Name: the use of sc1 (pluripotin) to support mesc self-renewal in the absence of lif
Uploaded: 2009-11-18T17:30:00
Duration: 5 min 58 s
Description: Watch this Scientific Journal Video about The use of SC1 Pluripotin to Support mESC Self-renewal in the Absence of LIF at JoVE.com

The authors wish to thank Dr. Sheng Ding of The Scripps Research Institute and Dr. Hongkui Deng of Peking University for their assistance and direction.

1. Preparation of MEF feeder plates
<ol>
<li>One day before culturing ES cells, thaw one vial of irradiated E14.5 CF-1 MEF feeder cells (see our procedure on how to thaw ES cells).</li>
<li>Plate the MEF feeder cells at a density of 10,000 cells/well in a 12 well plate. Incubate the cells overnight at 37&deg;C and 5% CO2. MEF cell media: DMEM supplemented with 10% ES-PBS, 1X glutamine and 1X non-essential amino acids.</li>
</ol>
2. Maintenance of Self-Renewal of Mouse ES Cells
<ol>
<li>Detach the ES cells using trypsin. Seed the cells at a density of 50,000 cells/well on the previously prepared MEF feeder plates in growth media (Knockout MEM supplemented with 15% KSR, 4 mM L-glutamine, 0.1 mM non-essential amino acids and 1X BME) supplemented with SC1 at the desired concentration (we used 100 nM, 300 nM, and 1 &mu;M). We also added a positive control well that was supplemented with 1000 &mu;/ml of LIF. Incubate the cells overnight at 37&deg;C and 5% CO2.</li>
<li>Culture the cells at 37&deg;C and 5% CO2 for 3-4 days in each passage. Refresh media every 48 hours.</li>
<li>Passage cells 1:10 to 1:15 with trypsin to maintain a similar density as the initial seeding density. After 5 passages, the cells can be examined for the expression of typical pluripotency markers using immunocytochemistry.</li>
</ol>
3. Immunocytochemical Examination of Pluripotency Markers
<ol>
<li>Wash the cells gently three times with PBS.</li>
<li>Fix the cells with 500 &mu;l of fixative for 20 minutes at room temperature.</li>
<li>Wash the cells gently three times with PBS.</li>
<li>Block non-specific binding with 500 &mu;l blocking buffer for one hour at room temperature.</li>
<li>Incubate the cells with 250 &mu;l of the specific primary antibody (we used Nanog, Sox2, SSEA1, and Oct4 at the following dilutions: 1:500, 1:2000, 1:500, and 1:500)</li>
<li>Wash the cells gently three times with PBS.</li>
<li>Incubate the cells with 250 &mu;l of secondary antibody for two hours at room temperature, keeping away from light (we used donkey anti-mouse conjugated with Alexa Fluor&reg; 555 at 1:2000 and donkey anti-rabbit conjugated with Alexa Fluor&reg; 488 at 1:2000).</li>
<li>Wash the cells gently three times with PBS.</li>
<li>Add DAPI (final concentration 1 &mu;g/ml) to the last wash and incubate 5 minutes to visualize nuclei.</li>
<li>Analyze the cells under a fluorescent microscope.</li>
</ol>
4. Representative Results:
1. Morphology results:
Mouse ES cells (ESC R1) were grown on MEF feeder cells in the presence of either LIF or SC1 to sustain self-renewal at an undifferentiated state. After the second passage, differentiation could be observed in cells without LIF or SC1 (negative control) and after 5 passages essentially no undifferentiated colonies were observed. LIF (positive control) treated colonies remained in an undifferentiated state throughout the 5 cell passages. Cells treated with SC1 at concentrations of 300 nM or 100 nM also remained undifferentiated after 5 passages, however, the cells treated with 1 &mu;M SC1 experienced cell death after the fourth passage. (fig 2 of app note) Table 1 summarizes the morphological observations.
2. Expression of Pluripotency Markers
Mouse ES cells maintained for 5 passages were fixed and examined by immunocytochemistry for SSEA1, Oct4, Nanog and Sox2. Marker expression was similar to cells maintained in LIF. (fig 3 and 4 of app note)
<img src="/files/ftp_upload/1550/1550fig2a.jpg" alt="Figure 1" /> Figure 1: Morphology comparison of cells maintained in LIF or SC1. No difference in morphology was observed.
<img src="/files/ftp_upload/1550/1550fig2c.jpg" alt="Figure 2 and 3" /> Figure 2 and Figure 3: Nanog, SSEA1, Sox2, and Oct4 staining of ES cells maintained with SC1 or LIF (fig 4 of app note). Cells maintained in SC1 show similar pluripotency marker expression to cells maintained in LIF.
Table 1
<table border="1" cellspacing="0" cellpadding="0">
<tbody>
 <tr>
 <td>&nbsp;</td>
 <td>Negative Control</td>
 <td>LIF Positive Control</td>
 <td>SC1 1uM</td>
 <td>SC1 300 nM</td>
 <td>SC1 100 nM</td>
 </tr>
 <tr>
 <td>1st Passage</td>
 <td>Normal ES cell morphology</td>
 <td>Normal ES cell morphology</td>
 <td>Normal ES cell morphology</td>
 <td>Normal ES cell morphology</td>
 <td>Normal ES cell morphology</td>
 </tr>
 <tr>
 <td>2nd Passage</td>
 <td>Some differentiated morphology</td>
 <td>Normal ES cell morphology</td>
 <td>Normal ES cell morphology</td>
 <td>Normal ES cell morphology</td>
 <td>Normal ES cell morphology</td>
 </tr>
 <tr>
 <td>3rd Passage</td>
 <td>Majority of cells differentiated</td>
 <td>Normal ES cell morphology</td>
 <td>Normal ES cell morphology</td>
 <td>Normal ES cell morphology</td>
 <td>Normal ES cell morphology</td>
 </tr>
 <tr>
 <td>4th Passage</td>
 <td>No ESC colonies</td>
 <td>Normal ES cell morphology</td>
 <td>Cell death observed</td>
 <td>Normal ES cell morphology</td>
 <td>Normal ES cell morphology</td>
 </tr>
 <tr>
 <td>5th Passage</td>
 <td>differentiated</td>
 <td>Normal ES cell morphology</td>
 <td>Cell death observed</td>
 <td>Normal ES cell morphology</td>
 <td>Normal ES cell morphology</td>
 </tr>
 </tbody>
</table>

<ol>
	<li>Williams, R. L., Hilton, D. J., Pease, S., Willson, T. A., Stewart, C. L., Gearing, D. P., Wagner, E. F., Metcalf, D., Nicola, N. A., Gough, N. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myeloid+leukaemia+inhibitory+factor+maintains+the+developmental+potential+of+embryonic+stem+cells.">Myeloid leukaemia inhibitory factor maintains the developmental potential of embryonic stem cells.</a> Nature. 336, 684-687 (1988).</li><li>Ying, Q. L., Wray, J., Nichols, J., Batlle-Morera, L., Doble, B., Woodgett, J., Cohen, P., Smith, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ground+state+of+embryonic+stem+cell+self-renewal.">The ground state of embryonic stem cell self-renewal.</a> Nature. 453, 519-523 (2008).</li><li>Chen, S., Do, J. T., Zhang, Q., Yao, S., Yan, F., Peters, E. C., Scholer, H. R., Schultz, P. G., Ding, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-renewal+of+embryonic+stem+cells+by+a+small+molecule.">Self-renewal of embryonic stem cells by a small molecule.</a> Proc. Natl. Acat. Sci. USA. 103, 17266-17271 (2006).</li></ol>

The authors of this article are employed by Stemgent that produces reagents and instruments used in this article.

The small molecule SC1 can be used to maintain self-renewal with an undifferentiated state in mouse ES cells. Before using on an untested cell line, however, it should be titrated to determine the optimal concentration. For example, we tested three concentrations on the mouse ES cell line ESC R1. 100 nM and 300 nM concentrations were able to sustain mouse ES cells, however, a 1 &mu;M concentration was toxic.
SC1 can also be used under feeder-free conditions.

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<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>SC1</td>
<td>&nbsp;</td>
<td>Stemgent</td>
<td>04-0011</td>
<td>&nbsp;</td>
<tr>
<td>LIF</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>ESG1107</td>
<td>&nbsp;</td>
<tr>
<td>SSEA-1 antibody</td>
<td>&nbsp;</td>
<td>Santa Cruz</td>
<td>21702</td>
<td>&nbsp;</td>
<tr>
<td>Nanog antibody</td>
<td>&nbsp;</td>
<td>Abcam</td>
<td>ab21603</td>
<td>&nbsp;</td>
<tr>
<td>Sox2 antibody</td>
<td>&nbsp;</td>
<td>Chemicon</td>
<td>Ab5603</td>
<td>&nbsp;</td>
<tr>
<td>Oct4 antibody</td>
<td>&nbsp;</td>
<td>Santa Cruz</td>
<td>SC-5279</td>
<td>&nbsp;</td>		</tbody>
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the use of sc1 (pluripotin) to support mesc self-renewal in the absence of lif

Mouse embryonic stem (ES) cells are conventionally cultured with Leukemia Inhibitory Factor (LIF) to maintain self-renewal.1 However, LIF is expensive and activation of the LIF/JAK/STAT3 pathway is not absolutely required to maintain the self-renewal state.2 The SC1 small molecule may be an economical alternative to LIF. SC1 functions through dual inhibition of Ras-GAP and ERK1.3 Illustration of its mechanism of action makes it a useful tool to study the fundamental molecular mechanism of self-renewal. Here we demonstrate the procedure for culturing mouse ES cells in the presence of SC1 and show that they are able to maintain self-renewal in the absence of LIF. Cells cultured with SC1 showed similar morphology compared to cells maintained with LIF. Both exhibited typical mouse ES morphology after five passages. Expression of typical pluripotency markers (Oct4, Sox2, Nanog, and SSEA1) was observed after five passages in the presence of SC1. Furthermore, SC1 caused no overt toxicity on mouse ES cells.

Watch this Scientific Journal Video about The use of SC1 Pluripotin to Support mESC Self-renewal in the Absence of LIF at JoVE.com

The use of SC1 Pluripotin to Support mESC Self-renewal in the Absence of LIF

SC1 functions through dual inhibition of Ras- GAP and ERK1. We tested the function of SC1 in supporting mouse ES cell self-renewal in the absence of LIF and showed that SC1 is able to maintain self-renewal of mouse ES cell cultures.

The use of SC1 (Pluripotin) to Support mESC Self-renewal in the Absence of LIF

Mouse embryonic stem (ES) cells are conventionally cultured with Leukemia Inhibitory Factor (LIF) to maintain self-renewal.1 However, LIF is expensive and ...

the-use-sc1-pluripotin-to-support-mesc-self-renewal-absence

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Biology

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The Protease Fluorescent Detection Kit provides ready-to-use reagents for detecting the presence of protease activity. This simple assay to detect protease activity uses casein labeled with fluorescein isothiocyanate (FITC) as the substrate.
Protease activity results in the cleavage of the FITC-labeled casein substrate into smaller fragments, which do not precipitate under acidic conditions. After incubation of the protease sample and substrate, the reaction is acidified with the addition of trichloroacetic acid (TCA). The mixture is then centrifuged with the undigested substrate forming a pellet and the smaller, acid soluble fragments remaining in solution. The supernatant is neutralized and the fluorescence of the FITC-labeled fragments is measured.
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Quantification of &gamma;H2AX Foci in Response to Ionising Radiation

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA lesions with respect to survival and preservation of genomic integrity. An early response to the induction of DSBs is phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX1. Following induction of DSBs, H2AX is rapidly phosphorylated by the phosphatidyl-inosito 3-kinase (PIKK) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Typically, only a few base-pairs (bp) are implicated in a DSB, however, there is significant signal amplification, given the importance of chromatin modifications in DNA damage signalling and repair. Phosphorylation of H2AX mediated predominantly by ATM spreads to adjacent areas of chromatin, affecting approximately 0.03% of total cellular H2AX per DSB2,3. This corresponds to phosphorylation of approximately 2000 H2AX molecules spanning ~2 Mbp regions of chromatin surrounding the site of the DSB and results in the formation of discrete &gamma;H2AX foci which can be easily visualized and quantitated by immunofluorescence microscopy2. The loss of &gamma;H2AX at DSB reflects repair, however, there is some controversy as to what defines complete repair of DSBs; it has been proposed that rejoining of both strands of DNA is adequate however, it has also been suggested that re-instatement of the original chromatin state of compaction is necessary4-8. The disappearence of &gamma;H2AX involves at least in part, dephosphorylation by phosphatases, phosphatase 2A and phosphatase 4C5,6. Further, removal of &gamma;H2AX by redistribution involving histone exchange with H2A.Z has been implicated7,8. Importantly, the quantitative analysis of &gamma;H2AX foci has led to a wide range of applications in medical and nuclear research. Here, we demonstrate the most commonly used immunofluorescence method for evaluation of initial DNA damage by detection and quantitation of &gamma;H2AX foci in &gamma;-irradiated adherent human keratinocytes9.

Quantification of &amp;#947;H2AX Foci in Response to Ionising Radiation

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Oct4GiP Reporter Assay to Study Genes that Regulate Mouse Embryonic Stem Cell Maintenance and Self-renewal

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Coupled with RNAi, the Oct4GiP reporter assay can be used to quickly identify and study regulators of ES cell maintenance and self-renewal 3,8. Compared to other methods for assaying self-renewal, it is more convenient, sensitive, quantitative, and of lower cost. It can be carried out in 96- or 384-well plates for large-scale studies such as high-throughput screens or genetic epistasis analysis. Finally, by using other lineage-specific reporter ES cell lines, the assay we describe here can also be modified to study fate specification during ES cell differentiation.

We describe a fluorescence reporter assay to quickly identify and characterize genes that regulate mouse embryonic stem cell maintenance and self-renewal.

Pluripotency and self-renewal are two defining characteristics of embryonic stem cells (ES cells). Understanding the underlying molecular mechanism will ...

The Use of Chemostats in Microbial Systems Biology

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Identification of Key Factors Regulating Self-renewal and Differentiation in EML Hematopoietic Precursor Cells by RNA-sequencing Analysis

Hematopoietic stem cells (HSCs) are used clinically for transplantation treatment to rebuild a patient&#39;s hematopoietic system in many diseases such as leukemia and lymphoma. Elucidating the mechanisms controlling HSCs self-renewal and differentiation is important for application of HSCs for research and clinical uses. However, it is not possible to obtain large quantity of HSCs due to their inability to proliferate in vitro. To overcome this hurdle, we used a mouse bone marrow derived cell line, the EML (Erythroid, Myeloid, and Lymphocytic) cell line, as a model system for this study.
RNA-sequencing (RNA-Seq) has been increasingly used to replace microarray for gene expression studies. We report here a detailed method of using RNA-Seq technology to investigate the potential key factors in regulation of EML cell self-renewal and differentiation. The protocol provided in this paper is divided into three parts. The first part explains how to culture EML cells and separate Lin-CD34+ and Lin-CD34- cells. The second part of the protocol offers detailed procedures for total RNA preparation and the subsequent library construction for high-throughput sequencing. The last part describes the method for RNA-Seq data analysis and explains how to use the data to identify differentially expressed transcription factors between Lin-CD34+ and Lin-CD34- cells. The most significantly differentially expressed transcription factors were identified to be the potential key regulators controlling EML cell self-renewal and differentiation. In the discussion section of this paper, we highlight the key steps for successful performance of this experiment.
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RNA-sequencing and bioinformatics analyses were used to identify significantly and differentially expressed transcription factors in Lin-CD34+ and Lin-CD34- subpopulations of mouse EMLcells. These transcription factors might play important roles in determining the switch between self-renewing Lin-CD34+ and partially differentiated Lin-CD34- cells.

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Use of Enzymatic Biosensors to Quantify Endogenous ATP or H2O2 in the Kidney

Enzymatic microelectrode biosensors have been widely used to measure extracellular signaling in real-time. Most of their use has been limited to brain slices and neuronal cell cultures. Recently, this technology has been applied to the whole organs. Advances in sensor design have made &#160;possible the measuring of&#160;cell signaling in blood-perfused in vivo kidneys. The present protocols list the steps needed to measure ATP and H2O2 signaling in the rat kidney interstitium. Two separate sensor designs are used for the ex vivo and in vivo protocols. Both types of sensor&#160;are coated with&#160;a thin enzymatic biolayer on top of a permselectivity layer to give fast responding, sensitive and selective biosensors. The permselectivity layer protects the signal from the interferents in biological tissue, and the enzymatic layer utilizes&#160;the sequential catalytic reaction of glycerol kinase and glycerol-3-phosphate oxidase in the presence of ATP to produce H2O2. The set of sensors used for the ex vivo studies further detected analyte by oxidation of H2O2 on a platinum/iridium (Pt-Ir) wire electrode. The sensors for the&#160;in vivo studies are instead based on the reduction of H2O2 on a mediator coated gold electrode designed for blood-perfused tissue. Final concentration changes&#160;are detected by real-time amperometry followed by calibration to known concentrations of analyte. Additionally, the specificity of the amperometric signal can be confirmed by the addition of enzymes such as catalase and apyrase that break down H2O2 and ATP correspondingly. These sensors also rely heavily on accurate calibrations before and after each experiment. The following two protocols establish the study of real-time detection of ATP and H2O2 in kidney tissues, and can be further modified to extend the described method for use in other biological preparations or whole organs.

Use of Enzymatic Biosensors to Quantify Endogenous ATP or H2O2 in the Kidney

Enzymatic microelectrode biosensors enable real-time measurements of extracellular cell signaling in biologically-relevant concentrations. The following protocols extend the applications of biosensors to the ex vivo and in vivo detection of ATP and H2O2 in the kidney.

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Modulation of renal epithelial ion transport allows organisms to maintain ionic and osmotic homeostasis in the face of varying external conditions. The Drosophila&#160;melanogaster Malpighian (renal) tubule offers an unparalleled opportunity to study the molecular mechanisms of epithelial ion transport, due to the powerful genetics of this organism and the accessibility of its renal tubules to physiological study. Here, we describe the use of the Ramsay assay to measure fluid secretion rates from isolated fly renal tubules, with the use of ion-specific electrodes to measure sodium and potassium concentrations in the secreted fluid. This assay allows study of transepithelial fluid and ion fluxes of ~20 tubules at a time, without the need to transfer the secreted fluid to a separate apparatus to measure ion concentrations. Genetically distinct tubules can be analyzed to assess the role of specific genes in transport processes. Additionally, the bathing saline can be modified to examine the effects of its chemical characteristics, or drugs or hormones added. In summary, this technique allows the molecular characterization of basic mechanisms of epithelial ion transport in the Drosophila tubule, as well as regulation of these transport mechanisms.

Use of the Ramsay Assay to Measure Fluid Secretion and Ion Flux Rates in the Drosophila melanogaster Malpighian Tubule

This protocol describes the use of the Ramsay assay to measure fluid secretion rates from isolated Malpighian (renal) tubules from Drosophila melanogaster. In addition, the use of ion-specific electrodes to measure sodium and potassium concentrations in the secreted fluid, allowing calculation of transepithelial ion flux, is described.

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The role of secreted soluble factors in the modification of cellular responses is a recurrent theme in the study of all tissues and systems. In an attempt to make straightforward the very complex relationships between the several cellular subtypes that compose multicellular organisms, in vitro techniques have been developed to help researchers acquire a detailed understanding of single cell populations. One of these techniques uses inserts with a permeable membrane allowing secreted soluble factors to diffuse. Thus, a population of cells grown in inserts can be co-cultured in a well or dish containing a different cell type for evaluating cellular changes following paracrine signaling in the absence of cell-cell contact. Such insert co-culture systems offer various advantages over other co-culture techniques, namely bidirectional signaling, conserved cell polarity and population-specific detection of cellular changes. In addition to being utilized in the field of inflammation, cancer, angiogenesis and differentiation, these co-culture systems are of prime importance in the study of the intricate relationships that exist between the different cellular subtypes present in the central nervous system, particularly in the context of neuroinflammation. This article offers general methodological guidelines in order to set up an experiment in order to evaluating cellular changes mediated by secreted soluble factors using an insert co-culture system. Moreover, a specific protocol to measure the neuroinflammatory effects of cytokines secreted by lipopolysaccharide-activated N9 microglia on neuronal PC12 cells will be detailed, offering a concrete understanding of insert co-culture methodology.

In multicellular organisms, secreted soluble factors elicit responses from different cell types as a result of paracrine signaling. Insert co-culture systems offer a simple way to assess the changes mediated by secreted soluble factors in the absence of cell-cell contact.

The role of secreted soluble factors in the modification of cellular responses is a recurrent theme in the study of all tissues and systems. In an attempt ...

The Use of Ex Vivo Whole-organ Imaging and Quantitative Tissue Histology to Determine the Bio-distribution of Fluorescently Labeled Molecules

Fluorescent labeling is a well-established process for examining the fate of labeled molecules under a variety of experimental conditions both in vitro and in vivo. Fluorescent probes are particularly useful in determining the bio-distribution of administered large molecules, where the addition of a small-molecule fluorescent label is unlikely to affect the kinetics or bio-distribution of the compound. A variety of methods exist to examine bio-distribution that vary significantly in the amount of effort required and whether the resulting measurements are fully quantitative, but using multiple methods in conjunction can provide a rapid and effective system for analyzing bio-distributions.
Ex vivo whole-organ imaging is a method that can be used to quickly compare the relative concentrations of fluorescent molecules within tissues and between multiple types of tissues or treatment groups. Using an imaging platform designed for live-animal or whole-organ imaging, fluorescence within intact tissues can be determined without further processing, saving time and labor while providing an accurate picture of the overall bio-distribution. This process is ideal in experiments attempting to determine the tissue specificity of a compound or for the comparison of multiple different compounds. Quantitative tissue histology on the other hand requires extensive further processing of tissues in order to create a quantitative measure of the labeled compounds. To accurately assess bio-distribution, all tissues of interest must be sliced, scanned, and analyzed relative to standard curves in order to make comparisons between tissues or groups. Quantitative tissue histology is the gold standard for determining absolute compound concentrations within tissues.
Here, we describe how both methods can be used together effectively to assess the ability of different administration methods and compound modifications to target and deliver fluorescently labeled molecules to the central nervous system1.

The Use of Ex Vivo Whole-organ Imaging and Quantitative Tissue Histology to Determine the Bio-distribution of Fluorescently Labeled Molecules

Ex vivo whole-organ imaging is a rapid method for determining the relative concentrations of fluorescently labeled compounds within and between tissues or treatment groups. Conversely, quantitative fluorescence histology, while more labor intensive, allows for the quantification of the absolute tissue levels of labeled molecules.

Fluorescent labeling is a well-established process for examining the fate of labeled molecules under a variety of experimental conditions both in vitro ...