人类胚胎干细胞的锰标签<ol><li> Trypsinize馈线免费5分钟的条件已在培养的人类胚胎茎。中加入文化传媒的胰蛋白酶。 </li><li>旋转下降的细胞，离心5分钟800转20摄氏度。重新悬浮的细胞沉淀后，通过台盼蓝染色计数细胞数量。根据获得的细胞计数，分为四个等分3万到锥形管的细胞的细胞。沉淀细胞再次离心。 </li><li>之前开始锰标签，氯化锰溶于0.9％氯化钠溶液做一个0.1mm的氯化锰溶液新鲜。 </li><li>为了观察钙离子通道的活动，4个样品的准备。第一个范例，是我们所能控制的，其中包含0.9％的氯化钠单独细胞。第二个样本含有细胞在0.1毫米MnCl 2。样品3和4包含在0.1毫米MnCl 2或者5μM的Ca 2 +通道激动剂孵育细胞，（S )-(-)-湾K8644，或250μM的Ca 2 +通道阻滞剂，维拉帕米。 4个样品在37摄氏度5％CO 2 30分钟。 </li><li> 30分钟后，降速为5分钟800转的细胞在20摄氏度。然后吸出上清液，用PBS洗两次标记的细胞。洗涤后，重新挂起用200μLPBS和转移到PCR管，每个颗粒。这些颗粒通常为白色。 </li></ol>铁人类胚胎干细胞的标记<ol><li>临床级硫酸鱼精蛋白与蒸馏​​水混合，得到1毫克/毫升的原液。 </li><li>在一个包含人类胚胎干细胞培养基的试管中，加增加12微克/毫升硫酸鱼精蛋白在100微克/毫升ferumoxides。混合的解决方案，大力为五分钟。 </li><li>混合溶液中加入等量的细胞培养介质，实现了终浓度为50微克/毫升的ferumoxides和6微克/毫升，硫酸鱼精蛋白标签细胞。在这12小时的解决方案，孵育细胞。 </li><li>两次用PBS清洗细胞，与去年洗含10U/ml肝素解散外ferumoxides硫酸鱼精蛋白复合物。洗涤后，trypsinize细胞获得单细胞悬液。 </li><li>计数细胞和分装到所需数量的样品。洗涤后，重新挂起用200μLPBS和转移到PCR管，每个颗粒。这些颗粒应该是深橙色棕色。 </li></ol>执行蜂窝磁共振成像<ol><li>设为一个幽灵，以稳定管细胞沉淀和从周围的空气，以避免在扫描文物。要做到这一点，琼脂0.8％和1％硫酸铜的蒸馏水混合，煮沸5-7分钟微波。至少1个小时前的扫描达到凝胶冷却该混合物。 </li><li>含有标记的细胞沉淀的试管放置在幽灵进行扫描。在体外细胞MRI Signa的HDX 3T MRI（GE医疗系统），通过临床膝关节线圈。 </li><li>扫描为每个使用自旋回波序列的三个样品的锰标记细胞：TR;重复时间= 800毫秒。 TE的回波时间=最低，视野，视野= 12 × 12厘米;矩阵= 256 × 256，NEX 1。 </li><li>使用梯度回波序列扫描铁标记的细胞。我们使用这种成像以下参数：TR = 100毫秒，TE = 10毫秒，足总杯;翻转角= 30 °; FOV = 12 × 12厘米;矩阵= 256 × 256，NEX 1。 </li></ol> MRI结果分析<ol><li>从4不同样品的锰标记细胞分析MRI扫描的图像： <ul><li>示例＃1是我们所能控制的，其中包含在0.9％氯化钠单独细胞。 </li><li>样品＃2包含0.1毫米MnCl 2细胞。 </li><li>样品＃3包含在0.1毫米MnCl 2 5微米的Ca 2 +通道激动剂，（S )-(-)-湾K8644孵育细胞。 </li><li>样品＃4包含在0.1毫米MnCl 2培养细胞的Ca 2 +通道阻滞剂，维拉帕米与250微米。 </li></ul></li><li>据预测，从锰标记的细胞信号强度的差异是可见的。标记与锰纯粹的细胞相比，信号强度明显增加的钙离子通道激动剂的存在和在钙离子通道拮抗剂的存在减少。锰标记的细胞面积增加信号强度测量用Image J（美国国立卫生研究院，美国马里兰州贝塞斯达，）。相同的软件，用于分析暗铁标记细胞的共振信号领域。黑暗从铁标记的细胞信号的大小取决于细胞或细胞中的铁粒子的数目。在这种情况下，一万个单元格显示的暗信号相比，从三百万细胞的信号较小的区域。 </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>

这些结果表明，锰输入通过电压门控性钙通道，锰可作为MRI造影剂的使用，还可以显示细胞活力。因此，我们建议，锰制导MRI可有用的监视后，人类胚胎干细胞移植到心肌细胞活力。

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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>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

人类胚胎干细胞（胚胎干细胞）已经证明能够恢复受伤的心肌。磁共振成像（MRI）已成为主要的成像方法之一来评估受伤的心肌恢复。此外，前体内标签剂，如氧化铁纳米粒子，已被用于跟踪和本地化移植的干细胞。但是，这种方法不监视一个基本的细胞生物学特性，细胞移植的可行性。它已经知道，氯化锰（MnCl 2）进入细胞，通过电压门控钙（ 钙离子）当细胞生物活性的渠道，并积累细胞内产生T 1缩短的效果。因此，我们建议，锰制导MRI可有用的监视后的人类胚胎干细胞移植到心肌细胞的活力。 在这段视频中，我们将展示如何与MnCl 2和如何使用MRI体外，这些细胞可以清楚地看到标签的人类胚胎干细胞。与此同时，生物活性的Ca 2 +渠道将调制利用双方的Ca 2 +通道激动剂和拮抗剂，以评估随之而来的信号变化。

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

在这段视频中，我们展示了如何标注人类胚胎干细胞（胚胎干细胞）与氯化锰（MnCl<sub&gt; 2</sub&gt;）可以通过电压门控钙离子通道进入细胞，当细胞生物活性。此外，我们将展示使用MnCl<sub&gt; 2</sub&gt;蜂窝磁共振造影剂，以确定人类胚胎干细胞在体外的生存能力。

在人类胚胎干细胞体外培养的标签用于磁共振成像

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 次观看.  Stanford University. 在这段视频中，我们展示了如何标注人类胚胎干细胞（胚胎干细胞）与氯化锰（MnCl 2）可以通过电压门控钙离子通道进入细胞，当细胞生物活性。此外，我们将展示使用MnCl 2蜂窝磁共振造影剂，以确定人类胚胎干细胞在体外的生存能力。

视频: 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

人类胚胎干细胞（ES细胞）的繁殖

科研

生物学

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.

人类胚胎干细胞的推导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 ...

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.

与非侵入性检测荧光染料标记的干细胞与光学成像

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.

在人类胚胎干细胞的线粒体复杂的活动初探

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.

在体外和体内生物发光记者人类胚胎干细胞的基因显像

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.

从人类胚胎干细胞的染色质免疫沉淀

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.

文化和维护人类胚胎干细胞

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.

为核型分析人类胚胎干细胞的染色体蔓延制备

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.

人类胚胎干细胞冷冻和解冻

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.

视频人类胚胎干细胞克隆生长的生物信息学分析

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 ...