1. Maintenance: Observing Cultures Under the Microscope
 <ol>
 <li>Remove one plate of hES cells from the 37&deg;C incubator and bring to the microscope.</li>
 <li>Observe the colonies at low power, using 2X or 5X magnification, to assess the overall culture quality. Note the following: normal medium color, absence of any visible contamination, overall colony size, quality and density, and any other observations.</li>
 <li>Observe medium color changes. Due to the change in pH and the depletion of nutrients in the medium after an overnight incubation with the hES cells, the medium will change from a red hue to a "straw" color. If the medium appears purple, a basic pH shift has occurred. If the medium appears extremely yellow, the medium has acidified. Very yellow medium may be the result of extremely overcrowded colonies in the well or contamination. The medium should appear clear at all times. Though a certain level of cell debris in the medium is normal, it should never appear cloudy. If a high level of cell debris is observed, the culture is not healthy and may be contaminated.</li>
 <li>If the cultures do not require maintenance or passaging, they can be taken to the biological safety cabinet to be fed (see section 2).</li>
 <li>If the cultures contain more than approximately 10% differentiating colonies, the cells should be "cleaned up" by manually removing the differentiated areas (see section 3). </li>
 <li>If the cultures contain large or dense colonies, or the MEF feeder layer is more than 12 days old, the cells should be passaged (see section 4).</li>
 </ol>
2. Feeding hES Cells
 <ol>
 <li>In a sterile biological safety cabinet, remove the lid of the culture dish and aspirate the spent medium. Do not allow the tip of the pipette to touch any part of the plate other than directly inside the wells. If there is any concern of contamination, change to a new, sterile pipette.</li>
 <li>Using a sterile glass pipet, add the appropriate amount of warmed hES cell culture medium to each well. For cultures in a 6-well plate, add 2.5 mL medium per well.</li>
 <li>Return the plate to the incubator to remain at 37&deg;C and 5% CO2.</li>
 </ol>
3. Removing Differentiation from hES Cell Cultures
 <ol>
 <li>Transform 9 inch glass Pasteur pipettes into picking tools by molding the tips over the controlled flame of an alcohol burner. Be sure that the tip of the pipette is sealed to prevent contamination and rounded to avoid scratching the plastic of the culture dish. Sterilize the picking tools before use using the UV light source in the biological safety cabinet or picking enclosure.</li>
 <li>Remove the differentiated areas. Differentiation often occurs in only a portion of an hES cell colony, usually along the edges or as isolated spots in the center. If only a section of a colony is differentiating, only the differentiated area needs to be removed. Differentiation can often lift off the plate in a "sticky" sheet, and it is helpful to first separate the differentiated cells from the rest of the colony by drawing a line through the colony with the end of the picking tool.</li>
 <li>Once the pieces of the colony are separated, gently glide the picking tool along the plate to detach the unwanted cells. Take care not to scrape away too much of the MEF feeder layer between the colonies in this process.</li>
 <li>When all of the differentiated cells are removed from the plate (and floating in the medium), return the culture to the biological safety cabinet. Aspirate the medium containing the differentiated cells and replace with fresh hES cell culture medium.</li>
</ol>
4. Passaging hES Cells
Enzyme Incubation
 <ol>
 <li>In a sterile biological safety cabinet, remove the lid of the 6-well culture dish and aspirate the spent medium from the wells to be passaged. Do not allow the tip of the pipette to touch any part of the plate other than directly inside the wells. </li>
 <li>Using a sterile glass pipet, add 1 mL of warmed collagenase IV to each well to be passaged.</li>
 <li>Return the plate to the 37&deg; C incubator for 5 minutes.</li>
 </ol>
Scraping and Pooling the hES Cells
 <ol start="4">
 <li>After incubating the cells with collagenase, observe the colonies under the microscope. The enzyme should cause a subtle but observable change in the colony edges.</li>
 <li>In the biological safety cabinet, gently aspirate the enzyme from each well and replace with 2.0 mL hES cell culture medium.</li>
 <li>Tip the culture plate slightly toward you and take up the 2.0 mL medium in the first well with a 5 mL glass pipet. Holding the pipet perpendicular to the bottom of the plate, gently glide the pipet tip across the well while slowly releasing the medium.</li>
 <li>Repeat the scraping and pipetting motion 3-4 times until all of the colonies have been removed from the well. Pipet gently to avoid breaking up the colonies into too small of pieces. When the cells have been removed from the first well, leave the contents in the well and begin scraping the cells off of the next well.</li>
 <li>When all of the wells to be passaged have been scraped, pool the medium containing the colony pieces from each well in a sterile 15 mL conical tube.</li>
 <li>Rinse the scraped wells by adding 1 mL of hES cell culture medium to each well. Collect this rinse and transfer to the conical tube.</li>
 <li>Pipet the cells gently in the tube to break up the colony pieces up to the desired size.</li>
 <li>Centrifuge the cells for 5 minutes at 200 x g.</li>
 </ol>
Plating the hES Cell Cells
 <ol start="12">
 <li>While the hES cells are in the centrifuge, prepare a new 6-well MEF feeder plate. Label the plate with the appropriate hES cell information: cell line, new passage number, and passage date. Aspirate the MEF medium from the wells and add 1.0 mL PBS to each well. Gently swirl the buffer around the wells and aspirate the PBS wash. Add 1.5 mL hES cell culture medium to each well and set the plate aside.</li>
 <li>When the hES cells are finished centrifuging, bring the tube back to the biological safety cabinet. Aspirate the supernatant, being careful not to disturb the loosely-packed cell pellet.</li>
 <li>Resuspend the cells in the pellet with enough medium for 1 mL hES cell culture medium per well to be plated. When splitting into 6 new wells, resuspend the pellet in 6 mL medium.</li>
 <li>Gently and evenly add 1 mL of the cell suspension to each prepared well of the MEF feeder plate.</li>
 <li>Place the plate into the 37&deg;C incubator and carefully slide the plate forward to back, and side to side to evenly distribute the cells throughout the well.</li>
 <li>Allow the cells to attach at 37&deg;C overnight.</li>
 </ol>
5. Representative Results:
Undifferentiated hES cells are small, tightly-packed, and usually consist of larger, more spread out cells. Differentiation can occur within a colony (Figure 1A) or between colonies (Figure 3B).
Different morhopologies can be seen under low magnification under the microscope. A good cell morphology, as seen in Figure 2A, contains small, tightly packed cells that grow in a monolayer. Cells should have clean, defined edges, with little to no differentiation. The cells shown in Figure 2B are ready for passaging, and the cells shown in the Figure 2C are overcrowded.
<img src="/files/ftp_upload/1427/1427fig1.jpg" alt="Figure 1" /> Figure 1. Differentiation in hES cultures. (A) differentiation of a colony (B)differentiation between colonies.
<img src="/files/ftp_upload/1427/1427fig2.jpg" alt="Figure 2" /> Figure 2. Morphologies seen in hES cultures. (A)good cell morphology (B) hES cells that are ready for passaging (C) overcrowded cells.

<ol>
	<li>Freshney, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Culture of Animal Cells: A Manual of Basic Technique. , (2005).</li></ol>

Sterility must be maintained at all times when working with hES cells. Clean and sterilize the biological safety cabinet and all equipment before use. All reagents must be filtered using a 0.22 &#956;m pore size filter prior to use. The use of antibiotics in hES cell culture is not necessary and should be avoided.
A separate environment should be set up as a designated picking station. The sterile enclosure for the station can be a static enclosure (such as a PCR workstation) with a UV light source, a laminar flow hood, or a biological safety cabinet. A dissecting microscope inside the picking station is needed to observe the colonies as the differentiated areas are removed. The front glass panel of a static enclosure or a biological safety cabinet must be modified to allow for the oculars of the microscope to extend through the panel without compromising proper airflow and sterility.
To maintain healthy hES cell cultures, cells must be passaged at the optimal time, typically every 4-7 days. At this time the colonies have reached their maximum size and may be beginning to merge. Merged colonies can increase the rate of differentiation in the culture. Split ratios generally fall between 1:3 and 1:5, depending on the number of colonies plated, expansion rate, and culture conditions. Overplated colonies (split ratio too low) will likely merge prematurely and need to be passaged before they reach their maximum size.
Cultures on an MEF feeder layer that is more than 2 weeks old must be passaged on to new MEFs that will support undifferentiated growth and proliferation.
Since differentiation naturally occurs in hES cell cultures, a small amount is expected. Frequent or excessive differentiation may occur if the cultures do not receive appropriate care. The cells must be fed every day, overgrowth between passages should be avoided. Always use fresh reagents. All of the media, MEFs and reagents should be used within 14 days to avoid undifferentiated hES cell growth. New lots of reagents, such as Knockout Serum Replacement, FBS and MEFs, should be screened prior to use. Remember that any differentiated cells not removed prior to passaging will be transferred to the new cultures. Also, try to keep the cells at 37&#176;C whenever possible. Minimize the time the cells spend outside of the incubator (e.g. on the microscope stage or in the biological safety cabinet.) A heated microscope stage can be very beneficial if the cells will be of the incubator for extended periods of time.
When observing the hES cell cultures under the microscope, first assess the overall quality and size of the colonies view using a low power (2x or 5x) objective. If potentially differentiated cells are observed at low power, confirm the differentiated morphology using a higher magnification objective (10X).
Immediately after removing differentiation, the edges of the colonies may appear jagged or damaged. Incubate the colonies overnight in fresh medium to allow the remaining undifferentiated cells to recover. Without the influence of differentiation in the culture, these remaining colonies will continue to proliferate normally.
If possible, begin experiments using low-passage hES cells. Karyotype the cells before and after all major experiments.

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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>D-MEM</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11965-092</td>
<td>For MEF medium</td>
</tr>
<tr>
<td>Fetal Bovine Serum (FBS), Certified</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>16000-044</td>
<td>Heat-inactivated 
For MEF medium</td>
</tr>
<tr>
<td>D-MEM/F-12</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11330-057</td>
<td>&nbsp;</td>
<tr>
<td>Knockout&trade; Serum Replacement</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>10828-028</td>
<td>Pre-screen to ensure support of hES cells</td>
</tr>
<tr>
<td>bFGF</td>
<td>&nbsp;</td>
<td>Stemgent</td>
<td>03-0002</td>
<td>Use at [4 ng/mL] final</td>
</tr>
<tr>
<td>Non-essential Amino Acids</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11140-050</td>
<td>&nbsp;</td>
<tr>
<td>L-glutamine</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>25030-081</td>
<td>200 mM (100X) 
Use at [1X] final
</td>
</tr>
<tr>
<td>PBS</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>14190-250</td>
<td>Without Ca2+ or Mg2+</td>
</tr>
<tr>
<td>Collagenase IV</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>17104-019</td>
<td>Make a fresh solution at 1 mg/mL in D-MEM/F-12</td>
</tr>
<tr>
<td>Gelatin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G1890</td>
<td>Type A, Porcine</td>
</tr>
<tr>
<td>Fetal Bovine Serum (FBS), Defined</td>
<td>&nbsp;</td>
<td>HyClone</td>
<td>SH300.70.01</td>
<td>For freezing medium</td>
</tr>
<tr>
<td>6-well plates</td>
<td>&nbsp;</td>
<td>Nunc</td>
<td>140675</td>
<td>For general culture</td>
</tr>
<tr>
<td>4-well plates</td>
<td>&nbsp;</td>
<td>Nunc</td>
<td>176740</td>
<td>For thawing</td>
</tr>
<tr>
<td>5 mL glass pipets</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>13-678-27E</td>
<td>Individually wrapped</td>
</tr>
<tr>
<td>15 mL conical tubes</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>430052</td>
<td>Polypropylene</td>
</tr>
<tr>
<td>Pasteur pipettes</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>13-678-20D</td>
<td>9&#x201D; glass</td>
</tr>		</tbody>
		</table>
		</div>

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.

Watch this Scientific Journal Video about Culture and Maintenance of Human Embryonic Stem Cells at JoVE.com

Culture and Maintenance of Human Embryonic Stem Cells

This protocol demonstrates how to maintain healthy, undifferentiated human embryonic stem (ES) cells.

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 ...

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47.6K Views.  Stemgent. Questo protocollo dimostra come mantenere sani, indifferenziate staminali embrionali umane (ES) cellule.

Video: Culture and Maintenance of Human Embryonic Stem Cells

Propagation of Human Embryonic Stem (ES) Cells

Propagation of Human Embryonic Stem ES Cells

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.

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.

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 ...

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.

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

Chromatin Immunoprecipitation from Human Embryonic Stem Cells

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are characterized by two fundamental properties: self-renewal and multipotency that allows a stem cell to differentiate into virtually any cell type 1. The progression stem cell to differentiated cell is characterized by loss of multipotency, structural and morphological changes and the hierarchic activity of transcription factors and signaling molecules, whose activities establish and maintain cell-type specific gene expression patterns. At the molecular level, cell differentiation involves dynamic changes of the structure and composition of chromatin and the detection of those dynamic changes can provide valuable insights into the functional features of stem cells and the cell differentiation process 2,3. Chromatin is a highly compacted DNA-protein complex that forms when cells package chromosomal DNA with proteins, mainly histones 4. Stemcellness and cell differentiation has been correlated with the presence of specific arrays of regulatory proteins such as epigenetic factors, histone variants, and transcription factors 2,3,5.
 Chromatin immunoprecipitation (ChIP) provides a valuable method to monitor the presence of RNA, proteins, and protein modifications in chromatin 6,7. The comparison of chromatin from different cell types can elucidate dynamic changes in protein-chromatin associations that occur during cell differentiation.
Chromatin immunoprecipitation involves the purification of in vivo cross-linked chromatin. The isolated chromatin is reduced to smaller fragments by enzymatic digestion or mechanical force. Chromatin fragments are precipitated using specific antibodies to target proteins or protein and DNA modifications. The precipitated DNA or RNA is purified and used as a template for PCR or DNA microarray based assays. Prerequisites for a successful ChIP are high quality antibodies to the desired antigen and the availability of chromatin from control cells that do not express the target molecule. ChIP can correlate the presence of proteins, protein and RNA modifications, and RNA with specific target DNA, and depending on the choice of outread tool, detects the association of target molecules at specific target genes or in the context of an entire genome. The comparison of the distribution of proteins in the chromatin of differentiating cells can elucidate the dynamic changes of chromatin composition that coincide with the progression of cells along a cell lineage.

The differentiation of ESC coincides with cell-type specific changes in the structure and composition of chromatin. The detection of those changes provides valuable insights into the mechanisms that define stemcellness and cell differentiation. Chromatin immunoprecipitation (ChIP) represents a valuable method to dissect the molecular mechanisms underlying stem cell differentiation.

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are ...

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.

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.

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.

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

Fate Mapping of Human Embryonic Stem Cells by Teratoma Formation

Human embryonic stem cells (hESCs) have an unlimited capacity for self-renewal, and the ability to differentiate into cells derived from all three embryonic germ layers (1). Directed differentiation of hESCs into specific cell types has generated much interest in the field of regenerative medicine (e.g., (2-5)), and methods for determining the in vivo fate of selected or manipulated hESCs are essential to this endeavor. We have adapted a highly efficient teratoma formation assay for this purpose. A small number of specifically selected hESCs is mixed with undifferentiated wild type hESCs and Phaseolus vulgaris lectin to form a cell pellet. This is grafted beneath the kidney capsule in an immunodeficient mouse. As few as 2.5 x 105 hESCs are needed to form a 16 cm3 teratoma within 8-12 weeks. The fate of the originally selected hESCs can then be determined by immunohistochemistry. This method provides a valuable tool for characterizing tissue-specific reagents for cell-based therapy.

Directed differentiation of hESCs into specific cells has generated much interest in regenerative medicine. We provide a concise, step-by-step protocol for determining the in vivo fate of selected hESCs that provides a valuable tool for characterizing tissue-specific reagents for cell-based therapy.

Human embryonic stem cells (hESCs) have an unlimited capacity for self-renewal, and the ability to differentiate into cells derived from all three ...

Adult and Embryonic Skeletal Muscle Microexplant Culture and Isolation of Skeletal Muscle Stem Cells

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a robust and reliable method for isolating relatively large numbers of proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. The authors have used micro-dissected explant cultures to analyse the growth characteristics of skeletal muscle cells in wild-type and dystrophic muscles. Each of the components of tissue growth, namely cell survival, proliferation, senescence and differentiation can be analysed separately using the methods described here. The net effect of all components of growth can be established by means of measuring explant outgrowth rates. The micro-explant method can be used to establish primary cultures from a wide range of different muscle types and ages and, as described here, has been adapted by the authors to enable the isolation of embryonic skeletal muscle precursors.
Uniquely, micro-explant cultures have been used to derive clonal (single cell origin) skeletal muscle stem cell (SMSc) lines which can be expanded and used for in vivo transplantation. In vivo transplanted SMSc behave as functional, tissue-specific, satellite cells which contribute to skeletal muscle fibre regeneration but which are also retained (in the satellite cell niche) as a small pool of undifferentiated stem cells which can be re-isolated into culture using the micro-explant method.

The micro-dissected explants technique is a robust and reliable method for isolating proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. Uniquely, these cells have been clonally derived to produce skeletal muscle stem cell lines used for in vivo transplantation.

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a ...