Name: collection of serum- and feeder-free mouse embryonic stem cell-conditioned medium for a cell-free approach
Uploaded: 2017-01-08T15:00:00.000+00:00
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This research was supported by the Basic Science Research Program (2013R1A1A2060930) and the Medical Research Center Program (2015R1A5A2009124) through the National Research Foundation of Korea (NRF), funded by the Ministry of Science, ICT, and Future Planning. This research is also supported by a Start-up Operating Grant from The Hospital for Sick Children (H. K. Sung). We would like to thank Laura Barwell and Sarah J. S. Kim for their excellent help in editing this manuscript and Dr. Andras Nagy for providing the G4 mESC line.

NOTE: A schematic of the serum- and feeder-free CM collection protocol is shown in Figure 1.
1. Materials (Preparation of MEFs, Medium, Plates, and Solutions)
<ol>
	<li>Prepare 500 ml of medium to culture the MEFs. Supplement Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM) with 10% FBS (ESC quality), 50 units/ml penicillin, and 50 mg/ml streptomycin.</li>
	<li>Isolate MEFs from embryos following an established routine protocol13 and maintain them in MEF medium.</li>
	<li>Prepare 500 ml of medium to culture the mESCs. DMEM is supplemented with 15% FBS and 2 mM L-glutamine, 100 &#181;M non-essential amino acids (NEAA), 100 &#181;M &#946;-mercaptoethanol, 100 units/ml leukemia inhibitory factor (LIF), 50 units/ml penicillin, and 50 mg/ml streptomycin.</li>
	<li>Prepare the gelatinized plates (5 gelatinized plates/1 mESC plate) by coating 10 cm cell culture dishes with 5 ml of 0.1% gelatin solution. Incubate for at least 10 min at room temperature.</li>
	<li>Prepare 500 ml of Reduced Serum Medium for a serum-free condition of mESCs. Supplement Reduced Serum Media with 1.2 g of sodium bicarbonate (pH 7.0). Filter through a 0.2 &#181;m bottle-top filter.</li>
	<li>Prepare the senescence-associated &#946;-galactosidase (SA &#946;-gal) staining solution for the detection of senescent cells: 1 mg/ml X-gal (dissolved in dimethylformamide, DMF), 40 mM citric acid/sodium phosphate buffer (pH 6.0), 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, 150 mM NaCl, and 2 mM MgCl214. 
	CAUTION: (Hazardous) DMF is a toxic and corrosive solution. Wear personal protective clothing (e.g., nitrile or latex gloves, a lab coat, and goggles) when handling solution. Use a fume hood.</li>
	<li>Prepare 500 ml of medium to culture human dermal fibroblasts (HDFs, NHDF-Ad-Der-Fibroblast). Supplement DMEM with 10% FBS and 100 units/ml penicillin and 100 mg/ml streptomycin.</li>
</ol>
2. Culture of Mouse Embryonic Stem Cells (Figure 1A and 2A)
NOTE: Carry out all steps in a cell culture biological safety hood.
<ol>
	<li>Treat the MEFs with 20 ml of MEF medium containing 10 &#181;g/ml of mitomycin C in a 15 cm cell culture dish. Incubate for 2 hr at 37 &#176;C and 5% CO2.</li>
	<li>Aspirate the medium from MEFs. Wash the cells with PBS three times. Add 3 ml of trypsin-EDTA (TE, 1x) and incubate for 3 min at 37 &#176;C and 5% CO2. After 3 min, neutralize the TE with 6 ml of MEF medium and centrifuge for 3 min at 300 x g.</li>
	<li>Resuspend in 5 ml of MEF medium. Determine the cell number in the resulting cell suspension using trypan blue staining and a hemocytometer. Plate inactivated MEFs (feeder) at a density of 2 x 106 cells per 10-cm cell culture dish in MEF medium. Incubate for 24 hr at 37 &#176;C and 5% CO2.</li>
	<li>Replace the MEF medium with mESC medium 24 hr after plating the feeder (on the following day).</li>
	<li>Plate the mESCs (G4 F1 hybrid ES cell) at a density of 2 x 106 cells on the feeder with mESC medium. Incubate for 48 hr at 37 &#176;C and 5% CO2. 
	NOTE: The anti-aging effect of mESC-CM is likely stronger when lower passage number mESCs are used15-17. We acquired a G4 mESC line from Dr. Andras Nagy in Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, 25 Orde Street, Toronto, ON, M5T 3H7, Canada.</li>
	<li>Keep the cells at a relatively high density and passage at a 70-80% sub-confluent state. Replace the medium with fresh mESC medium daily.</li>
</ol>
3. Collection of Serum- and Feeder-free Conditioned Medium (Figure 1B and 2B)
NOTE: Carry out all steps in a cell culture biological safety hood.
<ol>
	<li>Rinse the mESC plate with 5 ml of PBS. Add 1 ml of TE (2.5x) and incubate for 3 min at 37 &#176;C and 5% CO2. After 3 min, neutralize the TE with 2 ml of mESC medium and centrifuge for 3 min at 300 x g.</li>
	<li>Resuspend in 5 ml of mESC medium and plate 1 ml into each gelatin-coated culture dish (5 gelatinized plates/1 mESC plate) in mESC medium. Culture at 37 &#176;C and 5% CO2 until 80-85% confluency is reached.</li>
	<li>Wash mESCs with sufficient PBS to cover the cells (8 ml per 10 cm plate) for 10 min per wash, for a total of three washes. Incubate in Reduced Serum Medium for 24 hr at 37 &#176;C and 5% CO2. 
	NOTE: The washing step is important to prevent FBS contamination. It is important to follow the incubation time18,19.</li>
	<li>Collect mESC-CM into a 50 ml conical tube and centrifuge for 20 min at 2,500 x g. Collect the supernatant solution (CM) after the centrifugation. Filter through a 0.2 &#181;m bottle-top filter.</li>
</ol>
4. Effects of Mouse Embryonic Stem Cell-conditioned Medium (mESC-CM)
NOTE: The effects of mESC-CM were validated by several methods, such as SA &#946;-gal assay, cell cycle analysis, and qRT-PCR.
<ol>
	<li>SA &#946;-gal Assay (Figure 3A)
	<ol>
		<li>Seed HDFs at a density of 2 x 104 cells per well in 6-well plates in HDF medium. Incubate overnight at 37 &#176;C and 5% CO2.</li>
		<li>Following an overnight incubation, discard half of the HDF medium and add mESC-CM and control medium. Incubate for 72 hr at 37 &#176;C and 5% CO2. Control medium is derived from serum-free medium (Reduced Serum Media) in a gelatin-coated dish in the absence of mESCs.</li>
		<li>Wash the cells with sufficient PBS to cover the cells (2 ml per 6-well plate) for 30 sec per wash, for a total of two washes. Add 3.7% paraformaldehyde (PFA) for fixation. Incubate for 5 min at room temperature. 
		CAUTION: (Hazardous) Paraformaldehyde is a toxic and corrosive solution. Wear personal protective clothing (e.g., nitrile or latex gloves, a lab coat, and goggles) when handling the solution. Use a fume hood.</li>
		<li>Aspirate the fixation solution. Wash the fixed cells with PBS twice, as described in step 4.1.3.</li>
		<li>Add the SA &#946;-gal staining solution (1-2 ml per well in a 6-well plate). Incubate for 17.5 hr at 37 &#176;C. 
		NOTE: It is not to be incubated in a CO2 incubator.</li>
		<li>Aspirate the SA &#946;-gal staining solution and wash the cells with PBS twice, as described in step 4.1.3.</li>
		<li>Add the Eosin solution for counter-staining. Incubate for 5 min at room temperature. Wash the cells with PBS twice, as described in step 4.1.3.</li>
		<li>Image cells at 100X magnification using a light microscope and capture images using an attached digital camera for subsequent analysis. 
		NOTE: The total number of cells can be counted in a blind manner and the percentage of SA &#946;-gal positive blue cells can be calculated.</li>
	</ol>
	</li>
	<li>Cell Cycle Analysis (Figure 3B)
	<ol>
		<li>Seed HDFs at a density of 8 x 104 cells per well in a 6 cm cell culture dish in HDF medium. Incubate overnight at 37 &#176;C and 5% CO2.</li>
		<li>Following an overnight incubation, discard half of the HDF medium and add mESC-CM and control medium. Incubate for 24 hr at 37 &#176;C and 5% CO2.</li>
		<li>Trypsinize the cells, as described step in 3.1, and centrifuge for 5 min at 300 x g. Wash the cells with cold PBS solution (PBS with 0.5 mM CaCl2 and 2% FBS, 1 ml per 1.5 ml tube) twice and centrifuge for 3 min at 2,500 x g. Resuspend in 100 &#181;l of cold PBS solution.</li>
		<li>Fix the cells by dropping 200 &#181;l of cold ethanol while vortexing. Store at 4 &#176;C for at least 1 hr.</li>
		<li>Wash the cells with cold PBS solution twice, as described in step 4.2.3.</li>
		<li>Resuspend the cells in 250 &#181;l of sodium citrate buffer (1.12%, pH 8.5) containing 50 &#181;g/ml RNase. Incubate for 30 min at 37 &#176;C.</li>
		<li>Add 250 &#181;l of sodium citrate buffer containing 50 &#181;g/ml propidium iodide. Incubate for 20 min at room temperature.</li>
		<li>Measure the 10,000 cells in each sample using flow cytometry20.</li>
	</ol>
	</li>
	<li>qRT-PCR (Figure 3C)
	<ol>
		<li>Seed HDFs and add mESC-CM, as described in steps 4.1.1 and 4.1.2.</li>
		<li>Isolate total RNA from the HDFs using an RNA extraction kit according to the manufacturer&#39;s protocol. Quantify the extracted total RNA using a spectrophotometer21.</li>
		<li>Synthesize cDNA by adding 1 &#181;g of the total RNA to a 20 &#181;l of reaction mixture containing oligo (dT) primers and M-MLV reverse transcriptase, according to the manufacturer&#39;s protocol20.</li>
		<li>Measure the amplification of the cDNA with a real-time PCR machine, using Green PCR master mix and specific gene primers (Supplement Table 1). Normalize the data with GAPDH expression. Use the following PCR protocol: initial denaturation for 10 min at 95 &#176;C; 45 cycles for 15 sec at 95 &#176;C, 20 sec at 55 &#176;C, and 35 sec at 72 &#176;C; and the melting curve stage for 15 sec at 95 &#176;C, 1 min at 60 &#176;C, 30 sec at 95 &#176;C, and 5 sec at 60 &#176;C15.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

For the successful collection of serum- and feeder-free mESC-CM, the following suggestions should be taken into consideration. The most critical factor is using early passage mESCs for the collection of mESC-CM. Previously, it has been shown that early passage mESC-CM has better anti-aging effects compared to late passage mESCs. The passage number of mESCs has been reported to affect their developmental potential16 and pluripotency17.
While additional research is needed to analyze the specific factors of the mESC secretome, which induce anti-senescence effects, we can currently conclude that mESC-CM is sufficient to decrease senescence at the cellular level.
The identification of mESC-specific secretory factors that revert senescent cells back to young cells will be critical for future studies. For high-quality analyses on the secretory molecules, such as antibody array15 and secretome analysis, the washing step during the medium collection process (step 3) is critical. If the washing step is not properly conducted, the secretory molecules will be contaminated by serum (FBS) components18,19.
The serum- and feeder-free incubation time (24 hr) is very important in the medium collection process (step 3), as the longer incubation time (over 24 hr) may increase the possibility of cell autolysis or apoptosis by starvation under the serum- and feeder- depleted conditions18,19. The normal ESC culture condition requires a feeder layer for long-term culturing of undifferentiated cells, as the feeder secretes a large number of molecules22. The gelatin-coated plate prevents the possibility of contamination from the feeder cells.
The mESC-CM, harvested from serum- and feeder-free culture conditions, has an anti-senescence ability in senescent HDFs. Anti-senescence effects of mESC-CM have been demonstrated by senescence-associated multiple readouts, such as SA &#946;-gal activity; an enhanced proliferative potential (cell cycle analysis); and reduced p53, p21, p16, and IL-6 gene expression levels (Figure 3A-3C).
When human primary cells are treated with mESC-CM, xeno-contamination would be a critical issue for clinical application. Therefore, an investigation of the secretory factors from human ESCs or iPSCs would be an important future study for the clinical application of CM derived from human origins. The convergence of a cell-free approach based on a stem cells and an anti-senescence study is expected to expand the current understanding of senescence-associated diseases, resulting in greater insight into improvements on therapeutic approaches.

The goal of this protocol is to collect mouse embryonic stem cell (mESC)-conditioned medium (mESC-CM) from serum- and feeder-free culture conditions and to characterize its biological functions.
In general, embryonic stem cells (ESCs) have great potential for regenerative medicine and cell therapy due to their pluripotency and capacity for self-renewal1-3. However, the direct transplantation of stem cells has several limitations, such as immune rejection and tumor formation4,5. Therefore, a cell-free approach may provide an alternate therapeutic strategy for regenerative medicine and aging interventions6,7.
Senescence is viewed as a cellular counterpart to the aging of tissues and organs, characterized by a permanent state of growth arrest, altered cell physiology, and behaviors. Aging is the main risk factor for prevalent diseases including cancer, cardiovascular disease, type 2 diabetes, and neurodegeneration8. One of the obvious characteristics of aging is the decline in the regenerative potential of tissues, which is caused by stem cell aging and exhaustion9. Many significant studies have shown pharmacological molecules, such as rapamycin9, resveratrol10, and metformin11, and blood-borne systemic factors, namely GDF1112, that have the ability to consistently delay aging and extend life span.
In the present study, mESC-CM has been harvested without serum (fetal bovine serum, FBS) and feeder (mouse embryonic fibroblasts, MEFs) layers to exclude the contamination of serum factors and secretory factors from MEFs. These conditions allowed for a serum- and feeder-free CM that consequently enabled the accurate identification of mESC-specific secretory factors.
This proposed protocol is highly efficient, relatively cost effective, and easy to operate. This technique provides insights into the characterization of mESC-derived soluble factors that can mediate an anti-senescence effect, which may be used for the development of a safe and potentially advantageous cell-free therapeutic approach toward interventions for aging-associated diseases and other regenerative treatments.

<table><tbody><tr><td>DMEM</td><td>Invitrogen</td><td>#11960-044</td><td></td></tr><tr><td>FBS</td><td>Invitrogen</td><td>#30044333</td><td>20%, ES cell quality</td></tr><tr><td>Penicillin and streptomycin</td><td>Invitrogen</td><td>#15140</td><td>50 units/ml penicillin and 50&amp;nbsp;mg/ml streptomycin</td></tr><tr><td>L-glutamine</td><td>Invitrogen</td><td>#25030</td><td>2&amp;nbsp;mM</td></tr><tr><td>Nonessential amino acids (NEAA)</td><td>Invitrogen</td><td>#11140</td><td>100&amp;nbsp;&amp;micro;M</td></tr><tr><td>&amp;beta;-mercaptoethanol&amp;nbsp;</td><td>Sigma</td><td>#M3148</td><td>100&amp;nbsp;&amp;micro;M</td></tr><tr><td>Leukemia inhibitory factor&amp;nbsp;</td><td>Millipore</td><td>#ESG1107</td><td>100 units/ml</td></tr><tr><td>OPTI-MEM</td><td>Invitrogen</td><td>#22600</td><td></td></tr><tr><td>X-gal&amp;nbsp;</td><td>Sigma</td><td>#B4252</td><td>1 mg/ml</td></tr><tr><td>Paraformaldehyde (PFA)</td><td>Sigma</td><td>P6148</td><td>3.70%</td></tr><tr><td>Dimethylformamide (DMF)</td><td>Sigma</td><td>#D4551</td><td></td></tr><tr><td>Potassium ferricyanide</td><td>Aldrich</td><td>#455946</td><td>5 mM</td></tr><tr><td>potassium ferrocyanide</td><td>Aldrich</td><td>#455989</td><td>5 mM</td></tr><tr><td>NaCl</td><td>Sigma</td><td>#S7653</td><td>150 mM</td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma</td><td>#M2393</td><td>2 mM</td></tr><tr><td>Mitomycin C</td><td>Sigma</td><td>#M4287</td><td>10&amp;nbsp;&amp;micro;g/ml</td></tr><tr><td>Propidium iodide&amp;nbsp;</td><td>Sigma</td><td>#P4170</td><td>50&amp;nbsp;&amp;micro;g/ml</td></tr><tr><td>TRIzol</td><td>Ambion</td><td>#15596018</td><td></td></tr><tr><td>M-MLV reverse transcript-tase</td><td>Promega</td><td>#M170B</td><td></td></tr><tr><td>Power SYBR Green PCR master mix&amp;nbsp;</td><td>Applied Biosystems</td><td>#4367659</td><td></td></tr><tr><td>HDFs, NHDF-Ad-Der-Fibroblast&amp;nbsp;</td><td>LONZA</td><td>#CC-2511</td><td></td></tr><tr><td>Bottle top filter</td><td>Corning</td><td>#430513</td><td>0.2 &amp;mu;m</td></tr></tbody></table>

collection of serum- and feeder-free mouse embryonic stem cell-conditioned medium for a cell-free approach

The capacity of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) to generate various cell types has opened new avenues in the field of regenerative medicine. However, despite their benefits, the tumorigenic potential of ESCs and iPSCs has long been a barrier for clinical applications. Interestingly, it has been shown that ESCs produce several soluble factors that can promote tissue regeneration and delay cellular aging, suggesting that ESCs and iPSCs can also be utilized as a cell-free intervention method. Therefore, the method for harvesting mouse embryonic stem cell (mESC)-conditioned medium (mESC-CM) with minimal contamination of serum components (fetal bovine serum, FBS) and feeder cells (mouse embryonic fibroblasts, MEFs) has been highly demanded. Here, the present study demonstrates an optimized method for the collection of mESC-CM under serum- and feeder-free conditions and for the characterization of mESC-CM using senescence-associated multiple readouts. This protocol will provide a method to collect pure mESC-specific secretory factors without serum and feeder contamination.

Originally, mESCs are maintained on an MEF feeder in mESC medium with FBS and other supplements (Figures 1A and 2A). CM was collected from mESCs in Reduced Serum Media without a feeder layer, FBS, or other supplements (Figures 1B and 2B). This culture condition allows us to collect mESC-specific conditioned medium without potential contamination by the factors from the feeder, FBS, or other supplements. The control medium was collected under the same culture conditions, without mESCs.
mESCs show different morphologies between the two culture media: i) normal mESC culture conditions (Figure 2A) and ii) serum- and feeder-free culture conditions (Figure 2B). The mESC colonies grew on an MEF layer and demonstrated an oval and shiny appearance under the normal mESC culture conditions (Figure 2A). On the contrary, the mESCs in the serum- and feeder-free culture conditions showed a flattened and irregular morphology (Figure 2B).
The functional characterization of mESC-CM was achieved by senescence-associated methods, such as SA &#946;-gal assay (Figure 3A), cell cycle analysis (Figure 3B), and qPCR (Figure 3C). Treatment of senescent HDFs with mESC-CM decreased the number of positive SA &#946;-gal-positive cells, which is an indicator of cellular senescence (Figure 3A). Cell cycle analysis revealed that mESC-CM treatment dramatically increased the number of cells in the S and G2/M phase, whereas it reduced the number of cells in the G0/G1 phase (Figure 3B). In addition, mESC-CM treatment decreased the senescence-associated gene expression levels (namely, p53, p21, and p16) and the senescence-associated secretory phenotype (SASP) expression levels (IL-6).
<img alt="Figure 1" src="/files/ftp_upload/55035/55035fig1.jpg" /> 
Figure 1: Preparation and optimization of mESC-CM. Experimental strategy for the preparation and optimization of serum-free and feeder-free CM. (A) Normal mESC culture condition and (B) serum- and feeder-free mESC-CM culture condition. C: control medium without FBS and MEF; CM: conditioned medium without FBS and MEF. Modified with permission from Bae et al.15. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55035/55035fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/55035/55035fig2.jpg" /> 
Figure 2: Bright field images of mESCs. mESCs under (A) normal conditions and (B) serum- and feeder-free conditions. Yellow arrows indicate feeder cell (MEFs) in normal mESC culture conditions. Scale bars = 100 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55035/55035fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/55035/55035fig3.jpg" /> 
Figure 3: Characterization of the anti-aging effect of mESC-CM. (A) SA &#946;-gal activity staining and the percentage of SA &#946;-gal-positive cells. (B) Cell cycle analysis by flow cytometry. (C) Expression levels of senescence-associated gene expression levels (p53, p21, and p16) and senescence-associated secretory phenotype (SASP) expression levels (IL-6) by qRT-PCR. Values are the mean &#177; SD. Figures are representative of three independent experiments. Statistically-significant differences between groups were identified by one-way ANOVA and Tukey&#39;s post-hoc test. *p &#60;0.05, **p &#60;0.01. Y = non-senescent cells; S: senescent cells; C: control medium without FBS and MEF; CM: conditioned medium without FBS and MEF. Scale bars = 10 &#181;m. Modified with permission from Bae et al.15. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55035/55035fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Collection of Serum- and Feeder-free Mouse Embryonic Stem Cell-conditioned Medium for a Cell-free Approach at JoVE.com

Collection of Serum- and Feeder-free Mouse Embryonic Stem Cell-conditioned Medium for a Cell-free Approach

This protocol provides a method for the collection of mouse embryonic stem cell (mESC)-conditioned medium (mESC-CM) derived from serum (fetal bovine serum, FBS)- and feeder (mouse embryonic fibroblasts, MEFs)-free conditions for a cell-free approach. It may be applicable for the treatment of aging and aging-associated diseases.

The capacity of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) to generate various cell types has opened new avenues in the field ...

collection-serum-feeder-free-mouse-embryonic-stem-cell-conditioned

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

10.2K Views.  Yeungnam University.  Dit protocol verschaft een werkwijze voor het verzamelen van muis embryonale stamcellen (mES) -geconditioneerd medium (mES-CM) verkregen uit serum (foetaal bovine serum, FBS) - en feeder (muis embryonale fibroblasten MEF) vrij voorwaarden voor een cel -gratis aanpak. Het kan dienstig voor de behandeling van veroudering en veroudering-geassocieerde ziekten. 

Video: Collection of Serum- and Feeder-free Mouse Embryonic Stem Cell-conditioned Medium for a Cell-free Approach

Neural Differentiation of Mouse Embryonic Stem Cells in Serum-free Monolayer Culture

The ability to differentiate mouse embryonic stem cells (ESC) to neural progenitors allows the study of the mechanisms controlling neural specification as well as the generation of mature neural cell types for further study. In this protocol we describe a method for the differentiation of ESC to neural progenitors using serum-free, monolayer culture. The method is scalable, efficient and results in production of ~70% neural progenitor cells within 4 - 6 days. It can be applied to ESC from various strains grown under a variety of conditions. Neural progenitors can be allowed to differentiate further into functional neurons and glia or analyzed by microscopy, flow cytometry or molecular techniques. The differentiation process is amenable to time-lapse microscopy and can be combined with the use of reporter lines to monitor the neural specification process. We provide detailed instructions on media preparation and cell density optimization to allow the process to be applied to most ESC lines and a variety of cell culture vessels.

This protocol describes in detail the method for generating neural progenitors from embryonic stem cells using a serum-free monolayer method. These progenitors can be used to derive mature neural cell types or to study the process of neural specification and is amenable to multiwell format scaling for compound screening.

The ability to differentiate mouse embryonic stem cells (ESC) to neural progenitors allows the study of the mechanisms controlling neural specification as ...

Structure-function Studies in Mouse Embryonic Stem Cells Using Recombinase-mediated Cassette Exchange

Gene engineering in mouse embryos or embryonic stem cells (mESCs) allows for the study of the function of a given protein. Proteins are the workhorses of the cell and often consist of multiple functional domains, which can be influenced by posttranslational modifications. The depletion of the entire protein in conditional or constitutive knock-out (KO) mice does not take into account this functional diversity and regulation. An mESC line and a derived mouse model, in which a docking site for FLPe recombination-mediated cassette exchange (RMCE) was inserted within the ROSA26 (R26) locus, was previously reported. Here, we report on a structure-function approach that allows for molecular dissection of the different functionalities of a multidomain protein. To this end, RMCE-compatible mice must be crossed with KO mice and then RMCE-compatible KO mESCs must be isolated. Next, a panel of putative rescue constructs can be introduced into the R26 locus via RMCE targeting. The candidate rescue cDNAs can be easily inserted between RMCE sites of the targeting vector using recombination cloning. Next, KO mESCs are transfected with the targeting vector in combination with an FLPe recombinase expression plasmid. RMCE reactivates the promoter-less neomycin-resistance gene in the ROSA26 docking sites and allows for the selection of the correct targeting event. In this way, high targeting efficiencies close to 100% are obtained, allowing for insertion of multiple putative rescue constructs in a semi-high throughput manner. Finally, a multitude of R26-driven rescue constructs can be tested for their ability to rescue the phenotype that was observed in parental KO mESCs. We present a proof-of-principle structure-function study in p120 catenin (p120ctn) KO mESCs using endoderm differentiation in embryoid bodies (EBs) as the phenotypic readout. This approach enables the identification of important domains, putative downstream pathways, and disease-relevant point mutations that underlie KO phenotypes for a given protein.

Proteins often contain multiple domains that can exert different cellular functions. Gene knock-outs (KO) do not consider this functional diversity. Here, we report a recombination-mediated cassette exchange (RMCE)-based structure-function approach in KO embryonic stem cells that allows for the molecular dissection of various functional domains or variants of a protein.

Gene engineering in mouse embryos or embryonic stem cells (mESCs) allows for the study of the function of a given protein. Proteins are the workhorses of ...

Derivation of Stem Cell Lines from Mouse Preimplantation Embryos

Mouse embryonic stem cell (mESC) derivation is the process by which pluripotent cell lines are established from preimplantation embryos. These lines retain the ability to either self-renew or differentiate under specific conditions. Due to these properties, mESC are a useful tool in regenerative medicine, disease modeling, and tissue engineering studies. This article describes a simple protocol to obtain mESC lines with high derivation efficiencies (60-80%) by culturing blastocysts from permissive mouse strains on feeder cells in defined medium supplemented with leukemia inhibitory factor. The protocol can also be applied to efficiently derive mESC lines from non-permissive mouse strains, by the simple addition of a cocktail of two small-molecule inhibitors to the derivation medium (2i medium). Detailed procedures on the preparation and culture of feeder cells, collection and culture of mouse embryos, and derivation and culture of mESC lines are provided. This protocol does not require specialized equipment and can be carried out in any laboratory with basic mammalian cell culture expertise.

This article describes a protocol to efficiently derive and culture pluripotent stem cell lines from mouse embryos at the blastocyst stage.

Mouse embryonic stem cell (mESC) derivation is the process by which pluripotent cell lines are established from preimplantation embryos. These lines ...

CARIP-Seq and ChIP-Seq: Methods to Identify Chromatin-Associated RNAs and Protein-DNA Interactions in Embryonic Stem Cells

Embryonic stem (ES) cell self-renewal&#160;and differentiation is governed by extrinsic signals and intrinsic networks of transcription factors, epigenetic regulators, and post-translation modifications of histones that combinatorially influence the gene expression state of nearby genes. RNA has also been shown to interact with various proteins to regulate chromatin dynamics&#160;and gene expression. Chromatin-associated RNA immunoprecipitation (CARIP) followed by next-generation sequencing (CARIP-Seq) is a novel method to survey RNAs associated with chromatin proteins, while chromatin immunoprecipitation followed by next-generation sequencing (ChIP-Seq) is a powerful genomics technique to map the location of post-translational modification of histones, transcription factors, and epigenetic modifiers on a global-scale in ES cells. Here, we describe methods to perform CARIP-Seq and ChIP-Seq, including library construction for next-generation sequencing, to generate global chromatin-associated RNA and epigenomic maps in ES cells.

Here, we describe methods to perform ChIP-Seq and CARIP-Seq, including library preparation for next-generation sequencing, to generate global epigenomic and chromatin-associated RNA maps in ES cells.

Embryonic stem (ES) cell self-renewal&#160;and differentiation is governed by extrinsic signals and intrinsic networks of transcription factors, ...

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic progenitors of both mouse and human origin. It is now used to address a growing variety of complex genetic, cellular and molecular questions related to hematopoiesis, and is at the cutting edge of efforts to translate these basic findings to therapeutic applications. The procedures are straightforward and routinely yield robust results. However, achieving successful hematopoietic differentiation in vitro requires special attention to the details of reagent and cell culture maintenance. Furthermore, the protocol features technique sensitive steps that, while not difficult, take care and practice to master. Here we focus on the procedures for differentiation of T lymphocytes from mouse embryonic stem cells (mESC). We provide a detailed protocol with discussions of the critical steps and parameters that enable reproducibly robust cellular differentiation in vitro. It is in the interest of the field to consider wider adoption of this technology, as it has the potential to reduce animal use, lower the cost and shorten the timelines of both basic and translational experimentation.

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

Mouse embryonic stem cells can be differentiated to T cells in vitro using the OP9-DL1 co-culture system. Success in this procedure requires careful attention to reagent/cell maintenance, and key technique sensitive steps. Here we discuss these critical parameters and provide a detailed protocol to encourage adoption of this technology.

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic ...

Immunology and Infection

Differentiation of Mouse Embryonic Stem Cells into Cortical Interneuron Precursors

GABAergic cortical interneurons are a heterogeneous population of cells that play critical roles in regulating the output of excitatory pyramidal neurons as well as synchronizing the outputs of pyramidal neuron ensembles. Deficits in interneuron function have been implicated in a variety of neuropsychiatric disorders, including schizophrenia, autism, and epilepsy. The derivation of cortical interneurons from embryonic stem cells not only allows for the study of their development and function, but provides insight into the molecular mechanisms underlying the pathogenesis of cortical interneuron-related disorders. Interneurons also have the remarkable capacity to survive, migrate, and integrate into host cortical circuitry post-transplantation, making them ideal candidates for use in cell-based therapies. Here, we present a scalable, highly efficient, modified embryoid body-to-monolayer method for the derivation of Nkx2.1-expressing interneuron progenitors and their progeny from mouse embryonic stem cells (mESCs). Using a Nkx2.1::mCherry:Lhx6::GFP dual reporter mESC line, Nkx2.1 progenitors or their Lhx6-expressing post-mitotic progeny can be isolated via fluorescence-activated cell sorting (FACS) and subsequently used in a number of downstream applications. We also provide methods to enrich for parvalbumin (PV) or somatostatin (SST) interneuron subgroups, which may be helpful for studying aspects of fate determination or for use in therapeutic applications that would benefit from interneuron subgroup-enriched transplantations.

This protocol describes a method for generating cortical interneuron progenitors and post-mitotic interneuron precursors from mouse embryonic stem cells using a modified embryoid body-to-monolayer method. These progenitors/precursors can be used in vitro or fluorescently sorted and transplanted into neonatal neocortex for studying fate determination, or used in therapeutic applications.

GABAergic cortical interneurons are a heterogeneous population of cells that play critical roles in regulating the output of excitatory pyramidal neurons ...

Neuroscience

Preparation of Mouse Embryonic Fibroblast Cells Suitable for Culturing Human Embryonic and Induced Pluripotent Stem Cells

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it is not easy to establish an effective system for culturing these cells. Since the culture conditions can influence gene expression that confers pluripotency in hESCs and hiPSCs, the optimization and standardization of the culture method is crucial.
The establishment of hESC lines was first described by using MEFs as feeder cells and fetal bovine serum (FBS)-containing culture medium2. Next, FBS was replaced with knockout serum replacement (KSR) and FGF2, which enhances proliferation of hESCs3. Finally, feeder-free culture systems enable culturing cells on Matrigel-coated plates in KSR-containing conditioned medium (medium conditioned by MEFs)4. Subsequently, hESCs culture conditions have moved towards feeder-free culture in chemically defined conditions5-7. Moreover, to avoid the potential contamination by pathogens and animal proteins culture methods using xeno-free components have been established8.
To obtain improved conditions mouse feeder cells have been replaced with human cell lines (e.g. fetal muscle and skin cells9, adult skin cells10, foreskin fibroblasts11-12, amniotic mesenchymal cells13). However, the efficiency of maintaining undifferentiated hESCs using human foreskin fibroblast-derived feeder layers is not as high as that from mouse feeder cells due to the lower level of secretion of Activin A14. Obviously, there is an evident difference in growth factor production by mouse and human feeder cells.
Analyses of the transcriptomes of mouse and human feeder cells revealed significant differences between supportive and non-supportive cells. Exogenous FGF2 is crucial for maintaining self-renewal of hESCs and hiPSCs, and has been identified as a key factor regulating the expression of Tgf&beta;1, Activin A and Gremlin (a BMP antagonist) in feeder cells. Activin A has been shown to induce the expression of OCT4, SOX2, and NANOG in hESCs15-16.
For long-term culture, hESCs and hiPSCs can be grown on mitotically inactivated MEFs or under feeder-free conditions in MEF-CM (MEF-Conditioned Medium) on Matrigel-coated plates to maintain their undifferentiated state. Success of both culture conditions fully depends on the quality of the feeder cells, since they directly affect the growth of hESCs.
Here, we present an optimized method for the isolation and culture of mouse embryonic fibroblasts (MEFs), preparation of conditioned medium (CM) and enzyme-linked immunosorbent assay (ELISA) to assess the levels of Activin A within the media.

The quality of mouse embryonic fibroblasts (MEFs) is dictated by the right strain of mouse such as CF-1. Pluripotency-supportive MEFs and conditioned media (CM) obtained from these should contain optimal concentrations of Activin A, Gremlin and Tgf&beta;1 needed for the Activin/Nodal and FGF pathways to co-operatively maintain self-renewal and pluripotency.

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it ...

Biology

Mouse Embryonic Development in a Serum-free Whole Embryo Culture System

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in an oxygenated rolling bottle culture system. Mouse embryos at E10.5 were carefully isolated from the uterus with intact yolk sac and in a process involving precise surgical maneuver the embryos were gently exteriorized from the yolk sac while maintaining the vascular continuity of the embryo with the yolk sac. Compared to embryos prepared with intact yolk sac or with the yolk sac removed, these embryos exhibited superior survival rate and developmental progression when cultured under similar conditions. We show that these mouse embryos, when cultured in a defined medium in an atmosphere of 95% O2 / 5% CO2 in a rolling bottle culture apparatus at 37 &#176;&#8203;C for 16-40 hr, exhibit morphological growth and development comparable to the embryos developing in utero. We believe this method will be useful for investigators needing to utilize whole embryo culture to study signaling interactions important in embryonic organogenesis.

Serum utilized in embryo cultures contains unknown components that can affect the outcome of experiments especially in studies involving signaling interactions. Here we utilized a serum-free oxygenated culture system and show that mid-gestation mouse embryos cultured for 16-40 hr&#160;exhibit morphological development comparable to embryos developing in utero.

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in ...

Generating Cortical Interneuron Precursors From Mouse Embryonic Stem Cells

Source: Tischfield, D. J., et al <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/56358">Differentiation of Mouse Embryonic Stem Cells into Cortical Interneuron Precursors.</a> J. Vis. Exp. (2017)
This video presents a method for generating cortical interneuron precursors from mouse embryonic stem cells. The protocol entails culturing the stem cells in media containing specific small molecule inhibitors, which facilitate stem cell proliferation and aggregation into embryoid bodies (EBs). Subsequently, the EBs are dissociated with enzymes and plated on culture plate wells coated with adhesion proteins to promote their differentiation into cortical interneuron precursors.

1. Media Preparation
NOTE: Warm all media to 37 &#38;#176;C before use in cell culture.

	Mouse Embryonic Fibroblast (MEF) media (for preparing 500 mL)
	
	...

JoVE Encyclopedia of Experiments

Neuronal Culture Techniques

Advanced Neuronal Models

A Technique for Generating Neural Progenitor Cells from Mouse Embryonic Stem Cells

Source: Mao, X., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/61190">Neuronal Differentiation from Mouse Embryonic Stem Cells In vitro.</a> J. Vis. Exp. (2020).
This video demonstrates a method for the in vitro generation of neural progenitor cells (NPCs) from mouse embryonic stem cells (mESCs). The mESCs are cultured in suspension to form embryoid bodies (EBs), which are then guided to differentiate into NPCs via retinoic acid treatment in an adherent culture.

1. Mouse embryonic stem cell culture

	Prepare 0.1% gelatin coated cell culture dishes or plates.
	
		Add 2 mL of sterilized 0.1% gelatin (0.1% w/v in ...

Collection of Serum- and Feeder-free Mouse Embryonic Stem Cell-conditioned Medium for a Cell-free Approach

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