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>
	<li>Abe, A., Miyanohara, A., Friedmann, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+gene+transfer+with+fusogenic+liposomes+containing+vesicular+stomatitis+virus+G+glycoprotein.">Enhanced gene transfer with fusogenic liposomes containing vesicular stomatitis virus G glycoprotein.</a> J Virol. 72 (7), 6159-6163 (1998).</li><li>Dolatabadi, J., Valizadeh, H., Hamishehkar, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Solid+lipid+nanoparticles+as+efficient+drug+and+gene+delivery+systems:+recent+breakthroughs.">Solid lipid nanoparticles as efficient drug and gene delivery systems: recent breakthroughs.</a> Adv Pharm Bull. 5 (2), 151-159 (2015).</li><li>Torchilin, V. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multifunctional,+stimuli-sensitive+nanoparticulate+systems+for+drug+delivery.">Multifunctional, stimuli-sensitive nanoparticulate systems for drug delivery.</a> Nat Rev Drug Discov. 13 (11), 813-827 (2014).</li><li>Wolf, D. P., Mitalipov, N., Mitalipov, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+replacement+therapy+in+reproductive+medicine.">Mitochondrial replacement therapy in reproductive medicine.</a> Trends Mol Med. 21 (2), 68-76 (2015).</li><li>L&#243;pez-Otin, C., Blasco, M. A., Partridge, L., Serrano, M., Kroemer, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+hallmarks+of+aging.">The hallmarks of aging.</a> Cell. 153 (6), 1194-1217 (2013).</li><li>Plakkal, J., Paul, A. A., Goo, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+droplet-associated+proteins+in+atherosclerosis.">Lipid droplet-associated proteins in atherosclerosis.</a> Mol Med Rep. 13 (6), 4527-4534 (2016).</li><li>Hoppener, J. W. M., Ahren, B., Lips, C. J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Islet+amyloid+and+type+2+diabetes+mellitus.">Islet amyloid and type 2 diabetes mellitus.</a> N Engl J Med. 343 (6), 411-419 (2000).</li><li>Jagust, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Is+amyloid-&#946;+harmful+to+the+brain?+Insights+from+human+imaging+studies.">Is amyloid-&#946; harmful to the brain? Insights from human imaging studies.</a> Brain. 139 (Pt 1), 23-30 (2016).</li><li>Naidoo, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+endoplasmic+reticulum+stress+response+and+aging.">The endoplasmic reticulum stress response and aging.</a> Rev Neurosci. 20 (1), 23-37 (2009).</li><li>Cuervo, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+and+aging:+keeping+that+old+broom+working.">Autophagy and aging: keeping that old broom working.</a> Trends Genet. 24 (12), 604-612 (2008).</li><li>Bratic, A., Larsson, N. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+mitochondria+in+aging.">The role of mitochondria in aging.</a> J Clin Invest. 123 (3), 951-957 (2013).</li><li>Lin, H. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incorporation+of+VSV-G+produces+fusogenic+plasma+membrane+vesicles+capable+of+efficient+transfer+of+bioactive+macromolecules+and+mitochondria.">Incorporation of VSV-G produces fusogenic plasma membrane vesicles capable of efficient transfer of bioactive macromolecules and mitochondria.</a> Biomed Microdevices. 18 (3), 41 (2016).</li><li>Riekstina, U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+stem+cell+marker+expression+pattern+in+human+mesenchymal+stem+cells+derived+from+bone+marrow,+adipose+tissue,+heart+and+dermis.">Embryonic stem cell marker expression pattern in human mesenchymal stem cells derived from bone marrow, adipose tissue, heart and dermis.</a> Stem Cell Rev. 5 (4), 378-386 (2009).</li><li>Purandare, B., Teklemariam, T., Zhao, L. M., Hantash, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+HLA+profiling+and+immunomodulatory+effects+of+human+adult+bone+marrow-+and+adipose-derived+mesenchymal+stem+cells.">Temporal HLA profiling and immunomodulatory effects of human adult bone marrow- and adipose-derived mesenchymal stem cells.</a> Regen Med. 9 (1), 67-79 (2014).</li><li>Nemeth, K., Mayer, B., Sworder, B. J., Kuznetsov, S. A., Mezey, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+practical+guide+to+culturing+mouse+and+human+bone+marrow+stromal+cells.">A practical guide to culturing mouse and human bone marrow stromal cells.</a> Curr Protoc Immunol. 102, (2013).</li><li>Shahabipour, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosomes:+Nanoparticulate+tools+for+RNA+interference+and+drug+delivery.">Exosomes: Nanoparticulate tools for RNA interference and drug delivery.</a> J Cell Physiol. , (2017).</li><li>Lamichhane, T. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+roles+for+extracellular+vesicles+in+tissue+engineering+and+regenerative+medicine.">Emerging roles for extracellular vesicles in tissue engineering and regenerative medicine.</a> Tissue Eng B. 21 (1), 45-54 (2015).</li><li>Baumgart, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-scale+fluid/fluid+phase+separation+of+proteins+and+lipids+in+giant+plasma+membrane+vesicles.">Large-scale fluid/fluid phase separation of proteins and lipids in giant plasma membrane vesicles.</a> Proc Natl Acad Sci. 104 (9), 3165-3170 (2007).</li><li>Sezgin, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elucidating+membrane+structure+and+protein+behavior+using+giant+plasma+membrane+vesicles.">Elucidating membrane structure and protein behavior using giant plasma membrane vesicles.</a> Nat Protoc. 7 (6), 1042-1051 (2012).</li><li>Lingwood, D., Ries, J., Schwille, P., Simons, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasma+membranes+are+poised+for+activation+of+raft+phase+coalescence+at+physiological+temperature.">Plasma membranes are poised for activation of raft phase coalescence at physiological temperature.</a> Proc Natl Acad Sci. 105 (29), 10005-10010 (2008).</li><li>Pandey, A. P., Sawant, K. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polyethylenimine:+A+versatile,+multifunctional+non-viral+vector+for+nucleic+acid+delivery.">Polyethylenimine: A versatile, multifunctional non-viral vector for nucleic acid delivery.</a> Mater Sci Eng C Mater Biol Appl. 68, 904-918 (2016).</li></ol>

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 ...

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