Name: Video: Video Bioinformatics Analysis of Human Embryonic Stem Cell Colony Growth
Uploaded: 2010-05-20T17:00:00

تم تطوير هذا الأسلوب ممكنا بفضل تمويل من كاليفورنيا ذات الصلة بالتبغ برنامج بحوث الأمراض ، ومعهد كاليفورنيا للطب التجديدي ، وجبهة الخلاص الوطني منحة IGERT المعلوماتية الحيوية على الفيديو (# 0903667) في UCR ( <a href="http://www.cris.ucr.edu/IGERT/index.php">http://www.cris.ucr .edu / IGERT / أنشئ حسابا</a> ). معتمد من قبل سابرينا لين زمالة أطروحة من شعبة الدراسات العليا ، ويدعم من قبل شون Forteno زمالة مارك المعاهد الوطنية للصحة ، وذلك بدعم من قبل ساتيش Shruthi زمالة قسم الدراسات العليا. ونحن ممتنون لTrtchounian آنا لمساعدتها على إعداد الأرقام. ونحن نشكر أيضا وسام Alworth Jastromb نيد ليعلمنا كيفية استخدام البرنامج CL - كوانت.

الفيديو أدوات المعلوماتية الحيوية هي قوية لاستخراج البيانات بسرعة من صور الفيديو. بروتوكول لدينا لقياس النمو hESC مستعمرة يوضح تطبيق واحد من الفيديو المعلوماتية الحيوية لمشكلة بيولوجية. هذا الأسلوب الكمي ويحتوي على ميزة مثيرة للاهتمام لكشف البيانات من المستعمرات hESC...

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		<thead>
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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
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<tr>
<td>mTeSR1 Human Embryonic Stem Cell Maintenance Medium</td>
<td>&nbsp;</td>
<td>Stem Cell Technologies</td>
<td>05850</td>
<td>or any suitable medium for hESC culture.</td>
</tr>
<tr>
<td>BD Matrigel</td>
<td>&nbsp;</td>
<td>BD Bioscience</td>
<td>356234</td>
<td>or other suitable substrate.</td>
</tr>
<tr>
<td>DMEM/F12 Basal Medium</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11330-032</td>
<td>&nbsp;</td>
<tr>
<td>Phosphate Buffered Saline without Ca2+ and Mg2+</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Accutase Enzyme Cell Detachment Medium</td>
<td>&nbsp;</td>
<td>eBioscience</td>
<td>00-4555-56</td>
<td>or other suitable detachment enzyme.</td>
</tr>
<tr>
<td>3mm Glass beads</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>11-312A</td>
<td>optional.</td>
</tr>
<tr>
<td>12-well Tissue Culture Plates</td>
<td>&nbsp;</td>
<td>BD Falcon</td>
<td>353043</td>
<td>or any other plate format.</td>
</tr>
<tr>
<td>Nikon BioStation CT/IM</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>or other incubator/microscope suitable for collecting video data.</td>
</tr>
<tr>
<td>CL-Quant software (Nikon) and/or Photoshop (Adobe).</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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video bioinformatics analysis of human embryonic stem cell colony growth

لأن بيانات الفيديو معقدة وتتكون من العديد من الصور والمعلومات من مواد التعدين الفيديو هو من الصعب القيام به دون مساعدة من برامج الكمبيوتر. الفيديو المعلوماتية الحيوية هي طريقة فعالة لاستخراج الكمية المكانية والزمانية البيانات من صور الفيديو باستخدام برامج الحاسوب لتنفيذ التعدين وتاريخها والتحليل. في هذه المقالة ، ونحن نقدم طريقة الفيديو المعلوماتية الحيوية لقياس نمو الخلايا الجذعية الجنينية البشرية (hESC) عن طريق تحليل الوقت الفاصل بين أشرطة الفيديو التي تم جمعها في حاضنة نيكون CT BioStation مزودة كاميرا لتصوير الفيديو. في تجاربنا ، تم تصويره hESC المستعمرات التي كانت تعلق على Matrigel لمدة 48 ساعة في BioStation CT. لتحديد معدل نمو هذه المستعمرات ، وضعت وصفات باستخدام CL - كوانت البرمجيات التي تمكن المستخدمين من استخراج أنواع مختلفة من البيانات من صور الفيديو. لتقييم دقيق نمو مستعمرة ، أنشئت ثلاث وصفات. قياس أول صورة مجزأة الى مستعمرة والخلفية ، والثاني تعزيز صورة لتعريف المستعمرات في جميع أنحاء تسلسل الفيديو بدقة ، وثالثة لعدد البكسل في المستعمرة مع مرور الوقت. تم تشغيل وصفات ثلاثة في تسلسل على بيانات الفيديو التي تم جمعها في CT BioStation لتحليل معدل نمو المستعمرات hESC الفردية أكثر من 48 ساعة. للتحقق من صحة الوصفات CL - كوانت ، تم تحليل البيانات يدويا باستخدام نفس برنامج أدوبي فوتوشوب. وكانت النتائج التي تم الحصول عليها عندما تكون البيانات وصفات باستخدام CL - كوانت وقورنت فوتوشوب ، متطابقة تقريبا ، مما يدل على وصفات CL - كوانت كانوا صادقين. ويمكن تطبيق هذا الأسلوب هو موضح هنا إلى أية بيانات الفيديو لقياس معدلات نمو hESC أو الخلايا الأخرى التي تنمو في المستعمرات. بالإضافة ، يمكن وضع وصفات أخرى الفيديو المعلوماتية الحيوية في المستقبل لعمليات خلية أخرى مثل الهجرة ، وموت الخلايا المبرمج ، والتصاق الخلية.

Watch this Scientific Journal Video about Video Bioinformatics Analysis of Human Embryonic Stem Cell Colony Growth at JoVE.com

Video Bioinformatics Analysis of Human Embryonic Stem Cell Colony Growth

الفيديو المعلوماتية الحيوية هو التجهيز الآلي ، والتحليل ، والفهم ، واستخراج البيانات من البيانات المكانية والزمانية لالبيولوجي المستخرج من أشرطة الفيديو المجهرية. الغرض من هذه المقالة هو لشرح طريقة لقياس الخلايا الجذعية الجنينية البشرية نمو مستعمرة المعلوماتية الحيوية باستخدام طريقة الفيديو.

الفيديو المعلوماتية الحيوية تحليل النمو البشري الخلية الجذعية الجنينية كولوني

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 ...

video-bioinformatics-analysis-human-embryonic-stem-cell-colony

Research

JoVE Journal

Biology

12.7K Views.  University of California. الفيديو المعلوماتية الحيوية هو التجهيز الآلي ، والتحليل ، والفهم ، واستخراج البيانات من البيانات المكانية والزمانية لالبيولوجي المستخرج من أشرطة الفيديو المجهرية. الغرض من هذه المقالة هو لشرح طريقة لقياس الخلايا الجذعية الجنينية البشرية نمو مستعمرة المعلوما...

Video: Video Bioinformatics Analysis of Human Embryonic Stem Cell Colony Growth

Passaging HuES Human Embryonic Stem Cell-lines with Trypsin.

In this video we demonstrate how our lab routinely passages HuES human embryonic stem cell lines with trypsin.  Human embryonic stem cells are artifacts of cell culture, and tend to acquire karyotypic abnormalities with high population doublings.  Proper passaging is essential for maintaining a healthy, undifferentiated, karyotypically normal HuES human embryonic stem cell culture.  First, an expanding culture is washed in PBS to remove residual media and cell debris, then cells are overlaid with a minimal volume of warm 0.05% Trypsin-EDTA.  Trypsin is left on the cells for up to five minutes, then cells are gently dislodged with a 2mL serological pipette.  The cell suspension is collected and mixed with a large volume of HuES media, then cells are collected by gentle centrifugation.  The inactivated trypsin media mixture is removed, and cells resuspended in pre-warmed HuES media.  An appropriate split ratio is calculated (generally 1:10 to 1:20), and cells re-plated onto a 1-2 day old plate containing a monolayer of irradiated mouse embryonic fibroblast feeder cells.  The newly seeded HuES culture plate is left undisturbed for 48 hrs, then media is changed every day thereafter.  It is important not to trpsinize down to a single cell suspension, as this increases the risk of introducing karyotypic abnormalities.

In this video we demonstrate how our lab routinely passages HuES human embryonic stem cell lines with trypsin. Brought to you by JoVE.

الأشكال الركض الجذعية الجنينية البشرية خلية خطوط مع التربسين

In this video we demonstrate how our lab routinely passages HuES human embryonic stem cell lines with trypsin.  Human embryonic stem cells are artifacts ...

Propagation of Human Embryonic Stem (ES) Cells

Propagation of Human Embryonic Stem ES Cells

انتشار الخلايا الجنينية البشرية (ES) الجذعية

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.

اشتقاق خلايا الجذعية المكونة للدم من الفئران خلايا الجذعية الجنينية

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.

اشتقاق الخلايا الجذعية الجنينية البشرية التي 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 ...

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.

مناعي لونين من الخلايا الجذعية الجنينية البشرية

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.

الثقافة والمحافظة على الخلايا الجذعية الجنينية البشرية

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

Isolation and Large Scale Expansion of Adult Human Endothelial Colony Forming Progenitor Cells

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult human peripheral blood within a mean of 12 days. After large scale expansion &gt;1x108 ECFCs can be obtained for further tests. Advantageously by using pHPL the contact of human cells with bovine serum antigens can be excluded. By flow cytometry and immunohistochemistry the isolated cells can be characterized as ECFC and their in vitro functionality to form vascular like structures can be tested in a matrigel assay. Further these cells can be subcutaneously injected in a mouse model to form functional, perfused vessels in vivo. After long term expansion and cryopreservation proliferation, function and genomic stability appear to be preserved. 3,4
This animal-protein free isolation and expansion method is easily applicable to generate a large quantity of ECFCs.

Endothelial colony forming progenitor cells (ECFCs) are a promising tool to study vascular homeostasis and repair.1,2 This paper introduces a novel animal-serum free method for isolation and expansion of ECFC from heparinised adult human peripheral blood with pooled human platelet lysate (pHPL) diminishing the risk of anti-bovine immunisation.

العزلة وتوقعات بنمو كبير من الكبار الإنسان خلايا غشائي السلف تشكيل مستعمرة

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult ...

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells (MSCs) and Endothelial Colony Forming Progenitor Cells (ECFCs)

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal stem cells, MSCs) and endothelial colony forming progenitor cells (ECFCs). Both cell types are key players in maintaining the integrity of tissue and are probably also involved in regenerative processes and tumor formation.
To study their biology and function in a comparative manner it is important to have both cells types available from the same donor. It may also be beneficial for regenerative purposes to derive MSCs and ECFCs from the same tissue.
Because cellular therapeutics should eventually find their way from bench to bedside we established a new method to isolate and further expand progenitor cells without the use of animal protein. Pooled human platelet lysate (pHPL) replaced fetal bovine serum in all steps of our protocol to completely avoid contact of the cells to xenogeneic proteins.
This video demonstrates a methodology for the isolation and expansion of progenitor cells from one umbilical cord.
All materials and procedures will be described.

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells MSCs and Endothelial Colony Forming Progenitor Cells ECFCs

This protocol describes the isolation and subsequent expansion of mesenchymal stromal cells and endothelial colony forming cells without the use of animal serum to generate autologous pairs for experimental transplantation purposes.

العزلة والحيوان التوسع الحرة المصل الحبل السري الإنسان المستمدة الخلايا اللحمية الوسيطة (MSCS) وغشائي خلايا مستعمرة السلف التشكيل (ECFCs)

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal ...

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.

تجميد والذوبان الخلايا الجذعية الجنينية البشرية

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.

تمايز الخلايا الجذعية الجنينية في السلائف Oligodendrocyte

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