فإن الكتاب أن نعترف الدكتور فيليب H. شوارتز من العصبية الوطنية الجذعية البشرية الموارد خلية في مستشفى الأطفال في ليالي ، أورانج كاونتي معهد البحوث لتوفير hNSPC الثقافات.

ملاحظة : يرجى الرجوع إلى الإنسان الركض الخلايا الجذعية العصبية المادة ( <a href="/index/Details.stp?ID=263">https://www.jove.com/index/Details.stp؟ID=263</a> ) والإنسان العصبية العد المادة الخلايا الجذعية ( <a href="/index/Details.stp?ID=262">https://www.jove.com / مؤشر / Details.stp؟ ID = 262</a> ) لمعرفة كيفية resuspend hNSPCs والعد. إعداد <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تأكد من أن هناك الكثير من الخلايا المتاحة لترنسفكأيشن (أن عدة ملايين من الخلايا transfected لكل DNA). </li><li style=";text-align:right;direction:rtl"> معطف زراعة الأنسجة طبق ، الذي سيتم المصنف الخلايا transfected ، مع 10 ميكروغرام / مل فبرونيكتين الإنسان في الليلة التي سبقت ترنسفكأيشن. الحق قبل البدء في بروتوكول ترنسفكأيشن ، إزالة فبرونيكتين ، شطف الطبق مع برنامج تلفزيوني ، الثقافة والإعلام في مكان الطبق. يترك الطبق في 37 ° C زراعة الأنسجة حاضنة دافئة للوسائل الإعلام. أيضا ، ضع 1 مل من وسائل الإعلام الثقافة في حاضنة دافئة لمرحلة ما قبل إلى 37 درجة مئوية. </li><li style=";text-align:right;direction:rtl"> مكان الحمض النووي (ق) ليكون transfected ، والحل ترنسفكأيشن (من Amaxa) على الجليد. </li></ol> ترنسفكأيشن <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> يغسل عدد محدد من الخلايا التي تحتوي على برنامج تلفزيوني (ونحن عادة استخدام ~ 5 × 106 خلية لكل ترنسفكأيشن). بيليه الخلايا بواسطة الطرد المركزي (1000 دورة في الدقيقة (~ 200xg) لمدة 5 دقائق) ، وإزالة أكبر قدر ممكن من برنامج تلفزيوني ، وresuspend الكرية الخلية في 100 ميكرولتر من الحل ترنسفكأيشن قدمت مع عدة ترنسفكأيشن. تعليق نقل الخلية إلى أنبوب 1.7 مل إيبندورف. </li><li style=";text-align:right;direction:rtl"> إضافة 5 ميكرولتر من الحمض النووي (~ 50-10 ميكروغرام من الحمض النووي لكل ترنسفكأيشن) الى تعليق الخلية والمزيج بلطف. </li><li style=";text-align:right;direction:rtl"> باستخدام Amaxa مصغرة ماصة ، ونقل المزيج خلية الحمض النووي في كفيت ترنسفكأيشن Amaxa. تأكد أن لديك 1 مل من وسائل الاعلام ثقافة ما قبل تحسنت للخطوة التالية. أيضا ، إزالة الطبق الخلية مع وسائل الاعلام من حاضنة الثقافة ووضعه في نسيج الثقافة هود. </li><li style=";text-align:right;direction:rtl"> ضع كفيت في Nucleofector ، تدوير عجلة عقارب الساعة ، وحدد برنامج &quot;A - 033&quot;. أدخل الصحافة. </li><li style=";text-align:right;direction:rtl"> يشار إلى ترنسفكأيشن الناجحة التي قام بها وجود طبقة كثيفة جدا من الفقاعات على رأس الحل في كفيت. إضافة 500 وسائل الإعلام في الحارة ميكرولتر كفيت ، واستخدام Amaxa صغيرة ماصة ، ونقل الخلايا transfected في الطبق مع تحسنت قبل وسائل الإعلام. شطف كفيت مع وسائل الإعلام أكثر دافئ وإضافة إلى شطف الطبق نفسه. مكان الطبق مع الخلايا في زراعة الأنسجة 37 ° C الحاضنة. </li></ol>

<ol>
	<li>Gresch, O., Engel, F. B., Nesic, D., Tran, T. T., England, H. M., Hickman, E. S., Krner, I., Gan, L., Chen, S., Castro-Obregon, S., Hammermann, R., Wolf, J., M ller-Hartmann, H., Nix, M., Siebenkotten, G., Kraus, G., Lun, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+non-viral+method+for+gene+transfer+into+primary+cells.">New non-viral method for gene transfer into primary cells.</a> Methods. 33, 151-163 (2004).</li><li>Marchenko, S., Flanagan, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Passaging+Human+Neural+Stem+Cells.">Passaging Human Neural Stem Cells.</a> Journal of Visualized Experiments. 7, (2007).</li><li>Marchenko, S., Flanagan, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Counting+Human+Neural+Stem+Cells.">Counting Human Neural Stem Cells.</a> Journal of Visualized Experiments. 7, (2007).</li></ol>

يصف هذا البروتوكول إجراء ترنسفكأيشن سريع نسبيا وفعالة لhNSPCs. باستخدام هذا الإجراء ، لقد حصلنا على الكفاءة ترنسفكأيشن الأولي 35 ٪ ~. ويمكن استخدام الخلايا transfected بواسطة هذا الإجراء على المدى القصير دراسات (خلايا transfected عابر) أو على المدى الطويل إذا تم استخدام الدراسات عامل التحديد ?...

<table><tbody><tr><td>The Nucleofector Device</td><td>Tool</td><td>Amaxa</td><td>AAD-1001</td><td></td></tr><tr><td>Mouse neural stem cell nucleofector kit</td><td>Reagent</td><td>Amaxa</td><td>VPG-1004</td><td></td></tr></tbody></table>

transfecting human neural stem cells with the amaxa nucleofector

وقد أدى ترنسفكأيشن من الخلايا العصبية في الثدييات الأولية ، مثل الخلايا الجذعية البشرية / السلائف العصبي (hNSPCs) ، مع استخداما الموجبة الكواشف ترنسفكأيشن الدهون غالبا في بقاء الخلية الفقراء وذوي الكفاءة ترنسفكأيشن. محدودة أيضا الطرق الميكانيكية الأخرى لإدخال الجين في المصالح ، مثل مدفع الجينات أو microinjection ، من خلال بقاء الخلية الفقراء وانخفاض أعداد خلايا transfected. وقد تم تقييد استخدام استراتيجية يبني الفيروسية لإدخال جينة في الخلايا الخارجية الابتدائي بحلول المبلغ كل من الوقت والعمل المطلوب لإنشاء نواقل الفيروسية ومخاوف تتعلق بالسلامة المحتملة. نحن هنا وصف بروتوكول خطوة بخطوة لtransfecting hNSPCs باستخدام جهاز لAmaxa Nucleofector والتكنولوجيا مع معلمات التيار الكهربائي والحلول العازلة على وجه التحديد الأمثل لtransfecting الخلايا الجذعية العصبية. باستخدام هذا البروتوكول ، حققنا الكفاءة ترنسفكأيشن الأولي 35 ٪ ~ ~ و 70 ٪ بعد ترنسفكأيشن مستقرة. بروتوكول يستلزم الجمع بين عدد كبير من hNSPCs مع الحمض النووي ليكون transfected في المنطقة العازلة المناسبة تليها electroporation مع الجهاز Nucleofector.

Watch this Scientific Journal Video about Transfecting Human Neural Stem Cells with the Amaxa Nucleofector at JoVE.com

Transfecting Human Neural Stem Cells with the Amaxa Nucleofector

إدخال الجينات في المصالح في خلية هي طريقة فعالة لتوضيح وظيفتها في الجسم الحي. يصف هذا البروتوكول وسيلة فعالة لtransfecting ثقافة الخلايا الجذعية البشرية / السلائف العصبي (hNSPCs) باستخدام جهاز electroporation Nucleofector التي Amaxa.

Transfecting الإنسان الخلايا الجذعية العصبية مع Nucleofector Amaxa

Transfection of primary mammalian neural cells, such as human neural stem/precursor cells (hNSPCs), with commonly used cationic lipid transfection ...

transfecting-human-neural-stem-cells-with-the-amaxa-nucleofector

Research

JoVE Journal

Biology

23.5K Views.  University of California, Irvine (UCI). إدخال الجينات في المصالح في خلية هي طريقة فعالة لتوضيح وظيفتها في الجسم الحي. يصف هذا البروتوكول وسيلة فعالة لtransfecting ثقافة الخلايا الجذعية البشرية / السلائف العصبي (hNSPCs) باستخدام جهاز electroporation Nucleofector التي Amaxa.

Video: Transfecting Human Neural Stem Cells with the Amaxa Nucleofector

Direct Induction of Human Neural Stem Cells from Peripheral Blood Hematopoietic Progenitor Cells

Human disease specific neuronal cultures are essential for generating in vitro models for human neurological diseases. However, the lack of access to primary human adult neural cultures raises unique challenges. Recent developments in induced pluripotent stem cells (iPSC) provides an alternative approach to derive neural cultures from skin fibroblasts through patient specific iPSC, but this process is labor intensive, requires special expertise and large amounts of resources, and can take several months. This prevents the wide application of this technology to the study of neurological diseases. To overcome some of these issues, we have developed a method to derive neural stem cells directly from human adult peripheral blood, bypassing the iPSC derivation process. Hematopoietic progenitor cells enriched from human adult peripheral blood were cultured in vitro and transfected with Sendai virus vectors containing transcriptional factors Sox2, Oct3/4, Klf4, and c-Myc. The transfection results in morphological changes in the cells which are further selected by using human neural progenitor medium containing basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF). The resulting cells are characterized by the expression for neural stem cell markers, such as nestin and SOX2. These neural stem cells could be further differentiated to neurons, astroglia and oligodendrocytes in specified differentiation media. Using easily accessible human peripheral blood samples, this method could be used to derive neural stem cells for further differentiation to neural cells for in vitro modeling of neurological disorders and may advance studies related to the pathogenesis and treatment of those diseases.

A method was developed to directly derive human neural stem cells from hematopoietic progenitor cells enriched from peripheral blood cells.

الحث المباشر من العصبية الإنسان الخلايا الجذعية من خلايا الدم المحيطي المكونة للدم السلف

Human disease specific neuronal cultures are essential for generating in vitro models for human neurological diseases. However, the lack of access to ...

Developmental Biology

Transfection, Selection, and Colony-picking of Human Induced Pluripotent Stem Cells TALEN-targeted with a GFP Gene into the AAVS1 Safe Harbor

Targeted transgene addition can provide persistent gene expression while circumventing the gene silencing and insertional mutagenesis caused by viral vector mediated random integration. This protocol describes a universal and efficient transgene targeted addition platform in human iPSCs based on utilization of validated open-source TALENs and a gene-trap-like donor to deliver transgenes into a safe harbor locus. Importantly, effective gene editing is rate-limited by the delivery efficiency of gene editing vectors. Therefore, this protocol first focuses on preparation of iPSCs for transfection to achieve high nuclear delivery efficiency. When iPSCs are dissociated into single cells using a gentle-cell dissociation reagent and transfected using an optimized program, >50% cells can be induced to take up the large gene editing vectors. Because the AAVS1 locus is located in the intron of an active gene (PPP1R12C), a splicing acceptor (SA)-linked puromycin resistant gene (PAC) was used to select targeted iPSCs while excluding random integration-only and untransfected cells. This strategy greatly increases the chance of obtaining targeted clones, and can be used in other active gene targeting experiments as well. Two weeks after puromycin selection at the dose adjusted for the specific iPSC line, clones are ready to be picked by manual dissection of large, isolated colonies into smaller pieces that are transferred to fresh medium in a smaller well for further expansion and genetic and functional screening. One can follow this protocol to readily obtain multiple GFP reporter iPSC lines that are useful for in vivo and in vitro imaging and cell isolation.

TALEN-mediated gene editing at the safe harbor AAVS1 locus enables high-efficiency transgene addition in human iPSCs. This protocol describes the procedures for preparing iPSCs for TALEN and donor vector delivery, transfecting iPSCs, and selecting and isolating iPSC clones to achieve targeted integration of a GFP gene to generate reporter lines.

ترنسفكأيشن، اختيار، ومستعمرة قطف من صنع الإنسان المحفزة الخلايا الجذعية TALEN المستهدفة مع جين GFP في AAVS1 الملاذ الآمن

Targeted transgene addition can provide persistent gene expression while circumventing the gene silencing and insertional mutagenesis caused by viral ...

Generation of Neural Stem Cells from Discarded Human Fetal Cortical Tissue

Neural stem cells (NSCs) reside along the ventricular zone neuroepithelium during the development of the cortical plate. These early progenitors ultimately give rise to intermediate progenitors and later, the various neuronal and glial cell subtypes that form the cerebral cortex. The capacity to generate and expand human NSCs (so called neurospheres) from discarded normal fetal tissue provides a means with which to directly study the functional aspects of normal human NSC development 1-5. This approach can also be directed toward the generation of NSCs from known neurological disorders, thereby affording the opportunity to identify disease processes that alter progenitor proliferation, migration and differentiation 6-9. We have focused on identifying pathological mechanisms in human Down syndrome NSCs that might contribute to the accelerated Alzheimer's disease phenotype 10,11. Neither in vivo nor in vitro mouse models can replicate the identical repertoire of genes located on human chromosome 21. 
Here we use a simple and reliable method to isolate Down syndrome NSCs from aborted human fetal cortices and grow them in culture. The methodology provides specific aspects of harvesting the tissue, dissection with limited anatomical landmarks, cell sorting, plating and passaging of human NSCs. We also provide some basic protocols for inducing differentiation of human NSCs into more selective cell subtypes.

A simple and reliable method on isolation and culture of neural stem cells from discarded human fetal cortical tissue is described. Cultures derived from known human neurological disorders can be used for characterization of pathological cellular and molecular processes, as well as provide a platform to assess pharmacological efficacy.

جيل من الخلايا الجذعية العصبية من الأنسجة الجنينية البشرية المهملة القشرية

Neural stem cells (NSCs) reside along the ventricular zone neuroepithelium during the development of the cortical plate. These early progenitors ...

Neuroscience

Introducing Point Mutations into Human Pluripotent Stem Cells Using Seamless Genome Editing

Custom designed endonucleases, such as RNA-guided Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9, enable efficient genome editing in mammalian cells. Here we describe detailed procedures to seamlessly genome edit the hepatocyte nuclear factor 4 alpha (HNF4&#945;) locus as an example in human pluripotent stem cells. Combining a piggyBac-based donor plasmid and the CRISPR-Cas9 nickase mutant in a two-step genetic selection, we demonstrate correct and efficient targeting of the HNF4&#945; locus.

Here, we describe a detailed method for seamless gene editing in human pluripotent stem cells using a piggyBac-based donor plasmid and the Cas9 nickase mutant. Two point mutations were introduced into exon 8 of the hepatocyte nuclear factor 4 alpha (HNF4&#945;) locus in human embryonic stem cells (hESCs).

إدخال طفرات نقطية في الخلايا الجذعية البشرية متعددة القدرات باستخدام تحرير الجينوم السلس

Custom designed endonucleases, such as RNA-guided Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9, enable efficient genome editing ...

Genetics

Transfecting and Nucleofecting Human Induced Pluripotent Stem Cells

Genetic modification is continuing to be an essential tool in studying stem cell biology and in setting forth potential clinical applications of human embryonic stem cells (HESCs)1. While improvements in several gene delivery methods have been described2-9, transfection remains a capricious process for HESCs, and has not yet been reported in human induced pluripotent stem cells (iPSCs). In this video, we demonstrate how our lab routinely transfects and nucleofects human iPSCs using plasmid with an enhanced green fluorescence protein (eGFP) reporter. Human iPSCs are adapted and maintained as feeder-free cultures to eliminate the possibility of feeder cell transfection and to allow efficient selection of stable transgenic iPSC clones following transfection. For nucleofection, human iPSCs are pre-treated with ROCK inhibitor11, trypsinized into small clumps of cells, nucleofected and replated on feeders in feeder cell-conditioned medium to enhance cell recovery. Transgene-expressing human iPSCs can be obtained after 6 hours. Antibiotic selection is applied after 24 hours and stable transgenic lines appear within 1 week. Our protocol is robust and reproducible for human iPSC lines without altering pluripotency of these cells.

Despite recent advancements in genetic modification, transfection of human embryonic stem cells (HESCs) remains a capricious process. To our knowledge, systematic and efficient methods to transfect human induced pluripotent stem cells (iPSCs) have not been reported. Here, we describe robust protocols to efficiently transfect and nucleofect human iPSCs.

وبنقل العدوى إلى الإنسان Nucleofecting الجذعية المحفزة المستحثة خلايا

Genetic modification is continuing to be an essential tool in studying stem cell biology and in setting forth potential clinical applications of human ...

Zinc-finger Nuclease Enhanced Gene Targeting in Human Embryonic Stem Cells

One major limitation with current human embryonic stem cell (ESC) differentiation protocols is the generation of heterogeneous cell populations.&#160;These cultures contain the cells of interest, but are also contaminated with undifferentiated ESCs, non-neural derivatives and other neuronal subtypes.&#160; This limits their use in in vitro and in vivo applications, such as in vitro modeling for drug discovery or cell replacement therapy. To help overcome this, reporter cell lines, which offer a means to visualize, track and isolate cells of interest, can be engineered.&#160;However, to achieve this in human embryonic stem cells via conventional homologous recombination is extremely inefficient.&#160;This protocol describes targeting of the Pituitary homeobox 3 (PITX3) locus in human embryonic stem cells using custom designed zinc-finger nucleases, which introduce site-specific double-strand DNA breaks, together with a PITX3-EGFP-specific DNA donor vector. Following the generation of the PITX3 reporter cell line, it can then be differentiated using published protocols for use in studies such as in vitro Parkinson&#8217;s disease modeling or cell replacement therapy.

Reporter cell lines offer a means to visualize, track and isolate cells of interest from heterogeneous populations.&#160;However, gene-targeting using conventional homologous recombination in human embryonic stem cells is extremely inefficient. Herein, we describe targeting CNS midbrain specific transcription factor PITX3 locus with EGFP using zinc-finger nuclease enhanced homologous recombination.

الزنك الاصبع نوكلياز المحسن استهداف الجينات في الخلايا الجذعية الجنينية البشرية

One major limitation with current human embryonic stem cell (ESC) differentiation protocols is the generation of heterogeneous cell ...

<meta charset="utf8"></head><body>كفاءة النواة من NSCs المعزولة من منطقة البطين تحت البطين الفئران

Isolation, Expansion, and Nucleofection of Neural Stem Cells from Adult Murine Subventricular Zone

Isolation and expansion of neural stem cells (NSCs) from the subventricular zone (SVZ) of the adult mouse brain can be achieved in a medium supplemented with basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) as mitogens, producing clonal aggregates known as neurospheres. This in vitro system is a valuable tool for studying NSC potential. Transfection of siRNAs or genes carried in plasmids can be used to induce perturbations to gene expression and study NSC biology. However, the exogenous nucleic acid delivery to NSC cultures is challenging due to the low efficiency of central nervous system (CNS) cells transfection. Here, we present an improved nucleofection system that achieves high efficiency of gene delivery in expanded NSCs from adult murine SVZ. We demonstrate that this relatively simple method enhances gene perturbation in adult NSCs, surpassing traditional transfection protocols with survival rates exceeding 80%. Moreover, this method can also be applied in primary isolated NSCs, providing a crucial advancement in gene function studies through gene expression manipulation via knockdown or overexpression in neurosphere cultures.

<meta charset="utf8"></head><body>تسليط الضوء على المؤلف: تحسين النواة لتوصيل الجينات عالية الكفاءة في مزارع الخلايا الجذعية العصبية المشتقة من منطقة البطين تحت البطين

Here, we describe a nucleofection system designed to enhance gene delivery efficiency in expanded neural stem cells (NSCs) isolated from the adult murine subventricular zone. The findings demonstrate that this method significantly improves gene perturbation in NSCs, surpassing the effectiveness of traditional transfection protocols and enhancing cell survival rate.

عزل وتوسع ونواة الخلايا الجذعية العصبية من منطقة تحت البطين الفئران البالغة

Isolation and expansion of neural stem cells (NSCs) from the subventricular zone (SVZ) of the adult mouse brain can be achieved in a medium supplemented ...

Neuronal Transfection Methods

Transfection - the process of transferring genetic material into cells - is a powerful tool for the rapid and efficient manipulation of gene expression in cells. Because this method can be used to silence the expression of specific proteins or to drive the expression of foreign or modified proteins, transfection is an extremely useful tool in the study of the cellular and molecular processes that govern neuron function. However, mature neurons have a number of properties that make them difficult to transfect, so specialized techniques are required for the genetic manipulation of this cell type. 	This video reviews the principles and rationale behind transfecting neurons. Three common strategies for neuronal transfection are discussed, including nucleofection, gene-gun, and viral transduction. In addition to describing how each of these techniques overcomes the challenges associated with transfecting neurons, the presentation includes a description of how all three methods are performed. Finally, several applications of neuronal transfection are introduced, such as the expression of fluorescent tubulin proteins to visualize neuron morphology, and selective gene silencing to generate a cell culture model of Parkinson&#8217;s disease.

Neuronal Transfection and Nucleofection Methods

Transfection - the process of transferring genetic material into cells - is a powerful tool for the rapid and efficient manipulation of gene expression in ...

Transfecting الإنسان الخلايا الجذعية العصبية مع Nucleofector Amaxa

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الحث المباشر من العصبية الإنسان الخلايا الجذعية من خلايا الدم المحيطي المكونة للدم السلف

ترنسفكأيشن، اختيار، ومستعمرة قطف من صنع الإنسان المحفزة الخلايا الجذعية TALEN المستهدفة مع جين GFP في AAVS1 الملاذ الآمن

جيل من الخلايا الجذعية العصبية من الأنسجة الجنينية البشرية المهملة القشرية

إدخال طفرات نقطية في الخلايا الجذعية البشرية متعددة القدرات باستخدام تحرير الجينوم السلس

وبنقل العدوى إلى الإنسان Nucleofecting الجذعية المحفزة المستحثة خلايا

الزنك الاصبع نوكلياز المحسن استهداف الجينات في الخلايا الجذعية الجنينية البشرية

عزل وتوسع ونواة الخلايا الجذعية العصبية من منطقة تحت البطين الفئران البالغة

عزل وتوسع ونواة الخلايا الجذعية العصبية من منطقة تحت البطين الفئران البالغة

عزل وتوسع ونواة الخلايا الجذعية العصبية من منطقة تحت البطين الفئران البالغة

Neuronal Transfection Methods