팀 도일, 박사 및 bioluminescence 이미징과 지원 VIVO 이미징에 대한 스탠포드 센터에 감사드립니다. 또한 매트릭스 솔루션과 줄기 세포 공동 사출 그녀의 기술을 공유 Ngan 황, 박사 덕분에. 마지막으로, 스티브 덕분에 박사에게 펠트 동물 동물 보호와 지원.

<ol><li> 더블 퓨전 리포터 유전자의 건설 <ol><li> 인간 배아 줄기 세포의 bioluminescence 이미징을 수행하기 위해서는 먼저 안정 등 Ubiquitin이나 EF1a 같은 제정 발기인에 의한 반딧불 루시페라제로 루시페라제 리포터 유전자를 표현 세포를 얻기 위해 필요합니다. </li><li> 이 프로토콜의 초점은 기자의 유전자 응용 프로그램에, 자세한 절차를 여기에 제공되지 않습니다 때문에. 그러나, 우리 실험실의 일반적인 전략은 반딧불 루시페라제 (fluc)와 pCDNA 3.1 이내 스페이서 +로 구분하여 향상된 녹색 형광 단백질 (egfp)를 포함하는 이중 융합 구조 벡터를 사용하는 것입니다. </li><li> 간단히 우리 두 융합 유전자가 원래 pCDNA 3.1 사이토 메갈로 바이러스 프로 모터 (+)의 하류에 위치하고 있었기 때문에 3.3 kbp의 단편은 자체 운영을 중지시키지 (SIN의 여러 복제 사이트에 출혈도 잡았 NdeI와 노티 소화하고 무딘 엔드를 사용하여 excised되었습니다 Ubiquitin 모터의 제어하에) lentiviral 벡터. </li></ol></li><li> Lentiviral 형질 도입 <ol><li> hESCs 10의 감염의 다중성 (뫄) (106 전지 incubated 약 107의 바이러스 titer)의 통과 후 3~5일을 transduced 수 있습니다. </li><li> 해동 바이러스 주식은 신선한 hESC 미디어에 직접 추가할 수 있습니다. </li><li> 나중에 미디어 12 24 시간을 새로 고칩니다. </li><li> 48 H 후, 전달 효율에 의해 질적으로 형광 현미경을 사용하여 평가하실 수 있습니다. 그 후, 외과는 감염된 세포를 분리하는 데 사용할 수 있습니다. </li></ol></li><li> Culturing의 hESCs 및 설정 이미징 소개 <ol><li> 문화 귀하의 루시페라제 긍정적인 hESCs를 피더없는 조건입니다. 우리는 일반적으로 표준 WiCell 프로토콜에 따라 에어컨 미디어 또는 상업적 중 하나를 사용하여 Matrigel 감소 성장 인자에 코팅 6 자 접시, 피더없는 미디어를 사용합니다. </li><li> 루시페라제 긍정적인 세포를 감지하기 위해서는 기자 프로브 D - luciferin 이미 준비하셔야합니다. 45 MG / ML의 최종 농도에서 1-1.5 ML 준비에서, 나누어지는의 luciferin을 수행하려면. -20 ° C에서 aliquots을 유지하지 않을 때 종이 타월하지 않을 때 저장소에 함께 걸쳐 사용하고 조명에 노출을 피하 인치 </li><li> 루시페라제 긍정적인 세포를 시각화하기 위해, 우리는 IVIS 50 IVIS 200 이미징 시스템 (Xenogen 공사, 알라 메다, CA)을 사용합니다. 이 후자의 시스템은 작은 동물을 일시적으로 마취에 대한 통합 isoflourane 장치와 유도 챔버를 포함합니다. </li></ol></li><li> hESCs의 시험 관내 bioluminescence 이미징에 <ol><li> 시험 관내 bioluminescence 이미징의 경우, 그것은 멸균 상태를 유지하는 것이 중요합니다. 따라서, 영상 시스템은 선호 세포 배양 룸에서 멸균한다. </li><li> 이미징 전에 세포 미디어를 제거하고 세포를 충당하기 위해 충분 PBS를 추가합니다. 예를 들어, hESC 문화를 포함하는 6 자 판의 각 잘하는 1mL PBS를 추가합니다. </li><li> PBS로 D - Luciferin의 비율은 1:100해야하므로 6 잘 플레이트의 각 잘하는 해동 D - Luciferin 10 μL를 추가합니다. </li><li> 잠시만 기다려 다음 일초의 노출 시간을 사용하여 이미지를 가져가라. 신호가 약한 경우, 노출 간격을 증가하고 다시 시도하십시오. </li><li> 발광 생물 신호 휴대폰 번호를 반영, quantitation assays의 서로 다른 세포 숫자와 신호를 상관 관계 것을 수행할 수 있도록. </li></ol></li><li> 생체내 이미징에 대한 마우스 및 주입 세포의 준비 <ol><li> 리포터 유전자 영상은 일, 주, 심지어 몇 달 동안 이식 후 추적 세포에 대한 높은 처리량, 저렴하고 민감한 방법을 제공합니다. 이식 줄기 세포가 자주, 기형종 형성 숙주의 면역 체계에 의해 거부, 또는 단순히 죽고 때문에 이것은 특히 중요합니다. </li><li> 생체내 이미징에서 기형 종 형성을위한 가장 간단한 방법은 SCID 생쥐의 백업에 반딧불 루시페라제 리포터 유전자를 표현하고 IVIS 시스템 발광 생물 이미지를 획득 hESCs를 삽입하는 것입니다. </li><li> 당신이 세포를 이식하기 위해 준비가되면, 세포를 풀어 여러 번 씻어하고, 성장 인자 감소 - matrigel과 DMEM의 1:1 혼합물에서 그들을 정지 dispase 또는 collagenase를 사용합니다. 일반적으로 우리가 먼저 차게 DME​​M으로 세포를 섞어 다음 matrigel에 추가합니다. 각각의 피하 주입 사이트에, 우리는 matrigel / DMEM 혼합물의 50-100 UL 볼륨에서 정지 200,000 개 셀을 주입. 휴대폰 번호는 응용 프로그램에 따라 조정할 수 있습니다. 사전 주사로 얼음에있는 모든 유지되도록주의를 기울이십시오. </li><li> 세포가 준비가되면,이 켜져 isoflurane 흐름 유도 상자에 배치하여 마우스를 마취. 1-5분 후, 마우스 잠이이고 지속적인 isoflurane으로 수술대에 둘 수 있습니다. </li><li> 모피 자연스럽게 자동 fluoresces하고는 발광 생물 이미지를 모호한 수 있기 때문에, 우리는 마우스의 뒷면에서 머리를 제거해야합니다. 이 작업을 수행하는 가장 쉬운 방법은 전기 면도기를 사용하는 것입니다,하지만 당신은 머리 - 제거 젤을 사용할 수 있습니다. 소독에 알코올로 닦아나중에 피부 </li><li> 다음 주사기에 세포 현탁액을 그립니다. 그들은 세포와 방해할 않기 때문에 23-27 게이지 바늘은 최고의 작동합니다. 바늘이 (&lt;23 게이지) 너무 큰 경우 바늘의 넓이 세포가 다시 밖으로 누출 수있는을 통해 큰 구멍을 떠날 수 있습니다. </li><li> 마우스의 뒷면에 피부 아래에있는 세포를 주사. 피부가 매우 느슨한 때문에, 핀치에 엄지와 집게손가락을 사용하고 삽입하고자하는 영역을 스트레칭. 너무 깊이 주사로 치료하지 복용, 피부 아래에 주사를 막. 또한, 플런저를 우울 동안 사출 사이트의 밖으로 미끄러져에서 바늘을 유지하려고이 다시 주입하려고하면 세포가 누출 수있는을 통해 피부에 구멍을 만드는 것을 방지할 수 있습니다. </li><li> 세포 주입 후, 마우스 깨어있게하고 bioluminescence 이미징을위한 다시 anesthetizing 전에 주위를 실행하는 데 몇 시간을 기다립니다. 이렇게하면 isoflurane 독성을 방지합니다. </li></ol></li><li> 이식 세포의 생체내 bioluminescence 이미징에 <ol><li> 전체 동물 bioluminescence 이미징을 위해, 우리는 375 밀리그램 / kg 체중에서 D - luciferin의 intraperitoneal 주사를 않습니다. 사전 주사로 주사 량을 계산하기 위해 동물을 나가는거야. </li><li> 그냥 정중선에서, 복막에있는 D - luciferin을 주사. 너무 깊이 가지 않은 또는 내부 장기를 손상시킬 수주의하십시오. 마우스 깨어하기 시작하면, 녹아웃 상자에 다시 배치하고 몇 분만 기다립니다. 생쥐의 경우 최대 bioluminescence은 일반적으로 주사 후 15-40 분을 발생합니다. </li><li> hESCs가 배출되지 않은 빛을 흡수에 도움이되는 이미징 상자에 무광택 검은 종이를 삽입해야합니다. 검은 종이 위에, isoflurane과 이미징 챔버에서 마우스 (후면까지)을 놓으십시오. 10초의 노출 시간과 함께 시작합니다. 신호가 포화되면, 노출 시간을 감소 노력, 너무 약한 경우, 노출을 증가시킵니다. </li><li> 신호 노출 시간이 마우스에 최적화된되면, 신호가 최대에 도달할 때까지 이미지를 매 분 복용 시작합니다. 이것은 가장 사출 사이트를 커버 &quot;관심 영역 (ROI)&quot;을 선택 이미징 소프트웨어를 사용하여 이루어집니다. 각각의 투자 수익 (ROI)에서 신호 강도를 모니터링함으로써 쉽게 최대 신호 강도에 도달되었음을 나타내는 신호가 감소하기 시작하면 확인할 수 있습니다. 최대 신호는 최종 데이터로 사용해야합니다. </li><li> 당신이 이미지에 만족하는 경우, isoflurane에서 마우스를 제거하고는 새장에 깨어 수 있습니다. 보통 마우스는 15 분 이내에 깨어날 것입니다. </li><li> hESCs이 동물의 생존과 같은 Bioluminescence 기자의 유전자 이미징이있는 동안, 매일 주별 및 월별 반복 수 있습니다. hESCs가 세포 분열 따위에 의해 번식을 시작으로 발전 기형 종에서 신호는 일반적으로 주의 순서에, 시간이 지남에 따라 기하 급수적으로 증가합니다. </li><li> bioluminescence 이미지의 원하는 시간 코스 취득 후, 동물 희생 및 조직 섹션 조직학에 사용할 수 있습니다. </li></ol></li></ol>

<ol>
	<li>Passier, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+cardiomyocyte+differentiation+from+human+embryonic+stem+cells+in+serum-free+cultures.">Increased cardiomyocyte differentiation from human embryonic stem cells in serum-free cultures.</a> Stem Cells. 23, 772-780 (2003).</li><li>Laflamme, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiomyocytes+derived+from+human+embryonic+stem+cells+in+pro-survival+factors+enhance+function+of+infarcted+rat+hearts.">Cardiomyocytes derived from human embryonic stem cells in pro-survival factors enhance function of infarcted rat hearts.</a> Nature biotechnology. 25, 1015-1024 (2007).</li><li>Baharvand, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+differentiation+from+human+embryonic+stem+cells+in+a+defined+adherent+culture+conditi.">Neural differentiation from human embryonic stem cells in a defined adherent culture conditi.</a> Int J Dev Biol. 51, 371-378 (2007).</li><li>Schulz, T. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+neuronal+differentiation+of+human+embryonic+stem+cells.">Directed neuronal differentiation of human embryonic stem cells.</a> BMC Neurosci. 4, (2007).</li><li>Jiang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+Insulin-producing+Islet-like+Clusters+from+Human+Embryonic+Stem+Cells.">Generation of Insulin-producing Islet-like Clusters from Human Embryonic Stem Cells.</a> Stem Cells. 17, 17 (2007).</li><li>Swijnenburg, R. J., van der Bogt, K. E. A., Sheikh, A. Y., Cao, F., Wu, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+hurdles+for+the+transplantation+of+cardiomyocytes+derived+from+human+embryonic+stem+cells:+role+of+molecular+imaging.">Clinical hurdles for the transplantation of cardiomyocytes derived from human embryonic stem cells: role of molecular imaging.</a> Current Opinion in Biotechnology. 18, 38-45 (2007).</li><li>Herschman, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+imaging:+looking+at+problems,+seeing+solutions.">Molecular imaging: looking at problems, seeing solutions.</a> Science. 302, 605-608 (2003).</li><li>Lippincott-Schwartz, J., Patterson, G. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+use+of+fluorescent+protein+markers+in+living+cells.">Development and use of fluorescent protein markers in living cells.</a> Science. 300, 87-91 (2003).</li><li>Contag, P. R., Olomu, I. N., Stevenson, D. K., Contag, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioluminescent+indicators+in+living+mammals.">Bioluminescent indicators in living mammals.</a> Nature medicine. 4, 245-247 (1998).</li><li>Bhaumik, S., Gambhir, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+imaging+of+Renilla+luciferase+reporter+gene+expression+in+living+mice.">Optical imaging of Renilla luciferase reporter gene expression in living mice.</a> Proceedings of the National Academy of Sciences of the United States of America. 99, 377-382 (2002).</li><li>Tisi, L. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+thermostable+firefly+luciferase.">Development of a thermostable firefly luciferase.</a> Analyica Chimica Acta. 457, 115-123 (2002).</li><li>Ray, P., Tsien, R., Gambhir, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+and+validation+of+improved+triple+fusion+reporter+gene+vectors+for+molecular+imaging+of+living+subjects.">Construction and validation of improved triple fusion reporter gene vectors for molecular imaging of living subjects.</a> Cancer Res. 67, 3085-3093 (2007).</li><li>Cao, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+visualization+of+embryonic+stem+cell+survival,+proliferation,+and+migration+after+cardiac+delivery.">In vivo visualization of embryonic stem cell survival, proliferation, and migration after cardiac delivery.</a> . Circulation. 113, 1005-1014 (2006).</li><li>Cao, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+Imaging+of+Embryonic+Stem+Cell+Misbehavior+and+Suicide+Gene+Ablation.">Molecular Imaging of Embryonic Stem Cell Misbehavior and Suicide Gene Ablation.</a> Cloning and Stem Cells. 9, 107-117 (2007).</li><li>Ray, P., De, A., Min, J. J., Tsien, R. Y., Gambhir, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+tri-fusion+multimodality+reporter+gene+expression+in+living+subjects.">Imaging tri-fusion multimodality reporter gene expression in living subjects.</a> Cancer Res. 64, 1323-1330 (2004).</li><li>Wu, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+analysis+of+reporter+genes+for+molecular+imaging+of+transplanted+embryonic+stem+cells.">Proteomic analysis of reporter genes for molecular imaging of transplanted embryonic stem cells.</a> Proteomics. 6, 6234-6249 (2006).</li><li>Eiges, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+human+embryonic+stem+cell-transfected+clones+carrying+a+marker+for+undifferentiated+cells.">Establishment of human embryonic stem cell-transfected clones carrying a marker for undifferentiated cells.</a> Curr Biol. 11, 514-518 (2001).</li><li>Mohr, J. C., de Pablo, J. J., Palecek, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electroporation+of+human+embryonic+stem+cells:+Small+and+macromolecule+loading+and+DNA+transfection.">Electroporation of human embryonic stem cells: Small and macromolecule loading and DNA transfection.</a> Biotechnol Prog. 22, 825-834 (2006).</li><li>Siemen, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleofection+of+human+embryonic+stem+cells.">Nucleofection of human embryonic stem cells.</a> Stem Cells Dev. 14, 378-383 (2005).</li><li>Lakshmipathy, U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+transfection+of+embryonic+and+adult+stem+cells.">Efficient transfection of embryonic and adult stem cells.</a> Stem Cells. 22, 531-543 (2004).</li><li>Ma, Y., Ramezani, A., Lewis, R., Hawley, R. G., Thomson, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-Level+Sustained+Transgene+Expression+in+Human+Embryonic+Stem+Cells+Using+Lentiviral+Vectors.">High-Level Sustained Transgene Expression in Human Embryonic Stem Cells Using Lentiviral Vectors.</a> Stem Cells. 21, 111-117 (2003).</li><li>Menendez, P., Wang, L., Chadwick, K., Li, L., Bhatia, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retroviral+transduction+of+hematopoietic+cells+differentiated+from+human+embryonic+stem+cell-derived+CD45(neg)PFV+hemogenic+precursors.">Retroviral transduction of hematopoietic cells differentiated from human embryonic stem cell-derived CD45(neg)PFV hemogenic precursors.</a> Molecular therapy. 10, 1109-1120 (2004).</li><li>Thyagarajan, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Creation+of+engineered+human+embryonic+stem+cell+lines+using+phiC31+integrase.">Creation of engineered human embryonic stem cell lines using phiC31 integrase.</a> Stem cells. 26, 119-126 (2008).</li><li>Liew, C. -. G., Draper, J. S., Walsh, J., Moore, H., Andrews, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Transient and Stable Transgene Expression in Human Embryonic Stem Cell. , .</li></ol>

이러한 PET와 MRI와 같은 다른 modalities 비교, bioluminescence 제한된 공간 해상도와 방출되는 광자의 상대적으로 약한 에너지 (2-3 EV)으로 인해 감소 조직 침투력을 가지고, 이러한 이유로 그것은 지금까지 큰 동물에서 적용되지 않았습니다. 그러나, bioluminescence 작은 동물의 생체내 추적의 줄기 세포에 대해 그것이 매우 바람직 만들기, 저렴한 비용, 높은 처리량, 그리고 비침습되는 장점이 있습니다. 이러한 PET 및 형광 ...

<!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>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>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

in vitro and in vivo bioluminescence reporter gene imaging of human embryonic stem cells

인간 배아 줄기 세포 (hESCs)의 발견은 극적으로 재생 의료에 관심이 의료 과학자에 사용할 수있는 도구를 증가했습니다. 그러나, 살아있는 생물로, hESCs에서 차별 hESCs, 그리고 세포의 직접 분사는 지금까지 상당한 세포 사망, 기형 종의 형성, 및 호스트 면역 거부 반응에 의해 방해되었다. 이식 후 생체내 hESC 행동에을 이해하는 것은 새로운 이미징 기술은 길이 방향 hESC 현지화, 증식 및 생존을 모니터링해야합니다. 분자 영상은 조사에게 생체내 세포 일 동안 확산, 주, 심지어 개월 이내에 추적에 대한 높은 처리량, 저렴하고, 중요한 수단을 제공하고 있습니다. 이 발전은 크게 hESC의 engraftment, 확산, 생활 과목의 기형 종 - 형성 spatio - 시간적 동력학에 대한 이해를 증가하고 있습니다. 분자 이미징의 주요 사전은 생체내에서 다중 양상 이미징 플랫폼으로 분자 및 세포 생물학에서 비침 투 리포터 유전자의 assays의 확장되었습니다. 이 기자의 유전자는 숙주 세포의 transcriptional 기계를 활용 설계 발기인 및 강화의 통제하에, 벡터가 아닌 벡터 다양한 방법을 사용하여 세포에 도입하고 있습니다. 일단 세포에서 기자의 유전자를 사용 모터의 종류에 따라, 특정 생물 학적 또는 세포 조건 중 constitutively 또는 유일한 베꼈는데 수 있습니다. bioactive 단백질로 전사와 기자의 유전자의 번역은 다음 D - luciferin 같은 신호 생성 프로브를 사용하여 민감한, 비침 투 계측 (예 : CCD 카메라)와 검색입니다. 로 형광 이미징에 필요한 생체내에서 줄기 세포를 추적하는 흥분성의 조명의 필요성을 방지하기 위해, bioluminescence의 리포터 유전자 이미징 시스템은 발광을 유발에만 exogenously 관리 프로브가 필요합니다. 반딧불 Photinus pyralis에서 파생된 반딧불 루시페라제은, 광학 활성 대사, oxyluciferin에 D - luciferin을 catalyzes 효소를 인코딩합니다. 광학 활동 후 외부 CCD 카메라로 모니터링할 수 있습니다. 안정 기자 들고 세포들은 염색체 DNA 내에 구축 transduced 기자가 생체내에 hESC의 생존과 확산의 길이 모니터링있게 딸 세포에 DNA를 구성 전달합니다. 리포터 유전자 제품의 표현은 신호 생성을 위해 필요하기 때문에 또한, 오직 실용적 부모와 딸 세포는 bioluminescence 신호를 만들 것이다; apoptotic이나 죽은 세포가되지 않습니다. 이 동영상에서 특정 자료 및 bioluminescence 이미징과 함께 추적 줄기 세포 증식 및 기형 종의 형성에 필요한 방법은 설명한다.

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

줄기 세포 요법에 대한 관심이 점점으로, 분자 이미징 기술은 이식 후 모니터링 줄기 세포 행동에 이상적입니다. 루시페라제 리포터 유전자는 세포 생존, 위치 및 생체내의 확산의 비침습, 반복적인 평가를 활성화합니다. 이 동영상은 살아있는 마우스의 hESC 확산을 추적하는 방법을 보여줍니다 것입니다.

체외에서 인간 배아 줄기 세포의 생체내의 Bioluminescence 리포터 진 영상에

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

in-vitro-vivo-bioluminescence-reporter-gene-imaging-human-embryonic

Research

JoVE Journal

Biology

21.7K 조회수.  Stanford University School of Medicine. 줄기 세포 요법에 대한 관심이 점점으로, 분자 이미징 기술은 이식 후 모니터링 줄기 세포 행동에 이상적입니다. 루시페라제 리포터 유전자는 세포 생존, 위치 및 생체내의 확산의 비침습, 반복적인 평가를 활성화합니다. 이 동영상은 살아있는 마우스의 hESC 확산을 추적하는 방법을 보여줍니다 것입니다.

비디오: 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.

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 ...

연구

생물학

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.

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 ...

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 ...

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 ...

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.

작은 분자와 유도 Pluripotent 인간 배아 줄기 세포로부터 인간 심장 엽 성의 전구 물질과 Cardiomyocytes의 효율 유도

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.

GFP와 Retroviral 벡터를 사용하여 유도 Pluripotent 줄기 세포 (iPSCs)로 인간 체세포를 프로그래밍

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.

Ribosome 바운드 초기 Polypeptides의 분리<em&gt; 체외에서</em&gt; mRNA 함께 Translational 일시 중지 사이트를 식별하려면

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