung epithelial cell types through a process that faithfully recapitulates the stepwise events observed in vivo. From pluripotency, cells are differentiated to a definitive endoderm fate, followed by progression into anteriorized foregut endoderm that has the ability to give rise to both proximal and distal epithelial cells. Furthermore, this methodology allows for the study of lung dysfunction and disease modeling using patient-derived cells, as well as high-throughput pharmacological screening and eventually personalized therapies. Recently we were able to reproduce this protocol using the working cell bank of an hiPSC line made under current Good Manufacturing Practice (cGMP) conditions, a necessary step for the future clinical application of these cells. © 2019 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":53703,"journal":{"name":"Current Protocols in Stem Cell Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2019-04-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpsc.86","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37165081","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
Generation of ERK-Independent Human and Non-Human Primate Pluripotent Stem Cells 不依赖erk的人类和非人类灵长类多能干细胞的产生
Current Protocols in Stem Cell Biology Pub Date : 2019-04-17 DOI: 10.1002/cpsc.85
Alejandro De Los Angeles
{"title":"Generation of ERK-Independent Human and Non-Human Primate Pluripotent Stem Cells","authors":"Alejandro De Los Angeles","doi":"10.1002/cpsc.85","DOIUrl":"10.1002/cpsc.85","url":null,"abstract":"<p>The production of human organs inside human-animal interspecies chimeras might one day comprise a viable strategy for generating patient-specific organs, but such experiments will require human chimera-competent pluripotent stem (PS) cells. The stabilization of PS cell self-renewal in serum-free medium and ERK blockade might be critical for capturing primate chimera-competent pluripotency. It has recently been shown that shielding primate cells from the activation of ERK, WNT, and PKC signaling is crucial for deriving African green monkey ERK-independent PS cells. Here, I show that this principle is generalizable to human cells. In this chapter, methods are provided to reset conventional human PS cells to ERK-independence using histone deacetylase inhibitors and PGCX media comprised of N2B27 medium supplemented with LIF, PD0325901, Go6983, CHIR99021, and XAV939. The novel stem cells exhibit higher levels of KLF4 and manifest increased mitochondrial membrane depolarization. However, the author observed that not all PS cell lines are amenable to small molecule-mediated resetting. The ERK-independent PS cells described herein will provide a useful resource for testing interspecies organogenesis strategies. © 2019 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":53703,"journal":{"name":"Current Protocols in Stem Cell Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2019-04-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpsc.85","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37324404","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
A Method to Isolate CD34+ Mononuclear Cells from Canine Peripheral Blood 犬外周血CD34+单核细胞的分离方法
Current Protocols in Stem Cell Biology Pub Date : 2019-03-22 DOI: 10.1002/cpsc.84
Vanmathy Kasimanickam, Ramanathan Kasimanickam
{"title":"A Method to Isolate CD34+ Mononuclear Cells from Canine Peripheral Blood","authors":"Vanmathy Kasimanickam,&nbsp;Ramanathan Kasimanickam","doi":"10.1002/cpsc.84","DOIUrl":"10.1002/cpsc.84","url":null,"abstract":"<p>Embryonic stem cells are pluripotent whereas adult stem cells are multipotent in nature. In recent years, evidence suggests that adult stem cells not only differentiate into specific cell lineages but also transdifferentiate into multiple cell lineages. Progenitor cells are found in adult bone marrow, blood, and other organs and differentiate into numerous cell lineages regardless of origin. Identifying a subset that can differentiate into mature endothelial cells is essential. This article describes peripheral blood collection in adult beagle dogs, isolation of peripheral blood mononuclear cells (PBMNCs) from the cell fraction, separation of a subset of CD34+ cells using immunomagnetic principles, characterization of PBMNCs and CD34+ cells using flow cytometry, and evaluation of gene expression of CD34, KDR, and CD133 in CD34+ fractions. Efficient methods of isolation of endothelial progenitor cells (EPCs) will promote the ex vivo expansion and transplantation of EPCs in ischemic injury to enable neovascularization. © 2019 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":53703,"journal":{"name":"Current Protocols in Stem Cell Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2019-03-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpsc.84","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37243665","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
Rapid PCR Assay for Detecting Common Genetic Variants Arising in Human Pluripotent Stem Cell Cultures 检测人类多能干细胞培养中常见遗传变异的快速PCR方法
Current Protocols in Stem Cell Biology Pub Date : 2019-03-01 DOI: 10.1002/cpsc.83
Owen Laing, Jason Halliwell, Ivana Barbaric
{"title":"Rapid PCR Assay for Detecting Common Genetic Variants Arising in Human Pluripotent Stem Cell Cultures","authors":"Owen Laing,&nbsp;Jason Halliwell,&nbsp;Ivana Barbaric","doi":"10.1002/cpsc.83","DOIUrl":"10.1002/cpsc.83","url":null,"abstract":"<p>Human pluripotent stem cells (hPSCs) are prone to acquiring genetic changes upon prolonged culture. Particularly common are copy number changes, including gains of chromosomes 1q, 12p, 17q, and 20q, and/or loss of chromosomes 10p and 18q. The variant cells harboring common genetic changes display altered behaviors compared to their diploid counterparts, thus potentially impacting upon the validity of experimental results and safety of hPSC-derived cellular therapies. Hence, a critical quality attribute in hPSC maintenance should include frequent monitoring for genetic changes arising in cultures. This in turn places large demands on the genotyping assays for detection of genetic changes. Traditional methods for screening cells entail specialized cytogenetic analyses, but their high costs and a lengthy turnaround time make them impractical for high-throughput analyses and routine laboratory use. Here, we detail a protocol for a rapid, accessible, and affordable PCR-based method for detection of frequently occurring copy number changes in hPSCs. © 2019 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":53703,"journal":{"name":"Current Protocols in Stem Cell Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2019-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpsc.83","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37013045","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: 8
Isolation of Neural Stem Cells from Whole Brain Tissues of Adult Mice 成年小鼠全脑组织神经干细胞的分离
Current Protocols in Stem Cell Biology Pub Date : 2019-02-05 DOI: 10.1002/cpsc.80
Krutika Deshpande, Behnaz Saatian, Vahan Martirosian, Michelle Lin, Alex Julian, Josh Neman
{"title":"Isolation of Neural Stem Cells from Whole Brain Tissues of Adult Mice","authors":"Krutika Deshpande,&nbsp;Behnaz Saatian,&nbsp;Vahan Martirosian,&nbsp;Michelle Lin,&nbsp;Alex Julian,&nbsp;Josh Neman","doi":"10.1002/cpsc.80","DOIUrl":"10.1002/cpsc.80","url":null,"abstract":"<p>A population of neural stem cells exists in the adult mammalian central nervous system. Purification and characterization of neurospheres provide valuable tools to study the regulation and differentiation of neural stem cells both <i>in vitro</i> and <i>in vivo</i>. Successful stimulation and production of neurospheres can ultimately be used for therapeutic purposes. The currently available methods are limited by their poor yield and the large number of animals required to compensate for that. Here, we describe a procedure to purify neurospheres from adult mouse whole brain. We provide detailed steps on how to propagate, passage, and maintain the adult neurospheres, and how to differentiate the pure neurospheres into the lineage of interest. Using this method, neurospheres can be easily derived from adult mouse whole brain. The derived adult neurospheres maintain their homogenous undifferentiated status while retaining their differentiation potential. This new protocol facilitates adult neurospheres isolation, purification, maintenance, and differentiation. © 2019 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":53703,"journal":{"name":"Current Protocols in Stem Cell Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2019-02-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpsc.80","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"36978887","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
Autonomic Neurons with Sympathetic Character Derived From Human Pluripotent Stem Cells 具有交感特征的自主神经元来源于人多能干细胞
Current Protocols in Stem Cell Biology Pub Date : 2019-01-31 DOI: 10.1002/cpsc.78
Kenyi Saito-Diaz, Hsueh Fu Wu, Nadja Zeltner
{"title":"Autonomic Neurons with Sympathetic Character Derived From Human Pluripotent Stem Cells","authors":"Kenyi Saito-Diaz,&nbsp;Hsueh Fu Wu,&nbsp;Nadja Zeltner","doi":"10.1002/cpsc.78","DOIUrl":"10.1002/cpsc.78","url":null,"abstract":"<p>We describe an <i>in vitro</i> differentiation protocol to derive autonomic neurons of the peripheral nervous system with the character of postganglionic sympathetic neurons from human pluripotent stem cells. This protocol has been used to generate autonomic neurons from healthy embryonic stem cells as well as from patient-derived induced pluripotent stem cells, which were previously used to model familial dysautonomia, a genetic childhood disorder affecting the autonomic nervous system. Here, we describe each step in detail that is necessary to successfully derive these cells. First, we generate neural crest cells, which are purified using fluorescence-activated cell sorting. This is followed by intermediate culture as neural crest spheroids, where the cells can be expanded, and lastly long-term differentiation into neurons. The cells have morphological and molecular characteristics of autonomic neurons and thus can be employed to study diseases affecting the autonomic nervous system. © 2019 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":53703,"journal":{"name":"Current Protocols in Stem Cell Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2019-01-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpsc.78","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"36916260","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: 8
Extracellular-Vesicle Isolation from Different Biological Fluids by Size-Exclusion Chromatography 不同生物流体中细胞外囊泡的粒径排除色谱分离
Current Protocols in Stem Cell Biology Pub Date : 2019-01-30 DOI: 10.1002/cpsc.82
Marta Monguió-Tortajada, Miriam Morón-Font, Ana Gámez-Valero, Laura Carreras-Planella, Francesc E. Borràs, Marcella Franquesa
{"title":"Extracellular-Vesicle Isolation from Different Biological Fluids by Size-Exclusion Chromatography","authors":"Marta Monguió-Tortajada,&nbsp;Miriam Morón-Font,&nbsp;Ana Gámez-Valero,&nbsp;Laura Carreras-Planella,&nbsp;Francesc E. Borràs,&nbsp;Marcella Franquesa","doi":"10.1002/cpsc.82","DOIUrl":"10.1002/cpsc.82","url":null,"abstract":"<p>This unit describes how to isolate extracellular vesicles (EVs) from different biological fluids using size-exclusion chromatography (SEC) and how to prepare your starting sample and the EV product for downstream applications. EVs are membrane nanovesicles with specific content that reflects the phenotype and functions of the cell of origin, including protected proteins, lipids, metabolites, and nucleic acids. EVs are thus an excellent resource for noninvasive biomarker discovery in a number of pathological situations and are a promising nanotherapeutic tool to overcome the disadvantages associated with cellular therapy. However, there are still no standardized methods to isolate pure EV preparations, as many approaches do not guarantee proper EV purification, free of contaminating non-EV molecules. Currently, SEC is one of the most promising approaches to purify EVs from any biological fluid, as it avoids co-isolation of contaminants and is user friendly and scalable. © 2019 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":53703,"journal":{"name":"Current Protocols in Stem Cell Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2019-01-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpsc.82","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"36912328","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: 50
Efficient Generation of Trunk Neural Crest and Sympathetic Neurons from Human Pluripotent Stem Cells Via a Neuromesodermal Axial Progenitor Intermediate 通过神经中胚层轴向祖中间体从人多能干细胞高效生成主干神经嵴和交感神经元
Current Protocols in Stem Cell Biology Pub Date : 2019-01-28 DOI: 10.1002/cpsc.81
Thomas J. R. Frith, Anestis Tsakiridis
{"title":"Efficient Generation of Trunk Neural Crest and Sympathetic Neurons from Human Pluripotent Stem Cells Via a Neuromesodermal Axial Progenitor Intermediate","authors":"Thomas J. R. Frith,&nbsp;Anestis Tsakiridis","doi":"10.1002/cpsc.81","DOIUrl":"10.1002/cpsc.81","url":null,"abstract":"<p>The neural crest (NC) is a multipotent embryonic cell population that generates various cell types in an axial position-dependent manner. Cranial NC cells give rise to mesoectodermal derivatives, melanocytes, neurons, and glia whereas the vagal NC generates the enteric nervous system and trunk NC cells produce sympathetic neurons and neuroendocrine cells. An attractive approach for studying human NC biology and modeling NC-associated developmental disorders (neurocristopathies) involves the in vitro production of NC cells from human pluripotent stem cells (hPSCs). However, most conventional differentiation protocols generate predominantly cranial NC cells but fail to induce trunk NC cells. Here we describe a detailed protocol for the efficient in vitro generation of trunk NC cells and their derivatives from hPSCs. This relies on the induction of an intermediate cell population that exhibits neural and mesodermal potential, resembling the embryonic neuromesodermal progenitors, which generate the postcranial body axis in vivo. © 2019 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":53703,"journal":{"name":"Current Protocols in Stem Cell Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2019-01-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpsc.81","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"36903710","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: 26
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