Name: Video: Video Bioinformatics Analysis of Human Embryonic Stem Cell Colony Growth
Uploaded: 2010-05-20T17:00:00
Duration: 13 min 11 s

הפיתוח של שיטה זו התאפשרה הודות למימון של תוכנית הקשורה טבק קליפורניה מחקר מחלות, המכון קליפורניה עבור רפואה רגנרטיבית, ו-NSF IGERT מענק על וידאו ביואינפורמטיקה (# 0903667) ב UCR ( <a href="http://www.cris.ucr.edu/IGERT/index.php">http://www.cris.ucr .edu / IGERT / index.php</a> ). סברינה לין נתמך על ידי עמיתי דיסרטציה מאגף בוגר, שון Forteno נתמך על ידי מרק NIH המלגה, ואת Shruthi סאטיש נתמך על ידי מלגת בוגר החטיבה. אנו מודים אנה Trtchounian על עזרתה בהכנת הדמויות. אנו מודים גם סם Alworth ונד Jastromb עבור מלמד אותנו איך להשתמש בתוכנה CL-קוואנט.

וידאו כלי ביואינפורמטיקה חזקים עבור במהירות לחילוץ נתונים תמונות וידאו. פרוטוקול שלנו לכימות המושבה צמיחה hESC מדגים יישום אחד של וידאו ביואינפורמטיקה לבעיה ביולוגית. שיטה זו היא כמותית יש תכונה מעניינת של הנתונים מגלה ממושבות hESC בודדים. וידאו מתכונים ביואינפורמטיקה ניתן לפתח כדי לפקח על תהליכים תאיים אחרים כגון, אפופטוזיס התפשטות, הגירה מצורף התא למצע, וכן מצורף תא לתאים סמוכים. הדיוק של המתכונים פותח באמצעות תוכנת CL-קוואנט היה תוקף באמצעות Photoshop ו נמצאה אמת. לאחר מתכונים המתאימים מפותחים, נתונים ווידאו מכל מקור ניתן לנתח מהר מאוד. את הזמן הדרוש כדי לנתח את הנתונים וידאו באמצעות וידאו ביואינפורמטיקה כלים קטנה משמעותית מזו של הזמן הדרוש לניתוח שבוצע על ידי יד באמצעות Photoshop. יתר על כן, הניתוח נעשה על ידי המחשב הוא נוטה פחות שגיאות, כמו המחשב יהיה לנתח את הנתונים באותה דרך בכל פעם, ואילו אדם ביצוע ניתוח עלול לגרום לטעויות או פסקי דין שונה במקצת בכל פעם תמונה מנותח. למרות שלא נדון במסגרת פרוטוקול זה, קטעי וידאו ניתן גם לבחון שינויים מורפולוגיים במושבה. פרמטר זה יהיה שימושי במקרים בהם קבוצות הטיפול כלולות.

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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>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>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

video bioinformatics analysis of human embryonic stem cell colony growth

מאחר שהנתונים וידאו מורכבים מורכבות תמונות רבות, כריית מידע מתוך חומר וידאו קשה לעשות ללא סיוע של תוכנות מחשב. וידאו ביואינפורמטיקה היא גישה כמותית עוצמה לחילוץ במרחב ובזמן נתונים תמונות וידאו באמצעות תוכנת מחשב כדי לבצע כריית היכרויות וניתוח. במאמר זה, אנו מציגים שיטה וידאו ביואינפורמטיקה לכימות את הצמיחה של בתאי גזע עובריים אנושיים (hESC) על ידי ניתוח זמן לשגות קטעי וידאו שנאספו BioStation Nikon CT חממה מצויד במצלמת וידאו עבור הדמיה. בניסויים שלנו, מושבות hESC כי צורפו Matrigel צולמו במשך 48 שעות ב-CT BioStation. כדי לקבוע את קצב הגידול של מושבות אלו, המתכונים פותחו באמצעות CL-קוואנט תוכנה אשר מאפשרת למשתמשים להפיק סוגים שונים של נתוני תמונות וידאו. על מנת להעריך במדויק את הצמיחה המושבה, שלושה מתכונים שנוצרו. התמונה הראשונה מפולח לתוך המושבה ואת הרקע, התמונה השנייה משופרת להגדיר מושבות לאורך רצף וידאו במדויק, והשלישי מדדו את מספר הפיקסלים במושבה לאורך זמן. שלושת המתכונים נוהלו ברצף על נתונים שנאספו וידאו CT BioStation לנתח את קצב הגידול של מושבות hESC בודדים מעל 48 שעות. כדי לוודא את אמיתותם של CL-קוואנט מתכונים, נתונים באותו נותחו באופן ידני באמצעות תוכנת Adobe Photoshop. כאשר הנתונים שהתקבלו באמצעות CL-קוואנט מתכונים Photoshop הושוו, התוצאות היו כמעט זהים, המציין את CL-קוואנט המתכונים היו אמת. השיטה המתוארת כאן יכול להיות מיושם על כל הנתונים וידאו למדוד את שיעורי הצמיחה של hESC או תאים אחרים הגדלים במושבות. בנוסף, אחרים וידאו מתכונים ביואינפורמטיקה ניתן לפתח בעתיד תהליכים תאים אחרים כמו הגירה, אפופטוזיס, וכן הידבקות התא.

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

וידאו ביואינפורמטיקה הוא עיבוד וניתוח אוטומטי, הבנה, כריית נתונים של במרחב ובזמן נתונים ביולוגי המופק וידאו מיקרוסקופיים. מטרת מאמר זה היא להדגים שיטה למדידת תא גזע עובריים אנושיים צמיחה המושבה בשיטה ביואינפורמטיקה וידאו.

וידאו ביואינפורמטיקה ניתוח גדילה אנושי בתאי גזע עובריים קולוני

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 ...

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12.7K Views.  University of California. וידאו ביואינפורמטיקה הוא עיבוד וניתוח אוטומטי, הבנה, כריית נתונים של במרחב ובזמן נתונים ביולוגי המופק וידאו מיקרוסקופיים. מטרת מאמר זה היא להדגים שיטה למדידת תא גזע עובריים אנושיים צמיחה המושבה בשיטה ביואינפורמטיקה וידאו.

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.

גוונים Passaging האדם עובריים לתאי גזע קווי עם טריפסין

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

רביה של האדם גזע עובריים (ES) תאים

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.

גזירת בתאי גזע hematopoietic מ Murine בתאי גזע עובריים

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.

גזירת בתאי גזע עובריים אנושיים על ידי 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.

הכרומטין immunoprecipitation מן בתאי גזע עובריים אנושיים

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.

תרבות ותחזוקה של בתאי גזע עובריים אנושיים

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.

בידוד הרחבת בקנה מידה גדול של תאים אנושיים למבוגרים האנדותל המושבה יצירת אב

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.

בידוד בעלי חיים הרחבת חינם סרום של טבורי האדם נגזר תאים סטרומה mesenchymal (MSCs) ו האנדותל יצירת המושבה ובתאים (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.

מקפיא הפשרה בתאי גזע עובריים אנושיים

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.

התמיינות של תאים עובריים לתוך מבשרי Oligodendrocyte

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