وأيد هذا العمل جزئيا من خلال منحة المعاهد الوطنية للصحة K99EB009096 - 01A1 إلى JM - M.

<ol>
	<li>Melero-Martin, J. M., Bischoff, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+13.+An+in+vivo+experimental+model+for+postnatal+vasculogenesis.">Chapter 13. An in vivo experimental model for postnatal vasculogenesis.</a> Methods Enzymol. 445, 303-329 (2008).</li><li>Koike, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+engineering:+creation+of+long-lasting+blood+vessels.">Tissue engineering: creation of long-lasting blood vessels.</a> Nature. 428, 138-139 (2004).</li><li>Schechner, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+formation+of+complex+microvessels+lined+by+human+endothelial+cells+in+an+immunodeficient+mouse.">In vivo formation of complex microvessels lined by human endothelial cells in an immunodeficient mouse.</a> Proc Natl Acad Sci USA. 97, 9191-9196 (2000).</li><li>N&amp;ouml, r., E, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+and+characterization+of+functional+human+microvessels+in+immunodeficient+mice.">Engineering and characterization of functional human microvessels in immunodeficient mice.</a> Lab Invest. 81, 453-463 (2001).</li><li>Lin, Y., Weisdorf, D. J., Solovey, A., Hebbel, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Origins+of+circulating+endothelial+cells+and+endothelial+outgrowth+from+blood.">Origins of circulating endothelial cells and endothelial outgrowth from blood.</a> J Clin Invest. 105, 71-77 (2000).</li><li>Ingram, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+novel+hierarchy+of+endothelial+progenitor+cells+using+human+peripheral+and+umbilical+cord+blood.">Identification of a novel hierarchy of endothelial progenitor cells using human peripheral and umbilical cord blood.</a> Blood. 104, 2752-2760 (2004).</li><li>Melero-Martin, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+vasculogenic+potential+of+human+blood-derived+endothelial+progenitor+cells.">In vivo vasculogenic potential of human blood-derived endothelial progenitor cells.</a> Blood. 109, 4761-4768 (2007).</li><li>Au, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+in+vivo+potential+of+endothelial+progenitor+cells+from+human+umbilical+cord+blood+and+adult+peripheral+blood+to+form+functional+long-lasting+vessels.">Differential in vivo potential of endothelial progenitor cells from human umbilical cord blood and adult peripheral blood to form functional long-lasting vessels.</a> Blood. 111, 1302-1305 (2008).</li><li>Yoder, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Redefining+endothelial+progenitor+cells+via+clonal+analysis+and+hematopoietic+stem/progenitor+cell+principals.">Redefining endothelial progenitor cells via clonal analysis and hematopoietic stem/progenitor cell principals.</a> Blood. 109, 1801-1809 (2007).</li><li>Traktuev, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Robust+Functional+Vascular+Network+Formation+In+Vivo+by+Cooperation+of+Adipose+Progenitor+and+Endothelial+Cells.">Robust Functional Vascular Network Formation In Vivo by Cooperation of Adipose Progenitor and Endothelial Cells.</a> Circ Res. 104, 1410-1420 (2009).</li><li>Melero-Martin, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+robust+and+functional+vascular+networks+in+vivo+with+human+adult+and+cord+blood-derived+progenitor+cells.">Engineering robust and functional vascular networks in vivo with human adult and cord blood-derived progenitor cells.</a> Circ Res. 103, 194-202 (2008).</li><li>Melero-Martin, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Host+Myeloid+Cells+Are+Necessary+for+Creating+Bioengineered+Human+Vascular+Networks+In+Vivo.">Host Myeloid Cells Are Necessary for Creating Bioengineered Human Vascular Networks In Vivo.</a> Tissue Eng Part A. 16, 2457-2466 (2010).</li></ol>

الإعلان عن أي تضارب في المصالح.

هذا هو نموذج تجريبي لشبكات الهندسة الحيوية الأوعية الدموية البشرية. الخصائص الرئيسية لهذا النموذج هي : 1) تتكون من خلايا الإنسان microvessels معزولة عن الأنسجة بعد الولادة (أي والدم ونخاع العظام) ، 2) يتم تشكيل microvessels في حيوان بالغ ، و 3) microvessels لا تنشأ من قبل القائمة (المضيف) ولكن بدلا من السفن التي تتشكل فيها ، من جديد ، واحدة من خلايا معلقة في مادة هلامية مناسبة. الأوعية الدموية تلعب دورا هاما في هذا الاختبار لأن هناك حاجة إلى وصلات الأوعية الدموية الفئران لتحقيق الخلايا الحمراء المليئة الأوعية الدموية ، واحدة من وظيفية للقراءة في هذا الاختبار. بالإضافة إلى ذلك ، يتم تجنيد خلايا الدم النخاعي المضيف لعملية الزرع في زرع في وقت مبكر بعد أيام ، وأنها ضرورية لتكوين الأوعية الدموية الجديدة سرير 12. هذا النموذج التجريبي يقدم تنوعا ، ونظام نموذجي للقياس الكمي ، وبسيطة نسبيا لدراسة ما بعد الولادة تشكيل شبكات الأوعية الدموية في الجسم الحي الإنسان. بالإضافة إلى ذلك ، هذا الاختبار بسيط لأداء وأنها لا تحتاج إلى شق أو إجراء العمليات الجراحية. ويمكن استخدام النموذج لدراسة الامكانيات مكون للأوعية مصادر مختلفة من الخلايا البطانية والإنسان حول الوعاء. ويمكن استخدام هذا النموذج على الشاشة لمكافحة و- / أو مركبات الموالية للمكون للأوعية. أخيرا ، يمكن استخدام نموذج لدراسة دور (ق) من جينات معينة في تشكيل وظيفة الأوعية الدموية لشبكة تتألف من البطانة الإنسان.

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><th> اسم كاشف </th><th> شركة </th><th> فهرس العدد </th><th> تعليقات (اختياري) </th></tr><tr><td> بصل متوسطة البطانية ، EBM - 2 </td><td> Lonza </td><td> CC - 3156 </td><td></td></tr><tr><td> EGM - 2 ملاحق SingleQuot </td><td> Lonza </td><td> CC - 3162 </td><td></td></tr><tr><td> الجلوتامين ، البنسلين ، الستربتوميسين الحل ، 100X GPS </td><td> Mediatech ، وشركة </td><td> 30-009 - CI </td><td></td></tr><tr><td> الوسيطة نمو الخلايا الجذعية BulletKit متوسطة ، MSCBM / MSCGM </td><td> Lonza </td><td> PT - 3001 </td><td></td></tr><tr><td> ارتفاع الجلوكوز في Dulbecco التعديل النسر متوسطة ، 1X DMEM </td><td> Invitrogen </td><td> 11995-073 </td><td></td></tr><tr><td> MEM غير الضرورية حل الأحماض الأمينية ، 100X NEAA </td><td> Invitrogen </td><td> 11140-050 </td><td></td></tr><tr><td> ارتفاع الجلوكوز في Dulbecco التعديل النسر ، مسحوق DMEM </td><td> Invitrogen </td><td> 12100-046 </td><td></td></tr><tr><td> HEPES حل مؤقت </td><td> Invitrogen </td><td> 15630-080 </td><td></td></tr><tr><td> Cultrex الكولاجين البقري الحل الأول </td><td> Trevigen </td><td> 3442-050-01 </td><td></td></tr><tr><td> Cultrex الإنسان [فيبرونكتين] </td><td> Trevigen </td><td> 3420-001-01 </td><td></td></tr><tr><td> الفيبرينوجين من البلازما البقري </td><td> سيغما الدريخ </td><td> F8630 - 1G </td><td></td></tr><tr><td> الجيلاتين </td><td> الصياد </td><td> DF0143 - 17-9 </td><td></td></tr><tr><td> الفوسفات مخزنة في Dulbecco المالحة ، برنامج تلفزيوني </td><td> Invitrogen </td><td> 14190-250 </td><td></td></tr><tr><td> التربسين - EDTA الحل ، 1X </td><td> Invitrogen </td><td> 25300-054 </td><td></td></tr><tr><td> Isoflurane ، سائلة للاستنشاق </td><td> مؤسسة الرعاية الصحية Buxter </td><td> NDC 10019-360-40 </td><td></td></tr><tr><td> ثرومبين من البلازما البقري </td><td> سيغما الدريخ </td><td> T4648 - 1KU </td><td></td></tr><tr><td> محلول الفورمالين ، التخزين المؤقت متعادلة </td><td> سيغما الدريخ </td><td> HT501128 </td><td></td></tr><tr><td> وحيدة النسيلة المضادة للماوس جسم الإنسان CD31 </td><td> DakoCytomation </td><td> M0823 </td><td> استنساخ JC70A </td></tr></tbody></table>

bioengineering human microvascular networks in immunodeficient mice

فإن مستقبل هندسة الأنسجة والخلايا المستندة إلى علاجات لتجديد الأنسجة تعتمد على الأرجح على قدرتنا على توليد شبكات الأوعية الدموية وظيفية في الجسم الحي. في هذا الصدد ، والبحث عن نماذج تجريبية لبناء شبكات الأوعية الدموية في الجسم الحي هو في غاية الأهمية 1. وكان عرض لأول مرة في جدوى شبكات الاوعية الدموية الدقيقة الهندسة الحيوية في الجسم الحي باستخدام الأنسجة البشرية المستمدة من الخلايا البطانية ناضجة (ECS) 2-4 ، ولكن هذه الخلايا البطانية ذاتي المشاكل الحالية للاستخدام السريري واسعة ، لأنها من الصعب الحصول على كميات كافية وتتطلب حصاد من الأوعية الدموية الموجودة. هذه القيود قد حرض على البحث عن مصادر أخرى للECS. تحديد الخلايا التي تشكل بطانة الأوعية الدموية مستعمرة (ECFCs) في الدم قدمت فرصة لغير جراحية الحصول ECS 5-7. لقد أظهرنا والمؤلفين الأخرى التي ECFCs الدم المستمدة من البالغين والحبل لديها القدرة على تشكيل شبكات الأوعية الدموية في الجسم الحي وظيفية 7-11. الأهم من ذلك ، وقد أظهرت هذه الدراسات أيضا أن الحصول على شبكات الأوعية الدموية مستقرة ودائمة ، ECFCs تتطلب التعاون مع زرع الخلايا المحيطة بالأوعية. في مقايسة وصفنا هنا يوضح هذا المفهوم : كيف نظهر الإنسان المستمدة من دم الحبل السري ECFCs يمكن دمجها مع نخاع العظام المستمدة من الخلايا الجذعية الوسيطة (MSCS) على النحو تعليق خلية واحدة في هلام الكولاجين / فبرونيكتين / الفيزيولوجية لتشكيل الإنسان وظيفية شبكة الأوعية الدموية في غضون 7 أيام بعد زرع في ماوس العوز المناعي. ويمكن ملاحظة وجود شمعة ECFC مبطنة البشرية التي تحتوي على الكريات الحمراء المضيفة في جميع أنحاء يزرع يشير ليس فقط على تشكيل (دي نوفو) من شبكة الأوعية الدموية ، ولكن أيضا على تطوير anastomoses الفنية مع استضافة الدورة الدموية. هي مناسبة بشكل مثالي لهذا النموذج الفأري شبكة الأوعية الدموية البشرية الهندسة البيولوجية لإجراء دراسات عن الآليات الخلوية والجزيئية لتشكيل شبكة الأوعية الدموية البشرية وتطوير استراتيجيات ليوعي الأنسجة هندسيا.

Watch this Scientific Journal Video about Bioengineering Human Microvascular Networks in Immunodeficient Mice at JoVE.com

Bioengineering Human Microvascular Networks in Immunodeficient Mice

هنا ، نحن تصف منهجية لإيصال الدم الحبل الإنسان المستمدة من البطانية مستعمرة تشكيل الخلايا (ECFCs) ونخاع العظم المشتقة من الخلايا الجذعية الوسيطة (MSCS) ، جزءا لا يتجزأ من هلام الكولاجين / فبرونيكتين ، تحت الجلد في الفئران العوز المناعي. هذا المزيج خلية / هلام بإنشاء شبكة الأوعية الدموية التي تربط الإنسان مع الأوعية الدموية الماوس.

الهندسة الحيوية شبكات الاوعية الدموية الدقيقة الإنسان في الفئران العوز المناعي

The future of tissue engineering and cell-based therapies for tissue regeneration will likely rely on our ability to generate functional vascular networks ...

bioengineering-human-microvascular-networks-in-immunodeficient-mice

Research

JoVE Journal

Biology

12.2K Views.  Harvard Medical School. هنا ، نحن تصف منهجية لإيصال الدم الحبل الإنسان المستمدة من البطانية مستعمرة تشكيل الخلايا (ECFCs) ونخاع العظم المشتقة من الخلايا الجذعية الوسيطة (MSCS) ، جزءا لا يتجزأ من هلام الكولاجين / فبرونيكتين ، تحت الجلد في الفئران العوز المناعي. هذا المزيج خلية / هلام بإنشاء شب...

Video: Bioengineering Human Microvascular Networks in Immunodeficient Mice

Using Micro-Electro-Mechanical Systems (MEMS) to Develop Diagnostic Tools

Using Micro-Electro-Mechanical Systems MEMS to Develop Diagnostic Tools

باستخدام مايكرو سيستمز الكهربائية والميكانيكية (ممس) لتطوير أدوات التشخيص

CD4+ T-Lymphocyte Capture Using a Disposable Microfluidic Chip for HIV

CD4 + T - اللمفاويات التقاط باستخدام رقاقة ميكروفلويديك المتاح لفيروس نقص المناعة البشرية

Title Cell Encapsulation by Droplets

عنوان تغليف الخلية عن طريق الرذاذ

Cellular Encapsulation in 3D Hydrogels for Tissue Engineering 

The 3D encapsulation of cells within hydrogels represents an increasingly important and popular technique for culturing cells and towards the development of constructs for tissue engineering. This environment better mimics what cells observe in vivo, compared to standard tissue culture, due to the tissue-like properties and 3D environment. Synthetic polymeric hydrogels are water-swollen networks that can be designed to be stable or to degrade through hydrolysis or proteolysis as new tissue is deposited by encapsulated cells. A wide variety of polymers have been explored for these applications, such as poly(ethylene glycol) and hyaluronic acid. Most commonly, the polymer is functionalized with reactive groups such as methacrylates or acrylates capable of undergoing crosslinking through various mechanisms. In the past decade, much progress has been made in engineering these microenvironments - e.g., via the physical or pendant covalent incorporation of biochemical cues - to improve viability and direct cellular phenotype, including the differentiation of encapsulated stem cells (Burdick et al.).
The following methods for the 3D encapsulation of cells have been optimized in our and other laboratories to maximize cytocompatibility and minimize the number of hydrogel processing steps. In the following protocols (see Figure 1 for an illustration of the procedure), it is assumed that functionalized polymers capable of undergoing crosslinking are already in hand; excellent reviews of polymer chemistry as applied to the field of tissue engineering may be found elsewhere (Burdick et al.) and these methods are compatible with a range of polymer types. Further, the Michael-type addition (see Lutolf et al.) and light-initiated free radical (see Elisseeff et al.) mechanisms focused on here constitute only a small portion of the reported crosslinking techniques. Mixed mode crosslinking, in which a portion of reactive groups is first consumed by addition crosslinking and followed by a radical mechanism, is another commonly used and powerful paradigm for directing the phenotype of encapsulated cells (Khetan et al., Salinas et al.).

Cellular Encapsulation in 3D Hydrogels for Tissue Engineering

We present protocols for the 3-dimensional (3D) encapsulation of cells within synthetic hydrogels. The encapsulation procedure is outlined for two commonly used methods of crosslinking (michael-type addition and light-initiated free radical mechanisms), as well as a number of techniques for assessing encapsulated cell behavior.

تغليف الخلوية في الهلاميات المائية للهندسة الأنسجة 3D

The 3D encapsulation of cells within hydrogels represents an increasingly important and popular technique for culturing cells and towards the development ...

Visualization of the Interstitial Cells of Cajal (ICC) Network in Mice

The interstitial cells of Cajal (ICC) are mesenchymal derived "pacemaker cells" of the gastrointestinal (GI) tract that generate spontaneous slow waves required for peristalsis and mediate neuronal input from the enteric nervous system1. Different subtypes of ICC form distinct networks in the muscularis of the GI tract 2,3. Loss or injury to these networks is associated with a number of motility disorders4. ICC cells express the KIT receptor tyrosine kinase on the plasma membrane and KIT immunostaining has been used for the past 15 years to label the ICC network5,6. Importantly, normal KIT activity is required for ICC development5,6. Neoplastic transformation of ICC cells results in gastrointestinal stromal tumor (GIST), that frequently harbor gain-of-function KIT mutations7,8. We recently showed that ETV1 is a lineage-specific survival factor expressed in the ICC/GIST lineage and is a master transcriptional regulator required for both normal ICC network formation and for of GIST tumorigenesis9. We further demonstrate that it cooperates with activating KIT mutations in tumorigenesis. Here, we describe methods for visualization of ICC networks in mice, largely based on previously published protocols10,11. More recently, the chloride channel anoctamin 1 (ANO1) has also been characterized as a specific membrane marker of ICC11,12. Because of their plasma membrane localization, immunofluorescence of both proteins can be used to visualize the ICC networks. Here, we describe visualization of the ICC networks by fixed-frozen cyrosections and whole mount preparations.

Visualization of the Interstitial Cells of Cajal ICC Network in Mice

The interstitial cells of Cajal (ICC) are the pacemaker cells of the gastrointestinal (GI) tract. They form complex networks between smooth muscle cells and post-ganglionic neuronal fibers to regulate GI contractility. Here, we present immunofluorescence methods cross-sectional and whole-mount visualization of murine ICC networks.

تصور من خلايا خلالية لشبكة (ICC) في الفئران كاجال

The interstitial cells of Cajal (ICC) are mesenchymal derived "pacemaker cells" of the gastrointestinal (GI) tract that generate spontaneous slow waves ...

Endothelial Cell Co-culture Mediates Maturation of Human Embryonic Stem Cell to Pancreatic Insulin Producing Cells in a Directed Differentiation Approach

Embryonic stem cells (ESC) have two main characteristics: they can be indefinitely propagated in vitro in an undifferentiated state and they are pluripotent, thus having the potential to differentiate into multiple lineages. Such properties make ESCs extremely attractive for cell based therapy and regenerative treatment applications 1. However for its full potential to be realized the cells have to be differentiated into mature and functional phenotypes, which is a daunting task. A promising approach in inducing cellular differentiation is to closely mimic the path of organogenesis in the in vitro setting. Pancreatic development is known to occur in specific stages 2, starting with endoderm, which can develop into several organs, including liver and pancreas. Endoderm induction can be achieved by modulation of the nodal pathway through addition of Activin A 3 in combination with several growth factors 4-7. Definitive endoderm cells then undergo pancreatic commitment by inhibition of sonic hedgehog inhibition, which can be achieved in vitro by addition of cyclopamine 8. Pancreatic maturation is mediated by several parallel events including inhibition of notch signaling; aggregation of pancreatic progenitors into 3-dimentional clusters; induction of vascularization; to name a few. By far the most successful in vitro maturation of ESC derived pancreatic progenitor cells have been achieved through inhibition of notch signaling by DAPT supplementation 9. Although successful, this results in low yield of the mature phenotype with reduced functionality. A less studied area is the effect of endothelial cell signaling in pancreatic maturation, which is increasingly being appreciated as an important contributing factor in in-vivo pancreatic islet maturation 10,11.
The current study explores such effect of endothelial cell signaling in maturation of human ESC derived pancreatic progenitor cells into insulin producing islet-like cells. We report a multi-stage directed differentiation protocol where the human ESCs are first induced towards endoderm by Activin A along with inhibition of PI3K pathway. Pancreatic specification of endoderm cells is achieved by inhibition of sonic hedgehog signaling by Cyclopamine along with retinoid induction by addition of Retinoic Acid. The final stage of maturation is induced by endothelial cell signaling achieved by a co-culture configuration. While several endothelial cells have been tested in the co-culture, herein we present our data with rat heart microvascular endothelial Cells (RHMVEC), primarily for the ease of analysis.

The current study describes a directed differentiation approach in inducing pancreatic differentiation of human embryonic stem cells. Of great significance is the finding that endothelial cell co-culture mediates maturation of human embryonic stem cell derived pancreatic progenitors into insulin expressing cells.

البطانية نضوج خلية يتوسط المشارك ثقافة الإنسان الخلايا الجذعية الجنينية إلى خلايا البنكرياس المنتجة للأنسولين في نهج التفاضل إخراج

Embryonic stem cells (ESC) have two main characteristics: they can be indefinitely propagated in vitro in an undifferentiated state and they are ...

RNA-seq Analysis of Transcriptomes in Thrombin-treated and Control Human Pulmonary Microvascular Endothelial Cells

The characterization of gene expression in cells via measurement of mRNA levels is a useful tool in determining how the transcriptional machinery of the cell is affected by external signals (e.g. drug treatment), or how cells differ between a healthy state and a diseased state. With the advent and continuous refinement of next-generation DNA sequencing technology, RNA-sequencing (RNA-seq) has become an increasingly popular method of transcriptome analysis to catalog all species of transcripts, to determine the transcriptional structure of all expressed genes and to quantify the changing expression levels of the total set of transcripts in a given cell, tissue or organism1,2 . RNA-seq is gradually replacing DNA microarrays as a preferred method for transcriptome analysis because it has the advantages of profiling a complete transcriptome, providing a digital type datum (copy number of any transcript) and not relying on any known genomic sequence3.
Here, we present a complete and detailed protocol to apply RNA-seq to profile transcriptomes in human pulmonary microvascular endothelial cells with or without thrombin treatment. This protocol is based on our recent published study entitled "RNA-seq Reveals Novel Transcriptome of Genes and Their Isoforms in Human Pulmonary Microvascular Endothelial Cells Treated with Thrombin,"4 in which we successfully performed the first complete transcriptome analysis of human pulmonary microvascular endothelial cells treated with thrombin using RNA-seq. It yielded unprecedented resources for further experimentation to gain insights into molecular mechanisms underlying thrombin-mediated endothelial dysfunction in the pathogenesis of inflammatory conditions, cancer, diabetes, and coronary heart disease, and provides potential new leads for therapeutic targets to those diseases.
The descriptive text of this protocol is divided into four parts. The first part describes the treatment of human pulmonary microvascular endothelial cells with thrombin and RNA isolation, quality analysis and quantification. The second part describes library construction and sequencing. The third part describes the data analysis. The fourth part describes an RT-PCR validation assay. Representative results of several key steps are displayed. Useful tips or precautions to boost success in key steps are provided in the Discussion section. Although this protocol uses human pulmonary microvascular endothelial cells treated with thrombin, it can be generalized to profile transcriptomes in both mammalian and non-mammalian cells and in tissues treated with different stimuli or inhibitors, or to compare transcriptomes in cells or tissues between a healthy state and a disease state.

This protocol presents a complete and detailed procedure to apply RNA-seq, a powerful next-generation DNA sequencing technology, to profile transcriptomes in human pulmonary microvascular endothelial cells with or without thrombin treatment. This protocol is generalizable to various cells or tissues affected by different reagents or disease states.

RNA-يليها تحليل Transcriptomes في المعاملة والثرومبين الإنسان الرئوي مراقبة الخلايا البطانية الاوعية الدموية الدقيقة

The characterization of gene expression in cells via measurement of mRNA levels is a useful tool in determining how the transcriptional machinery of the ...

Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs include modeling pathogenesis of human genetic diseases, autologous cell therapy after gene correction, and personalized drug screening by providing a source of patient-specific and symptom relevant cells. However, there are several hurdles to overcome, such as eliminating the remaining reprogramming factor transgene expression after human iPSCs production. More importantly, residual transgene expression in undifferentiated human iPSCs could hamper proper differentiations and misguide the interpretation of disease-relevant in vitro phenotypes. With this reason, integration-free and/or transgene-free human iPSCs have been developed using several methods, such as adenovirus, the piggyBac system, minicircle vector, episomal vectors, direct protein delivery and synthesized mRNA. However, efficiency of reprogramming using integration-free methods is quite low in most cases.
Here, we present a method to isolate human iPSCs by using Sendai-virus (RNA virus) based reprogramming system. This reprogramming method shows consistent results and high efficiency in cost-effective manner.

Here, we present our established method to reprogram human somatic cells into transgene-free human iPSCs with Sendai virus, which shows consistent outcome and enhanced efficiency.

كفاءة الجيل المستحثة الإنسان افرة القوة الخلايا الجذعية من الخلايا الجسدية البشرية مع سينداي الفيروسات

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs ...

Determination of Sialic Acids in Liver and Milk Samples of Wild-type and CMAH Knock-out Mice.

CMAH (cytidine monophosphate-N-acetylneuraminic acid hydroxylase) is responsible for the oxidation of cytidine monophosphate-N-acetylneuraminic acids in mammals. However, humans cannot oxidize cytidine monophosphate-N-acetylneuraminic acid to cytidine monophosphate-N-glycolylneuraminic acid due to a primary exon deletion of the CMAH gene. To understand the effects and implications of the lack of CMAH activity in more detail, a Cmah knock-out model in mice is of keen interest in basic and applied research. The analysis method to determine the phenotype of this mouse model is herein described in detail, and is based on the detection of both N-acetylneuraminic acid and N-glycolylenuraminic acid in the liver and milk of wild-type and Cmah knock-out mice. Endogenous sialic acids are released and derivatized with o-phenylenediamine to generate fluorogenic derivatives, which can be subsequently analyzed by HPLC. The presented protocol can be also applied for the analysis of milk and tissue samples from various other origins, and may be of use to investigate the nutritional and health effects of N-glycolylneuraminic acid.

Determination of Sialic Acids in Liver and Milk Samples of Wild-type and CMAH Knock-out Mice.

We describe a HPLC-based method for the determination of N-acetylneuraminic acid and N-glycolylenuraminic acid in mouse liver and milk.

تحديد الأحماض السياليك في الكبد وعينات الحليب من البرية من نوع و<em&gt; CMAH</em&gt; خروج المغلوب الفئران.

CMAH (cytidine monophosphate-N-acetylneuraminic acid hydroxylase) is responsible for the oxidation of cytidine monophosphate-N-acetylneuraminic acids in ...

Quantitative Proteomics Workflow using Multiple Reaction Monitoring Based Detection of Proteins from Human Brain Tissue

The proteomic analysis of the human brain tissue over the last decade has greatly enhanced our understanding of the brain. However, brain related disorders continue to be a major contributor of deaths around the world, necessitating the need for even greater understanding of their pathobiology. Traditional antibody-based techniques like western blotting or immunohistochemistry suffer from being low-throughput besides being labor-intensive and qualitative or semi-quantitative. Even conventional mass spectrometry-based shotgun approaches fail to provide conclusive evidence to support a certain hypothesis. Targeted proteomics approaches are largely hypothesis driven and differ from the conventional shotgun proteomics approaches that have been long in use. Multiple reaction monitoring is one such targeted approach that requires the use of a special mass spectrometer called the tandem quadrupole mass spectrometer or triple quadrupole mass spectrometer. In the current study, we have systematically highlighted the major steps involved in performing a successful tandem quadrupole mass spectrometry-based proteomics workflow using human brain tissue with an aim to introduce this workflow to a broader research community.

The protocol aims to introduce the use of a triple quadrupole mass spectrometer for Multiple Reaction Monitoring (MRM) of proteins from clinical samples. We have provided a systematic workflow starting from sample preparation to data analysis for clinical samples with all the necessary precautions to be taken.