Teitell 연구소에서 인간 배아 줄기 세포 연구는 현재 재생 의료에 대한 캘리포니아 연구소 (CIRM) 씨앗 부여 RS1 - 00313에 의해 지원됩니다. 우리는 이러한 추가적인 연구에 조언과 통찰력을 제공하는 박사 칼라 쾰러 (UCLA)와 쾰러 실험실 구성원 감사합니다.

Mitochondrial 콤플렉스에 대한 고해상도 네이티브 클리어 젤 Electophoresis (hrCNE)가 인 - 젤 활동 Assays 인간 배아 줄기 세포 (hESCs)은 다음 표준 약관을 사용 MEF - 냉방 중간에 5 일 동안 Matrigel에서 성장으로 전환 ˠ - 조사 또는 mitomycin C - 대우 마우스 배아 섬유아 세포 (MEFs)의 monolayer에서 유지 관리되었습니다. MEFs 오염을 제거하려면, hESCs는 Matrigel에 다시 도금 및 추가 3 일간 MEF - 냉방 매체 성장했다. pluripotency 마커 월 4 SSEA - 4 Immunofluorescence 현미경은 hESC 차별 부족을 확인하는 데 사용되었다. <ol><li> 5 분 trypsinization (6 잘 플레이트에 따라 잘 1ml 1X의 트립신) 다음 따뜻한 1X PBS, pH를 7.4와 세포를 씻으십시오. </li><li> 신선한 트립신 억제제의 동일한 음량을 (1X PBS, pH를 7.4에서 1mg/ml) 추가합니다. 200g에 수확 세포와 원심 분리기, 5 분, 실온. </li><li> 신선한 1 &#39;테아제 억제제 칵테일과 함께, 1.7ml (세포 펠렛의 크기에 따라) 얼음 차가운 mitochondria 절연 버퍼 (6.6mM 이미다졸 / HCL, pH를 7.0 83mM 자당)의 - 0.8에서 뜨는 및 resuspend pelleted 세포를 폐기하십시오. 10 분 얼음에 품어. </li><li> 균 질기 (- 50 치기 ~ 40)와 세포를 다운스. 가벼운 현미경을 사용하여 균질의 완전성을 확인 trypan 파랑과 dounced 세포의 나누어지는 스테인. </li><li> 4 10 분 2만그램에서 원심 분리기 ° C 또한 핵 및 큰 세포 조각이 들어있는 원유 mitochondrial 펠렛을 얻을 수 있습니다. 가만히 따르다가와 뜨는을 버리고 시켰던 원유 mitochondrial 펠렛의 무게. 펠렛은 -80에 저장할 수 있습니다 ·이가 멈추는 지점있다면 C 액화 질소에 즉시 냉동에 따라. 참고 : 차등 원심 분리 또는 기타 그라디언트 기반 절차가 순수한 mitochondrial 막 분획을 분리하고 수집하기 위해 사용될 수 있습니다. 심판을 참조하십시오. 자세한 내용은 4. 이러한 대체 방법은 mitochondrial 단백질 대신 세포 중량 또는 휴대폰 번호의 MG 당 복잡한 활동의​​ MG 당 mitochondrial 복잡한 활동을 얻을 것입니다. mitochondrial 단백질의 농도는이 비교를하기 위해 정화 후 결정이 필요합니다. 또한, 다른 절연 버퍼이 접근 방식이 필요할 수 있습니다. 순수 mitochondrial 막 분획의 solubilization 들어, 아래의 단계 6 다음의 참고를 참조하십시오. </li><li> 얼음 차가운 solubilization 버퍼 (50mM NaCl, 50mM 이미다졸 / HCL 산도 7.0, 1mM EDTA (에틸렌 다이아 민 테트라 초산), 2mM 6 aminohexanoic 산성)과 vortexing하여 얼음에 원유 mitochondrial 펠렛을 Solubilize. 6 - aminohexanoic 산성은 1X 테아제 억제제 칵테일로 교체할 수 있습니다. 펠렛 중량의 20mg 당 solubilization 버퍼의 35μl를 사용하십시오. 10% 펠렛 중량 및 튜브를 flicking하여 믹스 20mg 당 W / V digitonin의 20μl를 추가합니다. 얼음 5-10 분 알을 품다. Dodecyl - BD - maltoside 또는 트리톤 X - 100은 단백질을 solubilize하는 digitonin에 대한 대체하실 수 있습니다. 세제 및 수량의 선택은 supercomplexes 또는 mitochondrial 단지의 multimeric 형태의 형성에 영향을 줄 수 있습니다. 자세한 내용은 ref.1를 참조하십시오. 참고 : 순수한 mitochondrial 막 분획을 solubilize에 필요한 세제의 수량 2~6그램 / G. 이르는 세제 / 단백질 무게 비율에 해당하는 mitochondrial 세포막을 solubilize하는 최적의 비율이 dodecyl - BD - maltoside에 대한 G / 2.5G이며, 대한 3g / G 트리톤 X - 100과 6g / digitonin에 대한 g. </li><li> 4 15 분 ° C.에 대한 10만그램에서 solubilized 펠릿을 원심 분리기 맑은 뜨는를 수집합니다. </li><li> 최종 5% W / V 글리세롤과 0.01 % W / V 개양귀비빛의 S (50 % W / V 글리세롤, 0.1 % W / V 개양귀비빛의 S 로딩 버퍼 재고 솔루션을 사용)에 버퍼를 로드할 추가합니다. </li><li> 레인 당 원유 mitochondrial 펠릿 20 - 40mg이 들어있는 볼륨 젤 우물을로드합니다. 3.5 %가 젤 스태킹있는 아크릴 아미드 기울기 젤 (4 -13 %)를 사용합니다. 참고 : 젤 준비와 전기 영동 조건 : bisacrylamide의 아크릴 아미드와 1.5g (49.5 % T, T는 AA와 bisAA와 C의 총 농도는 3% C.가의 48g : 겔 준비, 다음과 같은 아크릴 아미드 / 비스 - 아크릴 아미드 (AA / bisAA) 3 믹스를 사용하여 총 단량체로 crosslinker (bisAA)의 비율). 젤 버퍼 (3 &#39;) : 1.5M 6 - aminohexanoic 산성, 75mM 이미다졸, 산도 7.0 </li></ol><table style="padding-left: 60px;" border="0"><tbody><tr><td> 그라데이션 젤 : </td><td> 4퍼센트 </td><td> 13% </td><td> 젤 스태킹 : </td><td> 3.5 % </td></tr><tr><td> AA / bisAA 세 믹스 </td><td> 2 ML </td><td> 6.5 ML </td><td></td><td> 0.6 ML </td></tr><tr><td> 젤 버퍼 3 &#39; </td><td> 8.3 ML </td><td> 8.3 ML </td><td></td><td> 2.7 ML </td></tr><tr><td> 글리세린 </td><td></td><td> 5g </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> 총 부피 </td><td> 25 ML </td><td> 25 ML </td><td></td><td> 8 ML </td></tr><tr><td> 10 % APS </td><td> 139 μl </td><td> 125 μl </td><td></td><td> 67 μl </td></tr><tr><td> TEMED </td><td> 14 μl </td><td> 12.5 μl </td><td></td><td> 6.7 μl </td></tr></tbody></table> 양극 버퍼 : 25mM 이미다졸 / HCL, pH를 7.0 음극 버퍼 : 50mM Tricine, 7.5mM 이미다졸 / HCL, 0.02 % W / V dodecyl - BD - maltoside, 0.05 % W / V deoxycholate, 산도 7.0 기름을 붓고 감기 방 (4-7 ° C)에서 젤을 실행합니다. 사용되는 초기 전압은 100V입니다. 예제는 스태킹 젤 입력되면, 전압은 15mA (0.15 &#39;14&#39;14 cm 젤)로 현재 제한과 500V 증가합니다. 전기 영동은 붉은 개양귀비빛의 S 염료의 날카로운 라인이 젤 전면에 접근 3-4 H 후에 중지됩니다. 또는 젤은 70 100V에서 일정한 전압 밤새 실행할 수 있습니다. <ol><li> 인 겔 활동 assays. 각 분석에 대한 부화의 시간은 단백질 solubilization의 효율성과 잘 당 로드된 자료의 양에 따라 달라집니다. 단지 I, IV 및 V는 일반적으로 단지 II 및 III보다 강한 신호를 얻을 수 있습니다. </li></ol> 콤플렉스 I (CI) 몇 시간 동안 실온에서 CI 버퍼 (2.5mg/ml NBT,, 0.1mg/ml NADH 5mM 트리스 - HCL, pH를 7.4)로 겔 슬라이스를 품어. CI 버퍼 : 5mM 트리스 - HCL, pH를 7.4의 100ml 당 추가 : 0.01g NADH 0.25g NBT 15-45 분의 50 %와 10 %의 메탄올 초산에​​서 젤 수정. 고정 젤은 10% 초산에 보존되어 있습니다. 콤플렉스 II (CII) 몇 시간 동안 실온에서 CII 버퍼 (5mM 트리스 - HCL pH는 7.4, 0.2mM phenazine의 methasulfate,, 2.5mg/ml NBT succinate 20mM 나트륨)로 겔 슬라이스를 품어. CII 버퍼 : 5mM 트리스 - HCL, pH를 7.4의 100ml 당 추가 : 200μl 1M 나트륨 succinate 80μl의 phenazine의 methasulfate (250mM PMS는 DMSO에 용해) 0.25g NBT 처럼 복잡한에 대한 설명 젤 수정 콤플렉스 III (CIII) 상온에서 몇 시간 동안, CIII 버퍼 (0.05 % 3,3 &#39;- diaminobenzidine의 tetrahydrochloride (DAB) 50mM 인산 나트륨, 산도 7.2)로 겔 슬라이스를 품어. CIII 버퍼 : 50mM 인산 나트륨, 산도 7.2 100ml 당 추가 : 50mg의 diaminobenzidine의 tetrahydrochloride 처럼 복잡한에 대한 설명 젤 수정 콤플렉스 IV (문학) 몇 시간 동안 실온에서, 문학 버퍼 (0.05 % 3,3 &#39;- diaminobenzidine의 tetrahydrochloride (DAB), 50μM 말을 마음 시토크롬 C 50mM 인산 나트륨, 산도 7.2)로 겔 슬라이스를 품어. 문학 버퍼 : 50mM 인산 나트륨, 산도 7.2 100ml 당 추가 : 50mg의 diaminobenzidine의 tetrahydrochloride 0.1g 말을 마음 시토크롬 C 처럼 복잡한에 대한 설명 젤 수정 콤플렉스 V (CV) CV 버퍼 (35 밀리 트리스베이스, 270mM 글리신, 25 pH를 8.3 ° C, 14mM MgSO 4, 0.2 % W / V PB (NO 3) 2, 필름 ATP) 몇 시간 동안 실온에서.로 겔 슬라이스를 품어 CV 버​​퍼 : 솔루션의 당 100ml 추가 : <table border="0"><tbody><tr><td> 트리스베이스 </td><td> 0.424g </td></tr><tr><td> 글리신 </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> 산도를 (8.3 있어야합니다) 조정 필요 - 산성이나베이스를 사용하지 마십시오. 필터 추가 <table border="0"><tbody><tr><td> ATP </td><td> 0.441g </td></tr></tbody></table> 30 분 50 % 메탄올에서 젤를 수정하고 물을 전송할 수 있습니다.

<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>

이 비디오에서는, 우리는 인간의 배아 줄기 세포, 높은 해상도 명확한 기본 겔 전기 영동 (hrCNE)하여 분리하고, 인 겔 촉매 활동 assays에 의해 이후의 분석에서 mitochondrial 단백질 단지의 추출을 설명합니다. hrCNE 파란색 기본 겔 전기 영동의 비교 해상도 mitochondrial 막 단백질 단지를 해결 및 - 겔 활동 assays를위한 우수합니다. 이 기술은 mitochondrial 호흡 단지 IV를 정할뿐만 아니라 hESCs과 그들의 유도체 자손 모두에 어?...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<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

Mitochondria 세포질 세포의 대사 및 항상성에 주요 역할을 organelles, 세포 신호 네트워크의 규제 및 프로그램 세포 사망 있습니다. Mitochondria 생산 ATP는 세포질 산화 환원 상태와 Ca2 + 균형을 조절 지방산을 catabolize, 헴, 세포핵, 스테로이드 호르몬, 아미노산을 합성하고, 단백질의 철 - 유황 클러스터를 조립 도움이됩니다. Mitochondria는 열 생산에 필수적인 역할을했습니다. mitochondrial 게놈의 변이는 인간의 장애 여러 종류의 원인이됩니다. mtDNA 변이의 축적이 노화와 상호 및 암의 개발에 중요한 역할을 가지고 의심됩니다. 모든 세포 유형에서 극히 중요한 역할로 인해, mitochondria의 기능은 또한 줄기 세포에 중요합니다. 주요 진보는 줄기 세포 생존, 증식 및 분화 능력에 대한 우리의 이해에되었습니다. 그러나 줄기 세포의 기능 활동은, 특히 그들의 에너지 상태, 아직 자세히 연구되지 ​​않았습니다. 거의 아무것도 인간 배아 줄기 세포 (hESCs)와 그들의 차별화된 전구체 자손의 mitochondrial 속성에 대한 알려진되지 않습니다. hESC 기능과 발달 가능성에 mitochondria의 역할을 이해하고 평가하는 한 가지 방법은 직접 mitochondrial 호흡 단지의 활동을 측정하는 것입니다. 여기, 우리는 hESCs의 다섯 호흡기 체인 단지를 분석하는 방법으로 젤 활동 시각화의 고해상도 명확한 기본 겔 전기 영동과 이후에 대해 설명합니다.

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

이 비디오에서는, 우리는 인간의 배아 줄기 세포의 mitochondrial 호흡 체인 단지가 겔 활동 assays 사용하여 분석할 수있는 방법을 보여줍니다.

인간 배아 줄기 세포에서 Mitochondrial 복잡한 활동에 대한 탐색

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

Research

JoVE Journal

Biology

14.2K 조회수.  University of California, Los Angeles. 이 비디오에서는, 우리는 인간의 배아 줄기 세포의 mitochondrial 호흡 체인 단지가 겔 활동 assays 사용하여 분석할 수있는 방법을 보여줍니다.

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

Propagation of Human Embryonic Stem (ES) Cells

Propagation of Human Embryonic Stem ES Cells

인간의 배아 줄기 (ES) 세포의 전파

연구

생물학

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.

Immunosurgery에 의해 인간 배아 줄기 세포의 유도

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.

인간 배아 줄기 세포에서 염색질의 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.

자기 공명 영상을위한 인간 배아 줄기 세포의 체외 라벨에

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.

인간 배아 줄기 세포의 문화 및 유지 관리

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.

Karyotyping를위한 인간 배아 줄기 세포의 염색체 확산 준비

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.

인간 배아 줄기 세포를 동결 융해

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.

비디오 생물 정보학 인간의 배아 줄기 세포 콜로니 성장 분석

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.

Oligodendrocyte 엽 성의 전구 물질로 배아 줄기 세포의 분화

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.

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.