제출 작품은 FS하는 재생 의료에 대한 캘리포니아 연구소 (CIRM)에서 부여 (RS1 - 00477-1)에 의해 지원됩니다

<ol>
	<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>Boyer, L. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polycomb+complexes+repress+developmental+regulators+in+murine+embryonic+stem+cells.">Polycomb complexes repress developmental regulators in murine embryonic stem cells.</a> Nature. 441, 349-353 (2006).</li><li>Lee, T. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+developmental+regulators+by+Polycomb+in+human+embryonic+stem+cells.">Control of developmental regulators by Polycomb in human embryonic stem cells.</a> Cell. 125, 301-313 (2006).</li><li>Kornberg, R. D., Lorch, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Twenty-five+years+of+the+nucleosome,+fundamental+particle+of+the+eukaryotic+chromosome.">Twenty-five years of the nucleosome, fundamental particle of the eukaryotic chromosome.</a> Cell. 98, 285-294 (1999).</li><li>Guenther, M. G., Jenner, R. G., Chevalier, B., Nakamura, T., Croce, C. M., Canaani, E., Young, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+and+Hox-specific+roles+for+the+MLL1+methyltransferase.">Global and Hox-specific roles for the MLL1 methyltransferase.</a> Proc. Natl. Acad. Sci. U. S. A.. 102, 8603-8608 (2005).</li><li>Lee, T. I., Johnstone, S. E., Young, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatin+immunoprecipitation+and+microarray-based+analysis+of+protein+location.">Chromatin immunoprecipitation and microarray-based analysis of protein location.</a> Nat. Protoc. 1, 729-748 (2006).</li><li>Sanchez-Elsner, T., Gou, D., Kremmer, E., Sauer, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noncoding+RNAs+of+trithorax+response+elements+recruit+Drosophila+Ash1+to+Ultrabithorax.">Noncoding RNAs of trithorax response elements recruit Drosophila Ash1 to Ultrabithorax.</a> Science. 311, 1118-1123 (2006).</li></ol>

염색질 immunoprecipitation (칩)은 세포 분화 동안 염색질 기반 프로세스의 해부에 대한 귀중한 기술을 제공합니다. 이 방법의 성공을위한 전제 조건은 좋은 항체와 관심의 항원이 부족 제어 세포 또는 조직에서 염색질의 가용성입니다. DNA microarray 기술, 정보의 방대한와 칩을 결합하여 얻을 수 있습니다. 칩 결과의 검증은 세포 개발하는 동안 생물 학적 프로세스의 실행과 동적 단백질 협회, 단백질 수정 및 / 또는 RNA를 연결할 수있는 적합한 테스트 시스템의 가용성에 따라 달라집니다.

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<td>Poteinase-K</td>
<td>Reagent</td>
<td>Roche</td>
<td>#EO0491</td>
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<td>RNase-A</td>
<td>Reagent</td>
<td>Fermentas</td>
<td>#EN0531</td>
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<td>formaldehyde 37%</td>
<td>Reagent</td>
<td>EMD</td>
<td>FX040-13</td>
<td>&nbsp;</td>
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<td>Chloroform</td>
<td>Reagent</td>
<td>EMD</td>
<td>3150</td>
<td>&nbsp;</td>
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<td>Ethanol </td>
<td>Reagent</td>
<td>Goldshield</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>phenol:chloroform:isoamyl alcohol</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>15593-031</td>
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<td>SA, fraction V</td>
<td>Reagent</td>
<td>EMD</td>
<td>2930</td>
<td>&nbsp;</td>
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<td>Dubecco's Phosphate Buffered Saline</td>
<td>Reagent</td>
<td>Gibco</td>
<td>14190</td>
<td>&nbsp;</td>
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<td>HEPES</td>
<td>Reagent</td>
<td>EMD</td>
<td>5330</td>
<td>&nbsp;</td>
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<td>Sodium Chloride</td>
<td>Reagent</td>
<td>Fisher Scientific</td>
<td>S271-10</td>
<td>&nbsp;</td>
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<td>EDTA</td>
<td>Reagent</td>
<td>EMD</td>
<td>4050</td>
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<td>Triton X-100 </td>
<td>Reagent</td>
<td>EMD</td>
<td>9410</td>
<td>&nbsp;</td>
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<td>Sodium Dodecyl Sulfate</td>
<td>Reagent</td>
<td>J.T Baker</td>
<td>4095-02</td>
<td>&nbsp;</td>
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<td>Lithium chloride</td>
<td>Reagent</td>
<td>EMD</td>
<td>5910</td>
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<td>Tris-HCl</td>
<td>Reagent</td>
<td>EMD</td>
<td>9230</td>
<td>&nbsp;</td>
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<td>NP-40</td>
<td>Reagent</td>
<td>Calbiochem</td>
<td>492015</td>
<td>&nbsp;</td>
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<td>Deoxycholic acid, sodium salt</td>
<td>Reagent</td>
<td>Fisher Scientific</td>
<td>BP349-100</td>
<td>&nbsp;</td>
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<td>Ammonium acetate</td>
<td>Reagent</td>
<td>Applichem</td>
<td>631-61-8</td>
<td>&nbsp;</td>
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<td>Glycine</td>
<td>Reagent</td>
<td>EMD</td>
<td>4840</td>
<td>&nbsp;</td>
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<td>Glycine</td>
<td>Reagent</td>
<td>EMD</td>
<td>4840</td>
<td>&nbsp;</td>
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<td>dynalbeads, protein A</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>100.02D</td>
<td>&nbsp;</td>
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<td>dynalbeads, protein G</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>100.04D</td>
<td>&nbsp;</td>
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<td>1.5ml Low Retention Microtubes, Xtreme</td>
<td>Other</td>
<td>Phenix Research Products</td>
<td>MAX-815S</td>
<td>&nbsp;</td>
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<td>Phase Lock Gel Light, 2.0 ml</td>
<td>Reagent</td>
<td>Eppendorf</td>
<td>955154037</td>
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chromatin immunoprecipitation from human embryonic stem cells

metazoan 생물의 세포의 무수한의 기능 및 구조 복잡도는 줄기 세포의 소수에서 발생한다. 자기 갱신과 줄기 세포는 거의 모든 세포 1을 입력한 다음으로 차별화 수 있습니다 multipotency : 줄기 세포는 두 근본적인 속성을 특징으로하고 있습니다. 차별화된 세포에 진행의 줄기 세포가 활동을 설정하고 셀 타입 특정 유전자 발현 패턴을 유지 multipotency 구조 및 형태학의 변화와 전사 요소의 hierarchic 활동 및 신호 분자의 손실이 특징입니다. 분자 수준에서 세포 분화는 염색질의 구조와 조성의 역동적인 변화를 포함하고 이러한 역동적인 변화의 검출은 줄기 세포 및 세포 분화 과정 2,3의 기능 특징에 귀중한 통찰력을 제공할 수 있습니다. 염색질은 양식 고도로 압축된 DNA - 단백질 복합 때 단백질과 세포 패키지 염색체 DNA, histones 주로 4. Stemcellness 및 세포 분화는 이러한 epigenetic 요인, 히스톤 변형, 그리고 전사 요소 2,3,5와 같은 규제 단백질의 특정 배열의 존재와 상관되었습니다. 염색질 immunoprecipitation (칩)은 RNA의 존재, 단백질 및 염색질 6,7의 단백질 수정을 모니터하는 중요한 방법을 제공합니다. 다른 세포 유형에서 염색질의 비교는 세포 차별화하는 동안 발생하는 단백질 염색질 협회의 역동적인 변화를 명료하게하다 수 있습니다. 염색질 immunoprecipitation는 생체내 교차 연결된 염색질에의 정화를 포함한다. 절연 염색질은 효소 소화 또는 기계적 강제로 작은 조각으로 줄어 듭니다. 염색질 조각은 단백질 또는 단백질과 DNA의 수정을 대상으로 특정 항체를 사용하여 시켰던 있습니다. DNA 또는 RNA 시켰던은 정화와 PCR이나 DNA microarray 기반 assays에 대한 템플릿으로 사용됩니다. 성공을위한 전제 조건 칩 원하는 항원 및 대상 분자를 표현하지 컨트롤 세포에서 염색질의 가용성에 고품질의 항체입니다. 칩은 단백질, 단백질과 RNA 수정하고, 특정 타겟 DNA와 RNA의 존재를 상관 관계, 그리고 outread 도구의 선택에 따라 특정 타겟 유전자 또는 전체 게놈의 맥락에서 대상 분자의 연결을 감지하실 수 있습니다. 차별 세포의 염색질에 단백질의 분포의 비교는 세포 혈통을 따라 세포의 진행과 일치 염색질 구성의 역동적인 변화를 명료하게하다 수 있습니다.

Watch this Scientific Journal Video about Chromatin Immunoprecipitation from Human Embryonic Stem Cells at JoVE.com

Chromatin Immunoprecipitation from Human Embryonic Stem Cells

ESC의 차별은 셀 타입의 특정 구조의 변화와 염색질의 구성과 일치한다. 이러한 변화의 감지는 stemcellness 및 세포 분화를 정의하는 메커니즘에 중요한 통찰력을 제공합니다. 염색질 immunoprecipitation (칩)은 줄기 세포 분화를 기본 분자 메커니즘을 해부하다하는 소중한 방법을 나타냅니다.

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 ...

chromatin-immunoprecipitation-from-human-embryonic-stem-cells

Research

JoVE Journal

Biology

20.6K 조회수.  University of California - Riverside. ESC의 차별은 셀 타입의 특정 구조의 변화와 염색질의 구성과 일치한다. 이러한 변화의 감지는 stemcellness 및 세포 분화를 정의하는 메커니즘에 중요한 통찰력을 제공합니다. 염색질 immunoprecipitation (칩)은 줄기 세포 분화를 기본 분자 메커니즘을 해부하다하는 소중한 방법을 나타냅니다.

비디오: Chromatin Immunoprecipitation from Human Embryonic 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

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 ...

Culture and Maintenance of Human Embryonic Stem Cells  

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be  fed  every day by performing a complete medium change to replenish lost nutrients and to keep the cultures free of unwanted differentiation factors. Although a small amount of differentiation is normal and expected in stem cell cultures, the culture should be routinely cleaned up by manually removing, or "picking" differentiated areas. Identifying and removing excess differentiation from hES cell cultures are essential techniques in the maintenance of a healthy population of cells.

Culture and Maintenance of Human Embryonic Stem Cells

This protocol demonstrates how to maintain healthy, undifferentiated human embryonic stem (ES) cells.

인간 배아 줄기 세포의 문화 및 유지 관리

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be ...

Chromosomal Spread Preparation of Human Embryonic Stem Cells for Karyotyping

Although human embryonic stem cells (hESC) have been shown to present a stable diploid karyotype 1, many studies have reported that depending on culture conditions they become prone to acquire chromosomal anomalies such as addition of whole or parts of chromosomes. Indeed, during long-term culture, karyotypic alterations are observed when enzymatic or chemical dissociation are used 2,3,4, while manual dissection of colonies for passaging retains a stable karyotype 5. Besides, changes in the environment such as the removal of feeder cells also seem to compromise the genetic integrity of hESC 3,6. Once chromosomal alterations could affect cellular physiology, the characterization of the genetic integrity of hESC in vitro is crucial considering hESC as an essential tool in embryogenesis studies and drug testing. Furthermore, for future therapeutic purposes chromosomal changes are a real concern as it is frequently associated to carcinogenesis.
Here we show a simple and useful method to obtain high quality chromosome spreads for subsequent analysis of chromosome set by G-banding, FISH, SKY or CGH techniques 7,8. We recommend checking the chromosomal status routinely with intervals of 5 passages in order to monitor the appearance of translocations and aneuploidies
Priscila Britto and Rafaela Sartore contributed equally to the paper.

Karyotyping is a simple and useful technique widely used for detecting genetic alterations. Here we describe a step by step protocol for chromosome spread preparation of human embryonic stem cells for monitoring the chromosomal status of these cells maintained in culture.

Karyotyping를위한 인간 배아 줄기 세포의 염색체 확산 준비

Although human embryonic stem cells (hESC) have been shown to present a stable diploid karyotype 1, many studies have reported that depending on culture ...

Freezing and Thawing Human Embryonic Stem Cells

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

인간 배아 줄기 세포를 동결 융해

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells ...

Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant interest in deriving a pure population of lineage-committed oligodendrocyte precursor cells (OPCs) for transplantation. OPCs are characterized by the activity of the transcription factor Olig2 and surface expression of a proteoglycan NG2. Using the GFP-Olig2 (G-Olig2) mouse embryonic stem cell (mESC) reporter line, we optimized conditions for the differentiation of mESCs into GFP+Olig2+NG2+ OPCs. In our protocol, we first describe the generation of embryoid bodies (EBs) from mESCs. Second, we describe treatment of mESC-derived EBs with small molecules: (1) retinoic acid (RA) and (2) a sonic hedgehog (Shh) agonist purmorphamine (Pur) under defined culture conditions to direct EB differentiation into the oligodendroglial lineage. By this approach, OPCs can be obtained with high efficiency (&gt;80%) in a time period of 30 days. Cells derived from mESCs in this protocol are phenotypically similar to OPCs derived from primary tissue culture. The mESC-derived OPCs do not show the spiking property described for a subpopulation of brain OPCs in situ. To study this electrophysiological property, we describe the generation of spiking mESC-derived OPCs by ectopically expressing NaV1.2 subunit. The spiking and nonspiking cells obtained from this protocol will help advance functional studies on the two subpopulations of OPCs.

We describe a small molecule-based protocol for differentiation of mouse embryonic stem cells into oligodendrocyte precursor cells (OPCs). This protocol generates Olig2+NG2+ OPCs with high efficiency by 30 days of differentiation. We also describe a method to generate "spiking" OPCs that can fire action potentials.

Oligodendrocyte 엽 성의 전구 물질로 배아 줄기 세포의 분화

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant ...

Fate Mapping of Human Embryonic Stem Cells by Teratoma Formation 

Human embryonic stem cells (hESCs) have an unlimited capacity for self-renewal, and the ability to differentiate into cells derived from all three embryonic germ layers (1). Directed differentiation of hESCs into specific cell types has generated much interest in the field of regenerative medicine (e.g., (2-5)), and methods for determining the in vivo fate of selected or manipulated hESCs are essential to this endeavor. We have adapted a highly efficient teratoma formation assay for this purpose. A small number of specifically selected hESCs is mixed with undifferentiated wild type hESCs and Phaseolus vulgaris lectin to form a cell pellet. This is grafted beneath the kidney capsule in an immunodeficient mouse. As few as 2.5 x 105 hESCs are needed to form a 16 cm3 teratoma within 8-12 weeks. The fate of the originally selected hESCs can then be determined by immunohistochemistry. This method provides a valuable tool for characterizing tissue-specific reagents for cell-based therapy.

Fate Mapping of Human Embryonic Stem Cells by Teratoma Formation

Directed differentiation of hESCs into specific cells has generated much interest in regenerative medicine. We provide a concise, step-by-step protocol for determining the in vivo fate of selected hESCs that provides a valuable tool for characterizing tissue-specific reagents for cell-based therapy.

기형 종의 형성에 의해 인간 배아 줄기 세포의 운명 매핑

Human embryonic stem cells (hESCs) have an unlimited capacity for self-renewal, and the ability to differentiate into cells derived from all three ...

Chromatin Immunoprecipitation (ChIP) to Assay Dynamic Histone Modification in Activated Gene Expression in Human Cells

In response to a variety of extracellular ligands, the STAT (signal transducer and activator of transcription) transcription factors are rapidly recruited from their latent state in the cytoplasm to cell surface receptors where they are activated by phosphorylation at a single tyrosine residue1. They then dimerize and translocate to the nucleus to drive the transcription of target genes, affecting growth, differentiation, homeostasis and the immune response. Not surprisingly, given their widespread involvement in normal cell processes, dysregulation of STAT function contributes to human disease, particularly to cancers2 and autoimmune diseases3. It is well established that transcription is regulated by alterations to the chromatin template4,5. These alterations include the activities of ATP-dependent complexes, as well as covalent histone modifications and DNA methylation6. Because STAT activation of gene expression is both rapid and transient, it requires specific mechanisms for modulating the chromatin template at STAT-dependent gene loci. To define these mechanisms, we characterize the histone modifications and the enzymatic activities that generate them at gene loci that respond to STAT signaling. This protocol describes chromatin immunoprecipitation, a method that is valuable for the study of STAT signaling to chromatin in activated gene expression.

Chromatin Immunoprecipitation ChIP to Assay Dynamic Histone Modification in Activated Gene Expression in Human Cells

This protocol describes how chromatin immunoprecipitation (ChIP) is used to study the dynamic alterations to the chromatin template that regulate transcription induced by a signal transduction pathway.

인간의 세포에서 활성 유전자 발현의 어세 동적 히스톤 수정을 염색질 Immunoprecipitation (칩)

In response to a variety of extracellular ligands, the STAT (signal transducer and activator of transcription) transcription factors are rapidly recruited ...

Photobleaching Assays (FRAP &amp; FLIP) to Measure Chromatin Protein Dynamics in Living Embryonic Stem Cells

Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) enable the study of protein dynamics in living cells with good spatial and temporal resolution. Here we describe how to perform FRAP and FLIP assays of chromatin proteins, including H1 and HP1, in mouse embryonic stem (ES) cells. In a FRAP experiment, cells are transfected, either transiently or stably, with a protein of interest fused with the green fluorescent protein (GFP) or derivatives thereof (YFP, CFP, Cherry, etc.). In the transfected, fluorescing cells, an intense focused laser beam bleaches a relatively small region of interest (ROI). The laser wavelength is selected according to the fluorescent protein used for fusion. The laser light irreversibly bleaches the fluorescent signal of molecules in the ROI and, immediately following bleaching, the recovery of the fluorescent signal in the bleached area - mediated by the replacement of the bleached molecules with the unbleached molecules - is monitored using time lapse imaging. The generated fluorescence recovery curves provide information on the protein's mobility. If the fluorescent molecules are immobile, no fluorescence recovery will be observed. In a complementary approach, Fluorescence Loss in Photobleaching (FLIP), the laser beam bleaches the same spot repeatedly and the signal intensity is measured elsewhere in the fluorescing cell. FLIP experiments therefore measure signal decay rather than fluorescence recovery and are useful to determine protein mobility as well as protein shuttling between cellular compartments. Transient binding is a common property of chromatin-associated proteins. Although the major fraction of each chromatin protein is bound to chromatin at any given moment at steady state, the binding is transient and most chromatin proteins have a high turnover on chromatin, with a residence time in the order of seconds. These properties are crucial for generating high plasticity in genome expression1. Photobleaching experiments are therefore particularly useful to determine chromatin plasticity using GFP-fusion versions of chromatin structural proteins, especially in ES cells, where the dynamic exchange of chromatin proteins (including heterochromatin protein 1 (HP1), linker histone H1 and core histones) is higher than in differentiated cells2,3.

Photobleaching Assays FRAP & FLIP to Measure Chromatin Protein Dynamics in Living Embryonic Stem Cells

We describe photobleaching methods including Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) to monitor chromatin protein dynamics in embryonic stem (ES) cells. Chromatin protein dynamics, which is considered to be one of the means to study chromatin plasticity, is enhanced in pluripotent cells.

배아 줄기 세포 생활에서 염색질 단백질 역학을 측정하는 Assays (FRAP 및 플립)를 Photobleaching

Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) enable the study of protein dynamics in living cells with ...