Name: preparation of plasma membrane vesicles from bone marrow mesenchymal stem cells for potential cytoplasm replacement therapy
Uploaded: 2017-05-18T15:00:00
Description: Watch this Scientific Journal Video about Preparation of Plasma Membrane Vesicles from Bone Marrow Mesenchymal Stem Cells for Potential Cytoplasm Replacement Therapy at JoVE.com

This research was supported by the Li Ka Shing Foundation, the Guangdong High-Level University Project &#34;Green Technologies for Marine Industries,&#34; the Natural Science Foundation of China (http://www.nsfc.gov.cn/ Grant No. 30971665, 81172894, 81370925), and the Education Department of Guangdong (http://www.gdhed.edu.cn/ Grant No.cxzd1123).

8 to 12 week-old BALB/c mice were purchased from Shanghai Experimental Animal Center (Shanghai, China) and raised in a specific pathogen-free and air-conditioned animal facility. Animal care and experimental procedures were in compliance with the guidelines for the use and care of laboratory animals established by Shantou University.
1. Assembly of the Apparatus
<ol>
	<li>To ensure sterility, turn on the UV light of a tissue culture hood for 30 min before use.</li>
	<li>Unscrew a disposable ...

<ol>
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Cytoplasm replacement therapy as proposed in this manuscript has unique advantages over other reported approaches such as gene, molecular, and cell therapy. PMVs generated from BMSCs encapsulate not only the products of stemness genes but also intact cellular organelles, which are essential to remedy the ageing phenotypes associated with senescence. When young cytoplasm is delivered to senescent cells, the malfunctioning mechanisms may gain a brief relief; at the same time, the epigenome could be reprogrammed and invigor...

A tremendous amount of effort has been devoted to establishing approaches for gene, enzyme, and cell replacement therapies. This has resulted in great breakthroughs and even clinical applications1,2,3. Recently, a controversial mitochondria replacement therapy based on nucleus transfer technology was applied to in vitro fertilization for women of old age or carrying a lethal mitochondrial DNA mutation4. Defects found in age-related diseases, including atherosclerosis, type 2 diabetes, Alzheimer&#39;s disease, and cancer, are usually multi-faceted. It has been documented that the accumulation of lipid droplets; the deposition of amyloid protein; the retention of unfolded proteins in the endoplasmic reticulum; and defective proteasome, autophagosome, and mitochondria contribute to the development or aggravation of these diseases5,6,7,8,9,10,11. Presently, there is no available mechanism aimed at direct remediation of malfunction in the cytosol and organelles, which causes senescence and ageing phenotypes.
We have previously reported on the generation of plasma membrane vesicles (PMVs) through the mechanical extrusion of mammalian cells12. With the exception of the nucleus, components in the membrane or cytosol, including proteins and RNA, as well as the organelles, such as mitochondria, were found in PMVs. Essentially, a PMV can be regarded as a miniature enucleated cell. More importantly, the fusion of PMVs with mitochondria-deficient Rho0 cells restored mitotic activity under normal culture conditions. This is the first report on establishing a potentially efficient approach for cytoplasm replacement therapy.
Bone marrow mesenchymal stem cells (BMSCs) are multipotent progenitor cells that are routinely generated from the bone marrow and are readily expanded in culture. Embryonic stem cell markers Oct4, Nanog, and SOX2 have been detected at low levels in MSCs13. Telomerase activity is also measurable. In addition, the absence of co-stimulatory molecules and human leukocyte antigen (HLA) Class II molecules, as well as low HLA Class I expression on MSCs, make them ideal cells for allogeneic, or &#34;off-the-shelf,&#34; use in both regenerative medicine and immunomodulatory applications14.
Here, we describe how to prepare PMVs from mouse BMSCs via extrusion through a polycarbonate membrane with 3-&#181;m pores, determine the existence of mitochondria and examine the maintenance of membrane potential in PMVs using confocal microscopy, prepare concentrated but not aggregated PMVs by centrifugation, and carry out the in vivo injection of PMVs into the gastrocnemius muscle of mice.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>IsoporeTM membranes</td>
			<td>Millipore</td>
			<td>TSTP04700</td>
			<td>3 mm pore</td>
		</tr>
		<tr>
			<td>Disposable filter unit</td>
			<td>Xinya, Shanghai, China</td>
			<td>25 mm</td>
			<td>Medical grade polypropylene</td>
		</tr>
		<tr>
			<td>Insulin syringe</td>
			<td>BD</td>
			<td>328446</td>
			<td>1 ml</td>
		</tr>
		<tr>
			<td>pN1-EGFP</td>
			<td>Clontech</td>
			<td>&#160;6085-1</td>
			<td></td>
		</tr>
		<tr>
			<td>MitoTracker</td>
			<td>Molecular Probes</td>
			<td>M7514</td>
			<td>Green FM, 1 &#956;M</td>
		</tr>
		<tr>
			<td>JC-1</td>
			<td>Beyotime, Haimen, China</td>
			<td>C2006</td>
			<td>10 mg/ml</td>
		</tr>
		<tr>
			<td>CM-DiI</td>
			<td>Beyotime, Haimen, China</td>
			<td>C1036</td>
			<td>10 mM</td>
		</tr>
		<tr>
			<td>PEI</td>
			<td>Sigma</td>
			<td>P3143</td>
			<td>Mn = 75000</td>
		</tr>
		<tr>
			<td>Fluorescence Microscope</td>
			<td>Nikon</td>
			<td>Eclipse TE 2000</td>
			<td>With CCD camera</td>
		</tr>
		<tr>
			<td>Confocol Microscope</td>
			<td>Carl Zeiss</td>
			<td>LSM 510 Meta</td>
			<td></td>
		</tr>
		<tr>
			<td>PolyJet</td>
			<td>SigaGen</td>
			<td>SL100688</td>
			<td>For cell transfection</td>
		</tr>
	</tbody>
</table>

preparation of plasma membrane vesicles from bone marrow mesenchymal stem cells for potential cytoplasm replacement therapy

We have previously reported on the generation of plasma membrane vesicles (PMVs) through the mechanical extrusion of mammalian cells. The fusion of PMVs with mitochondrial deficient Rho0 cells restored mitotic activity under normal culture conditions. Atherosclerosis, type 2 diabetes, Alzheimer&#39;s disease, and cancer are age-related diseases that have been reported to be associated with multiple mechanical and functional defects in the cytosol and organelles of a variety of cell types. Bone marrow mesenchymal stem cells (BMSCs) represent a unique cell population from the bone marrow that possess self-renewal capabilities while maintaining their multipotency. The supplementation of senescence cells with young cytoplasm from autologous BMSCs via the fusion of PMVs provides a promising approach to ameliorate or even reverse age-associated phenotypes. This protocol describes how to prepare PMVs from BMSCs via extrusion through a polycarbonate membrane with 3 &#181;m pores, determine the existence of mitochondria and examine the maintenance of membrane potential within PMVs using a confocal microscope, concentrate PMVs by centrifugation, and carry out the in vivo injection of PMVs into the gastrocnemius muscle of mice.

The key to a successful preparation of PMVs depends heavily on the correct assembly of the filter unit (Figure 1), which can be tested by pushing 1 mL of PBS through the membrane. If leakage occurs, reassemble the filter unit and test again. However, the leakage can only be tested reliably when cells are pushed through the membrane. If only a few PMVs are detected under a regular microscope using the 10X objective, or if the size of the PMVs is mostly around 1 &#181;m, th...

Watch this Scientific Journal Video about Preparation of Plasma Membrane Vesicles from Bone Marrow Mesenchymal Stem Cells for Potential Cytoplasm Replacement Therapy at JoVE.com

Preparation of Plasma Membrane Vesicles from Bone Marrow Mesenchymal Stem Cells for Potential Cytoplasm Replacement Therapy

Age-related diseases are associated with multiple defects in components of the cytoplasm. Here, we present a protocol to prepare plasma membrane vesicles from bone marrow mesenchymal stem cells. This technique could potentially be used as a means of cytoplasm replacement therapy to ameliorate or even reverse age-associated phenotypes.

We have previously reported on the generation of plasma membrane vesicles (PMVs) through the mechanical extrusion of mammalian cells. The fusion of PMVs ...

The overall goal of this procedure is to deliver a novel cytoplasm replacement therapy to the gastrocnemius muscle of mice using plasma membrane vesicles, or PMVs derived from mouse bone marrow mesenchymal stem cells or BMSCs. This method can help answer questions with supplementation of cells with young cytoplasm from other BMSC via PMV fusion can ameliorate or even reverse the age associative phenotypes. The main advantage of this technique is that PMV encapsulation of BMSC cytosol and can be instantly induced by a mechanical extrusions.When you are demonstrating this method it is critical as you're extruding and constructing steps are difficult to learn by testing to actually owning. To generate the PMVs, first culture mouse BMSCs in complete DMEM culture medium in one well of a six well plate in a humidified incubator at 37 degrees Celsius and 5%carbon dioxide. When the cells have reached 90%confluency, rinse the well with 0.5 milliliters of calcium free PBS and detach the cells with 0.5 milliliters of 0.25%trypsin-EDTA.After three minutes at 37 degrees Celsius, tap the plate with the palm of the hand a few times to facilitate cell dissociation and stop the digestion with one milliliter of culture medium. Transfer the cells into a five milliliter conical tube for centrifugation and resuspend the pellet in four milliliters of fresh culture medium. Then divide the cells between two wells of a new six well plate and return the plate to the cell culture incubator for 24 hours.The next day, harvest the cells from one well by trypsin digestion as just demonstrated. Resuspending the pellet in 0.5 milliliters of fresh culture medium after collection by centrifugation. Load the cells into a one milliliter insulin syringe and attach the syringe to a filter unit.Quickly depress the plunger to squeeze the cells through the filter, and discard the extruded medium. Then load and dispense 0.5 milliliters of extra culture medium through the filter as just demonstrated, collecting the medium from the second extrusion. Seed 150 microliters of the collected medium into a 35 milliliter glass bottom dish and allow the vesicles to settle for about 10 minutes.Then confirm the presence of PMVs under an inverted phase contrast microscope equipped with a CCD camera using a 20 times objective lens. To trace the mitochondrial enclosure and membrane potential in the PMVs, stain a 90%confluent BMSC culture with an appropriate mitochondrial dye or a mitochondrial membrane potential probe respectively in fresh culture medium for 30 minutes at 37 degrees Celsius. Then rinse the cells three times with 0.5 milliliters of PBS per wash and harvest the cells by trypsin digestion for PMV generation as just demonstrated for their visualization by confocal microscopy.To trace the cytoplasmic localization of the PMV proteins, first dilute two micrograms of supercoiled plasmid DNA for an EGFP expression cassette and six microliters of cationic transfection reagent in individual 0.5 milliliter tubes containing 50 microliters of serum free DMEM. Vortex the tubes, followed by a brief centrifugation. Then add the transfection reagent drop wise into the tube containing the diluted DNA solution.Vortex the DNA transfection mixture robustly for one minute, followed by a brief spin in a 10 minute incubation at room temperature. Next, feed a 30%confluent BMSC culture in a six well plate with two milliliters of fresh culture medium and add the DNA mixture drop wise to the cells. 48 hours after their transfection, harvest the BMSC by trypsin digestion and prepare PMVs as just demonstrated.The PMVs can then be analyzed by confocal microscopy using a 100 times oil objective. Before injecting the PMVs, label a BMSC culture with a membrane staining fluorescent cell tracker dye in one milliliter of fresh culture medium at 37 degrees Celsius for 30 minutes and harvest the cells by trypsin digestion. Resuspend the labeled cells in 0.5 milliliters of fresh culture medium supplemented with one microgram of polyethylene amine and prepare PMVs as demonstrated.Then concentrate the PMVs by centrifugation and resuspend the pellet in 100 microliters of fresh culture medium with gentle swirling. Next, load the cells into a one milliliter insulin syringe equipped with a 30 gauge needle and disinfect the outside of the gastrocnemius muscle of an anesthetized BALB/c mouse with 75%ethanol. Then slowly inject the entire volume of PMVs into the muscle and allow the animal to recover under a heat lamp.After 12 hours, sprinkle 75%ethanol over the muscle, open the skin with scissors, and harvest the entire gastrocnemius muscle at both ends. Rinse the muscle with PBS and place it under a fluorescence microscope. Using the 20 times objective lens, remove the non-fluorescing muscle tissue pieces with a sharp razor blade and mince the remaining strongly red fluorescing pieces into about nine cubic millimeter cubes.Embed each cube in an optimal cutting temperature medium. Then submerge the cubes in liquid nitrogen for five minutes. And store the tissue samples at minus 20 degrees Celsius until their analysis.PMVs generated from BMSCs as demonstrated predominantly exhibit a size in the range of about three micrometers in diameter as examined under phase contrast microscopy using the 20 times objective. BMSC transfection with a fluorescent expression cassette for 48 hours illustrates the cytoplasmic encapsulation of the EGFP protein by the PMVs. Further, standing with a fluorescent mitochondrial dye reveals that a large fraction of the PMVs contain mitochondria.The majority of which stain positive for a mitochondrial membrane potential dye, indicating the functionality of the mitochondria. Polyethylene amine treatment prior to their en vivo administration prevents PMV aggregation during their centrifugal concentration for cellular component removal. Membrane dye labeling of the BMSCs before PMV generation facilitates the generation of fluorescence trackable PMVs.Indeed, visualization of frozen sections of the gastrocnemius muscle after intramuscular delivery of labeled PMVs indicates PMV delivery to the muscle cell cytoplasm probably via endocytosis. When attempting the PMV generation procedure it's important to remember to load the syringe with the appropriate number of cells to depress the plunger quickly when flushing the cells through the filter. After its development, this technique paved the way for researchers in the field of stem cell therapy to explore the treatment of age related diseases in mice.After watching this video, you should have a good understanding of how to prepare PMV from BMSC for the delivery of cytoplasm into the gastrocnemius muscle of mice.

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