وأيد هذا العمل من المنح المقدمة لJLRR من: التوحد يتحدث، نينا ايرلندا، مؤسسة الملاذات ويستون، NIMH R01 MH081880، وNIMH R37 MH049428. وأيد PRW من قبل زمالة من مجلس العلوم الوطني لتايوان. وأيد SFS التي كتبها F32 (MH103003).

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The authors declare that they have no competing financial interests.

استخدام GABAergic السلائف عصبون القشرية من الفضيلة العقدية الجنينية (غيس) للعلاجات الخلايا استنادا تبدو واعدة للعديد من الحالات 12 -1 4. وهناك حاجة إلى تقنيات جزيئية دقيقة لتتبع، والتعبير عن جينات الفائدة في مجموعات فرعية عصبون محددة. هنا نقدم بروتوكول مفصلة لوصفها ا...

تشمل GABAergic interneurons القشرية ~ 20-30٪ من الخلايا العصبية في القشرة المخية الحديثة الثدييات، في حين أن بقية تتوافق مع مثير، الخلايا العصبية الرئيسية glutamatergic. Interneurons ومتنوعة للغاية في الخصائص الكهربية، محور عصبي والتغصنات التشكل ومتشابك استهداف 1، وافترض الاختلالات في مثير لهجة / المثبطة لتكمن وراء بعض الظواهر من الاضطرابات العصبية والنفسية / العصبية بما في ذلك مرض التوحد والفصام والصرع 2. الهدف العام للبروتوكول الموصوفة هنا هو توفير وسيلة لتعديل بكفاءة وراثيا GABAergic الأسلاف عصبون القشرية قبل زرع في الجسم الحي التحليلات. ولدت interneurons القشرية GABAergic في الفضيلة العقدية وسطي والذيلية (MGE وفريق الخبراء الاستشاري، على التوالي) 3،4 وكذلك الباحة أمام البصرية 5. الأسلاف عصبون القشرية تخضع جاءت لمسافات طويلة الهجرة عرضيةالهجرة شعاعي للوصول إلى الأهداف النهائية. لدى وصولهم إلى وجهاتهم، يجب هذه interneurons القشرية تدمج بشكل صحيح في شبكة الخلايا العصبية الموجودة، وسيكون على كل عصبون فرعية فريدة تسهم في الدوائر القشرية بطرق محددة. أربع مجموعات فرعية رئيسية يمكن تمييزها بواسطة الواسمات الجزيئية: السوماتوستاتين MGE المشتقة (SST) + وparvalbumin (PV) + مجموعات فرعية، وفريق الخبراء الاستشاري المستمدة من الببتيد المعوي الفعال في الأوعية (VIP) + وريلين reelin +. SST - مجموعات فرعية 6. ولدت مختلفة مجموعات فرعية عصبون القشرية على أوقات مختلفة أثناء التطور الجنيني في MGE وفريق الخبراء الاستشاري 7 و 8. وقد استخدمت هذه وغيرها من القشرية GABAergic علامات عصبون لتوليد خطوط جنة المساواة العرقية سائق محددة للعديد من هذه المجموعات الفرعية 9-11. برزت زرع الأسلاف MGE كعلاج خلية القاعدة المحتملة لعلاج الاضطرابات التي قد تكون ناجمة عن اختلال التوازنفي الإثارة / تثبيط 12 - 24. قد تكون هذه الفوائد العلاجية يرجع ذلك جزئيا إلى قدرة فريدة من الأسلاف MGE (لتفريق، التفريق والاندماج في الدماغ المضيف)، أو يحتمل أن تكون لأنه يتم الحصول العديد من شبه جسدية PV المثبطة + الخلايا من MGE. خلايا MGE يمكن أن يكون أيضا بسرعة وكفاءة transduced مع lentiviruses قبل زرع 15، مما يسمح للخلايا التي يتم تعديلها وراثيا في المختبر ليتم دراستها في الجسم الحي. كان المبرر لتطوير هذا النهج للتغلب على الحواجز في دراسة GABAergic تطوير عصبون القشرية والنضج. على وجه الخصوص، يسمح زرع MGE الباحثين لدراسة تطوير خلايا متحولة في الجسم الحي، عندما الماوس متحولة، لولا ذلك لقوا حتفهم في نقطة الوقت المبكر. وعلاوة على ذلك، من خلال إدخال جينات من الفائدة قبل الزرع، يمكن تقييم آثار جينات معينة على النمط الظاهري متحولة في المؤسسة فكرةالطريقة icient. هنا، ونحن نقدم بروتوكول مفصلة لتنبيغ الخلايا MGE مع lentiviruses قبل الزرع. وبالإضافة إلى ذلك، وتبين لنا كيف يمكن تكييف هذه التقنية للتعبير عن الجينات في المصالح في مجموعات فرعية عصبون محددة من مجموعة غير متجانسة من السلائف عصبون القشرية، وذلك باستخدام مزيج من lentiviruses التعبير لجنة المساواة العرقية التي تعتمد على والمتاحة خطوط الماوس لجنة المساواة العرقية سائق. وعلاوة على ذلك، فإن هذا البروتوكول يقدم تقنيات ومنبرا للباحثين لتعديل وراثيا GABAergic السلائف عصبون القشرية لفي الدراسات المجراة بطريقة فريدة من نوعها. ميزة واحدة من هذه التقنية على النهج الحالية الأخرى هي أن الخلايا المزروعة MGE سوف تفريق بعيدا عن موقع الحقن. أيضا، على عكس الحقن الفيروسية التنسيق، بعد تفريق خلايا MGE التشكل هو أسهل لتقييم. هذا النهج يمكن أن تستخدم لدراسة تأثير إدخال جينات من الفائدة في النوع البري أو خلايا متحولة، وإدخال نوع من الخلايا المحددةمراسل لتقييم التشكل، أو يحتمل أن تكون لدراسة تأثير الأليلات المرض في الجسم الحي.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Reagents and equipment for MGE cell transplantation</td>
		</tr>
		<tr>
			<td>1 ml syringe</td>
			<td>REF 309602</td>
			<td colspan="2">BD, Becton Dickinson and company</td>
		</tr>
		<tr>
			<td>301/2 gauge needle</td>
			<td>305106</td>
			<td colspan="2">BD, Becton Dickinson and company</td>
		</tr>
		<tr>
			<td>Precision bore to deliver 5 &#181;l (comes with plunger)</td>
			<td>5-000-1005</td>
			<td colspan="2">Drummond scientific company</td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>PM-999</td>
			<td colspan="2">Polysciences, Inc.</td>
		</tr>
		<tr>
			<td>Stereo microscope with boomstand **</td>
			<td>MZ6</td>
			<td>Leica</td>
			<td></td>
		</tr>
		<tr>
			<td>Digital just for mice stereotaxic instrument **</td>
			<td>51725D</td>
			<td colspan="2">Stoelting company</td>
		</tr>
		<tr>
			<td>Single-axis oil hydraulic fine micromanipulator **</td>
			<td>MO-10</td>
			<td>Narishige</td>
			<td></td>
		</tr>
		<tr>
			<td>Diamond coated rotary beveler</td>
			<td>n.a.</td>
			<td colspan="2">made in house</td>
		</tr>
		<tr>
			<td>Needle pipette puller</td>
			<td>730</td>
			<td colspan="2">Kopf instruments</td>
		</tr>
		<tr>
			<td>Mineral oil</td>
			<td>n.a.</td>
			<td colspan="2">Any drug store or pharmacy</td>
		</tr>
		<tr>
			<td colspan="2">Any pipette and tips that can reliaby measure 1 &#181;l of volume</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="2">** These are the particular models we use, but many other setups should work</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Optional Reagents (for making Lentiviruses)</td>
		</tr>
		<tr>
			<td>25 mm, 0.45 &#181;m filter</td>
			<td>09-719B</td>
			<td colspan="2">Fisher Scientific</td>
		</tr>
		<tr>
			<td>Ultra-clear centrifuge tubes (25 x 89 mm)</td>
			<td>344058</td>
			<td colspan="2">Beckman Coulter&#174;</td>
		</tr>
		<tr>
			<td>Lipofectamine 2000 (1.5 ml size)</td>
			<td>11668019</td>
			<td colspan="2">Invitrogen</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Other commercially available supplies</td>
		</tr>
		<tr>
			<td>pMDLg/pRRE plasmid encoding gag/pol</td>
			<td>12251</td>
			<td>Addgene</td>
			<td></td>
		</tr>
		<tr>
			<td>pRSV-Rev plasmid encoding Rev</td>
			<td>12253</td>
			<td>Addgene</td>
			<td></td>
		</tr>
		<tr>
			<td>pMD2.G plasmid encoding VSVG</td>
			<td>12259</td>
			<td>Addgene</td>
			<td></td>
		</tr>
		<tr>
			<td>pAAV-Flex-GFP</td>
			<td>28304</td>
			<td>Addgene</td>
			<td></td>
		</tr>
	</tbody>
</table>

viral-mediated labeling and transplantation of medial ganglionic eminence (mge) cells for in vivo studies

GABAergic interneurons القشرية، والمستمدة من الإنسي الجنينية والفضيلة العقدية الذيلية (MGE وفريق الخبراء الاستشاري)، وظيفيا ومتنوعة شكليا. من إحراز تقدم في فهم أدوار متميزة مجموعات فرعية عصبون القشرية، ومع ذلك، لا يزال هناك العديد من الآليات التي عملت بها التي قد تسهم في تطوير ونضوج أنواع مختلفة من الخلايا GABAergic. وعلاوة على ذلك، يمكن أن تسهم تغيير GABAergic يشير إلى الظواهر التوحد والفصام والصرع. وقد بدأت لجنة المساواة العرقية خطوط سائق محددة للا يتجزأ من مهام فرعية عصبون فريدة من نوعها. وعلى الرغم من التقدم في نماذج الماوس، غالبا ما يكون من الصعب دراسة GABAergic الأسلاف عصبون القشرية بكفاءة مع النهج الجزيئية في الجسم الحي. أسلوب واحد الهامة المستخدمة لدراسة الخلية البرمجة مستقلة من هذه الخلايا هي زرع الخلايا MGE إلى القشور المضيف. هذه الخلايا المزروعة تهاجر على نطاق واسع، تمييز، وهوالثانية دمج وظيفيا. وبالإضافة إلى ذلك، وخلايا MGE يمكن transduced بكفاءة مع الفيروسة البطيئة السابقة على الفور لزراعة الأعضاء، مما يسمح للعديد من الأساليب الجزيئية. نحن هنا بالتفصيل بروتوكول لتنبيغ الخلايا MGE بكفاءة قبل زرع في الجسم الحي التحليل، وذلك باستخدام خطوط جنة المساواة العرقية سائق المتاحة وناقلات التعبير لجنة المساواة العرقية التي تعتمد على. هذا النهج هو مفيد لأنه يجمع بين التلاعب الجيني الدقيق مع قدرة هذه الخلايا لتفريق بعد الزرع، والسماح بمزيد من قرار معين الخلية من نوع في الجسم الحي.

منذ خلايا MGE لديها قدرة فريدة على الهجرة والاندماج عندما زرعها في القشرة المخية الحديثة المضيف 16، لأنها توفر نظام نموذجا ممتازا للتلاعب الجيني قبل الدراسات في الجسم الحي. هنا، وتبين لنا كيف يمكن للمرء أن عزل الأنسجة MGE من أجنة E13.5 (الشكلان 1 و 2)، وال...

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Viral-mediated Labeling and Transplantation of Medial Ganglionic Eminence MGE Cells for In Vivo Studies

GABAergic الأسلاف عصبون القشرية تفريق وتطوير وsynaptically الاندماج في القشرة المضيف بعد الزرع. هذه الخلايا يمكن transduced بسهولة قبل الزرع لفي الدراسات المجراة السلائف GABAergic المعدلة وراثيا. هنا، وتبين لنا تقنيات وضع العلامات الفيروسية لاستهداف مجموعات فرعية عصبون محددة باستخدام خطوط جنة المساواة العرقية القائمة وصحفيين لجنة المساواة العرقية التي تعتمد على.

وصفها والفيروسية بوساطة زرع الإنسي العقدية سماحة (MGE) خلايا ل<em&gt; في فيفو</em&gt; دراسات

GABAergic cortical interneurons, derived from the embryonic medial and caudal ganglionic eminences (MGE and CGE), are functionally and morphologically ...

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Developmental Biology

Viral-mediated Labeling and Transplantation of Medial Ganglionic Eminence (MGE) Cells for In Vivo Studies

11.4K Views.  University of California San Francisco.  GABAergic الأسلاف عصبون القشرية تفريق وتطوير وsynaptically الاندماج في القشرة المضيف بعد الزرع. هذه الخلايا يمكن transduced بسهولة قبل الزرع لفي الدراسات المجراة السلائف GABAergic المعدلة وراثيا. هنا، وتبين لنا تقنيات وضع العلامات الفيروسية لاسته?...

Video: Viral-mediated Labeling and Transplantation of Medial Ganglionic Eminence MGE Cells for In Vivo Studies

Structure-function Studies in Mouse Embryonic Stem Cells Using Recombinase-mediated Cassette Exchange

Gene engineering in mouse embryos or embryonic stem cells (mESCs) allows for the study of the function of a given protein. Proteins are the workhorses of the cell and often consist of multiple functional domains, which can be influenced by posttranslational modifications. The depletion of the entire protein in conditional or constitutive knock-out (KO) mice does not take into account this functional diversity and regulation. An mESC line and a derived mouse model, in which a docking site for FLPe recombination-mediated cassette exchange (RMCE) was inserted within the ROSA26 (R26) locus, was previously reported. Here, we report on a structure-function approach that allows for molecular dissection of the different functionalities of a multidomain protein. To this end, RMCE-compatible mice must be crossed with KO mice and then RMCE-compatible KO mESCs must be isolated. Next, a panel of putative rescue constructs can be introduced into the R26 locus via RMCE targeting. The candidate rescue cDNAs can be easily inserted between RMCE sites of the targeting vector using recombination cloning. Next, KO mESCs are transfected with the targeting vector in combination with an FLPe recombinase expression plasmid. RMCE reactivates the promoter-less neomycin-resistance gene in the ROSA26 docking sites and allows for the selection of the correct targeting event. In this way, high targeting efficiencies close to 100% are obtained, allowing for insertion of multiple putative rescue constructs in a semi-high throughput manner. Finally, a multitude of R26-driven rescue constructs can be tested for their ability to rescue the phenotype that was observed in parental KO mESCs. We present a proof-of-principle structure-function study in p120 catenin (p120ctn) KO mESCs using endoderm differentiation in embryoid bodies (EBs) as the phenotypic readout. This approach enables the identification of important domains, putative downstream pathways, and disease-relevant point mutations that underlie KO phenotypes for a given protein.

Proteins often contain multiple domains that can exert different cellular functions. Gene knock-outs (KO) do not consider this functional diversity. Here, we report a recombination-mediated cassette exchange (RMCE)-based structure-function approach in KO embryonic stem cells that allows for the molecular dissection of various functional domains or variants of a protein.

الدراسات هيكل الوظائف في الخلايا الجذعية الجنينية الماوس عن طريق بوساطة Recombinase-كاسيت تبادل

Gene engineering in mouse embryos or embryonic stem cells (mESCs) allows for the study of the function of a given protein. Proteins are the workhorses of ...

A Novel Mammary Fat Pad Transplantation Technique to Visualize the Vessel Generation of Vascular Endothelial Stem Cells

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper vessel development and homeostasis. However, studies on endothelial lineage hierarchy remain elusive due to the lack of tools to gain access as well as to directly evaluate their behavior in vivo. To address this shortcoming, a new tissue model to study angiogenesis using the mammary fat pad has been developed. The mammary gland develops mostly in the postnatal stages, including puberty and pregnancy, during which robust epithelium proliferation is accompanied by extensive vascular remodeling. Mammary fat pads provide space, matrix, and rich angiogenic stimuli from the growing mammary epithelium. Furthermore, mammary fat pads are located outside the peritoneal cavity, making them an easily accessible grafting site for assessing the angiogenic potential of exogenous cells. This work also describes an efficient tracing approach using fluorescent reporter mice to specifically label the targeted population of vascular endothelial stem cells (VESCs) in vivo. This lineage tracing method, coupled with subsequent tissue whole-mount microscopy, enable the direct visualization of targeted cells and their descendants, through which the proliferation capability can be quantified and the differentiation commitment can be fate-mapped. Using these methods, a population of bipotent protein C receptor (Procr) expressing VESCs has recently been identified in multiple vascular systems. Procr+ VESCs, giving rise to both new ECs and pericytes, actively contribute to angiogenesis during development, homeostasis, and injury repair. Overall, this manuscript describes a new mammary fat pad transplantation and in vivo lineage tracing techniques that can be used to evaluate the stem cell properties of VESCs.

This work demonstrates a novel approach to assess the proliferation, differentiation, and vessel-forming potential of vascular endothelial stem cells (VESCs) through mammary fat pad transplantation followed by whole-mount tissue preparation for microscopic observation. A lineage tracing strategy to investigate the behavior of VESCs in vivo is also presented.

تقنية زرع الثدي الثديية الرواية جديدة لتصور الجيل سفينة من الخلايا الجذعية البطانية الأوعية الدموية

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper ...

Horizontal Whole Mount: A Novel Processing and Imaging Protocol for Thick, Three-dimensional Tissue Cross-sections of Skin

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality microscopic images is not entirely dependent upon the quality of the microscope, but also upon the methods of tissue processing, which often involve multiple critical actions or steps. Furthermore, mesenchymal cell types in the skin and other tissues represent a new challenge for tissue preparation and imaging. Here, we present a complete process, from tissue harvest to microscopy. Our technique, called &#34;horizontal whole mount,&#34; is one that novices can quickly become proficient in and that allows for antigen preservation and detection in 60-300 &#181;m-thick sections cut with a cryostat. Sections of this thickness provide enhanced visualization of tissue microarchitecture in a three-dimensional environment. In addition, the protocol preserves mesenchymal cells in a manner that enhances image quality when compared to standard cryostat or paraffin sections, thereby increasing the efficacy and reliability of immunostaining. We believe that this protocol will benefit all laboratories that visualize skin, and possibly other tissues and organs.

This work presents a novel processing and imaging protocol for thick, three-dimensional tissue cross-section analysis that enables the full exploitation of confocal imaging modalities. This protocol preserves antigenicity and represents a robust system to analyze skin histology and potentially other tissue types.

أفقي جبل كامل: A معالجة الرواية والتصوير بروتوكول للثدي، ثلاثة-- الأبعاد الأنسجة عبر-- أقسام الجلد

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality ...

Immuno-fluorescent Labeling of Microtubules and Centrosomal Proteins in Ex Vivo Intestinal Tissue and 3D In Vitro Intestinal Organoids

The advent of 3D in vitro organoids that mimic the in vivo tissue architecture and morphogenesis has greatly advanced the ability to study key biological questions in cell and developmental biology. In addition, organoids together with recent technical advances in gene editing and viral gene delivery promises to advance medical research and development of new drugs for treatment of diseases. Organoids grown in vitro in basement matrix provide powerful model systems for studying the behavior and function of various proteins and are well suited for live-imaging of fluorescent-tagged proteins. However, establishing the expression and localization of the endogenous proteins in ex vivo tissue and in in vitro organoids is important to verify the behavior of the tagged proteins. To this end we have developed and modified tissue isolation, fixation, and immuno-labeling protocols for localization of microtubules, centrosomal, and associated proteins in ex vivo intestinal tissue and in in vitro intestinal organoids. The aim was for the fixative to preserve the 3D architecture of the organoids/tissue while also preserving antibody antigenicity and enabling good penetration and clearance of fixative and antibodies. Exposure to cold depolymerizes all but stable microtubules and this was a key factor when modifying the various protocols. We found that increasing the ethylenediaminetetraacetic acid (EDTA) concentration from 3 mM to 30 mM gave efficient detachment of villi and crypts in the small intestine while 3 mM EDTA was sufficient for colonic crypts. The developed formaldehyde/methanol fixation protocol gave very good structural preservation while also preserving antigenicity for effective labeling of microtubules, actin, and the end-binding (EB) proteins. It also worked for the centrosomal protein ninein although the methanol protocol worked more consistently. We further established that fixation and immuno-labeling of microtubules and associated proteins could be achieved with organoids isolated from or remaining within the basement matrix.

Immuno-fluorescent Labeling of Microtubules and Centrosomal Proteins in Ex Vivo Intestinal Tissue and 3D In Vitro Intestinal Organoids

We present protocols for isolation of intestinal 3D structures from in vivo tissue and in vitro basement matrix embedded organoids, and detail different fixation and staining protocols optimized for immuno-labeling of microtubule, centrosomal, and junctional proteins as well as cell markers including the stem cell protein Lgr5.

وسم المناعية-فلوري Microtubules والبروتينات سينتروسومال في 3D في المختبر أورجانويدس الأمعاء والأنسجة المعوية السابقين فيفو

The advent of 3D in vitro organoids that mimic the in vivo tissue architecture and morphogenesis has greatly advanced the ability to study key biological ...

Single-cell Photoconversion in Living Intact Zebrafish

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause disease when disrupted. Scientists have developed clever techniques to investigate characteristics and natural dynamics of these cells within intact tissue by expressing fluorescent proteins in subsets of cells. However, at times, experiments require more selected visualization of cells within the tissue, sometimes at the single-cell or population-of-cells manner. To achieve this and visualize single cells within a population of cells, scientists have utilized single-cell photoconversion of fluorescent proteins. To demonstrate this technique, we show here how to direct UV light to an Eos-expressing cell of interest in an intact, living zebrafish. We then image those photoconverted Eos+ cells 24 h later to determine how they changed in the tissue. We describe two techniques: single cell photoconversion and photoconversions of populations of cell. These techniques can be used to visualize cell-cell interactions, cell-fate and differentiation, and cell migrations, making it a technique that is applicable in numerous biological questions.

Here, we present a protocol to show how cell photoconversion is achieved through UV exposure to specific areas expressing the fluorescent protein, Eos, in living animals.

فوتوكونفيرسيون خلية واحدة في معيشة سليمة الزرد

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause ...

Intravenous and Intra-amniotic In Utero Transplantation in the Murine Model

In utero transplantation (IUT) is a unique and versatile mode of therapy that can be used to introduce stem cells, viral vectors, or any other substances early in the gestation. The rationale behind IUT for therapeutic purposes is based on the small size of the fetus, the fetal immunologic immaturity, the accessibility and proliferative nature of the fetal stem or progenitor cells, and the potential to treat a disease or the onset of symptoms prior to birth. Taking advantage of these normal developmental properties of the fetus, the delivery of hematopoietic stem cells (HSC) via an IUT has the potential to treat congenital hematologic disorders such as sickle cell disease, without the required myeloablative or immunosuppressive conditioning required for postnatal HSC transplants. Similarly, the accessibility of progenitor cells in multiple organs during development potentially allows for a more efficient targeting of stem/progenitor cells following an IUT of viral vectors for gene therapy or genome editing. Additionally, IUT can be used to study normal developmental processes including, but not limited to, the development of immunologic tolerance. The murine model provides a valuable and affordable means to understanding the potential and limitations of IUT prior to pre-clinical large animal studies and an eventual clinical application. Here, we describe a protocol for performing an IUT in the murine fetus through intravenous and intra-amniotic routes. This protocol has been used successfully to elucidate the necessary conditions and mechanisms behind in utero hematopoietic stem cell transplantation, tolerance induction, and in utero gene therapy.

Intravenous and Intra-amniotic In Utero Transplantation in the Murine Model

We describe a protocol for performing an in utero transplantation (IUT) through intravenous and intra-amniotic routes of injection in the murine model. This protocol can be used to introduce cells, viral vectors, and other substances into the unique immune-tolerant fetal environment.

عن طريق الحقن الوريدي وداخل السلي في الرحم زرع في نموذج مورين

In utero transplantation (IUT) is a unique and versatile mode of therapy that can be used to introduce stem cells, viral vectors, or any other substances ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give rise to multiple cell types, including the myotome (MYO), sclerotome (SCL), dermatome (D), and syndetome (SYN), which in turn develop into skeletal muscle, axial skeleton, dorsal dermis, and axial tendon/ligament, respectively. Therefore, the generation of SMs and their derivatives from human induced pluripotent stem cells (iPSCs) is critical to obtain pluripotent stem cells (PSCs) for application in regenerative medicine and for disease research in the field of orthopedic surgery. Although the induction protocols for MYO and SCL from PSCs have been previously reported by several researchers, no study has yet demonstrated the induction of SYN and D from iPSCs. Therefore, efficient induction of fully competent SMs remains a major challenge. Here, we recapitulate human SM patterning with human iPSCs in vitro by mimicking the signaling environment during chick/mouse SM development, and report on methods of systematic induction of SM derivatives (MYO, SCL, D, and SYN) from human iPSCs under chemically defined conditions through the presomitic mesoderm (PSM) and SM states. Knowledge regarding chick/mouse&#160;SM development was successfully applied to the induction of SMs with human iPSCs. This method could be a novel tool for studying human somitogenesis and patterning without the use of embryos and for cell-based therapy and disease modeling.

We present here a protocol for the differentiation of human induced pluripotent stem cells into each somite derivative (myotome, sclerotome, dermatome, and syndetome) in chemically defined conditions, which has applications in future disease modeling and cell-based therapies in orthopedic surgery.

في المختبر توليد المشتقات سميط من البشرية التي يسببها الخلايا الجذعية Pluripotent

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...

Controlled Cortical Impact Model of Mouse Brain Injury with Therapeutic Transplantation of Human Induced Pluripotent Stem Cell-Derived Neural Cells

Traumatic brain injury (TBI) is a leading cause of morbidity and mortality worldwide. Disease pathology due to TBI progresses from the primary mechanical insult to secondary injury processes, including apoptosis and inflammation. Animal modeling has been valuable in the search to unravel injury mechanisms and evaluate potential neuroprotective therapies. This protocol describes the controlled cortical impact (CCI) model of focal, open-head TBI. Specifically, parameters for producing a mild unilateral cortical injury are described. Behavioral consequences of CCI are analyzed using the adhesive tape removal test of bilateral sensorimotor integration. Regarding experimental therapy for TBI pathology, this protocol also illustrates a process for transplanting cultured cells into the brain. Neural cell cultures derived from human induced pluripotent stem cells (hiPSCs) were chosen for their potential to show superior functional restoration in human TBI patients. Chronic survival of hiPSCs in the host mouse brain tissue is detected using a modified DAB immunohistochemical process.

This protocol demonstrates methodologies for a mouse model of open-skull traumatic brain injury and transplantation of cultured human induced pluripotent stem cell-derived cells into the injury site. Behavioral and histologic tests of outcomes from these procedures are also described in brief.

نموذج تأثير القشرية الخاضعة للرقابة لإصابة الدماغ الماوس مع زرع العلاجية من الخلايا العصبية المستمدة من الخلايا الجذعية المتعددة القوى التي يسببها الإنسان

Traumatic brain injury (TBI) is a leading cause of morbidity and mortality worldwide. Disease pathology due to TBI progresses from the primary mechanical ...

Generation of Induced Neural Stem Cells from Peripheral Mononuclear Cells and Differentiation Toward Dopaminergic Neuron Precursors for Transplantation Studies

Parkinson&#39;s disease (PD) is caused by degeneration of dopaminergic (DA) neurons at the substantia nigra pars compacta (SNpc) in the ventral mesencephalon (VM). Cell replacement therapy holds great promise for treatment of PD. Recently, induced neural stem cells (iNSCs) have emerged as a potential candidate for cell replacement therapy due to the reduced risk of tumor formation and the plasticity to give rise to region-specific neurons and glia. iNSCs can be reprogrammed from autologous somatic cellular sources, such as fibroblasts, peripheral blood mononuclear cells (PBMNCs) and various other types of cells. Compared with other types of somatic cells, PBMNCs are an appealing starter cell type because of the ease to access and expand in culture. Sendai virus (SeV), an RNA non-integrative virus, encoding reprogramming factors including human OCT3/4, SOX2, KLF4 and c-MYC, has a negative-sense, single-stranded, non-segmented genome that does not integrate into host genome, but only replicates in the cytoplasm of infected cells, offering an efficient and safe vehicle for reprogramming. In this study, we describe a protocol in which iNSCs are obtained by reprogramming PBMNCs, and differentiated into specialized VM DA neurons by a two-stage method. Then DA precursors are transplanted into unilaterally 6-hyroxydopamine (6-OHDA)-lesioned PD mouse models to evaluate the safety and efficacy for treatment of PD. This method provides a platform to investigate the functions and therapeutic effects of patient-specific DA neural cells in vitro and in vivo.

The protocol presents the reprogramming of peripheral blood mononuclear cells to induce neural stem cells by Sendai virus infection, differentiation of iNSCs into dopaminergic neurons, transplantation of DA precursors into the unilaterally-lesioned Parkinson&#39;s disease mouse models, and evaluation of the safety and efficacy of iNSC-derived DA precursors for PD treatment.

توليد الخلايا الجذعية العصبية المستحثة من الخلايا أحادية النووية الطرفية والتمايز نحو سلائف الخلايا العصبية الدوبامين لدراسات زرع

Parkinson&#39;s disease (PD) is caused by degeneration of dopaminergic (DA) neurons at the substantia nigra pars compacta (SNpc) in the ventral ...

Isolation of Rat Adipose Tissue Mesenchymal Stem Cells for Differentiation into Insulin-producing Cells

Mesenchymal stem cells (MSCs)-especially those isolated from adipose tissue (Ad-MSCs)-have gained special attention as a renewable, abundant source of stem cells that does not pose any ethical concerns. However, current methods to isolate Ad-MSCs are not standardized and employ complicated protocols that require special equipment. We isolated Ad-MSCs from the epididymal fat of Sprague-Dawley rats using a simple, reproducible method. The isolated Ad-MSCs usually appear within 3 days post isolation, as adherent cells display fibroblastic morphology. Those cells reach 80% confluency within 1 week of isolation. Afterward, at passage 3-5 (P3-5), a full characterization was carried out for the isolated Ad-MSCs by immunophenotyping for characteristic MSC cluster of differentiation (CD) surface markers such as CD90, CD73, and CD105, as well as inducing differentiation of these cells down the osteogenic, adipogenic, and chondrogenic lineages. This, in turn, implies the multipotency of the isolated cells. Furthermore, we induced the differentiation of the isolated Ad-MSCs toward the insulin-producing cells (IPCs) lineage via a simple, relatively short protocol by incorporating high glucose Dulbecco&#39;s modified Eagle medium (HG-DMEM), &#946;-mercaptoethanol, nicotinamide, and exendin-4. IPCs differentiation was genetically assessed, firstly, via measuring the expression levels of specific &#946;-cell markers such as MafA, NKX6.1, Pdx-1, and Ins1, as well as dithizone staining for the generated IPCs. Secondly, the assessment was also carried out functionally by a glucose-stimulated insulin secretion (GSIS) assay. In conclusion, Ad-MSCs can be easily isolated, exhibiting all MSC characterization criteria, and they can indeed provide an abundant, renewable source of IPCs in the lab for diabetes research.

Adipose tissue-derived mesenchymal stem cells (Ad-MSCs) can be a potential source of MSCs that differentiate into insulin-producing cells (IPCs). In this protocol, we provide detailed steps for the isolation and characterization of rat epididymal Ad-MSCs, followed by a simple, short protocol for the generation of IPCs from the same rat Ad-MSCs.

عزل الخلايا الجذعية الوسيطة للأنسجة الدهنية للفئران للتمايز إلى خلايا منتجة للأنسولين

Mesenchymal stem cells (MSCs)-especially those isolated from adipose tissue (Ad-MSCs)-have gained special attention as a renewable, abundant source of ...