인간 배아 줄기 세포의 망간 라벨링 <ol><li> 피더없이 5 분 조건에서 양식되고있는 인간의 배아 줄기를 Trypsinize. 문화 미디어를 추가하여 트립신을 중화. </li><li> 20 5 분 800 rpm으로 centrifuging하여 세포를 스핀 다운 ° 섭씨. 다시 보류 세포 펠릿 후, trypan 파랑 얼룩에 의해 세포의 개수를 계산합니다. 얻은 세포 개수에 따라 원뿔 튜브에 3,000,000 세포 네 가지 aliquots으로 세포를 나눕니다. 원심 분리하여 다시 펠렛 세포. </li><li> 그냥 망간 상표를 시작하기 전에, 0.1mM 망간 염화 솔루션을 만들기 위해 0.9 %의 나트륨 염화물 솔루션 신선한 망간 염화을 풀다. </li><li> 칼슘 채널의 활동을 관찰하기 위해서는, 넷 샘플이 준비되었습니다. 첫 번째 예제는 혼자서 0.9 %의 나트륨 염화물에 incubated 세포를 포함 우리의 컨트롤입니다. 두 번째 예제 0.1 MM MnCl 2 incubated 세포가 포함되어 있습니다. 샘플 3 4 칼슘 2 + 채널 작용제 5 μm의 하나와 0.1 MM MnCl 2 incubated 세포 (S )-(-)- 베이 K8644, 또는 칼슘 2 + 채널 길항제, verapamil 250 μm의이 포함되어 있습니다. 37 네 개의 샘플을 품어 ° 30 분 5 % CO 2와 함께 섭씨. </li><li> 30 분 후에, 20 ° 섭씨에서 5 분 동안 800 rpm으로 세포를 스핀 다운. 그런 다음 뜨는을 대기음하고 PBS로 두 번 표시된 세포를 씻으십시오. 세척 후, PCR 튜브에 200 μL PBS 및 전송하여 각 펠렛을 다시 일시 중지합니다. 이 알약은 보통 흰색입니다. </li></ol> 인간 배아 줄기 세포의 이런 라벨링 <ol><li> 1 MG / ML 재고 솔루션을 위해 증류수와 임상 수준 프로타민 황산을 섞는다. </li><li> 인간 배아 줄기 세포 배양 매체를 포함하는 튜브에 12 μg / ML 프로타민 황산의 또한 다음에 100 μg / ML에서 ferumoxides를 추가합니다. 5 분 동안 적극적으로 솔루션을 섞는다. </li><li> 50 μg / ferumoxides의 ML 6 레이블 세포에 프로타민 황산의 μg / ML의 최종 농도를 달성하기위한 세포 배양 매체의 동일한 금액에 혼합 솔루션을 추가합니다. 12 시간이 솔루션에 세포를 품어. </li><li> 세포외 ferumoxides - 프로타민 황산 복합을 해산하기 위해 10U/ml 헤파린이 들어있는 마지막으로 세척과 함께 PBS로 두 번 세포를 씻으십시오. 세척 후, 세포가 하나의 세포 현탁액을 얻는 trypsinize. </li><li> 샘플 필요한 번호로 세포가 나누어지는 카운트. 세척 후, PCR 튜브에 200 μL PBS 및 전송하여 각 펠렛을 다시 일시 중지합니다. 이 알약은 색깔에서 짙은 주황색 갈색해야합니다. </li></ol> 세포 자기 공명 영상 공연 <ol><li> 셀 알약이 들어있는 튜브를 안정화하고 또한 스캔하는 동안 주위의 공기에서 유물을 피하기 위해 유령을 만듭니다. 이렇게하려면, 증류수에 한천의 0.8 %와 구리 황산의 1 %를 혼합하고 5~7분에 대한 microwaving하여 끓기 가져. 젤을 달성하기 위해 스캔 전에 최소한 1 시간 동안이 혼합물을 쿨. </li><li> 표시 셀 알약을 포함하는 튜브는 스캔에 대한 환상에 배치됩니다. 체외 세포 MRI 임상 무릎 코일을 사용하여 Signa HDx 3T MRI (GE 의료 시스템)에서 수행됩니다. </li><li> 스핀 에코 시퀀스를 사용 세 가지 샘플의 각 망간 전지 라벨 스캔 : TR을, 반복 시간 = 800 밀리초. TE, 에코 시간 = 최소, FOV,보​​기의 분야 = 12 X 12cm, 매트릭스 = 256x256, NEX 1. </li><li> 그라디언트 - 에코 시퀀스를 사용하여 철분 라벨 세포를 검사합니다. 이 영상에 대해 다음 매개 변수를 사용 : TR = 100 밀리초, TE = 10 밀리초, FA, 플립 각도 = 30 °, FOV = 12 X 12cm, 매트릭스 = 256 X 256, NEX 1. </li></ol> MRI 결과 분석 <ol><li> 망간 - 라벨 세포의 4 개의 샘플에서 MRI 스캔 이미지를 분석 : <ul><li> 샘플 # 1은 혼자 0.9 %의 나트륨 염화물에 incubated 세포를 포함 우리의 컨트롤입니다. </li><li> 샘플 # 2는 0.1 MM MnCl 2 incubated 세포가 포함되어 있습니다. </li><li> 샘플 # 3 칼슘 2 + 채널 작용제 5 μm의, (S )-(-)- 베이 K8644과 함께 0.1 MM MnCl 2 incubated 세포가 포함되어 있습니다. </li><li> 샘플 # 4 칼슘 2 + 채널 길항제, verapamil 250 μm의와 0.1 MM MnCl 2 incubated 세포가 포함되어 있습니다. </li></ul></li><li> 으로 예측, 망간 전지 라벨에서 신호 강도의 차이는 볼 수 있습니다. 망간과 순수라는 세포에 비해 신호 강도가 명확하게 칼슘 채널 작용제의 존재의 증가와 칼슘 채널 길항제의 존재에 줄어 듭니다. 신호 강도가 표시 망간 전지 증가 지역은 이미지 J을 (NIH, 베데스다, MD, 미국)를 사용하여 측정됩니다. 동일한 소프트웨어는 철 표시 세포의 어두운 오프 공명 신호의 영역을 분석하는 데 사용됩니다. 철 - 라벨 세포의 어두운 신호의 크기는 세포 또는 세포에 의해 포함된 철 입자의 숫자의 수에 따라 달라집니다. 이 경우에는, 백만 세포는 삼백만 세포의 신호에 비해 어두운 신호의 작은 영역을 보여줍니다. </li></ol>

<ol>
	<li>Bruvold, M., Nordhoy, W., Anthonsen, H. W., Brurok, H., Jynge, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Manganese-calcium+interactions+with+contrast+media+for+cardiac+magnetic+resonance+imaging:+a+study+of+manganese+chloride+supplemented+with+calcium+gluconate+in+isolated+Guinea+pig+hearts.">Manganese-calcium interactions with contrast media for cardiac magnetic resonance imaging: a study of manganese chloride supplemented with calcium gluconate in isolated Guinea pig hearts.</a> Invest Radiol. 40, 117-125 (2005).</li><li>Aoki, 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=Cell+labeling+for+magnetic+resonance+imaging+with+the+T1+agent+manganese+chloride.">Cell labeling for magnetic resonance imaging with the T1 agent manganese chloride.</a> NMR Biomed. 19, 50-59 (2006).</li><li>Arbab, A. S., 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=Efficient+magnetic+cell+labeling+with+protamine+sulfate+complexed+to+ferumoxides+for+cellular+MRI.">Efficient magnetic cell labeling with protamine sulfate complexed to ferumoxides for cellular MRI.</a> Blood. 104, 1217-1223 (2004).</li><li>Arbab, A. S., Frank, 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=Cellular+MRI+and+its+role+in+stem+cell+therapy.">Cellular MRI and its role in stem cell therapy.</a> Regen Med. 3, 199-215 (2008).</li></ol>

이러한 결과는 망간도 세포 생존을 보여줄 수 전압 - 게이 티드 칼슘 채널 및 망간은 MRI 대비 에이전트로 사용할 수를 통해 입력 않는다는 것을 나타냅니다. 따라서, 우리는 망간 - 안내 MRI는 myocardium로 인간 배아 줄기 세포의 이식 후 세포 생존을 모니터하는 데 유용할 수 있습니다하는 것이 좋습니다.

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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>Manganese chloride tetrahydrate</td>
<td>Reagent</td>
<td>Sigma</td>
<td>M8054</td>
<td>&nbsp;</td>
<tr>
<td>(S)-(-)-Bay K8644 </td>
<td>Reagent</td>
<td>Sigma</td>
<td>B133</td>
<td>&nbsp;</td>
<tr>
<td>(+)-Verapamil hydrochloride, minimum 99.0% titration </td>
<td>Reagent</td>
<td>Sigma</td>
<td>V4629</td>
<td>&nbsp;</td>
<tr>
<td>Sodium Chloride 0.85-0.90% W/V</td>
<td>Reagent</td>
<td>VWR</td>
<td>VW 3257-1</td>
<td>&nbsp;</td>
<tr>
<td>Feridex I.V. </td>
<td>Reagent</td>
<td>Berlex Laboratories</td>
<td>NDC 59338-7035-5</td>
<td>ferumoxides injectable solution 11.2mg iron/mL</td>
</tr>
<tr>
<td>Protamine sulfate</td>
<td>Reagent</td>
<td>American Pharmaceutical Partners</td>
<td>NDC 63323-229-05 </td>
<td>50 mg (10 mg/mL)</td>
</tr>
<tr>
<td>Knockout SR (Serum replacement for ES cells)</td>
<td>Reagent</td>
<td>Gibco</td>
<td>10828</td>
<td>&nbsp;</td>
<tr>
<td>DMEM/F12 (1:1)</td>
<td>Reagent</td>
<td>Gibco</td>
<td>11330</td>
<td>&nbsp;</td>
<tr>
<td>2- mercaptoethanol 98+%</td>
<td>Reagent</td>
<td>Sigma</td>
<td>M 3148</td>
<td>&nbsp;</td>
<tr>
<td>L-Glutamine 200mM 100x</td>
<td>Reagent</td>
<td>Gibco</td>
<td>25030</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin-Streptomyocin 10,000 units/ml</td>
<td>Reagent</td>
<td>Gibco</td>
<td>15140-122</td>
<td>&nbsp;</td>
<tr>
<td>MEM Non-Essential Amino Acids 100x solution</td>
<td>Reagent</td>
<td>Gibco </td>
<td>11140</td>
<td>&nbsp;</td>
<tr>
<td>Signa HDx 3T MRI</td>
<td>Tool</td>
<td>GE Medical Systems</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>clinical Knee coil</td>
<td>Tool</td>
<td>GE Medical Systems</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>DPBS</td>
<td>Reagent</td>
<td>Gibco</td>
<td>14190</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

in vitro labeling of human embryonic stem cells for magnetic resonance imaging

인간 배아 줄기 세포는 (hESC) 부상 myocardium를 복원하는 능력을 증명하고있다. 자기 공명 영상 (MRI)는 부상 myocardium의 복원을 평가하는 주된 이미징 modalities 중 하나로 떠오르고있다. 또한, 이러한 철 - 산화 nanoparticles으로 전 생체내 라벨 에이전트는, 추적하기 위해 고용 및 이식 줄기 세포를 집중하고 있습니다. 그러나,이 방법은 이식 세포의 생존에 관한 근본적인 세포 생물학 속성을 모니터하지 않습니다. 그것은 망간 염화 (MnCl 2) 전압 - 문이 칼슘 (칼슘 2 +) 세포 생물학적으로 활성화되어 채널을 통해 세포를 입력하고, T 1 단축 효과를 생성하는 intracellularly 축적되는 것으로 알려져있다. 따라서, 우리는 망간 - 안내 MRI는 myocardium에 hESC의 이식 후 세포 생존을 모니터하는 데 유용할 수 있습니다하는 것이 좋습니다. 이 비디오에서는, 우리는 MnCl 2 어떻게 이들 세포는 명확하게 체외에서 MRI를 사용하여 볼 수와 hESC 레이블을하는 방법을 보여줍니다. 동시에, CA 2 생물 학적 활동 + - 채널 수반하는 신호 변경을 평가하기 위해 칼슘 2 + 채널 작용제 및 길항제를 모두 활용 변조된 것이다.

Watch this Scientific Journal Video about In vitro Labeling of Human Embryonic Stem Cells for Magnetic Resonance Imaging at JoVE.com

In vitro Labeling of Human Embryonic Stem Cells for Magnetic Resonance Imaging

이 비디오에서는, 우리는 망간 염화와 인간의 배아 줄기 세포를 (hESC) (MnCl 라벨하는 방법을 보여주는데<sub&gt; 2</sub&gt;) 세포 생물학적으로 활성화된 경우 전압 - 문이 칼슘 채널을 통해 세포를 입력할 수있는. 또한, 우리는 MnCl의 사용을 보여<sub&gt; 2</sub&gt; 셀룰러 MRI 대비 에이전트로 hESC의에서 체외의 생존을 결정합니다.

자기 공명 영상을위한 인간 배아 줄기 세포의 체외 라벨에

Human embryonic stem cells (hESC) have demonstrated the ability to restore the injured myocardium. Magnetic resonance imaging (MRI) has emerged as one of ...

in-vitro-labeling-human-embryonic-stem-cells-for-magnetic-resonance

Research

JoVE Journal

Biology

10.1K 조회수.  Stanford University. 이 비디오에서는, 우리는 망간 염화와 인간의 배아 줄기 세포를 (hESC) (MnCl 라벨하는 방법을 보여주는데 2) 세포 생물학적으로 활성화된 경우 전압 - 문이 칼슘 채널을 통해 세포를 입력할 수있는. 또한, 우리는 MnCl의 사용을 보여 2 셀룰러 MRI 대비 에이전트로 hESC의에서 체외의 생존을 결정합니다.

비디오: In vitro Labeling of Human Embryonic Stem Cells for Magnetic Resonance Imaging

Propagation of Human Embryonic Stem (ES) Cells

Propagation of Human Embryonic Stem ES Cells

인간의 배아 줄기 (ES) 세포의 전파

연구

생물학

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 ...

Labeling Stem Cells with Fluorescent Dyes for non-invasive Detection with Optical Imaging

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo labeling of stem cells with a fluorescent dye, subsequent intravenous injection of the labeled cells and visualization of their accumulation in specific target organs or pathologies. The presented technique demonstrates how we label human mesenchymal stem cells (hMSC) by simple incubation with the lipophilic fluorescent dye DiD (C67H103CIN2O3S) and how we label human embryonic stem cells (hESC) with the FDA approved fluorescent dye Indocyanine Green (ICG). The uptake mechanism is via adherence and diffusion of the lypophilic dye across the phospholipid cell membrane bilayer. The labeling efficiency is usually improved if the cells are incubated with the dye in serum-free media as opposed to incubation in serum-containing media. Furthermore, the addition of the transfection agent Protamine Sulfate significantly improves contrast agent uptake.

This video shows techniques for labeling of human embryonic stem cells and mesenchymal stem cells with fluorescent dyes. This technique can be used for an in vivo tracking of transplanted stem cells with optical imaging and for histopathological correlations with fluorescence microscopy.

광학 이미징과 비침습 감지를위한 형광 염료와 함께 줄기 세포를 라벨링

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo ...

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 ...

In vitro and in vivo Bioluminescence Reporter Gene Imaging of Human Embryonic Stem Cells

The discovery of human embryonic stem cells (hESCs) has dramatically increased the tools available to medical scientists interested in regenerative medicine. However, direct injection of hESCs, and cells differentiated from hESCs, into living organisms has thus far been hampered by significant cell death, teratoma formation, and host immune rejection. Understanding the in vivo hESC behavior after transplantation requires novel imaging techniques to longitudinally monitor hESC localization, proliferation, and viability. Molecular imaging has given investigators a high-throughput, inexpensive, and sensitive means for tracking in vivo cell proliferation over days, weeks, and even months. This advancement has significantly increased the understanding of the spatio-temporal kinetics of hESC engraftment, proliferation, and teratoma-formation in living subjects.

A major advance in molecular imaging has been the extension of noninvasive reporter gene assays from molecular and cellular biology into in vivo multi-modality imaging platforms. These reporter genes, under control of engineered promoters and enhancers that take advantage of the host cell s transcriptional machinery, are introduced into cells using a variety of vector and non-vector methods. Once in the cell, reporter genes can be transcribed either constitutively or only under specific biological or cellular conditions, depending on the type of promoter used. Transcription and translation of reporter genes into bioactive proteins is then detected with sensitive, noninvasive instrumentation (e.g., CCD cameras) using signal-generating probes such as D-luciferin.

To avoid the need for excitatory light to track stem cells in vivo as is required for fluorescence imaging, bioluminescence reporter gene imaging systems require only an exogenously administered probe to induce light emission. Firefly luciferase, derived from the firefly Photinus pyralis, encodes an enzyme that catalyzes D-luciferin to the optically active metabolite, oxyluciferin. Optical activity can then be monitored with an external CCD camera. Stably transduced cells that carry the reporter construct within their chromosomal DNA will pass the reporter construct DNA to daughter cells, allowing for longitudinal monitoring of hESC survival and proliferation in vivo. Furthermore, because expression of the reporter gene product is required for signal generation, only viable parent and daughter cells will create bioluminescence signal; apoptotic or dead cells will not. 

In this video, the specific materials and methods needed for tracking stem cell proliferation and teratoma formation with bioluminescence imaging will be described.

With the growing interest in stem cell therapies, molecular imaging techniques are ideal for monitoring stem cell behavior after transplantation. Luciferase reporter genes have enabled non-invasive, repetitive assessment of cell survival, location, and proliferation in vivo. This video will demonstrate how to track hESC proliferation in a living mouse.

체외에서 인간 배아 줄기 세포의 생체내의 Bioluminescence 리포터 진 영상에

The discovery of human embryonic stem cells (hESCs) has dramatically increased the tools available to medical scientists interested in regenerative ...

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 ...

Video Bioinformatics Analysis of Human Embryonic Stem Cell Colony Growth

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer software.  Video bioinformatics is a powerful quantitative approach for extracting spatio-temporal data from video images using computer software to perform dating mining and analysis. In this article, we introduce a video bioinformatics method for quantifying the growth of human embryonic stem cells (hESC) by analyzing time-lapse videos collected in a Nikon BioStation CT incubator equipped with a camera for video imaging. In our experiments, hESC colonies that were attached to Matrigel were filmed for 48 hours in the BioStation CT.  To determine the rate of growth of these colonies, recipes were developed using CL-Quant software which enables users to extract various types of data from video images.  To accurately evaluate colony growth, three recipes were created. The first segmented the image into the colony and background, the second enhanced the image to define colonies throughout the video sequence accurately, and the third measured the number of pixels in the colony over time.  The three recipes were run in sequence on video data collected in a BioStation CT to analyze the rate of growth of individual hESC colonies over 48 hours. To verify the truthfulness of the CL-Quant recipes, the same data were analyzed manually using Adobe Photoshop software. When the data obtained using the CL-Quant recipes and Photoshop were compared, results were virtually identical, indicating the CL-Quant recipes were truthful. The method described here could be applied to any video data to measure growth rates of hESC or other cells that grow in colonies. In addition, other video bioinformatics recipes can be developed in the future for other cell processes such as migration, apoptosis, and cell adhesion.  

Video bioinformatics is the automated processing, analysis, understanding, and data mining of biological spatio-temporal data extracted from microscopic videos. The purpose of this article is to demonstrate a method for measuring human embryonic stem cell colony growth using a video bioinformatics method.

비디오 생물 정보학 인간의 배아 줄기 세포 콜로니 성장 분석

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer ...