Tim Doyle, Doktora ve Stanford biyoparlaklık görüntüleme ile yardım için Vivo Görüntüleme Merkezi sayesinde. Ayrıca matris çözümü ile kök hücre ko-enjeksiyon tekniği paylaşmak için Ngan Huang, doktora teşekkür eder. Son olarak, Steve sayesinde Keçe, Doktora veterinerlik hayvan bakımı ile ilgili yardım için.

<ol><li> Double Fusion Reporter Gen İnşaatı <ol><li> Insan embriyonik kök hücreleri biyoparlaklık görüntüleme gerçekleştirmek için, ilk stably ateşböceği lusiferaz Ubiquitin veya EF1a gibi bir kurucu organizatörü tarafından tahrik gibi bir lusiferaz muhabiri gen ifade hücreleri elde etmek için gerekir. </li><li> Bu protokolün odak muhabiri gen uygulamaları, ayrıntılı yordamlar burada verilmez. Ancak, bizim laboratuvar genel stratejisi ateşböceği lusiferaz (fluc) ve pCDNA 3.1 içinde bir boşluk + ayrılmış gelişmiş yeşil flüoresan protein (egfp) içeren bir çift füzyon inşa vektör kullanmaktır. </li><li> Kısaca, çift füzyon geninin ilk pCDNA 3.1 sitomegalovirüs organizatörü (+) akış aşağısında bulunan, bu yüzden 3.3 kbç parçası bir öz-inaktive (SIN çoklu klonlama siteye bağlandı NdeI ve Noti sindirim ve künt uç kullanılarak eksize edildi Ubiquitin promotör kontrolü altında) lentiviral vektör. </li></ol></li><li> Lentiviral İletimi <ol><li> hESC 10 enfeksiyon bir çokluk (İçişleri Bakanlığı) (106 hücreleri ile inkübe yaklaşık 107 viral titresi) 3-5 gün geçtikten sonra transduced olabilir. </li><li> Çözülmüş viral stok taze hESC medya doğrudan eklenebilir. </li><li> Medya 12 ve 24 saat sonra yenileyin. </li><li> 48 saat sonra, transdüksiyon verimliliği niteliksel floresan mikroskopi kullanılarak değerlendirildi. Daha sonra, FACS enfekte hücreleri izole etmek için kullanılabilir. </li></ol></li><li> Kültürleme hESC ve Set-up Görüntüleme için giriş <ol><li> Kültür lusiferaz olumlu hESC besleyici özgür koşullar. Biz genellikle standart WiCell protokolü takip ve klimalı ortam veya ticari ya Matrigel azalır büyüme faktörü kaplı 6-kuyucuğu, besleyici ücretsiz medya kullanın. </li><li> Lusiferaz pozitif hücrelerin saptamak için, muhabir prob D-luciferase şimdiden hazırlıklı olması gerekir. 1-1.5 mL hazırlıklarında son bir 45 mg / ml konsantrasyonda bu kısım luciferase yapmak için. -20 ° C&#39;de alikotları depolama zaman kağıt havlu ile kaplayarak kullanmak ve ışığa maruz kalmasını önlemek. </li><li> Lusiferaz pozitif hücrelerin görselleştirmek için, biz IVIS 50 ve IVIS 200 Görüntüleme Sistemleri (Xenogen Corporation, Alameda, CA) kullanın. Bu ikinci sistem, küçük hayvanlar geçici anestezi için entegre bir isoflourane cihaz ve indüksiyon odası içerir. </li></ol></li><li> HESC in vitro biyoparlaklık görüntüleme <ol><li> In vitro biyoparlaklık görüntüleme için, steril koşullarda muhafaza etmek önemlidir. Bu nedenle, görüntüleme sistemi, tercihen bir hücre kültürü odada, steril olmalıdır. </li><li> Görüntüleme önce, hücre medyayı çıkartın ve hücreleri kapsayacak şekilde yeterli PBS ekleyin. Örneğin, her bir kuyunun hESC kültürleri içeren 6-plaka 1ml PBS ekleyin. </li><li> D-luciferase PBS oranı 1:100 olmalıdır, bu nedenle 6 plaka her iyi çözülmüş D-luciferase 10 mcL ekleyin. </li><li> Bir dakika bekleyin, sonra 1 saniye pozlama süresi kullanarak bir görüntü alır. Sinyal zayıfsa, pozlama aralığı artırmak ve tekrar deneyin. </li><li> Bioluminescent sinyali hücre sayısı yansıtır, farklı hücre sayıları ile sinyal korele olduğu kantitatif testleri yapılabilir. </li></ol></li><li> In vivo görüntüleme için fare ve hücreleri enjekte hazırlanması <ol><li> Muhabir gen görüntüleme, günler, haftalar, ve hatta aylar boyunca transplantasyonu sonrası izleme hücreler için yüksek verimli, ucuz ve duyarlı bir yöntem sağlar. Nakledilen kök hücreler sık ​​teratom formu konağın bağışıklık sistemi tarafından reddedilir, ya da sadece ölmek bu yana bu özellikle önemlidir. </li><li> In vivo görüntüleme teratom oluşumu için basit yöntem, SCID farelere sırtında ateşböceği lusiferaz muhabiri gen ifade ve IVIS sistemi ile bioluminescent görüntüler elde hESC enjekte etmek. </li><li> Hücre nakli için hazır olduğunuzda, hücreler gevşetmek için birkaç kez yıkayın ve büyüme faktörü azaltılmış matrigel ve DMEM 1:1 karışımı askıya dispase veya kollajenaz kullanın. Genelde ilk soğutulmuş DMEM hücrelerin karışımı ve sonra matrigel ekleyin. Her subkutan enjeksiyon site için, biz matrigel / DMEM karışımı 50-100 ul hacmi askıya 200.000 hücreler enjekte edilir. Hücre sayısı Kullandığınız uygulamaya bağlı olarak ayarlanabilir. Enjeksiyondan önce buz üzerinde her şeyi tutmak için unutmayın. </li><li> Hücrelerin hazır edildikten sonra, açık izofluran akışı ile indüksiyon kutusuna koyarak fare uyutmak. 1-5 dakika sonra, fare uyuyor ve sürekli izofluran ile ameliyat masasına yerleştirilebilir. </li><li> Kürk doğal otomatik fluoresces ve bioluminescent görüntü gizleyebilir, çünkü biz farenin arka tarafında saç kaldırmak gerekir. Bunu yapmanın en kolay yolu, bir elektrikli tıraş makinesi kullanmak için, ama aynı zamanda saç kaldırılması jelleri kullanabilirsiniz. Sterilize etmek için alkol ile silin.Daha sonra deriyi </li><li> Sonra, bir şırınga hücre süspansiyonu çizin. Hücrelerle birlikte tıkamaz yılından bu yana 23 - 27 gauge iğne en iyi şekilde çalışır. Iğne çok büyükse (23 gauge) iğne yolu hücreleri geri sızıntı olabilir büyük bir delik bırakabilir. </li><li> Hücreler farenin arkasına deri altına enjekte edilir. Cildi oldukça gevşek olduğundan, çimdik, başparmak ve işaret parmağı kullanmak ve enjekte etmek istediğiniz bölgeyi germek. Ponksiyon çok derinden umurumda değil alarak, sadece deri altına enjekte edilir. Ayrıca, Medyumu sırasında enjeksiyon yerinde dışarı kayar iğne tutmaya çalışın: Bu cilt hücreleri tekrar enjekte etmeye çalışırsanız sızıntı olabilir bir delik oluştururken önleyecektir. </li><li> Hücreleri enjekte edildikten sonra, fare uyanmak için izin ve biyoparlaklık görüntüleme için yeniden anestezi önce çalıştırmak için bir kaç saat bekleyin. Bunu yapmak izofluran toksisite önler. </li></ol></li><li> Nakledilen hücrelerin in vivo biyoparlaklık görüntülemede <ol><li> Tüm hayvan biyoparlaklık görüntüleme için, D-luciferase intraperitoneal olarak 375 mg / kg vücut ağırlığı. Enjeksiyondan önce dozu hesaplamak için hayvan tartılır. </li><li> Sadece orta hat kapalı, periton D-luciferase enjekte edilir. Çok derin gitmek ya da iç organlara zarar verebilir dikkatli olun. Fare uyanmak başlarsa, nakavt kutusuna geri koyun ve bir ya da iki dakika bekleyin. Fareler için, maksimum biyoparlaklık genellikle enjeksiyondan sonra 15-40 dakika oluşur. </li><li> HESC tarafından yayılan herhangi bir ışık absorbe yardımcı olmak için mat siyah kağıt görüntüleme kutusunda yer unutmayın. Siyah kağıt üstünde, izofluran ile görüntüleme odasında fare (arka kadar) yerleştirin. 10 saniyelik bir pozlama süresi ile başlayın. Sinyal doymuş ise, pozlama süresini azaltmayı deneyin, çok zayıf, pozlama artırmak. </li><li> Farenizi sinyal maruz kalma süresi için optimize edildikten sonra, sinyal maksimum ulaşıncaya kadar görüntüler her dakika başlar. Bu en iyi enjeksiyon yerlerini gizlemeleri &quot;ilgi alanları&quot; (ROI) seçmek için görüntüleme yazılımı kullanılarak yapılır. Her ROI sinyal yoğunluğu izleyerek kolayca maksimum sinyal yoğunluğu ulaşıldı belirten sinyal azalmaya başladığında belirleyebilirsiniz. Maksimum sinyal son veri olarak kullanılması gerekir. </li><li> Görüntüleri memnun olduğunuzda, izofluran fare kaldırmak ve onun kafes uyanmak için izin. Genellikle, fare, 15 dakika içinde uyanmak gerekir. </li><li> HESC hayvan hayatta Biyoparlaklık muhabiri gen görüntüleme sürece, günlük, haftalık ve aylık tekrar edilebilir. Gelişmekte olan bir teratom Sinyal hESC sayısı hızla artmaya başlar, genellikle hafta sipariş üzerine, zaman içinde katlanarak artacaktır. </li><li> Biyoparlaklık görüntülerin istenilen zaman içerisinde elde edildikten sonra, hayvan kurban ve histoloji için kullanılan doku kesitlerinde olabilir. </li></ol></li></ol>

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PET ve MRI gibi diğer yöntemler ile karşılaştırıldığında, biyoparlaklık sınırlı mekansal çözünürlükte ve nispeten zayıf fotonların enerji (2-3 eV) nedeniyle azalmış doku penetrasyonu; bu nedenden dolayı şimdiye kadar büyük hayvanlara uygulanabilir değil. Ancak, biyoparlaklık in vivo kök hücre küçük hayvanlar izleme için son derece cazip, düşük maliyetli, yüksek verim ve non-invaziv olma avantajına sahiptir. PET ve floresan yapıları gibi Sigara biyoparlaklık muhabiri genler lusiferaz ile birlikte, b...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>Dulbecco's Modified Eagle's Medium (DMEM)</td>
<td>&nbsp;</td>
<td>HyClone</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>BD Matrigel&#x2122; Basement Membrane Matrix</td>
<td>Growth factor reduced (optional: phenol-red free)</td>
<td>BD Biosciences</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>mTeSR1 Maintenance Medium for Human Embryonic Stem Cells</td>
<td>&nbsp;</td>
<td>StemCell Technologies</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Phosphate Buffered Saline (PBS)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>D-Luciferin Firefly, potassium salt</td>
<td>&nbsp;</td>
<td>Biosynth AG</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Collagenase IV solution</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Dissolve 30 mg Collagenase Type IV in 30 mL DMEM-F12 media. Sterile filter and store at 4 degrees (Celsius).</td>
</tr>
<tr>
<td>Baked Pasteur pipets</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>6-well tissue culture-treated plates</td>
<td>&nbsp;</td>
<td>TPP</td>
<td>92006</td>
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in vitro and in vivo bioluminescence reporter gene imaging of human embryonic stem cells

Insan embriyonik kök hücreleri (hESC) keşif dramatik rejeneratif tıp ilgilenen tıp bilim adamları, mevcut araçları arttı. Ancak, canlı organizmaların içine hESC ayırt hESC ve hücrelerin doğrudan enjeksiyon şimdiye kadar önemli hücre ölümü, teratom oluşumu ve konağın bağışıklık ret engel olmuştur. Naklinden sonra in vivo hESC davranışı anlamak roman görüntüleme teknikleri boyuna hESC lokalizasyonu, proliferasyon ve canlılığı izlemek için gereklidir. Moleküler görüntüleme araştırmacılar, gün boyunca in vivo hücre çoğalması, haftalar ve hatta aylar izlemek için yüksek verimli, ucuz ve duyarlı bir araç vermiştir. Bu gelişme, uzay-zamansal hESC engraftman, yayılması ve yaşayan kişilerde teratom oluşumu kinetiği anlayışı önemli ölçüde artmıştır. Moleküler görüntüleme alanında önemli bir ilerleme noninvaziv raportör gen deneyleri, in vivo ve çok amaçlı görüntüleme platformlar moleküler ve hücre biyolojisi uzantısı olmuştur. Bu muhabir genler, konak hücre s transkripsiyonel makine yararlanmak mühendislik rehberleri ve arttırıcılar kontrolü altında, vektör ve vektörel olmayan yöntemleri kullanarak çeşitli hücre içine tanıtıldı. Muhabiri genler hücre kullanılan organizatörü türüne göre, biyolojik veya hücresel belirli şartlar altında ya yapısal ya da sadece transkripsiyonu olabilir. Biyoaktif proteinlerin içine muhabiri genlerin transkripsiyon ve tercümesi sonra D-luciferase gibi sinyal üreten problar kullanılarak hassas, noninvaziv enstrümantasyon (örneğin, CCD kamera) ile tespit edilir. Floresan görüntüleme için gerekli olan kök hücrelerin in vivo olarak izlemek için eksitatör ışık için ihtiyaç önlemek için, biyoparlaklık muhabiri gen görüntüleme sistemleri, ışık emisyon ikna etmek için sadece bir Eksojen olarak uygulanan prob gerektirir. Firefly lusiferaz, ateşböceği Photinus pyralis türetilen, optik olarak aktif metaboliti, oxyluciferin D-luciferase katalize bir enzim kodlar. Optik faaliyet, daha sonra harici bir CCD kamera ile takip edilebilir. Stabil muhabir taşıyan hücreler kendi kromozomal DNA&#39;da inşa transduced muhabiri hESC hayatta kalma ve in vivo çoğalması boyuna izlenmesi için izin kızı hücrelerinin DNA inşa geçecek. Ayrıca, muhabir gen ürünü ifade sinyal üretimi için gerekli olduğundan, tek uygun anne-baba ve kızı hücreleri biyoparlaklık sinyal yaratacak, apoptotik ya da ölü hücrelerden olmaz. Bu video, özel malzemeler ve biyoparlaklık görüntüleme izleme kök hücre çoğalması ve teratom oluşumu için gerekli yöntemler ele alınacaktır.

Watch this Scientific Journal Video about In vitro and in vivo Bioluminescence Reporter Gene Imaging of Human Embryonic Stem Cells at JoVE.com

In vitro and in vivo Bioluminescence Reporter Gene Imaging of Human Embryonic Stem Cells

Giderek artan bir ilgi ile kök hücre tedavileri, moleküler görüntüleme teknikleri transplantasyonu sonrası izleme kök hücre davranışı için idealdir. Lusiferaz muhabiri genler hücre sağkalım, konumu ve in vivo çoğalması non-invaziv, tekrarlayan değerlendirme sağladı. Bu video izlemek için yaşayan bir fare hESC çoğalması gösterecektir.

In vitro ve in vivo Biyoparlaklık Reporter Gen Görüntüleme İnsan Embriyonik Kök Hücreleri

The discovery of human embryonic stem cells (hESCs) has dramatically increased the tools available to medical scientists interested in regenerative ...

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Research

JoVE Journal

Biology

21.7K Görüntüleme.  Stanford University School of Medicine. Giderek artan bir ilgi ile kök hücre tedavileri, moleküler görüntüleme teknikleri transplantasyonu sonrası izleme kök hücre davranışı için idealdir. Lusiferaz muhabiri genler hücre sağkalım, konumu ve in vivo çoğalması non-invaziv, tekrarlayan değerlendirme sağladı. Bu video izlemek için yaşayan bir fare hESC çoğalması gösterecektir.

Video: In vitro and in vivo Bioluminescence Reporter Gene Imaging of Human Embryonic Stem Cells

Derivation of Human Embryonic Stem Cells by Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass. 


The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass.

Immunosurgery İnsan Embriyonik Kök Hücreleri türetilmesi

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both ...

Biyoloji

Probing for Mitochondrial Complex Activity in Human Embryonic Stem Cells

Mitochondria are cytoplasmic organelles that have a primary role in cellular metabolism and homeostasis, regulation of the cell signaling network, and programmed cell death. Mitochondria produce ATP, regulate the cytoplasmic redox state and Ca2+ balance, catabolize fatty acids, synthesize heme, nucleotides, steroid hormones, amino acids, and help assemble iron-sulfur clusters in proteins. Mitochondria also have an essential role in heat production. Mutations of the mitochondrial genome cause several types of human disorder. The accumulation of mtDNA mutations correlates with aging and is suspected to have an important role in the development of cancer. Due to their vitally important role in all cell types, the function of mitochondria must also be critical for stem cells. Key advances have been made in our understanding of stem cell viability, proliferation, and differentiation capacity. But the functional activity of stem cells, in particular their energy status, was not yet been studied in detail. Almost nothing is known about the mitochondrial properties of human embryonic stem cells (hESCs) and their differentiated precursor progeny. One way to understand and evaluate the role of mitochondria in hESC function and developmental potential is to directly measure the activity of mitochondrial respiratory complexes. Here, we describe high resolution clear native gel electrophoresis and subsequent in gel activity visualization as a method for analyzing the five respiratory chain complexes of hESCs.

In this video, we will show you how the mitochondrial respiratory chain complexes of human embryonic stem cells can be analyzed using in gel activity assays.

İnsan Embriyonik Kök Hücreleri Mitokondrial Kompleksi Aktivite araştırmayı

Mitochondria are cytoplasmic organelles that have a primary role in cellular metabolism and homeostasis, regulation of the cell signaling network, and ...

In vitro Labeling of Human Embryonic Stem Cells for Magnetic Resonance Imaging

Human embryonic stem cells (hESC) have demonstrated the ability to restore the injured myocardium. Magnetic resonance imaging (MRI) has emerged as one of the predominant imaging modalities to assess the restoration of the injured myocardium. Furthermore, ex-vivo labeling agents, such as iron-oxide nanoparticles, have been employed to track and localize the transplanted stem cells. However, this method does not monitor a fundamental cellular biology property regarding the viability of transplanted cells. It has been known that manganese chloride (MnCl2) enters the cells via voltage-gated calcium (Ca2+) channels when the cells are biologically active, and accumulates intracellularly to generate T1 shortening effect. Therefore, we suggest that manganese-guided MRI can be useful to monitor cell viability after the transplantation of hESC into the myocardium.
In this video, we will show how to label hESC with MnCl2 and how those cells can be clearly seen by using MRI in vitro. At the same time, biological activity of Ca2+-channels will be modulated utilizing both Ca2+-channel agonist and antagonist to evaluate concomitant signal changes.

In this video, we are showing how to label human embryonic stem cells (hESC) with manganese chloride (MnCl2) which can enter cells via voltage-gated calcium channels when the cells are biologically active. Additionally, we show the use of MnCl2 as cellular MRI contrast agent to determine the in vitro viability of hESC.

İnsan Embriyonik Kök Hücreleri Manyetik Rezonans Görüntüleme için in vitro Etiketleme

Human embryonic stem cells (hESC) have demonstrated the ability to restore the injured myocardium. Magnetic resonance imaging (MRI) has emerged as one of ...

Culture and Maintenance of Human Embryonic Stem Cells  

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be  fed  every day by performing a complete medium change to replenish lost nutrients and to keep the cultures free of unwanted differentiation factors. Although a small amount of differentiation is normal and expected in stem cell cultures, the culture should be routinely cleaned up by manually removing, or "picking" differentiated areas. Identifying and removing excess differentiation from hES cell cultures are essential techniques in the maintenance of a healthy population of cells.

Culture and Maintenance of Human Embryonic Stem Cells

This protocol demonstrates how to maintain healthy, undifferentiated human embryonic stem (ES) cells.

İnsan Embriyonik Kök Hücre Kültürü ve Bakım

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be ...

Freezing and Thawing Human Embryonic Stem Cells

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

İnsan Embriyonik Kök Hücreler Donma ve Çözülme

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells ...

Video Bioinformatics Analysis of Human Embryonic Stem Cell Colony Growth

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer software.  Video bioinformatics is a powerful quantitative approach for extracting spatio-temporal data from video images using computer software to perform dating mining and analysis. In this article, we introduce a video bioinformatics method for quantifying the growth of human embryonic stem cells (hESC) by analyzing time-lapse videos collected in a Nikon BioStation CT incubator equipped with a camera for video imaging. In our experiments, hESC colonies that were attached to Matrigel were filmed for 48 hours in the BioStation CT.  To determine the rate of growth of these colonies, recipes were developed using CL-Quant software which enables users to extract various types of data from video images.  To accurately evaluate colony growth, three recipes were created. The first segmented the image into the colony and background, the second enhanced the image to define colonies throughout the video sequence accurately, and the third measured the number of pixels in the colony over time.  The three recipes were run in sequence on video data collected in a BioStation CT to analyze the rate of growth of individual hESC colonies over 48 hours. To verify the truthfulness of the CL-Quant recipes, the same data were analyzed manually using Adobe Photoshop software. When the data obtained using the CL-Quant recipes and Photoshop were compared, results were virtually identical, indicating the CL-Quant recipes were truthful. The method described here could be applied to any video data to measure growth rates of hESC or other cells that grow in colonies. In addition, other video bioinformatics recipes can be developed in the future for other cell processes such as migration, apoptosis, and cell adhesion.  

Video bioinformatics is the automated processing, analysis, understanding, and data mining of biological spatio-temporal data extracted from microscopic videos. The purpose of this article is to demonstrate a method for measuring human embryonic stem cell colony growth using a video bioinformatics method.

Video Biyoinformatik İnsan Embriyonik Kök Hücre Colony Büyüme Analizi

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer ...

Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant interest in deriving a pure population of lineage-committed oligodendrocyte precursor cells (OPCs) for transplantation. OPCs are characterized by the activity of the transcription factor Olig2 and surface expression of a proteoglycan NG2. Using the GFP-Olig2 (G-Olig2) mouse embryonic stem cell (mESC) reporter line, we optimized conditions for the differentiation of mESCs into GFP+Olig2+NG2+ OPCs. In our protocol, we first describe the generation of embryoid bodies (EBs) from mESCs. Second, we describe treatment of mESC-derived EBs with small molecules: (1) retinoic acid (RA) and (2) a sonic hedgehog (Shh) agonist purmorphamine (Pur) under defined culture conditions to direct EB differentiation into the oligodendroglial lineage. By this approach, OPCs can be obtained with high efficiency (&gt;80%) in a time period of 30 days. Cells derived from mESCs in this protocol are phenotypically similar to OPCs derived from primary tissue culture. The mESC-derived OPCs do not show the spiking property described for a subpopulation of brain OPCs in situ. To study this electrophysiological property, we describe the generation of spiking mESC-derived OPCs by ectopically expressing NaV1.2 subunit. The spiking and nonspiking cells obtained from this protocol will help advance functional studies on the two subpopulations of OPCs.

We describe a small molecule-based protocol for differentiation of mouse embryonic stem cells into oligodendrocyte precursor cells (OPCs). This protocol generates Olig2+NG2+ OPCs with high efficiency by 30 days of differentiation. We also describe a method to generate "spiking" OPCs that can fire action potentials.

Embriyonik Kök Hücreler farklılaşma oligodendrosit Öncüleri

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant ...

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic9-11. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.

Küçük Molekül İndüksiyon Pluripotent İnsan Embriyonik Kök Hücreleri İnsan Kardiyak Öncüleri ve kardiyomiyositlerde Verimli türetilmesi

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells (iPSCs) Using Retroviral Vector with GFP

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has been previously shown that human somatic cells can be reprogrammed to pluripotency by ectopic expression of four transcription factors (Oct4, Sox2, Klf4 and Myc) and become induced pluripotent stem cells (iPSCs)2-4 . Like hESCs, human iPSCs are pluripotent and a potential source for autologous cells. Here we describe the protocol to reprogram human fibroblast cells with the four reprogramming factors cloned into GFP-containing retroviral backbone4. Using the following protocol, we generate human iPSCs in 3-4 weeks under human ESC culture condition. Human iPSC colonies closely resemble hESCs in morphology and display the loss of GFP fluorescence as a result of retroviral transgene silencing. iPSC colonies isolated mechanically under a fluorescence microscope behave in a similar fashion as hESCs. In these cells, we detect the expression of multiple pluripotency genes and surface markers.

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells iPSCs Using Retroviral Vector with GFP

A method to generate human induced pluripotent stem cells (iPSCs) via retrovirus-mediated ectopic expression of OCT4, SOX2, KLF4 and MYC is described. A practical way to identify human iPSC colonies based on GFP expression is also discussed.

GFP Retroviral Vector kullanarak İsteyerek Pluripotent Kök Hücrelerinin (iPSCs) içine İnsan somatik hücreleri yeniden programlama

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has ...

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

Ribozom Bağlı Doğan Polipeptitler İzolasyonu<em&gt; In vitro</em&gt; MRNA boyunca Translasyonel Duraklat Siteleri Belirleme

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...