Name: collection of serum- and feeder-free mouse embryonic stem cell-conditioned medium for a cell-free approach
Uploaded: 2017-01-08T15:00:00
Description: 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

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...

<ol>
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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 t...

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.

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		<col span="4" />
	</colgroup>
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:64px;">DMEM</td>
			<td style="width:64px;">Invitrogen</td>
			<td style="width:64px;">#11960-044</td>
			<td style="width:64px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:64px;">FBS</td>
			<td style="width:64px;">Invitrogen</td>
			<td style="width:64px;">#30044333</td>
			<td style="width:64px;">20%, ES cell quality</td>
		</tr>
		<tr height="126">
			<td height="148" rowspan="2" style="height:148px;width:64px;">Penicillin and streptomycin&#160;</td>
			<td rowspan="2" style="width:64px;">Invitrogen</td>
			<td rowspan="2" style="width:64px;">#15140</td>
			<td style="width:64px;">50units/ml penicillin and 50mg/ml strepto</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:64px;">-mycin.</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:64px;">L-glutamine&#160;</td>
			<td style="width:64px;">Invitrogen</td>
			<td style="width:64px;">#25030</td>
			<td style="width:64px;">2mM</td>
		</tr>
		<tr height="106">
			<td height="106" style="height:106px;width:64px;">Nonessential amino acids (NEAA)&#160;</td>
			<td style="width:64px;">Invitrogen</td>
			<td style="width:64px;">#11140</td>
			<td style="width:64px;">100uM</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:64px;">&#946;-mercaptoethanol&#160;</td>
			<td style="width:64px;">Sigma</td>
			<td style="width:64px;">#M3148</td>
			<td style="width:64px;">100uM</td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:64px;">Leukemia inhibitory factor&#160;</td>
			<td style="width:64px;">Millipore</td>
			<td style="width:64px;">#ESG1107</td>
			<td style="width:64px;">100units/ml</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:64px;">OPTI-MEM</td>
			<td style="width:64px;">Invitrogen</td>
			<td style="width:64px;">#22600</td>
			<td style="width:64px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:64px;">X-gal&#160;</td>
			<td style="width:64px;">Sigma</td>
			<td style="width:64px;">#B4252</td>
			<td style="width:64px;">1mg/ml</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:64px;">Paraformaldehyde (PFA)</td>
			<td style="width:64px;">Sigma</td>
			<td style="width:64px;">P6148</td>
			<td style="width:64px;">3.70%</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:64px;">Dimethylformamide (DMF)</td>
			<td style="width:64px;">Sigma</td>
			<td style="width:64px;">#D4551</td>
			<td style="width:64px;"></td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:64px;">Potassium ferricyanide&#160;</td>
			<td style="width:64px;">Aldrich</td>
			<td style="width:64px;">#455946</td>
			<td style="width:64px;">5mM</td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:64px;">potassium ferrocyanide&#160;</td>
			<td style="width:64px;">Aldrich</td>
			<td style="width:64px;">#455989</td>
			<td style="width:64px;">5mM</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:64px;">NaCl&#160;</td>
			<td style="width:64px;">Sigma</td>
			<td style="width:64px;">#S7653</td>
			<td style="width:64px;">150mM</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:64px;">MgCl2&#160;</td>
			<td style="width:64px;">Sigma</td>
			<td style="width:64px;">#M2393</td>
			<td style="width:64px;">2mM</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:64px;">Mytomycin C&#160;</td>
			<td style="width:64px;">Sigma</td>
			<td style="width:64px;">#M4287</td>
			<td style="width:64px;">10ug/ml</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:64px;">Propidium iodide&#160;</td>
			<td style="width:64px;">Sigma</td>
			<td style="width:64px;">#P4170</td>
			<td style="width:64px;">50ug/ml</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:64px;">TRIzol</td>
			<td style="width:64px;">Ambion</td>
			<td style="width:64px;">#15596018</td>
			<td style="width:64px;"></td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:64px;">M-MLV reverse transcript-tase</td>
			<td style="width:64px;">Promega</td>
			<td style="width:64px;">#M170B</td>
			<td style="width:64px;"></td>
		</tr>
		<tr height="127">
			<td height="127" style="height:127px;width:64px;">Power SYBR Green PCR master mix&#160;</td>
			<td style="width:64px;">Applied Biosystems</td>
			<td style="width:64px;">#4367659</td>
			<td style="width:64px;"></td>
		</tr>
		<tr height="106">
			<td height="106" style="height:106px;width:64px;">HDFs, NHDF-Ad-Der-Fibroblast&#160;</td>
			<td style="width:64px;">LONZA</td>
			<td style="width:64px;">#CC-2511</td>
			<td style="width:64px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:64px;">Bottle top filter,&#160;</td>
			<td style="width:64px;">Corning</td>
			<td style="width:64px;">#430513</td>
			<td style="width:64px;">0.2&#956;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...

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 ...

The overall goals of this protocol are to collect conditioned medium from mouse embryonic stem cells under serum and feeder free culture conditions and to characterize the biological functions of this medium. This procedure provides insights into the anti senescence activity of soluble factors secreted from mouse embryonic stem cells for the development of safe and cell free therapeutic approach for age related diseases. Demonstrating this procedure will be Dr.Yun-Ui Bae a research professor of my laboratory.In a biological safety hood, treat a mouse embryonic fibroblast or MEF cell culture with 20 milliliters of MEF medium supplemented with mitomycin C in a 15 centimeter cell culture dish for two hours at 37 degrees celsius and 5%carbon dioxide. At the end of the incubation, wash the cells three times with PBS and detach them with three milliliters of trypsin EDTA for three minutes at 37 degrees celsius. Neutralizing the trypsin with six milliliters of fresh MEF medium when the cells have lifted from the bottom of the plate.After collecting the cells by centrifugation, re-suspend the pellet in five milliliters of MEF medium and plate two times 10 to the sixth inactivated MEFs per 10 centimeter culture dish in fresh MEF medium. Culture the feeder cells for 24 hours. Then replace the MEF medium with mouse embryonic stem cell medium and seed two times 10 to the sixth mouse embryonic stem cells onto the feeders for at least 48 hours of co culture.In a biological safety hood, rinse a plate of mouse embryonic stem cells with five milliliters of PBS and detach the cells with trypsin plus EDTA as just demonstrated. After collecting the cells by centrifugation, re-suspend the stem cell pellet in five milliliters of fresh stem cell medium and plate one milliliter of cells per dish in fresh embryonic stem cell medium onto five gelatin coated cell culture dishes. Return the cells to the incubator until the cultures reach 80-85%confluency.Then rinse the cells in at least eight milliliters of PBS per plate for three 10 minute washes followed by a 24 hour incubation in reduced serum medium. After no more than 24 hours, transfer the supernatant into a 50 milliliter conical tube for centrifugation. Followed by filtration through a 0.2 micrometer bottle top strainer.To test the effects of the embryonic stem cell conditioned medium by a senescence associated beta galactosidase assay, seed human dermal fibroblasts in a six well plate in a human dermal fibroblast medium. After an overnight incubation at 37 degrees celsius and 5%carbon dioxide, replace half of the medium with fresh mouse embryonic stem cell conditioned medium for another 72 hour culture. On the third day of culture, rinse the cells in two milliliters of PBS per well for two 30 second washes.Then, fix the cells in 3.7%paraformaldehyde for five minutes at room temperature in a fume hood. After fixing, wash the cells two times with two milliliters of PBS, as just demonstrated and label the cells with one to two milliliters of SA beta gal solution for 17 and half hours at 37 degrees celsius without carbon dioxide. At the end of the staining period, wash the cells two times with PBS as just demonstrated.Then, counter stain with Eosen for five minutes at room temperature. After another set of PBS washes, image the cells at a 100 times magnification by light microscopy. For cell cycle analysis, seed eight times 10 to the fourth human dermal fibroblast per well in a six centimeter cell culture dish in fresh human dermal fibroblast medium for an overnight incubation at 37 degrees celsius and 5%carbon dioxide.The next morning, replace half of the medium with mouse embryonic stem cell conditioned medium and return the cells to the incubator. After 24 hours, harvest the cells by trypsin EDTA dissociation followed by two washes in one milliliter of PBS solution. Re-suspend the pellet in 100 microliters of cold PBS solution after the second wash.Then, fix the cells with the drop wise application at 200 microliters of cold ethanol while vortexing. Store the cells at four degrees celsius for at least an hour. Then, wash them in fresh PBS solution.After the second centrifugation, treat the pellet with 250 microliters of sodium citrate buffer supplemented with RNase for 30 minutes at 37 degrees celsius without carbon dioxide. At the end of the incubation, label the cells with 250 microliters of sodium citrate buffer containing propidium iodide for 20 minutes. Then, assess the cell cycle stages of each sample by flow cytometry.For quantitative reverse transcriptase PCR analysis, use an RNA extraction kit to isolate the total RNA from mouse embryonic stem cell conditioned medium treated human dermal fibroblasts according to the manufacturer's protocol. Use a spectrophotometer to quantify the total extracted RNA. Then, formulate reaction mixture containing the oligo deoxythymidine primers and M-MLV reverse transcriptase according to the manufacturer's protocol.Then add one microgram of the total RNA to the PCR tube containing the reaction mixture. Finally, measure the amplification of the CDNA with a real time PCR machine using the green PCR master mix and the appropriate gene primers. Mouse embryonic stem cells grown under normal mouse embryonic stem cell culture conditions on a MEF feeder cell layer display an oval shape and a shiny appearance.While mouse embryonic stem cells grown under serum and feeder free culture conditions demonstrate a flattened and irregular morphology. The treatment of the senescent human dermal fibroblast with mouse embryonic stem cell conditioned medium decreases the number of senescent associated betagalactosidase positive cells. Further, cell cycle analysis of these cells reveals that mouse embryonic stem cell conditioned medium treatment dramatically increases the number of cells in the S and G2/M phases while reducing the number of cells in the G0/G1 phase.In addition, mouse embryonic stem cell conditioned medium treatment decreases the expression of senescence associated genes and of secretary cytokines associated with the senescent phenotype. While attempting this procedure, it is very important to remember to collect the serum and feeder free medium after 24 hours, as a long incubation increases the possibility of cell autolysis or apoptosis by starvation. After watching this video, you should have a good understanding of how to collect serum and feeder free mouse embryonic stem cell conditioned medium for a cell free approach.Don't forget that working with reagent such as paraformaldehyde can be extremely hazardous, and that precaution, such as handling this reagent in a fume hood, should always be taken while performing this procedure.

Research

JoVE Journal

Developmental Biology

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 ...

Generation of Induced Pluripotent Stem Cells from Human Peripheral T Cells Using Sendai Virus in Feeder-free Conditions

Recently, iPSCs have attracted attention as a new source of cells for regenerative therapies. Although the initial method for generating iPSCs relied on ...

Generating CRISPR/Cas9 Mediated Monoallelic Deletions to Study Enhancer Function in Mouse Embryonic Stem Cells

Enhancers control cell identity by regulating tissue-specific gene expression in a position and orientation independent manner. These enhancers are often ...

Feeder-free Derivation of Melanocytes from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) represent a platform to study human development in vitro under both normal and disease conditions. Researchers can ...

Reprogramming Mouse Embryonic Fibroblasts with Transcription Factors to Induce a Hemogenic Program

This protocol details the induction of a hemogenic program in mouse embryonic fibroblasts (MEFs) via overexpression of transcription factors (TFs). We ...

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 ...

Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing ...

Defined Xeno-free and Feeder-free Culture Conditions for the Generation of Human iPSC-derived Retinal Cell Models

The production of specialized cells from pluripotent stem cells provides a powerful tool to develop new approaches for regenerative medicine. The use of ...

Serum Free Production of Three-dimensional Human Hepatospheres from Pluripotent Stem Cells

The development of renewable sources of liver tissue is required to improve cell-based modelling, and develop human tissue&#160;for transplantation. Human ...

A Static Self-Directed Method for Generating Brain Organoids from Human Embryonic Stem Cells

Human brain organoids differentiated from embryonic stem cells offer the unique opportunity to study complicated interactions of multiple cell types in a ...