저자는 어린이 병원 parafilm의 얼룩 기술의 초기 교육에 대해 에모리 대학에서 hNSPC 문화와 박사 게리 Bassell를 제공하는 오렌지 카운티 연구소 국립 인간 신경 줄기 세포 자원의 박사 필립 H. 슈워츠을 인정하고 싶습니다.

1 일 : 독일어 유리 Coverslips 준비 및 심는 세포 참고 : 독일어 유리 coverslips 자주 culturing 주요 신경 줄기 세포 뉴런에 바람직합니다. 우리는 세포 접착 및 확산을 개선하기 위해 다음과 같은 청소 프로토콜을 사용합니다. coverslip 양쪽에 액체 액세스를 허용하는 작은 랙에 위치 coverslips. 첫째, 10 분 1 % Liquinox에서 품어의 coverslips는 탈이온수 3 씻는다 다음. 어떤 비누 거품이 남아도는지 확인합니다. 다음, 30 분 1M HCL에 품어 전표는 탈이온수 3 씻는다 다음. 65 드라이 coverslips ° C 하룻밤. 유리 접시와 소독을 압력솥에 coverslips을 전송. <ol><li> 조직 문화 후드에서 적절하게 크기 잘 플레이트에 살균 coverslips를 배치합니다. </li><li> 세포 유착에 대한 laminin과 외투 coverslips. 10 μg / 실온에서 5 분간 살균 물에 ML 폴리 D - 라이신 (PDL)의 부화의 coverslips. 37 ° C 조직 문화 인큐베이터에서 하루 4 시간 또는 EMEM 20 μg / ML의 laminin의 PDL과 부화 coverslips를 제거합니다. <ol><li> 참고 : Passaging 인간 신경 줄기 세포 문서 (참조 <a href="/index/Details.stp?ID=263">https://www.jove.com/index/Details.stp?ID=263</a> ) 및 계산 인간 신경 줄기 세포 문서 ( <a href="/index/Details.stp?ID=262">https://www.jove.com / 색인 / Details.stp? ID = 262</a> hNSPCs을 resuspend하고 계산하는 방법을 알아보려면). </li></ol></li><li> 각 coverslip 함유 특정 밀도 (우리는 종종 우리가 immunostaining에 대한 도금하는 세포에 대한 세포 100,000-300,000 / ML의 초기 도금 밀도를 사용)에서 잘으로 PBS 및 종자 세포와 laminin - 코팅 coverslips 린스. </li><li> 37 플레이스 플레이트는 세포가 coverslips (보통 필요 4 시간 최소)을 준수 ° C 조직 문화와 보육을 허용합니다. </li></ol> 일 2 : 고정, Permeabilizing, 차단하고, 세포 차 항체를 추가 참고 : 우리는 세포 형태를 유지하기 위해 다음과 같은 4 % paraformaldehyde의 정착액을 사용합니다. 조리법은 paraformaldehyde이 솔루션으로 갈 수 있도록하는 산도의 변화를 포함합니다. 높은 기온이 형광을 사용하면 배경 얼룩을 높일 수 산성 개미의 생성으로 이어질 수 있기 때문에 우리는이 방법보다는 paraformaldehyde를 해산하기 위해 열을의 사용을 선호합니다. 다른 전반적인 형태 (자당)을 유지하는 데 도움이 동안 정착액 도움의 일부 구성 요소, cytoskeletal 구조 (MgCl 2 EGTA)를 보존합니다. 전지 4 % Paraformaldehyde의 정착액 <table style="border-collapse: collapse; border: medium none" border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> 시약 및 단계 </td><td> 정착액 100 ML에 대한 금액 : </td></tr><tr><td> MilliQ 물 </td><td> 75 ML </td></tr><tr><td> Paraformaldehyde (펌 후드에서 달다) </td><td> 4g </td></tr><tr><td> 10N NaOH는 저어 및 솔루션이 나올 때까지 dropwise 추가 </td><td> 필요에 </td></tr><tr><td> 10X PBS </td><td> 10 ML </td></tr><tr><td> 1M MgCl 2 </td><td> 0.5 ML </td></tr><tr><td> 0.5 M EGTA </td><td> 2 ML </td></tr><tr><td> 자당 </td><td> 4g </td></tr><tr><td> 6N HCL, pH를 7.4로 적정하다 </td><td> 필요에 </td></tr><tr><td> MilliQ 물 </td><td> 100 ML 가져다 </td></tr><tr><td colspan="2"> 최대 1 주일 4 ° C에 보관하십시오. 37 따뜻한 ° C 사용하기 전에 </td></tr></tbody></table><ol><li> 벤치에서 미디어를 제거하고 prewarmed 정착액을 추가합니다. 5-15분 (감지하는 항원에 따라, 우리가 일반적으로 10 분 사용)을 품어. 이것을 실온에서 다음 단계를 수행합니다. </li><li> 많은 기관들이 유해 폐기물로 paraformaldehyde를 처리 등 적절한 용기에 정착액 폐기하십시오. PBS로 세포를 3 번​​, 5 분 때마다 씻으십시오. 세포에서 솔루션을 제거하는 경우, 흡입은 세포를 건조하게 만들지 않는 것을주의하십시오. 또한, 항상 외투 그들은 너무 세포가 건조 왼쪽으로하지 않는 솔루션을 제거하기 전에 세포에 추가 반면에 다음 해결책을 가지고 있습니다. </li><li> 관심의 항원은 세포 내부, 세포막은 항체의 진입을 허용 투과하여야하는 경우. 세포를 permeabilize하기 위해 PBS에 신선한 0.3 % 트리톤 X - 100 용액을 사용하십시오. 피펫 천천히 Triton은 점성이며, 세제가 완전히 세포에 추가하기 전에 PBS에 용해되어 있는지 확인 때문입니다. PBS 5 분마다있는 세포에게 3 번 씻는 다음 5 분 세포를 Permeabilize. </li><li> 세포는 항체가 아닌 특정 바인딩을 방지하기 위해 차단 에이전트로 치료해야합니다. 우리는 차단 요원으로 (BSA) 소 혈청 알부민을 사용합니다. (이것은 용해 시간이 걸립니다 때문에 락커 또는 회전)를 차단 솔루션 PBS로 5 % BSA를 용해 만든 후 솔 필터링0.45 μm의 주사기 필터로 ution. 실온에서 1 시간 5% BSA / PBS 용액에 세포를 차단합니다. </li></ol> 1 % 안에 미리 정해진 농도 기본 항체를 (관심의 단백질에 대한 구체적인 희망되는) 희석 BSA / PBS (5 % BSA / PBS에서 희석하여 준비). 장소 parafilm 덮여 유리 접시에 기본 항체를 (25mm의 coverslip 30 μl, 18mm의 coverslip 당 20 μl, 12mm의 coverslip 당 15 μl) 희석. 세포가 다운 얼굴, 그리고 세포가 항체에 접촉되도록 항체 솔루션을 통해 그것을하다​​ 이러한 반전, 고정 세포로 coverslip을 선택하십시오. 전체 구조를 통해 두 번째 parafilm 스트립를 놓습니다. 4 ° C 하룻밤에 품어. 3 일 : 세탁, 보조 항체 보육, 원자력 스테인 및 장착 <ol><li> 조심스럽게 세포가 직면 있도록 최고 parafilm 스트립을 제거하고 coverslip을 받아 그것을 반전하고, 세척 각 우물에 PBS와 함께 접시에 돌려 놓으십시오. </li><li> PBS로 세포를 3 번​​, 5 분 때마다 씻으십시오. </li><li> , 기본 항체를 검출하는 기본 항체가 토끼로 만들어진 경우 예를 들어, 보조 항체는 토끼 IgG를 인식해야 보조 항체를 선택합니다. 또한 (IgG, IgM 등) 항체 isotype에 맞게 처리해. 여러 차 항체를 사용하는 경우, 그들은 서로 다른 종 또는 isotypes에서, 그리고 보조 항체에 연결된 다른 마커와 감지하는 계획을 확인하십시오. 예를 들어, 녹색 형광단에 방지 마우스 이차 결합과 빨간색 형광단에 방지 토끼 보조 결합을 사용할 수 있습니다. 기본과 동일한 기술을 사용하여 상온에서 어둠 속에서 2 시간 동안 1 % BSA에 적절한 농도로 희석 보조 항체 용액에 세포를 품어 (동영상에 표시되지 단계). 보조가 글리세롤 솔루션 (25mm의 coverslip 40 μl, 18mm의 coverslip 30 μl, 12mm의 coverslip 당 25 μl)으로 저장되는 경우 부화에 필요한 보조 항체의 볼륨 차 항체에 대한보다 약간 클 수 있습니다 . <ul><li> 참고 : 보조 항체를 시각화하는 형광 분자를 사용하는 경우 예제는 보조 항체가 추가되었습니다 일단 빛으로부터 보호되어야합니다. 어둠 속에서, 또는 호일 커버 플레이트에 품어. </li></ul></li><li> 조심스럽게로 기본 항체에 대한 설명 parafilm에서 coverslips를 제거합니다. 세포에게 2 μg / ML Hoechst 핵 PBS에 희석 얼룩 (핵 counterstain 선호하는 경우)에서 1 분 부화 다음 PBS로 2 회, 각 시간 5 분, 씻으십시오. 얼룩 Hoechst 오래된이고 신호가 너무 희미있다면, 배양 시간 및 / 또는 농도를 증가시킬 수 있습니다. Hoechst와 부화 후 5 분 PBS로 1 회 씻는다. </li><li> coverslips는 현미경의 시각화를위한 설치 미디어와 슬라이드에 장착해야합니다. 형광 분자가 이차 항체의 시각화에 사용되는 경우에는 설치 미디어가 photobleaching 최소화하기 위해 에이전트를 포함해야합니다. 우리는 형광에 대한 설치 매체로 Vectashield를 사용합니다. 현미경 슬라이드 (25mm의 coverslip 당 12 μl, 18mm의 coverslip 당 6 μl, 12mm의 coverslip 당 3 μl)에 Vectashield의 ​​적절한 크기 드롭를 놓습니다. </li><li> 조심스럽게 coverslip을 선택하고 소금을 (PBS에서) 제거 탈이온수로 다시 씻어. kimwipe이나 종이 타월에 과도한 물에서 DAB하고 아래로 향하게 전지 Vectashield의 ​​드롭에 누워. 각도에 coverslip을 놓고 트래핑 기포를 피하기 위해 천천히 내려 수 있습니다. 날짜 및 샘플 정보로 슬라이드를 라벨. </li><li> coverslips와 슬라이드 coverslip 가장자리 주변에서 (어둠)을 초과하는 Vectashield에서 다음 흡입을 건조하도록 허용합니다. 원하는 경우 coverslip의 가장자리는 현재 (coverslips가 거꾸로 현미경에서 볼 수있는 경우 특히 유용합니다), 매니큐어와 봉인 수 있습니다. 우리는 일반적으로 -20 ° C의 냉장고에있는 슬라이드를 저장합니다. </li></ol>

<ol>
	<li>Flanagan, L. A., Chou, J., Falet, H., Neujahr, R., Hartwig, J. H., Stossel, T. P., Filamin, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Filamin+A,+the+Arp2/3+complex,+and+the+morphology+and+function+of+cortical+actin+filaments+in+human+melanoma+cells.">Filamin A, the Arp2/3 complex, and the morphology and function of cortical actin filaments in human melanoma cells.</a> J. Cell Biol. 155, 511-518 (2001).</li><li>Flanagan, L. A., Rebaza, L. M., Derzic, S., Schwartz, P. H., Monuki, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+human+neural+precursor+cells+by+laminin+and+integrins.">Regulation of human neural precursor cells by laminin and integrins.</a> J. Neurosci. Res. 83, 845-856 (2006).</li><li>Flanagan, L. A., Ziaeian, B., Palmer, T., Schwartz, P. H., Loring, J. F., Wesselschmidt, R. W., Schwartz, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunocytochemical+analysis+of+stem+cells.">Immunocytochemical analysis of stem cells.</a> In Human Stem Cell Manual: A Laboratory Guide. , (2007).</li><li>Marchenko, S., Flanagan, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Passaging+Human+Neural+Stem+Cells.">Passaging Human Neural Stem Cells.</a> Journal of Visualized Experiments. 7, (2007).</li><li>Marchenko, S., Flanagan, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Counting+Human+Neural+Stem+Cells.">Counting Human Neural Stem Cells.</a> Journal of Visualized Experiments. 7, (2007).</li></ol>

이 프로토콜은 필요한 항체의 볼륨을 최소화하고 안정적​​인 세포 얼룩을 제공 hNSPCs위한 immunostaining 절차를 설명합니다. 설명된 절차는 세포내 항원 최선이지만, 세포 표면 분자를 얼룩이나 cytoskeleton의 얼룩을 향상시키기 위해 수정할 수 있습니다.

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<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>German glass (Deutsche Spiegelglas) coverslips</td>
<td>Tool</td>
<td>Carolina Biological Supply Company</td>
<td>WW-63-3029</td>
<td>12,15,18, 22, 25 mm coverslips available</td>
</tr>
<tr>
<td>Liquinox phosphate-free detergent</td>
<td>Reagent</td>
<td>Sigma</td>
<td>Z273279</td>
<td>very viscous</td>
</tr>
<tr>
<td>Poly-D-lysine hydrobromide</td>
<td>Reagent</td>
<td>Sigma</td>
<td>P7280</td>
<td>&nbsp;</td>
<tr>
<td>Laminin, natural mouse</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>23017-015</td>
<td>&nbsp;</td>
<tr>
<td>EMEM (1X)</td>
<td>Reagent</td>
<td>Mediatech</td>
<td>MT-10-010-CV</td>
<td>Distributed by Fisher under the indicated catalog #</td>
</tr>
<tr>
<td>24 well plate</td>
<td>Tool</td>
<td>Fisher</td>
<td>08-772-1</td>
<td>&nbsp;</td>
<tr>
<td>Triton X-100</td>
<td>Reagent</td>
<td>Fisher</td>
<td>BP151-500</td>
<td>very viscous</td>
</tr>
<tr>
<td>Paraformaldehyde</td>
<td>Reagent</td>
<td>Sigma</td>
<td>P6148</td>
<td>potential carcinogen, use caution when using it in powder and solution form</td>
</tr>
<tr>
<td>Bovine Serum Albumin, IgG-free</td>
<td>Reagent</td>
<td>Jackson Immunoresearch</td>
<td>001-000-161</td>
<td>&nbsp;</td>
<tr>
<td>Hoechst 33342</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>H-1399</td>
<td>&nbsp;</td>
<tr>
<td>Vectashield</td>
<td>Reagent</td>
<td>Vector Laboratories</td>
<td>H-1000</td>
<td>viscous</td>
</tr>		</tbody>
		</table>
		</div>
			<div>
					</div>

immunocytochemistry: human neural stem cells

Immunocytochemistry은 존재를 결정하는 매우 강력하고 매우 간단 방법, subcellular 지방화, 그리고 가장 일반적으로 흥미, 교양 세포에있는 단백질의 항원의 상대적인 풍부합니다. 이 프로토콜은 선물 쉬운 따라 자신의 얼룩 밖으로 고품질 재현할 질적 및 양적 데이터를 가져오는 동안 연구자 기본 및 보조 항체를 보존하기 위해 사용됩니다 단계 시리즈. 얼룩에 필요한 항체의 볼륨을 보존하는 데 도움이 프로토콜의 두 측면이 있습니다. 하나의 세포는 조직 문화 판의 우물에 배치됩니다 작은 원형 coverslips에 성장하고 있습니다. 고정 후 coverslips에있는 세포는 접시의 우물에서 제거할 수 있습니다. 항체 얼룩 경우, 세포와 coverslip은 parafilm에 대한 항체 솔루션의 작은 방울로 반전하고 건조 방지하기 위해 parafilm의 두 번째 조각으로 덮여있다. 이 방법을 사용하여 항체 솔루션만이 ~ 25 μl가 물들다 각 coverslip (또는 샘플) 필요합니다. 이 프로토콜은 인간의 신경 줄기 / 전구체 세포 (hNSPCs)의 immunostaining 설명하지만, 많은 다른 세포 유형에 사용할 수 있습니다.

Watch this Scientific Journal Video about Immunocytochemistry: Human Neural Stem Cells at JoVE.com

Immunocytochemistry: Human Neural Stem Cells

Immunocytochemistry은 존재, subcellular 지방화 및 교양 세포에 대한 관심의 항원의 상대적인 풍부를 결정하는 강력한 방법입니다. 이 프로토콜은 선물 쉬운 따라 하나가 항체를 절약 하나의 얼룩을 최대한 활용 수있게 단계 시리즈.

Immunocytochemistry : 인간 신경 줄기 세포

Immunocytochemistry is a very powerful and fairly straightforward method for determining the presence, subcellular localization, and relative abundance of ...

immunocytochemistry-human-neural-stem-cells

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JoVE Journal

Biology

27.3K 조회수.  University of California, Irvine (UCI). Immunocytochemistry은 존재, subcellular 지방화 및 교양 세포에 대한 관심의 항원의 상대적인 풍부를 결정하는 강력한 방법입니다. 이 프로토콜은 선물 쉬운 따라 하나가 항체를 절약 하나의 얼룩을 최대한 활용 수있게 단계 시리즈.

비디오: Immunocytochemistry: Human Neural Stem Cells

Propagation of Human Embryonic Stem (ES) Cells

Propagation of Human Embryonic Stem ES Cells

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

인간의 배아 줄기 (ES) 세포의 전파

연구

생물학

Transfecting Human Neural Stem Cells with the Amaxa Nucleofector

Transfection of primary mammalian neural cells, such as human neural stem/precursor cells (hNSPCs), with commonly used cationic lipid transfection reagents has often resulted in poor cell viability and low transfection efficiency.  Other mechanical methods of introducing a gene of interest, such as a &#8220;gene gun&#8221; or microinjection, are also limited by poor cell viability and low numbers of transfected cells.  The strategy of using viral constructs to introduce an exogenous gene into primary cells has been constrained by both the amount of time and labor required to create viral vectors and potential safety concerns.  We describe here a step-by-step protocol for transfecting hNSPCs using Amaxa's Nucleofector device and technology with electrical current parameters and buffer solutions specifically optimized for transfecting neural stem cells.  Using this protocol, we have achieved initial transfection efficiencies of ~35% and ~70% after stable transfection.  The protocol entails combining a high number of hNSPCs with the DNA to be transfected in the appropriate buffer followed by electroporation with the Nucleofector device.

Introducing a gene of interest into a cell is a powerful method for elucidating its function in vivo. This protocol describes an efficient method of transfecting a culture of human neural stem/precursor cells (hNSPCs) using the Nucleofector electroporation apparatus made by Amaxa.

Amaxa Nucleofector과 인간 신경 줄기 세포를 Transfecting

Transfection of primary mammalian neural cells, such as human neural stem/precursor cells (hNSPCs), with commonly used cationic lipid transfection ...

Counting Human Neural Stem Cells

Knowledge of the exact number of viable cells in a given volume of a cell suspension is required for many routine tissue culture manipulations, such as plating cells for immunocytochemistry or for cell transfections. This protocol describes a straightforward and fast method for differentiating between live and dead cells and quantifying the cell concentration and total cell number using a hemacytometer. This procedure first requires detaching cells from a growth surface and resuspending them in media. Next, the cells are diluted in a solution of Trypan blue (ideally to a concentration that will give 20-50 cells per quadrant) and placed in the hemacytometer. Finally, averaging the counts of viable cells in several randomly selected quadrants, dividing the average by the volume of one 1 mm2 quadrant (0.1 &mu;l) and multiplying by the dilution factor gives the number of cells per l. Multiplying this cell concentration by the total volume in &mu;l gives the total cell number. This protocol describes counting human neural stem/precursor cells (hNSPCs), but can also be used for many other cell types.

Knowledge of the exact number of viable cells is required for many tissue culture manipulations. This protocol describes how to differentiate between live and dead cells and quantify cells using a hemacytometer. Although it describes counting human neural stem/precursor cells (hNSPCs), it can be used for other cell types.

인간 신경 줄기 세포를 계산

Knowledge of the exact number of viable cells in a given volume of a cell suspension is required for many routine tissue culture manipulations, such as ...

Passaging Human Neural Stem Cells

The ability to manipulate human neural stem/precursor cells (hNSPCs) in vitro provides a means to investigate their utility as cell transplants for therapeutic purposes as well as to explore many fundamental processes of human neural development and pathology.  This protocol presents a simple method of culturing and passaging hNSPCs in hopes of standardizing this technique and increasing reproducibility of human stem cell research.  The hNSPCs we use were isolated from cadaveric postnatal brain cortices by the National Human Neural Stem Cell Resource and grown as adherent cultures on flasks coated with fibronectin (Palmer et al., 2001; Schwartz et al., 2003).  We culture our hNSPCs in a DMEM:F12 serum-free media supplemented with EGF, FGF, and PDGF and passage them 1:2 approximately every seven days.  Using these conditions, the majority of the cells in the culture maintain a bipolar morphology and express markers of undifferentiated neural stem cells (such as nestin and sox2).

The ability to manipulate human neural stem/precursor cells (hNSPCs) in vitro allows to investigate their utility as cell transplants for therapeutic purposes and to explore human neural development. This protocol presents a method of culturing and passaging hNSPCs in hopes of increasing reproducibility of human stem cell research.

인간 신경 줄기 세포를 Passaging

The ability to manipulate human neural stem/precursor cells (hNSPCs) in vitro provides a means to investigate their utility as cell transplants for ...

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 ...

Mouse Epidermal Neural Crest Stem Cell (EPI-NCSC) Cultures

EPI-NCSC are remnants of the embryonic neural crest in an adult location, the bulge of hair follicles. They are multipotent stem cells that have the physiological property to generate a wide array of differentiated cell types, including neurons, nerve supporting cells, smooth muscle cells, bone/cartilage cells and melanocytes. EPI-NCSC are easily accessible in the hairy skin and can be isolated as a highly pure population of stem cells. This video provides a detailed protocol for preparing mouse EPI-NCSC cultures from whisker follicles. The whisker pad of an adult mouse is removed, and whisker follicles dissected. The follicles are then cut longitudinally and subsequently transversely above and below the bulge region. The bulge is removed from the collagen capsule and placed in a culture plate. EPI-NCSC start to emigrate from the bulge explants 3 to 4 days later.

Mouse Epidermal Neural Crest Stem Cell EPI-NCSC Cultures

Here we show our method to isolate mouse epidermal neural crest stem cells (EPI-NCSC). Technique involves micro-dissecting whisker follicles, isolating the bulge and placeing it into tissue culture. EPI-NCSC start to emigrate from bulge explants onto the substratum within 3 - 4 days.

마우스 표피 신경 크레스트 줄기 세포 (에피 NCSC) 문화

EPI-NCSC are remnants of the embryonic neural crest in an adult location, the bulge of hair follicles. They are multipotent stem cells that have 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 ...

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 ...

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 ...

Transfecting and Nucleofecting Human Induced Pluripotent Stem Cells

Genetic modification is continuing to be an essential tool in studying stem cell biology and in setting forth potential clinical applications of human embryonic stem cells (HESCs)1. While improvements in several gene delivery methods have been described2-9, transfection remains a capricious process for HESCs, and has not yet been reported in human induced pluripotent stem cells (iPSCs). In this video, we demonstrate how our lab routinely transfects and nucleofects human iPSCs using plasmid with an enhanced green fluorescence protein (eGFP) reporter. Human iPSCs are adapted and maintained as feeder-free cultures to eliminate the possibility of feeder cell transfection and to allow efficient selection of stable transgenic iPSC clones following transfection. For nucleofection, human iPSCs are pre-treated with ROCK inhibitor11, trypsinized into small clumps of cells, nucleofected and replated on feeders in feeder cell-conditioned medium to enhance cell recovery. Transgene-expressing human iPSCs can be obtained after 6 hours. Antibiotic selection is applied after 24 hours and stable transgenic lines appear within 1 week. Our protocol is robust and reproducible for human iPSC lines without altering pluripotency of these cells.

Despite recent advancements in genetic modification, transfection of human embryonic stem cells (HESCs) remains a capricious process. To our knowledge, systematic and efficient methods to transfect human induced pluripotent stem cells (iPSCs) have not been reported. Here, we describe robust protocols to efficiently transfect and nucleofect human iPSCs.

인간 유도 만능의 줄기 세포를 Transfecting 및 Nucleofecting

Genetic modification is continuing to be an essential tool in studying stem cell biology and in setting forth potential clinical applications of human ...