AEC는 의료 연구 및 연구 머크 연구소에 대한 제인 코핀 차일즈 기념 기금의 동료입니다. 우리는 실험적인 조언과 토론 D. 에글리과 C. 카원 감사드립니다.

<html> 인간의 배아 줄기 (hES) 세포 유도에 사용되는 냉동 인간 배아는 하버드 대학에서 인간 과목의 사용에위원회와 배아 줄기 세포 연구 감독위원회 모두의 검토와 승인되었습니다 연구 프로토콜에 따라 적절한 정보를 동의를 다음과 같은 연구를 위해 기증했다 . 아래 서면 프로토콜 카원 외 의해 보고된 출판 매개 변수를 기반으로합니다. 2004 년 약간의 수정. 배아 문화 개발까지 확장 blastocyst 단계에 15 % Plasmanate로 보충 글로벌 매체의 방울로 퀸의 장점 해동 키트와 문화를 사용하여 인간 배아를 해동. 내부 세포 질량 Immunosurgery 기반 격리 준비 : <ol><li> 10cm 플레이트의 각 솔루션의 방울을 설정하여 조나 pellucida 및 immunosurgery의 제거 (아래 참조) 접시를 준비합니다. 증발을 방지하기 위해 미네랄 오일과 중첩. 파생위한 배아마다 하나의 행을 준비합니다. <ul><li> EmbryoMax 산성 Tyrodes는 그대로 사용합니다. </li><li> hES 세포 유도 매체 : 75 % KO - DMEM 10 % KO - 세럼 교체, 10 % Plasmanate, 2.5 % ES 세포 검사 태아 소 혈청 (FBS), 2mM Glutamax - I, 1 %가 아닌 필수 아미노산, 50 대 / ML 페니실린, 스트렙토 마이신의 50μg/ml, 0.055mM B - 메르 캅 토 에탄올, 5ng/ml bFGF. </li><li> 토끼 안티 - 인간의 적혈구 세포 일차 항체는 hES 세포 유도 매체와 1시 10분에 희석 제조 업체의 제안에 따라 재구성. </li><li> 기니 돼지 보완 혈청, hES 세포 유도 매체와 1시 10분에 희석 제조 업체의 제안에 따라 재구성. <img src="/files/ftp_upload/old/574_2008_9_18_6/574_Figure-01.png" alt="574_Figure - 01" width="600" height="166" /> </li></ul></li><li> ° C 조직 문화 인큐베이터에서 사용하기 전에 37 모든 번호판을 평형. </li><li> 전송 배아에 사용하기 위해 50μl - 100μl 모세관 pipettes (VWR)을 당기세요. 다이아몬드 펜과 0.2μm 필터를 갖춘 입 피펫 튜브와 함께 사용 끝을 잘라. </li></ol> Immunosurgery 절차 : 참고 : <ul><li> 이 절차에서는 trophectoderm의 세포는 보완 활동과 협력하여 인간의 세포에 대한 감독 항체에 대한 간단한 노출에 의해 파괴됩니다. blastocyst만을 구조적 무결성 또한 면역 반응에 민감한되는 ICM을 방지 등 따라서, 프로토콜, 손상 trophectoderm와 함께 고품질의 배아와 함께 잘 작동됩니다. </li><li> 모든 절차가 열띤 무대를 갖춘 절개 범위를 사용하여 수행했습니다. </li></ul> 단계 : <ol><li> 고집에서 배아를 방지하기 위해 FBS와 배아 전송을 위해 사용할 수 입 피펫을 씻어. </li><li> 문화 접시에서 요리 AT의 hES 개최 드롭에 blastocyst을 전송. </li><li> 방울 AT 시리즈를 통해 hES 드롭에서 blastocyst를 전송하여 절차를 시작합니다. 셋째 드롭, 조나 pellucida (보통 5~30초)의 해산에 대한 감시. 아니여 - 치료 또는 배아 무결성에주의하는 것은 위험 수 있습니다. <img src="/files/ftp_upload/old/574_2008_7_21_2/574_Figure-02.png" alt="574_Figure - 02" width="551" height="273" /> </li><li> hES 세포 유도 매체의 시리즈를 통해 전송 blastocyst은 하차 린스로 방울. 미디어와 Preloading 피펫은 즉시 반응 AT 무력화하는 것이 좋습니다. 모두 - 처리 AT 때까지 Blastocysts 여기 개최 수 있습니다. </li><li> 37 부화 30 분 제 3 드롭 ° 조직 문화 인큐베이터에서 C에두고, 기본 항체 방울의 시리즈를 통해 blastocyst을 전송합니다. </li><li> hES 세포 유도 매체의 시리즈를 통해 전송 blastocyst은 기본을 린스로 방울. </li><li> trophectoderm 세포의 용해를 위해 볼 셋째 드롭 떠나, 보수 방울의 시리즈를 통해 blastocyst을 전송합니다. 그들이 lyse로 trophectoderm 세포의 &quot;버블링&quot;에 대한 무대에서 처음으로 30 초 동안 blastocyst을 관찰. 용해이 기간 내에 관찰되면 완료 때까지 반응을 모니터하기 위해 현미경의 무대에서 요리를 계속. 더 버블링이 최초 30 초 동안 관찰하지 않으면 trophectoderm 대부분 lysed 때까지 5 분마다 확인, 인큐베이터으로 돌아갑니다. 이 5~15분 소요될 수 있습니다. 반응 시간과 내부 세포 덩어리 잠재적인 피해를 최소화하기 위해 밀접하게 반응을 모니터링합니다. <img src="/files/ftp_upload/old/574_2008_7_21_3/574_Figure-03.png" alt="574_Figure - 03" width="571" height="273" /> </li><li> hES 세포 유도 매체의 시리즈를 통해 전송 blastocyst은 보완 작업을 씻어 방울. </li><li> blastocyst (10μl VWR 모세관 튜브에서 나온)보다 약간 작은 직경 유리 피펫을 사용하여 lysed trophectoderm의 대부분을 벗겨 아래로 blastocyst을 그려합니다. G &quot;고도 =&quot;574_Figure - 0 &quot;너비 =&quot;577 &quot;높이 =&quot;270 &quot;/&gt; </li><li> 플레이트 ICM은 mitotically inactivated 마우스 배아 섬유아 세포 (MEFs)의 피더 세포로 분리. MEFs는 파생 전날을 준비과 hES 세포 유도 미디어 하루에 전환해야합니다. 4 잘 NUNC 요리 유도의 초기 단계를 잘 작동합니다. </li><li> 첫째 1~2일에 대한 번호판을 방해하지 마십시오. 이후 배아 줄기 세포 식민지 (보통 명백한 1~2주 이내)의 가지에 대해 매일 확인합니다. 미디어는 3 일에있는 모든 다른 하루를 시작 이분의 일 볼륨에서 변경해야합니다. </li></ol> ES 세포 파생물 및 passaging hES 세포 가지가 약 0.5mm 이상에 도달하면 기계 수확하여 확장 준비가되었습니다. <ol><li> 기계식 가지의 절반을 분산 신선한 피더를 포함하는 새로운 접시에 전송할 수 있습니다. 배아의 줄기 세포 대단히 짧은 시간은 약 30-50 전지 각각해야합니다. 백업 최초의 통로 (P1)에 같은 가지의 다른 절반을 둡니다. 이 확장을 계속하고 같이 파생물 (p0)이 여러 번 포착에서하실 수 있습니다. <img src="/files/ftp_upload/old/574_2008_7_21_5/574_Figure-05.png" alt="574_Figure" width="639" height="219" /> </li><li> 보조 식민지는 마찬가지로 분산하고 새로운 세포 라인이 효소 트립신과 passaging 적응 수있는 시간 구절 3-5 때까지 큰 문화 요리에 걸쳐 확장하실 수 있습니다. 문화 미디어의 혈청 내용도 점차 ES 세포 차별을 최소화하기 위해 이러한 초기 구절 중에 1퍼센트 0 %로 감소한다. </li><li> 모든 기존 배아 줄기 세포 라인은 향후 확장을위한 초기 구절의 일부를 냉동하여 틀었해야합니다. </li><li> 또한, 각각의 세포 라인은 핵형 분석에 의해 특징이어야하며 체외 및 생체내에 pluripotency와 분화 연구에 의해 확인되었습니다. </li></ol>

<ol>
	<li>Cowan, C. A., Klimanskaya, I., McMahon, J., Atienza, J., Whitmyer, J., Zucker, J. P., Wang, S. P., Morton, C. C., McMahon, A. P., Powers, D., Melton, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Derivation+of+embryonic+stem-cell+lines+from+human+blastocysts.">Derivation of embryonic stem-cell lines from human blastocysts.</a> N ENGL J MED. 350 (13), 1353-1356 (2004).</li></ol>

The authors have nothing to disclose.

프로토콜 여기에 소개가와 연구자를 제공하는 간결한, 쉬운 따라 내부 세포 질량 immunosurgical 절연에 의해 인간 배아 줄기 세포 라인을 도출하는 방법에 대한 개요.

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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>Quinn's Advantage Thaw Kit </td>
<td>&nbsp;</td>
<td>Sage</td>
<td>ART-8016</td>
<td>&nbsp;</td>
<tr>
<td>Global medium</td>
<td>&nbsp;</td>
<td>Life Global</td>
<td>GMGB-050</td>
<td>&nbsp;</td>
<tr>
<td>Plasmanate</td>
<td>&nbsp;</td>
<td>Talecris</td>
<td>61325</td>
<td>&nbsp;</td>
<tr>
<td>EmbryoMax Acidic Tyrodes</td>
<td>&nbsp;</td>
<td>Chemicon/Millipore</td>
<td>MR-004-D</td>
<td>&nbsp;</td>
<tr>
<td>KO-DMEM</td>
<td>&nbsp;</td>
<td>Invitrogen/Gibco</td>
<td>10829-018</td>
<td>&nbsp;</td>
<tr>
<td>KO-Serum Replacement</td>
<td>&nbsp;</td>
<td>Invitrogen/Gibco</td>
<td>10828-028</td>
<td>&nbsp;</td>
<tr>
<td>ES cell-tested fetal bovine serum, Defined</td>
<td>&nbsp;</td>
<td>Hyclone</td>
<td>SH30070.03</td>
<td>&nbsp;</td>
<tr>
<td>Glutamax-I</td>
<td>&nbsp;</td>
<td>Invitrogen/Gibco</td>
<td>35050-079</td>
<td>&nbsp;</td>
<tr>
<td>Non-essential amino acids</td>
<td>&nbsp;</td>
<td>Invitrogen/Gibco</td>
<td>11140-076</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin/Streptomycin</td>
<td>&nbsp;</td>
<td>Invitrogen/Gibco</td>
<td>15070-063</td>
<td>&nbsp;</td>
<tr>
<td>Recombinant human basic fibroblast growth factor</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>13256-029</td>
<td>&nbsp;</td>
<tr>
<td>Beta-mercaptoethanol </td>
<td>&nbsp;</td>
<td>Invitrogen/Gibco</td>
<td>21985-023</td>
<td>&nbsp;</td>
<tr>
<td>Rabbit anti-human red blood cell primary antibody</td>
<td>&nbsp;</td>
<td>Rockland</td>
<td>109-4139</td>
<td>&nbsp;</td>
<tr>
<td>Guinea-pig complement serum</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S-1639</td>
<td>&nbsp;</td>
<tr>
<td>Capillary pipettes in 10 microL, 50 microL, 100 microL sizes</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Mouth pipettes with tubing</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>&nbsp;</td>
<td>supplied with capillary pipettes from VWR</td>
</tr>
<tr>
<td>0.2 microm Acrodisc syringe filter </td>
<td>&nbsp;</td>
<td>Pall Life Sciences</td>
<td>PN4192</td>
<td>&nbsp;</td>
<tr>
<td>0.5% Trypsin-EDTA</td>
<td>&nbsp;</td>
<td>Invitrogen/Gibco</td>
<td>25300-120</td>
<td>&nbsp;</td>
<tr>
<td>Tissue culture dishes</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Tissue culture dishes</td>
<td>&nbsp;</td>
<td>NUNC</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Nikon SMZ1500 dissection scope equipped with Nikon Tokai Hit ThermoPlate warming stage </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Mouse embryonic fibroblasts (E12.5)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

derivation of human embryonic stem cells by immunosurgery

자기 갱신과 신체의 모든 세포 유형으로 차별화하는 인간 배아 줄기 세포의 능력은 그들이 모두 의료 어플 리케이션을위한 개발과 질병의 근본적인 질문을 해결하기위한 연구 도구로 큰 약속을 개최하는 것이 좋습니다. 여기서 우리는 내부 세포 질량 immunosurgical 절연에 의해 배아에서 인간 배아 줄기 세포의 유도에 대한 간결한, 단계별 절차를 제공합니다.

Watch this Scientific Journal Video about Derivation of Human Embryonic Stem Cells by Immunosurgery at JoVE.com

Derivation of Human Embryonic Stem Cells by Immunosurgery

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 ...

derivation-of-human-embryonic-stem-cells-by-immunosurgery

Research

JoVE Journal

Biology

33.7K 조회수.  Harvard. 자기 갱신과 신체의 모든 세포 유형으로 차별화하는 인간 배아 줄기 세포의 능력은 그들이 모두 의료 어플 리케이션을위한 개발과 질병의 근본적인 질문을 해결하기위한 연구 도구로 큰 약속을 개최하는 것이 좋습니다. 여기서 우리는 내부 세포 질량 immunosurgical 절연에 의해 배아에서 인간 배아 줄기 세포의 유도에 대한 간결한, 단계별 절차를 제공합니다.

비디오: Derivation of Human Embryonic Stem Cells by Immunosurgery

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 ...

Probing for Mitochondrial Complex Activity in Human Embryonic Stem Cells

Mitochondria are cytoplasmic organelles that have a primary role in cellular metabolism and homeostasis, regulation of the cell signaling network, and programmed cell death. Mitochondria produce ATP, regulate the cytoplasmic redox state and Ca2+ balance, catabolize fatty acids, synthesize heme, nucleotides, steroid hormones, amino acids, and help assemble iron-sulfur clusters in proteins. Mitochondria also have an essential role in heat production. Mutations of the mitochondrial genome cause several types of human disorder. The accumulation of mtDNA mutations correlates with aging and is suspected to have an important role in the development of cancer. Due to their vitally important role in all cell types, the function of mitochondria must also be critical for stem cells. Key advances have been made in our understanding of stem cell viability, proliferation, and differentiation capacity. But the functional activity of stem cells, in particular their energy status, was not yet been studied in detail. Almost nothing is known about the mitochondrial properties of human embryonic stem cells (hESCs) and their differentiated precursor progeny. One way to understand and evaluate the role of mitochondria in hESC function and developmental potential is to directly measure the activity of mitochondrial respiratory complexes. Here, we describe high resolution clear native gel electrophoresis and subsequent in gel activity visualization as a method for analyzing the five respiratory chain complexes of hESCs.

In this video, we will show you how the mitochondrial respiratory chain complexes of human embryonic stem cells can be analyzed using in gel activity assays.

인간 배아 줄기 세포에서 Mitochondrial 복잡한 활동에 대한 탐색

Mitochondria are cytoplasmic organelles that have a primary role in cellular metabolism and homeostasis, regulation of the cell signaling network, and ...

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 ...

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 ...

Isolation and Derivation of Mouse Embryonic Germinal Cells

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum (dpc) mouse embryos.  Developing gonadal ridges from mouse embryos (C57BL6J) were dissociated by mechanical disruption with collagenase, then plated in a mouse embryo fibroblast feeder layer (MEF-CF1) that was previously mitotically inactivated with mitomycin C in the presence of knockout media and supplemented with Leukemia Inhibitor Factor (LIF), basic Fibroblast Growth Factor (bFGF), and Stem Cell Factor (SCF).  Using these optimized methods for PCG identification, isolation, and establishment of culture conditions permits long term cultures of EG cells for more than 40 days.  The embryonic germinal cell lines showed embryonic phenotype and expression of common used markers of the pluripotent state.  Isolation and derivation of germinal cells in culture provide a tool to understand their development in vitro and offer the opportunity to monitor cumulative damage at genetic and epigenetic levels after exposure to oxidative stress.

The ability of embryonic germinal cells to differentiate into primordial germinal cells during early development stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum mouse embryos.

절연 및 마우스 배아 새싹 세포의 유도

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental ...

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 ...

Derivation of Mouse Trophoblast Stem Cells from Blastocysts

Specification of the trophectoderm is one of the earliest differentiation events of mammalian development. The trophoblast lineage derived from the trophectoderm mediates implantation and generates the fetal part of the placenta. As a result, the development of this lineage is essential for embryo survival. Derivation of trophoblast stem (TS) cells from mouse blastocysts was first described by Tanaka et al. 1998. The ability of TS cells to preserve the trophoblast specific property and their expression of stage- and cell type-specific markers after proper stimulation provides a valuable model system to investigate trophoblast lineage development whereby recapitulating early placentation events. Furthermore, trophoblast cells are one of the few somatic cell types undergoing natural genome amplification. Although the molecular pathways underlying trophoblast polyploidization have begun to unravel, the physiological role and advantage of trophoblast genome amplification remains largely elusive. The development of diploid stem cells into polyploid trophoblast cells in culture makes this ex vivo system an excellent tool for elucidating the regulatory mechanism of genome replication and instability in health and disease. Here we describe a protocol based on previous reports with modification published in Chiu et al. 2008.

In this video, we demonstrate the isolation of mouse blastocysts and the derivation of trophoblast stem cells from blastocysts. We also describe conditions for maintenance of the stem cell property as well as induction of differentiation in culture.

Blastocysts에서 마우스 Trophoblast의 줄기 세포의 유도

Specification of the trophectoderm is one of the earliest differentiation events of mammalian development. The trophoblast lineage derived from the ...