Teitell laboratuvar İnsan embriyonik kök hücre çalışmaları henüz California Rejeneratif Tıp Enstitüsü (CIRM) tohum hibe RS1-00313 tarafından desteklenmektedir. Biz bu ve ek çalışmalar tavsiye ve anlayışlar sağlamak için Dr. Carla Köhler (UCLA) ve Köhler, laboratuvar üyelerine teşekkür ederim.

Mitokondrial Kompleksi için Yüksek Çözünürlüklü Temizle Doğal Jel Electophoresis (hrCNE)-Gel Aktivitesi Tahliller İnsan embriyonik kök hücreleri (hESC) standart koşullar kullanarak MEF-klimalı ortamda 5 gün sonra Matrigel büyüme geçiş ˠ ışınlanmış veya mitomisin C ile tedavi edilen fare embriyonik fibroblastlar (MEFS), bir tek tabaka üzerinde tutuldu. MEFS kirlenmesine neden kaldırmak için, hESC Matrigel yeniden kaplama ve ek olarak 3 gün boyunca MEF-klimalı ortamda yetişen. Pluripotency belirteçlerinin Ekim-4 ve SSEA-4 İmmünofloresan mikroskopi hESC farklılaşma olmaması onaylamak için kullanılır oldu. <ol><li> , 5 dakika boyunca trypsinization (6 plaka kişi başına 1ml 1x tripsin) takip ılık 1x PBS, pH 7.4, hücreleri yıkayın. </li><li> Taze tripsin inhibitörü eşit hacmi (1x PBS, pH 7.4 1mg/ml) ekleyin. Hasat hücreleri ve 200g az santrifüj, 5 dakika, oda sıcaklığında. </li><li> Taze hazırlanmış 1 &#39;Proteaz İnhibitör Kokteyl; 1.7ml (hücre pelet boyutu dayalı), buz gibi mitokondri izolasyon tamponu (6.6mM imidazol / HCl, pH 7.0 83mm sakkaroz) - 0.8 supernatant ve tekrar süspansiyon pelet hücreleri atın. 10 dakika buz üzerinde inkübe edin. </li><li> Homojenizatör (- 50 vuruş ~ 40) ile hücreler Dounce. Tripan mavisi ile dounced hücrelerinin ışık mikroskobu kullanılarak homojenizasyon tamlığı kontrol etmek için bir kısım Leke. </li><li> 4 10 dakika 20.000 g Santrifüj ° C çekirdek ve daha büyük hücre parçaları da içeren bir ham mitokondriyal pelet elde etmek için. Durusu ve süpernatantı atmak ve çöktürülmüş ham mitokondriyal pelet tartmak. Pelet -80 saklanabilir ° Bu bir durak noktası ise C sıvı azot anlık dondurma. NOT: Diferansiyel santrifüj veya diğer gradyan tabanlı işlemleri saf mitokondriyal membran fraksiyonu yalıtma ve toplamak için kullanılabilir. Bkz. Ürün. 4 Ayrıntılı bilgi için. Bu alternatif bir yaklaşım yerine hücre ağırlığı veya hücre sayısını mg başına karmaşık aktivite mitokondriyal protein mg başına mitokondriyal karmaşık aktivite verecektir. Mitokondriyal proteinlerin konsantrasyonu, bu karşılaştırma yapmak için arıtma sonra tespit edilmesi gerekecektir. Ayrıca bu yaklaşım, farklı bir izolasyon tampon için gerekli olabilir. Saf mitokondriyal membran fraksiyonu çözünürleştirme için, adım 6&#39;da aşağıdaki nota bakınız. </li><li> Buz üzerinde buz çözünürleştirme tamponu (50mm NaCl, 50mm imidazol / HCl pH 7,0, 1mm EDTA, 2mm 6-aminohexanoic asit) ile vorteks ham mitokondriyal pelet çözünür. 6-aminohexanoic asit 1x Proteaz İnhibitör Kokteyl ile değiştirilebilir. 35μl pelet ağırlığı 20 mg başına çözünürleştirme tampon kullanın. % 10 w / v digitonin pelet ağırlığı ve tüp hafifçe vurarak karıştırın 20mg başına 20μl ekleyin. Buz üzerinde 5-10 dakika inkübe. Dodesil-BD-maltoside veya triton X-100 proteinleri çözünür digitonin ikame edilebilir. Deterjan ve miktarı seçim supercomplexes veya mitokondriyal komplekslerinin multimer formları oluşumunu etkileyebilir. Daha fazla bilgi için, ref.1 bkz. NOT: saf mitokondriyal membran fraksiyonu çözünür için gerekli deterjan miktarı, 2-6g / g arasında değişen bir deterjan / protein ağırlık oranına karşılık Mitokondrial membranlar çözünür optimal oranı dodesil-BD-maltoside g / 2.5g için 3g / g triton X-100 ve 6g / digitonin g. </li><li> 15 dakika 4 ° C 100.000 g çözündüğünü pelet Santrifüj Net süpernatant toplayın. </li><li> Son% 5 w / v gliserol ve% 0.01 w / v Ponceau S (% 50 w / v gliserol,% 0.1 w / v Ponceau S yükleme tamponu stok solüsyonu kullanmak) tampon yükleme ekleyin. </li><li> 20-40mg şeritli başına ham mitokondriyal pelet içeren bir hacmi ile jel kuyu yükleyin. % 3.5 jel istifleme ile bir akrilamid gradyan jel (4 -13%) kullanın. NOT: Jel hazırlanması ve elektroforez koşulları: Bisacrylamide akrilamid ve 1.5g (% 49.5 T, T, AA ve bisAA ve C toplam konsantrasyonu% 3 C 48g: jel hazırlanması için, aşağıdaki gibi bir akrilamid / bis-akrilamid (AA / bisAA) 3 karışımı kullanın toplam monomer, çapraz bağlayıcı (bisAA) yüzde olarak). Jel tampon (3): 1.5M 6-aminohexanoic asit; 75mm imidazol, pH 7.0 </li></ol><table style="padding-left: 60px;" border="0"><tbody><tr><td> Gradient jel: </td><td> % 4 </td><td> % 13 </td><td> Jel İstifleme: </td><td> % 3.5 </td></tr><tr><td> AA / bisAA 3 karışımı </td><td> 2 ml </td><td> 6,5 ml </td><td></td><td> 0,6 ml </td></tr><tr><td> Jel tampon 3 &#39; </td><td> 8.3 ml </td><td> 8.3 ml </td><td></td><td> 2.7 ml </td></tr><tr><td> Gliserin </td><td></td><td> 5 g </td><td></td><td></td></tr><tr><td> H 2 O </td><td> 14.7 ml </td><td> 5 ml </td><td></td><td> 4.7 ml </td></tr><tr><td> Toplam hacim </td><td> 25 ml </td><td> 25 ml </td><td></td><td> 8 ml </td></tr><tr><td> 10% APS </td><td> 139 ul </td><td> 125 ul </td><td></td><td> 67 ul </td></tr><tr><td> TEMED </td><td> 14 ul </td><td> 12.5 ul </td><td></td><td> 6.7 ul </td></tr></tbody></table> Anot tampon: 25mm imidazol / HCl, pH 7.0 Katot tamponu: 50mm Tricine, 7.5mm imidazol / HCl,% 0,02 w / v dodesil-BD-maltoside,% 0.05, pH 7.0 w / v deoksikolatın Dökün ve soğuk bir oda (4-7 ° C) jeller çalıştırmak. Kullanılan ilk gerilimi 100V. Örnek istifleme jel girdiğinde, voltaj 15mA (0.15 &#39;14&#39; 14 cm jeller), akım sınırlı 500V yükseltilir. Elektroforez keskin bir çizgi, kırmızı Ponceau S boya jel ön yaklaşımlar 3-4 saat sonra durdurulur. Alternatif olarak, bir jel 70-100V sabit gerilim ile gece çalıştırmak olabilir. <ol><li> Jel aktivite testleri. Protein çözünmüş verimlilik ve iyi ortalama yüklenen malzeme miktarı her bir deney için inkübasyon süresi bağlıdır. I, IV ve V kompleksler genellikle kompleksleri II ve III daha güçlü sinyaller verir. </li></ol> Kompleks I (CI) Birkaç saat oda sıcaklığında jel dilim CI tamponu (2.5mg/ml NBT; 0.1mg/ml NADH 5mM Tris-HCl, pH 7.4) ile inkübe edin. CI tamponu: 5mM Tris-HCl, pH 7.4 100ml başına ekleyin: 0.01g NADH 0.25g NBT 15-45 dakika için% 50 metanol ve% 10 asetik asit jel sabitleyin. Sabit% 10 asetik asit jel korunur. Kompleks II (CII) CII tampon (0.2mm phenazine methasulfate;; 5mM Tris-HCl pH: 7.4; 2.5mg/ml NBT süksinat 20mm sodyum) ile oda sıcaklığında birkaç saat için jel dilim inkübe edin. CII tamponu: 5mM Tris-HCl, pH 7.4 100ml başına ekleyin: 200μl 1M sodyum süksinat 80μl phenazine methasulfate (250mm PMS DMSO içinde çözünmüş) 0.25g NBT Fix gibi Kompleksi için jel Kompleks III (CIII) , Oda sıcaklığında birkaç saat için; CIII tamponu (0.05% 3,3 &#39;-diaminobenzidin tetrahydrochloride (DAB), 50mm sodyum fosfat, pH 7.2) ile jel dilim inkübe edin. CIII tampon: 50mm sodyum-fosfat, pH 7,2 100ml başına ekleyin: 50mg diaminobenzidin tetrahydrochloride Fix gibi Kompleksi için jel Kompleks IV (CIV) Birkaç saat süreyle oda sıcaklığında; CIV tampon (0.05% 3,3 &#39;-diaminobenzidin tetrahydrochloride (DAB), 50μM at kalp sitokrom c 50mm sodyum fosfat, pH 7.2) ile jel dilim inkübe edin. CIV tampon: 50mm sodyum-fosfat, pH 7,2 100ml başına ekleyin: 50mg diaminobenzidin tetrahydrochloride 0.1g at kalp sitokrom c Fix gibi Kompleksi için jel Karmaşık V (CV) Jel dilim CV tampon (35mm Tris-taban, 270mm glisin pH 8.3, 25 ° C, 14mm MgSO 4,% 0.2 w / v Pb (NO 3) 2, 8mm ATP) birkaç saat oda sıcaklığında inkübe CV tampon başına çözüm 100ml eklemek: <table border="0"><tbody><tr><td> Tris-baz </td><td> 0.424g </td></tr><tr><td> Glisin </td><td> 2.026g </td></tr><tr><td> MgSO 4 </td><td> 0.345g </td></tr><tr><td> Pb (NO 3) 2 </td><td> 0.29g </td></tr></tbody></table> PH (8.3 olmalıdır) ayarlamak gerek yok - asit veya baz kullanmayın. Filtre ve ekle <table border="0"><tbody><tr><td> ATP </td><td> 0.441g </td></tr></tbody></table> 30 dakika süreyle% 50 metanol Fix jel ve su aktarmak.

<ol>
	<li>Wittig, I., Braun, H. -. P., Sch&#747;gger, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blue+native+PAGE.">Blue native PAGE.</a> Nature Protocols. 1, 418-428 (2006).</li><li>Wittig, I., Karas, M., Sch&#747;gger, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+Resolution+Clear+Native+Electrophoresis+for+In-gel+Functional+Assays+and+Fluorescence+Studies+of+Membrane+Protein+Complexes.++6.">High Resolution Clear Native Electrophoresis for In-gel Functional Assays and Fluorescence Studies of Membrane Protein Complexes. 6.</a> Molecular &amp; Cellular Proteomics. 6, 1215-1225 (2007).</li><li>Wittig, I., Carrozzo, R., Santorelli, F. M., Sch&#747;gger, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+assays+in+high-resolution+clear+native+gels+to+quantify+mitochondrial+complexes+in+human+biopsies+and+cell+lines.">Functional assays in high-resolution clear native gels to quantify mitochondrial complexes in human biopsies and cell lines.</a> Electrophoresis. 28, 3811-3820 (2007).</li><li>Castle, J. D., Coligan, J. E., Dunn, B. M., Speicher, D. W., Wingfield, P. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+Organelles+from+Mammalian+Cells.">Purification of Organelles from Mammalian Cells.</a> Current Protocols in Protein Science. 4.2, 4.2.1-4.2.57 (2007).</li></ol>

Bu video, insan embriyonik kök hücreleri, yüksek çözünürlükte net yerli jel elektroforezi (hrCNE) ayrılması, ve jel katalitik etkinlik analizleri ile sonraki analiz mitokondriyal protein kompleksleri çıkarma tarif ettiler. hrCNE mavi yerel jel elektroforezi ile karşılaştırılabilir bir çözünürlükte mitokondrial membran protein kompleksleri giderir ve jel faaliyet deneyleri için üstündür. Bu teknik, mitokondriyal solunum kompleksleri IV ölçmek için değil, aynı zamanda hESC ve bunların türev döl hem de herhangi bir mitokondrial membran protein kompleksi altbirim kompozisyon analiz etmek değil sadece istihdam edilebilirler.

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>0.5M EDTA</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>BP120-1</td>
<td>adjust pH to 8.0 with NaOH </td>
</tr>
<tr>
<td>1M NaCl</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S3014-500MG</td>
<td>&nbsp;</td>
<tr>
<td>1M Sodium succinate</td>
<td>&nbsp;</td>
<td>Fisher Scientific </td>
<td>S413-500</td>
<td>&nbsp;</td>
<tr>
<td>0.1M Sodium phosphate buffer pH 7.2</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>BP332-1 and BP329-1</td>
<td>Mix 68.4 ml of 1M Na2HPO4 and 31.6 ml of 1M NaH2PO4 to prepare 0.1M sodium phosphate buffer with pH 7.2 at 25 C </td>
</tr>
<tr>
<td>1M Tricine, pH 7.0 (adjusted with NaOH)</td>
<td>&nbsp;</td>
<td>EMD</td>
<td>9010</td>
<td>store at 4 C </td>
</tr>
<tr>
<td>1M imidazole/HCl, pH 7.0</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>I-2399</td>
<td>store at 4 C</td>
</tr>
<tr>
<td>1M Tris-HCl, pH 7.4</td>
<td>&nbsp;</td>
<td>Fisher Scientific </td>
<td>BP153-1</td>
<td>&nbsp;</td>
<tr>
<td>20% (wt/vol) dodecyl-b-D-maltoside (DDM)</td>
<td>&nbsp;</td>
<td>Fluka</td>
<td>44205-500MG</td>
<td>Dissolved in water. Store 1 ml aliquots at &#x2013;20 C.</td>
</tr>
<tr>
<td>10% (wt/vol) digitonin (>50% purity, used without recrystallization)</td>
<td>&nbsp;</td>
<td>Fluka</td>
<td>37008-1G</td>
<td>Dissolved in water. Store 0.1&#x2013;1 ml aliquots at &#x2013;20 C. Should be warmed up to 95 C before use.</td>
</tr>
<tr>
<td>2M 6-aminohexanoic acid</td>
<td>&nbsp;</td>
<td>Fluka</td>
<td>07260-100G</td>
<td>store at 4 C</td>
</tr>
<tr>
<td>10x Ponceau S/glycerol stock solution</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P-3504 and G6279-1L</td>
<td>0.1% Ponceau S, 50% glycerol, (wt/vol) in water.</td>
</tr>
<tr>
<td>10% w/v sodium deoxycholate</td>
<td>&nbsp;</td>
<td>Fluka </td>
<td>30970-25G</td>
<td>Dissolved in water. Must be protected from light. Adjust pH to 7.0 with HCl, filter and store at room temperature.</td>
</tr>
<tr>
<td>250 mM phenazine methasulfate (PMS)</td>
<td>&nbsp;</td>
<td>TCI America</td>
<td>P0083</td>
<td>Dissolved in DMSO and aliquoted. Store at &#x2013;20 C.</td>
</tr>
<tr>
<td>Nitro-blue tetrazolium (NBT)</td>
<td>&nbsp;</td>
<td>Amresco </td>
<td>0329-1G</td>
<td>&nbsp;</td>
<tr>
<td>3,3&#x2019;-diaminobenzidine tetrahydrochloride (DAB)</td>
<td>&nbsp;</td>
<td>TCI America </td>
<td>D0078</td>
<td>&nbsp;</td>
<tr>
<td>Pb(NO3)2</td>
<td>&nbsp;</td>
<td>Fisher Scientific </td>
<td>L62-100</td>
<td>&nbsp;</td>
<tr>
<td>MgSO4</td>
<td>&nbsp;</td>
<td>Fisher Scientific </td>
<td>BP213-1</td>
<td>&nbsp;</td>
<tr>
<td>Tris base </td>
<td>&nbsp;</td>
<td>Fisher Scientific </td>
<td>BP152-10</td>
<td>&nbsp;</td>
<tr>
<td>Glycine</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>G45-212</td>
<td>&nbsp;</td>
<tr>
<td>Adenosine 5&#x2019;-triphosphate disodium salt (ATP)</td>
<td>&nbsp;</td>
<td>Sigma </td>
<td>A2383-5G</td>
<td>Grade I, minimum 99%</td>
</tr>
<tr>
<td>beta-nicotineamide adenine dinucleotide (NADH) reduced form </td>
<td>&nbsp;</td>
<td>Sigma </td>
<td>N-8129</td>
<td>&nbsp;</td>
<tr>
<td>Cytochrome c From horse heart</td>
<td>&nbsp;</td>
<td>Sigma </td>
<td>C2506-250MG</td>
<td>&nbsp;</td>
<tr>
<td>Sucrose </td>
<td>&nbsp;</td>
<td>Fisher Scientific </td>
<td>BP220-212</td>
<td>&nbsp;</td>
<tr>
<td>100x protease inhibitor cocktail</td>
<td>&nbsp;</td>
<td>Sigma </td>
<td>P8340</td>
<td>Store at &#x2013; 20 C.</td>
</tr>
<tr>
<td>Dimethyl sulfoxide (DMSO)</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>D128-500</td>
<td>&nbsp;</td>
<tr>
<td>Trypan blue Stain 0.4%</td>
<td>&nbsp;</td>
<td>Gibco </td>
<td>15250-061</td>
<td>&nbsp;</td>
<tr>
<td>10x Trypsin (0.5%)</td>
<td>&nbsp;</td>
<td>Gibco </td>
<td>15400-054</td>
<td>&nbsp;</td>
<tr>
<td>Trypsin Inhibitor </td>
<td>&nbsp;</td>
<td>Gibco </td>
<td>17075-029</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

probing for mitochondrial complex activity in human embryonic stem cells

Mitokondri, sitoplazma, hücre metabolizması ve homeostasis birincil rolü var organelleri, hücre sinyal ağının düzenlenmesi, ve programlı hücre ölümü. Mitokondri üretmek ATP, sitoplazmik redoks devlet ve Ca2 + dengesini düzenleyen yağ asitleri catabolize, hem, nükleotidler, steroid hormonlar, amino asitler, sentez ve proteinlerin demir-kükürt kümeleri bir araya yardımcı. Mitokondri, aynı zamanda ısı üretimi önemli bir rolü var. Mitokondriyal genom mutasyonları insan bozukluğunun çeşitli neden olur. MtDNA mutasyonlarının birikmesi, yaşlanma ile ilişkilidir ve kanser gelişiminde önemli bir role sahip şüphelenilen. Tüm hücre tipleri hayati önemli rol nedeniyle, mitokondri fonksiyonu da kök hücreler için kritik öneme sahip olmalıdır. Kök hücre canlılığı, proliferasyon ve farklılaşma kapasitesi anlayışımızın önemli ilerlemeler yapılmıştır. Ancak kök hücrelerin fonksiyonel aktivite, özellikle enerji durumu, henüz detaylı bir şekilde incelenmemiştir değildi. Insan embriyonik kök hücreleri (hESC) ve farklılaştırılmış habercisi döl mitokondriyal özellikleri hakkında neredeyse hiçbir şey bilinir. HESC işlev ve gelişim potansiyeli mitokondri rolünü anlamak ve değerlendirmek için tek yolu doğrudan mitokondriyal solunum kompleksleri aktivitesini ölçmek için. Burada, yüksek çözünürlüklü hESC beş solunum zinciri kompleksleri analiz etmek için bir yöntem olarak jel faaliyet görselleştirme net yerli jel elektroforezi ve sonraki açıklar.

Watch this Scientific Journal Video about Probing for Mitochondrial Complex Activity in Human Embryonic Stem Cells at JoVE.com

Probing for Mitochondrial Complex Activity in Human Embryonic Stem Cells

Bu video, insan embriyonik kök hücreleri mitokondriyal solunum zinciri kompleksleri jel faaliyet deneyleri kullanarak nasıl analiz edilebilir size gösterecektir.

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 ...

probing-for-mitochondrial-complex-activity-human-embryonic-stem

Universidad San Sebastian

Research

JoVE Journal

Biology

14.2K Görüntüleme.  University of California, Los Angeles. Bu video, insan embriyonik kök hücreleri mitokondriyal solunum zinciri kompleksleri jel faaliyet deneyleri kullanarak nasıl analiz edilebilir size gösterecektir.

Video: Probing for Mitochondrial Complex Activity in Human Embryonic Stem Cells

Propagation of Human Embryonic Stem (ES) Cells

Propagation of Human Embryonic Stem ES Cells

Yeah, so I'm Lauren Deon. I'm in charge of the Human Amgen stem cell co facility at MGH. Yeah, so I'm Lauren, I'm in charge of the Human AM stem cell co facility at MGH.So I've been working with human Amgen stem cells for years now since I was a postdoc in George De Lab before, from four, four years. And so right now we have two cell lines, H one and H nine, which are both from Y cell. And so the way we are expanding or maintaining these cells in culture is by, at least the way we are splitting these cells is by using collagenous.So what I usually do is for the first few passage after throwing the cells, I use a technical, the picking colonies. So in this case, I can check under the microscope which colonies are good and differentiate and nice shape, and I will collect only these colonies and I will cut them in few pieces and put them back in, in a fresh flat. And the, the colonies that look kind of differentiate it are just left them alone.The other way to pass the cells is by using trypsin. So I don't use this technique here. I think collagenase is a little bit safer because human being stem cells don't like to be individualized or they don't like to be alone.So if you do trypsin, if you use trypsin for a little bit too long, if you use it for like three to four minutes, which is the way we use strips in for other type of cells, human stem cells will be individualized and most of them will die or just differentiate or will not attach to the myth. So the idea is you have to keep the cells as clumps. You don't want them to be alone in the in, in the flask.So using collagenase, the advantage is to keep cells as plants. Using chipsy, you just select for cells that will survive this very, very harsh process of g opsonization. So these cells are usually very highly proliferative.So you get, you can adapt your cells to this tripsin process. The problem is that you have cells that they behave differently, they go much faster. And one risk is that you can get some commercial abnormality to, to this, to this very high division process.So you say that ization can cause selection of cells in culture for most proliferative cells.Right. More ative and probably also higher survival rates than other cells. So after proliferation, basically culture after proliferation in tripsin, after several passages become a different culture, these different properties.So it's, it's hard to figure out if they are exactly identical. If you do the test, the usual test, let's say you check markers, regular markers of end differentiated cells like OC four, nano one 60 SC four C, C3, all this molecule might still be expressed. If you do TER formation, this cells might still differentiate and be formed like al like normal cells.If you look at the karyotype, you might have some abnormality, but you might still have a normal karyotype. The, the, the selective advantage might be due to just a mutation that you cannot see by doing a karyotype. So the fact that they grow much faster is by itself I think a little bit worser.And if we want to use these cells for transplantation at some point, I would definitely not use this type of cells.Okay. So would you like to tell me what are the main technical problems with using this technique for, for passages of cells? So using S, yes.It's just a little bit longer and also it's, since the cells are not going as fast, it's just much longer to get a high number of cells to do some experiment. So it's just less convenient I would say. Would you like to tell me about few words about the goals of your experiment?How do you use CS cells? Okay, so my main project here is to get endothelial cells from human AB stem cells. And the idea is to be able to make blood.And we are working a lot with tissue bioengineering labs and one of their goal is to make tissue in vitro. But one of the limitation to this is the lack of blood vessels going through this tissues. So if we can bring endothelial cells or put endothelial cells in this tissue, we would have better growth of this tissue.So the idea few sources of cells are known for endothelial cells like cold blood or pressure blood, but it would be interesting to have another source of cells like human years. And the advantage is since human years can be expanded and most infinitely, you could modify these cells, make some inducible system and be able to use it. Then in your final product, your material cells, in this case.

İnsan Embriyonik Kök Hücre (ES) yayılması

Biyoloji

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 ...

Chromatin Immunoprecipitation from Human Embryonic Stem Cells

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are characterized by two fundamental properties: self-renewal and multipotency that allows a stem cell to differentiate into virtually any cell type 1. The progression stem cell to differentiated cell is characterized by loss of multipotency, structural and morphological changes and the hierarchic activity of transcription factors and signaling molecules, whose activities establish and maintain cell-type specific gene expression patterns. At the molecular level, cell differentiation involves dynamic changes of the structure and composition of chromatin and the detection of those dynamic changes can provide valuable insights into the functional features of stem cells and the cell differentiation process 2,3. Chromatin is a highly compacted DNA-protein complex that forms when cells package chromosomal DNA with proteins, mainly histones 4. Stemcellness and cell differentiation has been correlated with the presence of specific arrays of regulatory proteins such as epigenetic factors, histone variants, and transcription factors 2,3,5.
 Chromatin immunoprecipitation (ChIP) provides a valuable method to monitor the presence of RNA, proteins, and protein modifications in chromatin 6,7. The comparison of chromatin from different cell types can elucidate dynamic changes in protein-chromatin associations that occur during cell differentiation.
Chromatin immunoprecipitation involves the purification of in vivo cross-linked chromatin. The isolated chromatin is reduced to smaller fragments by enzymatic digestion or mechanical force. Chromatin fragments are precipitated using specific antibodies to target proteins or protein and DNA modifications. The precipitated DNA or RNA is purified and used as a template for PCR or DNA microarray based assays. Prerequisites for a successful ChIP are high quality antibodies to the desired antigen and the availability of chromatin from control cells that do not express the target molecule. ChIP can correlate the presence of proteins, protein and RNA modifications, and RNA with specific target DNA, and depending on the choice of outread tool, detects the association of target molecules at specific target genes or in the context of an entire genome. The comparison of the distribution of proteins in the chromatin of differentiating cells can elucidate the dynamic changes of chromatin composition that coincide with the progression of cells along a cell lineage.

The differentiation of ESC coincides with cell-type specific changes in the structure and composition of chromatin. The detection of those changes provides valuable insights into the mechanisms that define stemcellness and cell differentiation. Chromatin immunoprecipitation (ChIP) represents a valuable method to dissect the molecular mechanisms underlying stem cell differentiation.

İnsan Embriyonik Kök Hücre Kromatin Immunoprecipitation

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are ...

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 ...

Chromosomal Spread Preparation of Human Embryonic Stem Cells for Karyotyping

Although human embryonic stem cells (hESC) have been shown to present a stable diploid karyotype 1, many studies have reported that depending on culture conditions they become prone to acquire chromosomal anomalies such as addition of whole or parts of chromosomes. Indeed, during long-term culture, karyotypic alterations are observed when enzymatic or chemical dissociation are used 2,3,4, while manual dissection of colonies for passaging retains a stable karyotype 5. Besides, changes in the environment such as the removal of feeder cells also seem to compromise the genetic integrity of hESC 3,6. Once chromosomal alterations could affect cellular physiology, the characterization of the genetic integrity of hESC in vitro is crucial considering hESC as an essential tool in embryogenesis studies and drug testing. Furthermore, for future therapeutic purposes chromosomal changes are a real concern as it is frequently associated to carcinogenesis.
Here we show a simple and useful method to obtain high quality chromosome spreads for subsequent analysis of chromosome set by G-banding, FISH, SKY or CGH techniques 7,8. We recommend checking the chromosomal status routinely with intervals of 5 passages in order to monitor the appearance of translocations and aneuploidies
Priscila Britto and Rafaela Sartore contributed equally to the paper.

Karyotyping is a simple and useful technique widely used for detecting genetic alterations. Here we describe a step by step protocol for chromosome spread preparation of human embryonic stem cells for monitoring the chromosomal status of these cells maintained in culture.

Karyotipik İnsan Embriyonik Kök Hücreleri kromozom Yayılı Hazırlık

Although human embryonic stem cells (hESC) have been shown to present a stable diploid karyotype 1, many studies have reported that depending on culture ...

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 ...

Fate Mapping of Human Embryonic Stem Cells by Teratoma Formation 

Human embryonic stem cells (hESCs) have an unlimited capacity for self-renewal, and the ability to differentiate into cells derived from all three embryonic germ layers (1). Directed differentiation of hESCs into specific cell types has generated much interest in the field of regenerative medicine (e.g., (2-5)), and methods for determining the in vivo fate of selected or manipulated hESCs are essential to this endeavor. We have adapted a highly efficient teratoma formation assay for this purpose. A small number of specifically selected hESCs is mixed with undifferentiated wild type hESCs and Phaseolus vulgaris lectin to form a cell pellet. This is grafted beneath the kidney capsule in an immunodeficient mouse. As few as 2.5 x 105 hESCs are needed to form a 16 cm3 teratoma within 8-12 weeks. The fate of the originally selected hESCs can then be determined by immunohistochemistry. This method provides a valuable tool for characterizing tissue-specific reagents for cell-based therapy.

Fate Mapping of Human Embryonic Stem Cells by Teratoma Formation

Directed differentiation of hESCs into specific cells has generated much interest in regenerative medicine. We provide a concise, step-by-step protocol for determining the in vivo fate of selected hESCs that provides a valuable tool for characterizing tissue-specific reagents for cell-based therapy.