1. 해동 hES 세포 사전 실험 설정 <ol><li> 하루 전에 해동 hES 세포로, 플레이트 MEF 문화 매체에 젤라틴 코팅 6 잘 조직 배양 플레이트 (D - 멤 기초 매체와 보충 잘 하나에 조사 마우스 배아 섬유아 세포 (MEF), CF1 변형의 피더 층 10% 열 inactivated FBS, 1 %가 아닌 필수 아미노산). 37 밤새 품어 ° C와 5% CO 2. </li><li> 액체 질소에서 hES 세포를 제거하기 전에, 장소에 필요한 모든 장비와 시약을 가지고 있는지 확인하고 효율적이고 성공적으로 해동을 위해 사용할 수 있습니다. </li></ol> hES 세포를 해동 <ol start="3"><li> 금속 집게를 사용하여 액체 질소 저장 탱크에서 hES 세포의 극저온 약병을 제거합니다. </li><li> 서리를 제거하는 3-5초 위해 장갑 낀 손 사이의 병을 롤. </li><li> 유리병의 라벨에 정보를 기록합니다. </li><li> 금속 집게를 사용하여 37 ° C의 물 목욕으로 병을 담가. 소용돌이 부드럽게 유리병과 얼음 결정의 크기를 확인하기 위해 조명에 병을를 개최하여 (그러나 빨리!) 자주 해동의 진행을 관찰. 잠수함 물 목욕에있는 유리병의 뚜껑을이 세포를 오염시킬 수 있으므로하지 마십시오. </li><li> 단지 작은 얼음 결정이 남아 잠수함 소독에 에탄올의 전체 유리병. </li><li> 무균 생물 학적 안전 캐비닛에서 직접 15 ML 원뿔 관의 하단에 극저온 약병의 내용을 전송합니다. </li><li> 천천히 hES 세포 배양 매체 4 ML (D-MEM/F-12 기초 매체 20 % 넉아웃 혈청 교체, 1 %가 아닌 필수 아미노산, 1 % L - 글루타민, 0.2 % β - 메르 캅 토 에탄올과 4 NG /로 보충을 추가 튜브에 ML bFGF). 부드럽게 새로운 매체가 튜브에 추가로 지속적으로 세포를 섞어 바위. </li><li> 200 X G.에서 5 분 동안 세포를 원심 분리기 </li></ol> hES 세포를 도금 <ol start="11"><li> 셀 라인 통로 번호 및 해동 날짜 : hES 세포는 원심 분리기에있는 동안, 해당 hES 세포 정보를 라벨로 MEF 피더 플레이트를 준비합니다. </li><li> 스핀 시간에 남아있는 약 2 분이되면, MEF 문화 매체를 대기음하고 잘하는 PBS의 1.0 ML를 추가합니다. </li><li> 생물 학적 안전 캐비닛 다시 pelleted hES 세포를 가지고, 그리고 신중하게 뜨는을 대기음. 이 솔루션은 냉동 매체에서 DMSO가 들어 있으므로 세포 펠렛을 대기음지만, 가능 한한 많이 뜨는 제거하지 않도록주의하십시오. </li><li> 펠렛은 5 ML 유리 pipet을 사용하여 hES 세포 배양 매체 2.5 ML를 추가하고 3-4 회를 pipetting하여 매우 부드럽게 다시 일시 중지합니다. </li><li> 잘 MEF 피더에서 PBS를 대기음 천천히 6 잘 접시 잘 준비에 hES 세포 현탁액의 2.5 ML를 추가합니다. </li><li> 37 ° C 배양기에 접시를 놓고 신중하게 고르게 잘 내내 세포를 배포하는쪽으로 뒤로 앞으로 판과 측면을 슬라이드. 중앙의 식민지를 집중 방지하기 원형 패턴에 소용돌이 접시를하지 마십시오. </li><li> 세포 ° C와 5% CO 2 밤새 37 첨부할 수 있습니다. </li><li> 해동 후 하루 매체를 제거합니다 (임의의 부동 세포)와 잘 신선한 hES 세포 배양 매체 2.5 ML를 추가합니다. </li><li> 옵션 : 중간 제거하고 세포가 별도의 준비 MEF 피더 플레이트로 전송하실 수 있습니다. 이 단계는 종종 원래 해동의 세포와 병렬로 양식 수있는 새로운 식민지를 생성합니다. </li><li> 37 ° C와 5% CO 2 밤새에서 접시를 품어. </li></ol> 2. hES 세포를 동결 <ol><li> hES 세포는 원심 분리기에있는 동안, 세포 라인 통로 번호 및 동결 기간을 포함한 생물 학적 안전 캐비닛 내부의 극저온 튜브를 라벨. 냉동 hES 세포에 대한 전형적인 밀도는 극저온 유리병 당 세포 (6 잘 판에서) 잘 하나입니다. </li><li> hES 세포가 centrifuging 완료되면, 생물 안전 캐비닛 다시 튜브를 가져와. 느슨하게 - 포장 세포 펠렛을 방해하지 않도록주의하고, 튜브의 표면에 뜨는를 대기음. </li><li> 극저온 유리병 당 0.5 ML hES 세포 배양 매체 : hES 세포 배양 매체의 적절한 금액으로 세포 펠렛을 Resuspend. 비교적 큰 콜로니 조각에 얼어 붙 때 hES 세포보다 회복으로, pipetting 할 때 조심. </li><li> 천천히, 부드럽게 튜브를 흔들어 동안 세포 2X hES 세포 냉동 매체 (60 % 정의된 FBS, 20 % hES 세포 배양 매체, 20 % DMSO)의 적절한 금액 (cryovial 당 0.5 ML)를 추가합니다. 일단 냉동 매체 pipet 믹스에 1-2 시간은 매우 부드럽게, 솔루션에 추가됩니다. </li><li> 신속하게하지만 천천히, 각 극저온 약병에 세포 현탁액 1 ML를 추가합니다. </li><li> 에 이소프로판올 냉동 컨테이너와 장소에 튜브를 전송하룻밤 -80 ° C 냉동고. 세포는 1 ° C 이소프로판올 냉동 컨테이너에 분당 정지됩니다. </li><li> 그 다음날, 신속하게 금속 집게를 사용하여 액체 질소 저장 탱크에 냉동 튜브를 전송합니다. </li></ol> 대표 결과 인간 ES 세포를 해동하는 첫째날 이후 작은 식민지가 투명하게 나타날 수 있고 현미경으로 볼 수 어려울 수 있습니다. 새로 해동​​ ES 세포가 서서히 분아 따위에 의해 번식하는 경향이 있기 때문에, 그들은 식민지 설립 (그림 1)로 표시 며칠이 걸릴 수 있습니다. 그림 1. 핸드폰 복구 및 성장. hES 세포 액체 질소에서 해동이 몇 군데 있었다 1, 7 11일 해동 후.

<ol>
	<li>Freshney, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Culture of Animal Cells: A Manual of Basic Technique. , (2005).</li><li>Thomson, J. A., Itskovitz-Eldor, J., Shapiro, S. S., Waknitz, M. A., Swiergiel, J. J., Marshall, V. S., Jones, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+stem+cell+lines+derived+from+human+blastocysts.">Embryonic stem cell lines derived from human blastocysts.</a> Science. 282 (5391), 1145-1147 (1998).</li></ol>

함께 비디오 hES 세포를 해동 및 냉동하는 방법을 보여줍니다. 액화 질소에 냉동 세포는 최상의 복구를 얻기 위해 최대한 빨리 목욕을 해동한다. 해동 세포를 pipetting 할 때 매우 조심해야하는 기억 세포의 취급을 최소화하고 부드럽게 피펫. hES 세포 중 하나 유리병은 6 자 판, 또는 4 자 판의 모든 우물을 잘 둘 중 누구에 도금 즉시 인큐베이터에 배치하실 수 있습니다. 네 별도의 우물에 culturing은 오염에 대한 전체 문화를 잃는의 가능성을 감소하고, 쉽게 작은 표면적에 hES 세포 식민지를 찾을 수 있습니다 이후 처음으로 hES 세포를 해동하면, 4 - 음 판을 권장합니다. hES 세포를 해동 후 첫날, 식민지 작은 있으며, 투명 수 있습니다. 그것이 설립 식민지로 표시 hES 세포에 대한 문화에 며칠이 걸릴 수 있습니다 이후 식민지가 현미경으로 명확하지 않더라도, 매일 hES 세포 배양 매체를 보충. 식민지가 자주 해동 후 10 12일까지 passaging에 적절한 크기에 도달하지 않으면 그것은 세포를 해동하면 갓 도금 MEF 피더 레이어를 사용하는 것이 중요합니다. hES 세포의 낮은 통로의 약병을 해동하면, 그것은 미래의 문화와 실험에 대한 낮은 통과 세포의 재고를 유지하기 위해 추가 튜브를 확장하고 동결하는 것이 좋습니다. 또한 정기적으로 냉동 주식을 유지하기 위해 모든 &quot;추가&quot;건강한 hES 세포를 동결하는 것이 좋습니다. 가장 성공적인 녹아서 이후 문화는 고품질, undifferentiated, 적극적으로 나누어 hES 세포 식민지로 동결했다. 큰 세포 집계로 부드럽게 처리하는 경우 hES 세포가보다 효율적으로 정지에서 복구합니다. 최종 집계 크기는 일상적인 통로 후 도금 집계의 크기 (또는보다 약간 큰)과 유사해야합니다.

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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>D-MEM</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11965-092</td>
<td>For MEF mediumjavascript:void(0);</td>
</tr>
<tr>
<td>Fetal Bovine Serum (FBS), Certified</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>16000-044</td>
<td>Heat-inactivated 
For MEF medium</td>
</tr>
<tr>
<td>D-MEM/F-12</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11330-057</td>
<td>&nbsp;</td>
<tr>
<td>Knockout Serum Replacement</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>10828-028</td>
<td>Pre-screen to ensure support of hES cells</td>
</tr>
<tr>
<td>bFGF</td>
<td>&nbsp;</td>
<td>Stemgent</td>
<td>03-0002</td>
<td>Use at [4 ng/mL] final</td>
</tr>
<tr>
<td>Non-essential Amino Acids</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11140-050</td>
<td>&nbsp;</td>
<tr>
<td>L-glutamine</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>25030-081</td>
<td>200 mM (100X) 
Use at [1X] final</td>
</tr>
<tr>
<td>PBS</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>14190-250</td>
<td>Without Ca2+ or Mg2+</td>
</tr>
<tr>
<td>Collagenase IV</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>17104-019</td>
<td>Make a fresh solution at 1 mg/mL in D-MEM/F-12</td>
</tr>
<tr>
<td>Gelatin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G1890</td>
<td>Type A, Porcine</td>
</tr>
<tr>
<td>DMSO</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>D2560</td>
<td>For freezing medium</td>
</tr>
<tr>
<td>Fetal Bovine Serum (FBS), Defined</td>
<td>&nbsp;</td>
<td>HyClone</td>
<td>SH300.70.01</td>
<td>For freezing medium</td>
</tr>
<tr>
<td>6-well plates</td>
<td>&nbsp;</td>
<td>Nunc</td>
<td>140675</td>
<td>For general culture</td>
</tr>
<tr>
<td>4-well plates</td>
<td>&nbsp;</td>
<td>Nunc</td>
<td>176740</td>
<td>For thawing</td>
</tr>
<tr>
<td>5 mL glass pipettes</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>13-678-27E</td>
<td>Individually wrapped</td>
</tr>
<tr>
<td>15 mL conical tubes</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>430052</td>
<td>Polypropylene</td>
</tr>
<tr>
<td>Cryogenic vials</td>
<td>&nbsp;</td>
<td>Nunc</td>
<td>5000-1020</td>
<td>1.5 mL capacity</td>
</tr>
<tr>
<td>Freezing container</td>
<td>&nbsp;</td>
<td>Nalgene</td>
<td>5100-0001</td>
<td>&#x201C;Mr. Frosty&#x201D;</td>
</tr>		</tbody>
		</table>
		</div>

freezing and thawing human embryonic stem cells

제임스 톰슨 이후<em&gt; 외</em&gt; 문화 hES을 분리하고 성장 1998 년 기술을 개발, 나중에 사용하고 냉동 주식에서 세포를 해동 및 확장을위한 냉동 세포는 일상 hES 세포 배양에서 수행한 중요한 절차가있다. hES 세포는 동결 융해의 스트레스에 민감하고 있으므로 특별한주의가 있어야합니다. 여기 우리는 신속하게, 액체 질소 주식에서 hES 세포를 해동 마우스 배아 피더 세포를 도금하고, 천천히 장기 저장을 위해 그들을 오해해서 적절한 방법을 보여줍니다.

Watch this Scientific Journal Video about Freezing and Thawing Human Embryonic Stem Cells at JoVE.com

Freezing and Thawing Human Embryonic Stem Cells

제임스 톰슨 외 문화에 hES을 분리하고 성장 1998 년 기술을 개발 때문에, 나중에 사용하기 위해 및 냉동 주식에서 세포를 해동 및 확대 냉동 세포는 일상 hES 세포 배양에서 수행한 중요한 절차가있다. hES 세포는 동결 융해의 스트레스에 민감하고 있으므로 특별한주의가 있어야합니다. 여기 우리는 신속하게, 액체 질소 주식에서 hES 세포를 해동 마우스 배아 피더 세포를 도금하고, 천천히 장기 저장을 위해 그들을 오해해서 적절한 방법을 보여줍니다.

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 ...

freezing-and-thawing-human-embryonic-stem-cells

Research

JoVE Journal

Biology

45.6K 조회수.  Stemgent. 제임스 톰슨 외 문화에 hES을 분리하고 성장 1998 년 기술을 개발 때문에, 나중에 사용하기 위해 및 냉동 주식에서 세포를 해동 및 확대 냉동 세포는 일상 hES 세포 배양에서 수행한 중요한 절차가있다. hES 세포는 동결 융해의 스트레스에 민감하고 있으므로 특별한주의가 있어야합니다. 여기 우리는 신속하게, 액체 질소 주식에서 hES 세포를 해동 마우스 배아 피더...

비디오: Freezing and Thawing Human Embryonic Stem Cells

Freezing Human ES Cells

Here we demonstrate how our lab freezes HuES human embryonic stem cell lines.  A healthy, exponentially expanding culture is washed with PBS to remove residual media that could otherwise quench the Trypsin reaction.  Warmed 0.05% Trypsin-EDTA is then added to cover the cells, and the plate allowed to incubate for up to 5 mins at room temperature.  During this time cells can be observed rounding, and colonies lifting off the plate surface.  Gentle repeated pipetting will remove cells and colonies from the plate surface.  Trypsinized cells are placed in a standard conical tube containing pre-warmed hES cell media to quench remaining trypsin, and then spun.  Cells are resuspended growth media at a concentration of approximately one million cells in one mL of media, a concentration such that one frozen aliquot is sufficient to resurrect a culture on a 10cm plate.  After cells are adequately resuspended, ice cold freezing media is added at equal volume.  Cell suspensions are mixed thoroughly, aliquoted into freezing vials, and allowed to slowly freeze to -80C over 24 hours.  Frozen cells can then moved to the vapor phase of liquid nitrogen for long term storage, or remain at -80 for approximately six months.

Here we demonstrate how our lab freezes HuES human embryonic stem cell lines.

냉동 인간 ES 세포

Here we demonstrate how our lab freezes HuES human embryonic stem cell lines.  A healthy, exponentially expanding culture is washed with PBS to remove ...

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생물학

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.

인간의 배아 줄기 (ES) 세포의 전파

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 ...

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 ...

Freezing, Thawing, and Packaging Cells for Transport

Cultured mammalian cells are used extensively in cell biology studies. It requires a number of special skills in order to be able to preserve the structure, function, behavior, and biology of the cells in culture. This video describes the basic skills required to freeze and store cells and how to recover frozen stocks. 

Cultured mammalian cells are used extensively in cell biology studies. This video describes the basic skills required to freeze and store cells and how to recover frozen stocks.

, 동결 해동 및 운송을위한 포장 셀

Cultured mammalian cells are used extensively in cell biology studies. It requires a number of special skills in order to be able to preserve the ...

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 ...

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 ...