이 작품은 일부 보건 국립 연구소 (RO1 NS059043 및 RO1 ES015988), 국립 다중 경화증 학회, 빈혈 연구, Feldstein 의료 재단 로체 재단, 어린이를위한 이너스 병원에서 WD로 보조금에 의해 지원되었다. 우리는 제안 데이빗 플레져와 제니퍼 비행기를 감사하고 싶습니다. 우리는이 문서에 관한 더 경쟁 이익을 선언하지 않습니다.

GFP - Olig2 마우스 배아 줄기 세포 (mESCs)에서 oligodendrocyte 전구체 세포 (OPCs)를 생성하는 자세한 절차 : <ol><li> 마우스 ES 세포 라인 GFP - Olig2 (G - Olig2)은 미국 유형 문화 수집 (ATCC)에서 구입. mESCs는 정기적으로 조사 마우스 배아 fibroblast (MEF) 피더 계층에 삼일마다 passaged 있습니다. MEF 세포는 젤라틴 코팅 여섯 잘 조직 문화 판 ESCs을 passaging하기 전에 최소한 1 일 이상 0.1 %로 도금입니다. mESC 문화 매체 20 % 태아 소 혈청 (GIBCO), 2 MM L - 글루타민, 1 ㎜의 나트륨 pyruvate, 0.1 MM β - 메르 캅 토 에탄올 /, 1 % 불필요한 아미노산 (와 보충 Dulbecco의 수정 이글 배지 (DMEM) (GIBCO)입니다 NEAA), 1000 U / ML 백혈병 억제 인자. </li><li> MES의 식민지가 하나의 셀에 (TrypLE, 5 분, 37 ° C) trypsinized 및 녹아웃 혈청 대체 (KSR) 중간에 중단되며, 다음 세포는 세포에서 코스타 초저 첨부 여섯 잘 플레이트 (코닝)으로 전송됩니다 50,000 세포 / cm 2 밀도. 이러한 조건에서 mESCs는 embryoid기구 (EBS)를 형성합니다. KSR 매체 20 % KSR, 1 MM 나트륨 pyruvate, 1 % NEAA, 그리고 0.1 MM β - 메르 캅 토 에탄올 /과 보완의 - 최소한의 필수적인 매체를 (- 멤)로 구성되어 있습니다. </li><li> 일 4 일 7, retinoic 산 (RA, 0.2 M)와 purmorphamine (PUR, 1 M)은 같은 그림과 같이 포함되어 있습니다. KSR 또는 N2 매체에서 1A. N2 매체 1X의 N2의 보완, 1 MM 나트륨 pyruvate, 1 % NEAA, 그리고 0.1 MM β - 메르 캅 토 에탄올 /를 포함하는 - 멤입니다. 매체는 매일 변경됩니다. </li><li> 일 8시, EBS는 N2 매체와 fibroblast 구성되어 OPC 매체에서 0.01 % polyornithine - 코팅 요리 TrypLE (5 분, 37 ° C) 및 도금을 사용하여 disaggregated 아르 성장 인자 - 2 (FGF - 2, 20, NG / ML). 중간 이틀마다 변경됩니다. </li><li> 세포 trypsinized (TrypLE, 3 분, 37 ° C)과 그들이 합류이되면 매주 약을 replated 있습니다. 30 일, 세포의 80 %가 GFP/Olig2 표현을 표시하고, NG2 - 긍정적인 OPCs의 특징입니다. </li><li> 급상승 OPCs를 생성하려면, BacMam 나 + 채널 키트 이러한 mESC - 파생 OPCs에 나 V 1.2 subunit을 소개하는 데 사용됩니다. BacMam 시약 (1 ML, 구성 요소가) 5 ML의 최종 볼륨 PBS와 혼합됩니다. 다음, 혼합 BacMam 시약 전에 PBS로 rinsing하기 (50~70%의 합류에서) 60mm 문화 접시에 mESC - 파생 OPCs 문화로 추가됩니다. 2 시간 보육 후, 혼합 BacMam 시약은 1X 확장기와 함께 보충 OPC 매체로 제거하고 교체합니다. 확장기는 구성 요소 B DMSO (구성 요소 C)에서 재구성됩니다. 다음, 추가 2 시간 보육 후 확장기와 매체 전체 OPC 매체로 제거하고 교체합니다. 세포는 24 시간 후에 assayed 있습니다. </li></ol> 대표 결과 : 그림에 표시된 차별 프로토콜을 다음. 1A, G - Olig2의 mESCs는 처음 embryoid 단체 (EBS) (그림의 1B)를 형성 KSR 매체에서 4 일간 정지되었습니다. 그렇다면, 우리는 순차적으로 RA와 PUR와 EBS를 취급. EBS는 4 일 모든 GFP의 형광을 표시하고 D8 (그림의 1B)에 강한 GFP의 형광을 표명하지 않았다. 이 시점에서, EBS는 trypsinized되었으며 성장 요인 FGF - 2와 함께 보충 N2 매체에 도금. 22일 (D30)에 대한 자세한 문화 후, GFP + 세포의 비율은 80.3에 도달 ± 0.6 % 우리의 유동세포계측법 결과에 따라. 마찬가지로 그림에 표시됩니다. 1C, GFP + 세포가 지속적으로 NG2 얼룩 (96.4 ± GFP 양성 세포의 1.3 % 긍정적인 NG2 및 94.8 ± NG2 긍정적인 세포의 1.5 %가 긍정적인 GFP 쓰셨)와 overlapped. 따라서, 이러한 전지는 OPCs으로 그들을 정의 GFP/Olig2 및 NG2을 표현. mESC 파생 OPCs (76 셀)는 탈분극 (그림 2A)에 따라 행동 잠재력을 해고하지 못했습니다. 나 V 1.2 subunit를 들고 baculovirus와 transducing 후 다음 mESC 파생 OPCs (17 세포의 94.1 %, 16)는 (그림 2B)로 분류 급상승 수 있습니다. <img alt="그림 1" src="/files/ftp_upload/1960/1960fig1.jpg" /> 그림 1. embryoid 신체의 프로토콜을 보여주는 OPCs로 GOlig - mESC의 분화. (A) 계획 (EB) 기반 및 작은 분자 기반 차별화. D8에서는 EBS가 disaggregated 및 도금했다. 그들이 합류되었을 때 세포는 주 당 한 번 passaged했다. (B) D8 아니지만 D4 EBS, GFP 발현을 보여주었다. (C) NG2 (빨간색)의 Immunostaining는 D30에서 GFP (녹색) 표현식과 일치했다. DAPI (파란색)은 핵을 식별하는 데 사용되었다. 스케일 바 : 20mm. <img alt="그림 2" src="/files/ftp_upload/1960/1960fig2.jpg" /> 그림 2. 에 의해 mESC - 파생 OPCs를 nonspiking에서 난리 OPCs의 생성 바이러스 중재 셀 0으로 depolarized 때도 나 V 채널 표현. (A) 멤브레인 가능성은 -60 뮤직 비디오와 mESC 파생 OPCs에서 개최 되었음, 액션 잠재력을 발사하는 데 실패 MV. (B) mESC 파생 OPC의 예바이러스 전달 후 행동 잠재력을 (빨간색으로 강조) 발사.

<ol>
	<li>Xian, H., Gottlieb, D. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dividing+Olig2-expressing+progenitor+cells+derived+from+ES+cells.">Dividing Olig2-expressing progenitor cells derived from ES cells.</a> Glia. 47 (1), 88-101 (2004).</li><li>Clair, A., Gottlieb, D. 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+subset+of+ES-cell-derived+neural+cells+marked+by+gene+targeting.">A subset of ES-cell-derived neural cells marked by gene targeting.</a> Stem Cells. 21 (1), 41-49 (2003).</li><li>Evans, M. J., Kaufman, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+in+culture+of+pluripotential+cells+from+mouse+embryos.">Establishment in culture of pluripotential cells from mouse embryos.</a> Nature. 292, 154-156 (1981).</li><li>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=Embryonic+stem+cell+lines+derived+from+human+blastocysts.">Embryonic stem cell lines derived from human blastocysts.</a> Science. 282, 1145-1147 (1998).</li><li>Shin, S., Xue, H., Mattson, M. P., Rao, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stage-dependent+Olig2+expression+in+motor+neurons+and+oligodendrocytes+differentiated+from+embryonic+stem+cells.">Stage-dependent Olig2 expression in motor neurons and oligodendrocytes differentiated from embryonic stem cells.</a> Stem Cells Dev. 16, 131-141 (2007).</li><li>Billon, N., Jolicoeur, C., Ying, Q. L., Smith, A., Raff, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normal+timing+of+oligodendrocyte+development+from+genetically+engineered,+lineage-selectable+mouse+ES+cells.">Normal timing of oligodendrocyte development from genetically engineered, lineage-selectable mouse ES cells.</a> J Cell Sci. 115, 3657-3665 (2002).</li><li>Nishiyama, A., Lin, X. H., Giese, N., Heldin, C. H., Stallcup, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Co-localization+of+NG2+proteoglycan+and+PDGF+alpha-receptor+on+O2A+progenitor+cells+in+the+developing+rat+brain.">Co-localization of NG2 proteoglycan and PDGF alpha-receptor on O2A progenitor cells in the developing rat brain.</a> J Neurosci Res. 43, 299-314 (1996).</li><li>Ligon, K. L. <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+NG2+neural+progenitor+cells+requires+Olig+gene+function.">Development of NG2 neural progenitor cells requires Olig gene function.</a> Proc Natl Acad Sci U S A. 103, 7853-7858 (2006).</li><li>Dawson, M. R., Polito, A., Levine, J. M., Reynolds, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NG2-expressing+glial+progenitor+cells:+an+abundant+and+widespread+population+of+cycling+cells+in+the+adult+rat+CNS.">NG2-expressing glial progenitor cells: an abundant and widespread population of cycling cells in the adult rat CNS.</a> Mol Cell Neurosci. 24, 476-488 (2003).</li><li>Chittajallu, R., Aguirre, A., Gallo, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NG2-positive+cells+in+the+mouse+white+and+grey+matter+display+distinct+physiological+properties.">NG2-positive cells in the mouse white and grey matter display distinct physiological properties.</a> J Physiol. 561, 109-122 (2004).</li><li>Karadottir, R., Hamilton, N. B., Bakiri, Y., Attwell, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spiking+and+nonspiking+classes+of+oligodendrocyte+precursor+glia+in+CNS+white+matter.">Spiking and nonspiking classes of oligodendrocyte precursor glia in CNS white matter.</a> Nat Neurosci. 11, 450-456 (2008).</li></ol>

관심 없음 충돌 선언하지 않습니다.

blastocyst의 배아에서 분리된 배아 줄기 세포 (ESCs)는, oligodendrocyte 사양을 포함하여 초기 포유류의 개발 연구를위한 체외 모델 시스템을 제공하는 유기체 3, 4의 모든 세포 lineages로 구별하실 수 있습니다. mESCs는 소닉 헤지 호그 (쉬) 5의 치료 oligodendrocyte 전구체 세포 (OPCs)로 차별화하는 표시되었습니다. 또한, mESCs에서 쉬 유발 OPC의 차별화는 배아 개발 6 관찰 올바른 타이밍을 유지합니다. 따라서 mESCs에서 체외 OPC의 분화에의 성격은 생체내 개발에 배운되었습니다 것과 일치로 간주됩니다. 여기, 작은 분자 RA와 쉬 작용제의 PUR 사용, 우리는 성공적으로 고효율 GFP + Olig2 + NG2 + OPCs에 G - Olig2 mESCs을 차별. 그들은 전체 세포의 상당한 비율 (~ 5 %)를 구성하고 어디 proteoglycan NG2 7 표현과 나선형 루프 - 나선의 전사 인자 Olig2 8 시까지 특징 OPCs는, 개발하고 성숙한 CNS에 oligodendrocytes를 생성 주요 proliferating 세포 유형 9. V 채널 나 보여준 근 20 년 연구가들은 OPCs의 subpopulation 표현하고 탈분극 10, 11시 활성화할 수 있습니다. 또한, 최근 인상적인 관찰 9 전압 - 게이 티드 나트륨 (나 V) 채널의 표현에 따라 언제 depolarized 흰 물질 현장 쥐 CNS에서에서 OPCs는 (~ 50 %) 액션 잠재력을 생성하기 때문에 난리로 세분화 될 수 있다고했습니다 와 subpopulations을 nonspiking. 그러나 이러한 요동 치고 속성의 기능은 여전히​​ 대부분 알 수 있습니다. 우리는 electrophysiologically, 쉬 - 의존 프로토콜 차별화된 mESC 파생 OPCs는 현장 뇌 OPCs에서 같은이 아니라, 사실을 발견했습니다. subunit 나 V 1.2를 소개하면 다음과 침묵 mESC 파생 OPCs가 튀어 오르고 수 있었다. 따라서 요동 치고 / mESC - 파생 OPCs과 차별화 프로토콜 여기에 설명된 내용을 nonspiking (1) OPCs 춤을 추는과 nonspiking 사이의 기능적 차이, (2) mESC 파생 OPCs에 나트륨 채널 표현을 촉진 수도 선별 새로운 요소를 공부하고 용이하게 수 있습니다 ( 3) 개발 및 인간 ESC 또는 유도 pluripotent 줄기 세포 (iPSCs)에서 OPCs의 차별 프로토콜을 최적화.

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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>MEFs</td>
<td>&nbsp;</td>
<td>GlobalStem </td>
<td>GSC-6001G</td>
<td>&nbsp;</td>
<tr>
<td>TrypLE Express</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>12604</td>
<td>&nbsp;</td>
<tr>
<td>Retinoic acid</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>R-2625</td>
<td>&nbsp;</td>
<tr>
<td>Purmorphamine</td>
<td>&nbsp;</td>
<td>Cayman Chemical</td>
<td>10009634</td>
<td>&nbsp;</td>
<tr>
<td>FGF-2</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>GF003</td>
<td>&nbsp;</td>
<tr>
<td>BacMam NaV 1.2</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>B10341</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

differentiation of embryonic stem cells into oligodendrocyte precursors

Oligodendrocytes은 중추 신경계의 myelinating 세포 수 있습니다. demyelinating 질병의 재생 세포 치료, 이식을위한 혈통 - 최선을 다하고 oligodendrocyte 전구체 세포 (OPCs)의 순수한 인구 파생에 상당한 관심이 있습니다. OPCs는 전사 인자의 Olig2 및 proteoglycan의 NG2의 표면 표현의 활동에 의해 특징입니다. GFP - Olig2 (G - Olig2) 마우스 배아의 줄기 세포 (mESC) 기자 라인을 사용하여, 우리는 GFP + Olig2 + NG2 + OPCs에 mESCs의 차별 화를위한 조건을 최적화. 우리의 프로토콜에서는, 우리는 먼저 mESCs에서 embryoid 기관의 세대 (EBS)를 설명합니다. 둘째, 우리는 작은 분자로 mESC - 파생 EBS의 치료를 설명 : (1) retinoic 산 (RA)와 정의 문화 조건 (2) 소닉 헤지 호그 (쉬) 작용제의 purmorphamine (PUR)는 oligodendroglial 혈통에 EB 차별을 직접합니다. 이 방법으로 OPCs 30 일 기간에 높은 효율 (&gt; 80 %)로 구할 수 있습니다. 이 프로토콜의 mESCs에서 파생된 세포는 기본 조직 문화에서 파생된 OPCs에 phenotypically 비슷합니다. mESC 파생 OPCs는 현장에서 뇌 OPCs의 subpopulation에 대한 설명 급상승 속성을 표시하지 않습니다. 이 electrophysiological 속성을 연구하기 위해, 우리는 ectopically 나 표현하여 mESC - 파생 OPCs 춤을 추는의 생성을 설명<sub&gt; V</sub&gt; 1.2 subunit. 이 프로토콜에서 얻은 요동 치고 및 nonspiking 세포가 OPCs의 두 subpopulations에 사전 기능 공부를하는 데 도움이됩니다.

Watch this Scientific Journal Video about Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors at JoVE.com

Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

우리는 작은 분자 기반 oligodendrocyte 전구체 세포 (OPCs)에 마우스 배아 줄기 세포의 차별 화를위한 프로토콜을 설명합니다. 이 프로토콜은 차별화 후 30 일에 의해 고효율 Olig2 + NG2 + OPCs를 생성합니다. 우리는 또한 행동 잠재력을 불 수있다 &quot;급상승&quot;OPCs를 생성하는 방법을 설명합니다.

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 ...

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JoVE Journal

Biology

15.3K 조회수.  School of Medicine, University of California, Davis. 우리는 작은 분자 기반 oligodendrocyte 전구체 세포 (OPCs)에 마우스 배아 줄기 세포의 차별 화를위한 프로토콜을 설명합니다. 이 프로토콜은 차별화 후 30 일에 의해 고효율 Olig2 + NG2 + OPCs를 생성합니다. 우리는 또한 행동 잠재력을 불 수있다 "급상승"OPCs를 생성하는 방법을 설명합니다.

비디오: Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

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

Culture of Mouse Neural Stem Cell Precursors

Primary neural stem cell cultures are useful for studying the mechanisms underlying central nervous system development. Stem cell research will increase our understanding of the nervous system and may allow us to develop treatments for currently incurable brain diseases and injuries. In addition, stem cells should be used for stem cell research aimed at the detailed study of mechanisms of neural differentiation and transdifferentiation and the genetic and environmental signals that direct the specialization of the cells into particular cell types.  This video demonstrates a technique used to disaggregate cells from the embryonic day 12.5 mouse dorsal forebrain. The dissection procedure includes harvesting E12.5 mouse embryos from the uterus, removing the "skin" with fine dissecting forceps and finally isolating pieces of cerebral cortex. Following the dissection, the tissue is digested and mechanically dissociated. The resuspended dissociated cells are then cultured in "stem cell" media that favors growth of neural stem cells.

This video describes the method used for isolation of neuroprecursors from the developing cortex of embryonic mice. The procedure for removing embryos from the uterus, dissecting the cortical tissue, and digesting the isolated cerebral cortex is shown.

마우스 신경 줄기 세포 엽 성의 전구 물질의 문화

Primary neural stem cell cultures are useful for studying the mechanisms underlying central nervous system development. Stem cell research will increase ...

Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are derived from the blastocyst of the early embryo and are pluripotent in differentiative ability.  Their vast differentiative potential has made them the focus of much research centered on deducing how to coax them to generate clinically useful cell types.  The successful derivation of hematopoietic stem cells (HSC) from mouse ESC has recently been accomplished and can be visualized in this video protocol.  HSC, arguably the most clinically exploited cell population, are used to treat a myriad of hematopoietic malignancies and disorders.  However, many patients that might benefit from HSC therapy lack access to suitable donors.  ESC could provide an alternative source of HSC for these patients.  The following protocol establishes a baseline from which ESC-HSC can be studied and inform efforts to isolate HSC from human ESC.  In this protocol, ESC are differentiated as embryoid bodies (EBs) for 6 days in commercially available serum pre-screened for optimal hematopoietic differentiation.  EBs are then dissociated and infected with retroviral HoxB4.  Infected EB-derived cells are plated on OP9 stroma, a bone marrow stromal cell line derived from the calvaria of  M-CSF-/- mice, and co-cultured in the presence of hematopoiesis promoting cytokines for ten days.  During this co-culture, the infected cells expand greatly, resulting in the generation a heterogeneous pool of 100s of millions of cells.  These cells can then be used to rescue and reconstitute lethally irradiated mice.

This protocol details the derivation of transplantable hematopoietic stem cells from mouse embryonic stem cells (ESC) and their subsequent injection into lethally irradiated recipient mice. Briefly, ESC are differentiated as embryoid bodies, which are then infected with retroviral HoxB4 and co-cultured with OP9 stromal cells and hematopoietic cytokines.

Murine 배아 줄기 세포로부터 조혈 줄기 세포의 유도

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are ...

Derivation of Human Embryonic Stem Cells by Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass. 


The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass.

Immunosurgery에 의해 인간 배아 줄기 세포의 유도

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both ...

Chromatin Immunoprecipitation from Human Embryonic Stem Cells

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

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

인간 배아 줄기 세포에서 염색질의 Immunoprecipitation

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

In vitro Differentiation of Mouse Embryonic Stem (mES) Cells Using the Hanging Drop Method

Stem cells have the remarkable potential to develop into many different cell types. When a stem cell divides, each new cell has the potential to either remain a stem cell or become another type of cell with a more specialized function, This promising of science is leading scientists to investigate the possibility of cell-based therapies to treat disease.

When culture in suspension without antidifferentiation factors, embryonic stem cells spontaneously differentiate and form three-dimensional multicellular aggregates. These cell aggregates are called embryoid bodies(EB). Hanging drop culture is a widely used EB formation induction method. The rounded bottom of hanging drop allows the aggregation of ES cells which can provide mES cells a good environment for forming EBs. The number of ES cells aggregatied in a hanging drop can be controlled by varying the number of cells in the initial cell suspension to be hung as a drop from the lid of Petri dish. Using this method we can reproducibly form homogeneous EBs from a predetermined number of ES cells. 

In vitro Differentiation of Mouse Embryonic Stem mES Cells Using the Hanging Drop Method

This video demonstrates how to conduct in vitro differentiation of mouse embryonic stem cells to embryoid bodies using the hanging drop method.

공중 드롭 방법을 사용하여 마우스 배아 줄기 (MES) 세포의 체외 차별에

Stem cells have the remarkable potential to develop into many different cell types. When a stem cell divides, each new cell has the potential to either ...

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 ...

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 ...

Efficient Differentiation of Mouse Embryonic Stem Cells into Motor Neurons

Direct differentiation of embryonic stem (ES) cells into functional motor neurons represents a promising resource to study disease mechanisms, to screen new drug compounds, and to develop new therapies for motor neuron diseases such as spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS). Many current protocols use a combination of retinoic acid (RA) and sonic hedgehog (Shh) to differentiate mouse embryonic stem (mES) cells into motor neurons1-4. However, the differentiation efficiency of mES cells into motor neurons has only met with moderate success. We have developed a two-step differentiation protocol5 that significantly improves the differentiation efficiency compared with currently established protocols. The first step is to enhance the neuralization process by adding Noggin and fibroblast growth factors (FGFs). Noggin is a bone morphogenetic protein (BMP) antagonist and is implicated in neural induction according to the default model of neurogenesis and results in the formation of anterior neural patterning6. FGF signaling acts synergistically with Noggin in inducing neural tissue formation by promoting a posterior neural identity7-9. In this step, mES cells were primed with Noggin, bFGF, and FGF-8 for two days to promote differentiation towards neural lineages. The second step is to induce motor neuron specification. Noggin/FGFs exposed mES cells were incubated with RA and a Shh agonist, Smoothened agonist (SAG), for another 5 days to facilitate motor neuron generation. To monitor the differentiation of mESs into motor neurons, we used an ES cell line derived from a transgenic mouse expressing eGFP under the control of the motor neuron specific promoter Hb91. Using this robust protocol, we achieved 51&plusmn;0.8% of differentiation efficiency (n = 3; p &lt; 0.01, Student's t-test)5. Results from immunofluorescent staining showed that GFP+ cells express the motor neuron specific markers, Islet-1 and choline acetyltransferase (ChAT). Our two-step differentiation protocol provides an efficient way to differentiate mES cells into spinal motor neurons.

We developed a new protocol to improve efficiency of in vitro differentiation of mouse embryonic stem cells into motor neurons. The differentiated ES cells acquired motor neurons features as evidenced by expression of neuronal and motor neuron markers using immunohistochemical techniques.

모터 뉴런에 마우스 배아 줄기 세포의 효율적인 분화

Direct differentiation of embryonic stem (ES) cells into functional  motor neurons represents a promising resource to study disease mechanisms, to  screen ...

Differentiation of Newborn Mouse Skin Derived Stem Cells into Germ-like Cells In vitro

Studying germ cell formation and differentiation has traditionally been very difficult due to low cell numbers and their location deep within developing embryos. The availability of a "closed" in vitro based system could prove invaluable for our understanding of gametogenesis. The formation of oocyte-like cells (OLCs) from somatic stem cells, isolated from newborn mouse skin, has been demonstrated and can be visualized in this video protocol. The resulting OLCs express various markers consistent with oocytes such as Oct4 , Vasa , Bmp15, and Scp3. However, they remain unable to undergo maturation or fertilization due to a failure to complete meiosis. This protocol will provide a system that is useful for studying the early stage formation and differentiation of germ cells into more mature gametes. During early differentiation the number of cells expressing Oct4 (potential germ-like cells) reaches ~5%, however currently the formation of OLCs remains relatively inefficient. The protocol is relatively straight forward though special care should be taken to ensure the starting cell population is healthy and at an early passage.

Differentiation of Newborn Mouse Skin Derived Stem Cells into Germ-like Cells In vitro

In recent years the formation of germ cells using in vitro culture methods has been demonstrated using several different types of somatic stem cells. This article will visually present the differentiation of newborn mouse derived stem cells to early oocyte-like cells.

세균과 같은 세포로 신생아 마우스 피부 유래 줄기 세포의 분화<em&gt; 체외</em

Studying  germ cell formation and differentiation has traditionally been very difficult  due to low cell numbers and their location deep within developing ...