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 25 mm filter unit and submerge the cap and bottom of the unit into a 200 mL glass cup filled with 75% ethanol for 30 min. The unit is made of medical-grade polypropylene.</li>
	<li>Pick up the cap and bottom of the unit using forceps, shake off the remaining ethanol by hand, and allow the parts to air dry for 10 min in the hood.</li>
	<li>Wet a 19 mm polycarbonate membrane in PBS and place it carefully on top of the supporting matrix of the bottom of the unit. The polymer film has a smooth, flat surface and track-etched 3 &#181;m pores.</li>
	<li>Reassemble the unit by screwing the cap tightly against the bottom. Make sure that the membrane is not dislodged from the center.</li>
	<li>Remove the needle of a 1 mL insulin syringe and draw 1 mL of PBS. Attach the syringe to the filter unit and then push PBS through the unit to wet the assembly and to test if there is any leakage.</li>
</ol>
2. Generation of Plasma Membrane Vesicles (PMVs)
<ol>
	<li>Establish a BMSC culture, as reported by Nemeth et al.15, in DMEM culture medium containing 15% FBS and 1% penicillin/streptomycin. Cultivate the cells in a 6-well plate in a humidified incubator containing 5% CO2 at 37 &#176;C.</li>
	<li>To propagate the cells, remove the culture medium and rinse the well with 0.5 mL of calcium-free PBS.</li>
	<li>Add 0.5 mL of 0.25% trypsin-EDTA and incubate the cells at 37 &#176;C for 3 min. Tap the plate against the palm of the hand a couple times to facilitate cell detachment. Stop digestion by adding 1 mL of culture medium.</li>
	<li>Collect the cells in a 15 mL conical tube and spin at 200 x g for 2 min in a bench top centrifuge. Resuspend the cells in 4 mL of culture medium and aliquot the cells into two wells of a 6-well plate. 
	NOTE: Cells usually reach confluence at around 24 h.</li>
	<li>To generate PMVs, harvest the cells from one well (about 5 x 105 cells) of a 6-well plate by trypsin digestion and monodisperse the cells in 0.5 mL of culture medium.</li>
	<li>Load the cells into the insulin syringe, attach it to the filter unit, and push the plunger quickly to squeeze the cells through the filter. Discard the extruded medium because there should only be a few PMVs inside it.</li>
	<li>Load an additional 0.5 mL of medium into the syringe and quickly push it through the filter. Collected the medium of the second extrusion, which contains PMVs of various size.</li>
	<li>Load 150 &#181;L of the collected medium into a 35-mm glass-bottom dish and allow the PMVs to settle to the bottom for about 10 min. Examine the PMVs under an inverted phase contrast microscope equipped with a CCD camera using the 20X objective lens.</li>
</ol>
3. Examination of the Content within the PMVs Using Confocal Microscopy
<ol>
	<li>To perform transfection in the BMSCs, dilute 2 &#181;g of supercoiled plasmid DNA and 6 &#181;L of cationic transfection reagent (e.g., PolyJet) in 50 &#181;L of serum-free DMEM. Vortex to mix well and spin briefly to collect all liquid from the sides of the tube.</li>
	<li>Add the diluted solution (from step 3.1) dropwise into diluted DNA solution, vortex strongly for 1 min, spin briefly, and incubate at room temperature for 10 min.</li>
	<li>Replace the old medium on the BMSCs, which have been seeded at about 30% confluence overnight, with 2 mL of fresh culture medium and add DNA transfection mixture dropwise.</li>
	<li>Replace with 2 mL of fresh medium after 12 h of transfection.</li>
	<li>To trace cytoplasmically localized proteins, transfect the cells with the plasmid containing an EGFP expression cassette (pEGFP-N1), as described above. Harvest the cells by trypsin digestion 48 h after transfection for PMV generation, as described above.</li>
	<li>To trace the mitochondria, stain the cells with mitochondrial dye (1 &#181;M, MitoTracker) in fresh culture medium for 30 min at 37 &#176;C.</li>
	<li>To detect the membrane potential of the mitochondria, stain the cells with the cyanine dye JC-1 (5,5&#39;,6,6&#39;-tetrachloro-1,1&#39;,3,3&#39;-tetraethylbenzimi-dazolylcarbocyanine iodide) (10 &#181;g/mL) in fresh culture medium for 30 min at 37 &#176;C.
	<ol>
		<li>Wash the cells with 0.5 mL of PBS three times and then harvest the cells by trypsin digestion for PMV generation, as described above.</li>
		<li>Load 150 &#181;L of the collected medium into a 35-mm glass-bottom dish and allow the PMVs to settle to the bottom for about 10 min. Examine the PMVs under a confocal microscope using the 100X oil objective lens.</li>
	</ol>
	</li>
	<li>To detect EGFP or the mitochondrial dye, turn on the 488-nm laser and collect signal at 505-530 nm. To detect JC-1, turn on the 488-nm and 543-nm lasers and collect signals at 505-530 nm and 560-615 nm. Set the pinhole value to around 1.4.</li>
</ol>
4. Preparation of Concentrated PMVs by Centrifugation
<ol>
	<li>Harvest the cells by trypsin digestion and generate PMVs, as described above, in 0.5 mL of culture medium.</li>
	<li>Spin the PMVs at 1,200 x g in a benchtop centrifuge for 5 min at room temperature. Carefully aspirate the supernatant and resuspend the pellet in 100 &#181;L of culture medium with gentle swirling.</li>
	<li>Load 50 &#181;L of PMVs into a 35 mm glass-bottom dish and allow them to settle at room temperature for about 10 min. Examine the PMVs under a confocal microscope using the 40X oil objective lens.</li>
	<li>Add the series of diluted polyethylenimine (PEI) solution to the BMSC culture for 1 h and check for signs of cytotoxicity. 
	NOTE: Here, the PEI at 2 &#181;g/mL was chosen since no sign of toxicity was detected in BMSCs.</li>
	<li>Harvest the cells by trypsin digestion and add PEI at 2 &#181;g/mL for 1 h to charge the cell membrane as a means of preventing aggregation. Generate PMVs, as described above, in 0.5 mL of culture medium. Concentrate the PMVs and examine them under a confocal microscope, as described above.</li>
</ol>
5. Injection of PMVs into the Gastrocnemius Muscle
<ol>
	<li>To stain the membrane, add CM-DiI (chloromethyl dialkyl-indocarbocyanine) (10 &#956;M) dye in 1 mL of fresh culture medium to the BMSCs for 30 min.</li>
	<li>Harvest the cells (about 5 x 105) by trypsin digestion and resuspend them in 0.5 mL of culture medium. Add PEI (2 &#181;g/mL) for 1 h. Generate and concentrate the PMVs in 100 &#181;L of culture medium, as described above.</li>
	<li>Anaesthetize a BALB/c mouse by injecting sodium barbital (10 mg/kg) after 12 h of fasting.</li>
	<li>Clean the outside of the gastrocnemius muscle with 75% ethanol. Slowly inject the PMVs (100 &#181;L) into the gastrocnemius muscle using a 30 G insulin syringe.</li>
	<li>After administering the anesthesia, place a wet gauze over the eyes for the duration of the experiment and warm the mouse with an incandescent light until it wakes up. House the mouse alone in an individual ventilation cage.</li>
	<li>To harvest the gastrocnemius muscle 12 h post-operation, kill the mouse by cervical dislocation.</li>
	<li>Sprinkle 75% ethanol over the gastrocnemius muscle, open the skin with scissors, and cut out the entire gastrocnemius muscle at both ends.</li>
	<li>Rinse the muscle with PBS and examine it under a fluorescence microscope using the 20X objective lens.</li>
	<li>Trim the muscle with a sharp razor blade to remove of the parts with no fluorescence. Cut the parts with strong red fluorescence into about 9 mm3 cubes.</li>
	<li>Embed each cube in optimal cutting temperature medium (OCT), submerge it in liquid nitrogen for 5 min, and store it in a -20 &#176;C freezer until use.</li>
	<li>Cut the frozen sections into pieces 20 &#181;m thick using a cryostat and adhere each section to a glass slide. Rinse the section once with PBS.</li>
	<li>Draw a circle around the section with a stain circle pen and add 100 &#181;L of DAPI (1 &#181;g/mL). Aspirate the DAPI solution after 10 min of incubation at room temperature in the dark and wash three times with PBS.</li>
	<li>Drop mounting oil over the specimen, cover it with a glass slip, and seal the slide with nail polish. Examine the section under a confocal microscope using the 100X oil objective lens.</li>
	<li>To detect CM-DiI, turn on the 543 nm laser and collect signal at 560-615 nm. To detect DAPI, turn on the 405 nm laser and collect signal at 420-480 nm. Set the pinhole value to about 1.4.</li>
</ol>

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The authors have nothing to disclose.

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 invigorated to provide a long-lasting effect. Additionally, exogenous mitochondria might have a faster proliferation rate, as indicated in our previous study12. The replacement of damaged mitochondria is essential because mitochondrial dysfunction has been linked to many senescent diseases11. In addition, unlike stem cells, the enucleated PMVs are not be able to proliferate; thus, it is unlikely to raise the concern of cancer development. Lastly, the lack of nuclear DNA in PMVs also reduces the risk of genome damage by DNA random integration. 
Cultivated cells produce a variety of membranous extracellular vesicles (EV) under normal or stress conditions. Exosomes originate from late endosomes and are released by most mammalian cells as extracellular vesicles of 30-120 nm in diameter16. Larger EVs, called microvesicles, have a size ranging from 0.1-1 &#181;m and are directly shed from the plasma membranes of cells17. Microvesicles and exosomes may have overlapping cellular constituents and functionalities in tissue repair. On the other hand, giant plasma membrane vesicles18,19 and similarly, plasma membrane spheres20 about 30 &#181;m in size can be generated from multiple cell types by chemical induction or cell swelling. However, the application of these cell-size vesicles in therapeutics is still largely undeveloped. In any event, the production of EVs are time consuming, and, unlike PMVs, the contents within EVs are limited by the obviously absent cellular organelles. 
This manuscript describes the use of PEI to prevent the aggregation of PMVs after centrifugation. PEI is a potent non-viral gene delivery carrier due to its capability of condensing DNA using multiple positively charged amino groups. A concentration of 2 &#181;g/mL of PEI, which showed no sign of cytotoxic effect, was chosen to positively charge the cell surface and consequently resulted in the superior dispersion of PMVs. Moreover, the binding of PEI to the surface might help with the endocytosis of PMVs. Once inside of the cells, the further protonation of the amino groups in low pH can lead to endosomal swelling and bursting21. 
However, the PMVs generated here are far from perfect. The 10-fold reduction in diameter, for example, from 30 &#181;m (BMSCs) to 3 &#181;m (PMVs) could theoretically lead to a 90% loss of cellular content. Even worse, a large fraction of PMVs have a diameter smaller than 3 &#181;m. Furthermore, substantial cellular debris was detected during the squeezing process, probably due to the stiffness of the cell membrane, which was caused by the presence of integrated membrane proteins and lipids such as cholesterol. It is possible, however, that the flexibility of the cell membranes could be improved by incorporating fatty acids with short side chains, such as Octyl-beta-Glucoside. 
Another reason for the generation of cellular debris is the presence of the cytoskeleton, which may also hinder the inclusion of organelles into the PMVs. This might explain that some of the PMVs, even those that have a diameter of 3 &#181;m or larger, have no detectable mitochondria. Since most of the organelles are attached to the polymerized cytoskeleton, it would be interesting to test if blocking the polymerization of the cytoskeleton could improve the encapsulation of organelles. 
In addition, the in vivo application of PMVs must overcome a myriad of hurdles. For example, to target hepatocytes, PMVs must avoid being captured, not only by the scavenger system in the circulation, but also by Kupffer cells that line the sinus of the liver. A method to facilitate the migration of PMVs from the circulation to target tissues may also be required. Nonetheless, in the face of these many caveats, the advantage of using PMVs for therapy is outstanding. We showed that PMVs were readily endocytosed into the myofiber of the gastrocnemius muscle after an intramuscular injection. We are currently testing to see if a dystrophic gastrocnemius muscle induced by hydrogen peroxide can be recused by PMV medication.

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><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>&amp;nbsp;6085-1</td><td></td></tr><tr><td>MitoTracker</td><td>Molecular Probes</td><td>M7514</td><td>Green FM, 1 &amp;mu;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 = 75,000</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, that would indicate that most of the cells are trapped inside the unit due to incorrect assembly. PMVs with a size in the range of about 3 &#181;m in diameter were predominant when examined under a phase contrast microscope using the 20X objective (Figure 2). Since PMVs of nanometer-size were not easily visualized, the percentage of large PMVs in terms of number was low. It was found to be helpful to press the syringe quickly to reduce the amount of membrane debris, which apparently fails to form spherical PMVs. Furthermore, PMVs of smaller sizes (less than 1 &#181;m) were found in the first extrusion medium, with larger PMVs recovered in the second extrusion.
The most unique aspect of the PMVs was the enclosing of cellular components aside from the nucleus12. BMSCs were transfected with an expression cassette of EGFP for 48 h before being used for PMV preparation. The results show that cytoplasmically localized EGFP protein can be encapsulated in PMVs (Figure 3A). The enclosure of mitochondria was evidenced by the staining of mitochondrial dye, showing that a large fraction of, but not all, PMVs contain green fluorescent dots, indicative of mitochondria (Figure 3). Some of the PMVs, even those with a diameter as large as 3 &#181;m, did not show evidence of containing mitochondria, suggesting that some mechanical constraints might prevent the encapsulation of mitochondria into PMVs. The membrane potential of mitochondria was detected by JC-1 staining, which emits red fluorescence when mitochondria have a normal potential. The result show that red fluorescence indeed was detected in PMVs (Figure 3), while the green fluorescence was not detectable (data not shown), suggesting that the mitochondria within the PMVs were functional.
A concentration process for PMVs is needed, especially for in vivo application to reduce the volume as well as to remove the cellular components released during extrusion. After centrifugation at 1,200 x g for 5 min at room temperature, very few PMVs of smaller size were found in the supernatant. To facilitate the resuspension of the pellet, a 2-mL round-bottom tube was used for the centrifugation. However, most of the PMVs were aggregated in the pellet, especially PMVs of smaller sizes (Figure 4). This is probably due to adhesion molecules on the membrane. The medium was therefore supplemented with PEI (2 &#181;g/mL), whose concentration was tailored to have no noticeable cytotoxicity. The positive charge of PEI attached to the cell membrane prevented the aggregation of PMVs during centrifugation (Figure 4).
Finally, the membranes of BMSCs were labeled with CM-DiI dye before the preparation of the PMVs (Figure 5). After concentration, PMVs were injected into the gastrocnemius muscles of mice, which were harvested 12 h post-operation and examined under a confocal microscope. Frozen sections of the gastrocnemius muscle were examined for the presence of red fluorescence, which was detected within the myofiber (Figure 5), demonstrating that PMVs were delivered to the cytoplasm, probably via endocytosis. Of note, red fluorescence was found around the periphery of the myofiber (data not shown), indicating that a large fraction of PMVs were either not endocytosed or were just at the beginning of endocytosis at 12 h post-injection.
<img alt="Figure 1" src="/files/ftp_upload/55741/55741fig1.jpg" /> 
Figure 1: Assembly of the filter unit. (A) Commercially available polycarbonate membranes. (B) Track-etched pores of 3 &#181;m in diameter, viewed under an optical microscope with a 20X objective. Scale bar = 50 &#181;m. (C) The membrane was placed on top of the supporting matrix of a disposable filter unit. (D) The assembled filter unit with a membrane inside. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55741/55741fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/55741/55741fig2.jpg" /> 
Figure 2: Generation of PMVs from BMSCs. (A) BMSCs from mice were cultivated in a 6-well plate. (B) BMSCs (5 x 105) were harvested by trypsin digestion and monodispersed in 500 &#181;L of culture medium. Images of attached and resuspended BMSCs were taken under an optical microscope with a 20X objective. (C) Cells were loaded into an insulin syringe before being attached to the filter unit. (D) PMVs generated from BMSCs were loaded into a 35 mm glass-bottom dish and examined under an inverted phase contrast microscope using a 20X objective lens. Scale bars = 40 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55741/55741fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/55741/55741fig3.jpg" /> 
Figure 3: Detection of PMV content by confocal microscopy. PMVs were generated from (A) BMSCs transfected with pEGFP-N1 for 48 h to trace the cytoplasmically localized EGFP protein, (B) BMSCs stained with mitochondrial dye (1 &#181;M) for 30 min to trace the existence of mitochondria, or (C) BMSCs staining with JC-1 (10 &#181;g/mL) for 30 min to confirm the membrane potential of mitochondria. PMVs generated from BMSCs were loaded onto a 35 mm glass-bottom dish and examined by confocal microscopy using a 100X oil objective lens. Scale bars = 10 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55741/55741fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/55741/55741fig4.jpg" /> 
Figure 4: Concentration of PMVs by centrifugation. BMSCs (5 x 105) were harvested by trypsin digestion and resuspended in 0.5 mL of culture medium, which was supplemented with or without 2 &#181;g/mL of PEI. Generated PMVs were spun at 1,200 g for 5 min, resuspended in 100 &#181;L of culture medium, and examined under a confocal microscope with a 40X oil objective. (A) Schematic of PMV aggregation probably caused by the interaction of adhesion molecules after centrifugation. (B) Schematic of PMVs maintaining monodispersion after being charged with PEI. (C) Aggregated PMVs after centrifugation. (D) PMVs show less aggregation after centrifugation due to positive charging with PEI. Scale bars = 10 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55741/55741fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/55741/55741fig5.jpg" /> 
Figure 5: Delivery of CM-DiI-labeled PMVs to the gastrocnemius muscle. PMVs were generated from 5 x 105 BMSCs, concentrated by centrifugation into a volume of 100 &#181;L, and injected slowly into the gastrocnemius muscle of a BALB/c mouse. (A) Schematic of PMV injection. (B) BMSCs labeled with CM-DiI; Scale bar = 20 &#181;m. (C) PMVs generated from CM-DiI-labeled BMSCs and examined by confocal microscopy; Scale bar = 10 &#181;m. (D) Gastrocnemius muscle harvested 12 h post-operation; Scale bar = 20 &#181;m. (E-H) Frozen section of the gastrocnemius muscle examined by confocal microscopy; Scale bar = 10 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55741/55741fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

preparation-plasma-membrane-vesicles-from-bone-marrow-mesenchymal

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

7.5K Views.  Shantou University.  Leeftijdgerelateerde aandoeningen zijn geassocieerd met meerdere defecten in componenten van het cytoplasma. Hier presenteren we een protocol om plasmamembraan vesicles uit mesmermale stamcellen van beenmerg te bereiden. Deze techniek kan potentieel gebruikt worden als middel voor het vervangen van cytoplasma ter verbetering van de leeftijd-geassocieerde fenotypen. 

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

Isolation of Human Mesenchymal Stem Cells and their Cultivation on the Porous Bone Matrix

Mesenchymal stem cells (MSCs) have a differentiation potential towards osteoblastic lineage when they are stimulated with soluble factors or specific biomaterials. This work presents a novel option for the delivery of MSCs from human amniotic membrane (AM-hMSCs) that employs bovine bone matrix Nukbone (NKB) as a scaffold. Thus, the application of MSCs in repair and tissue regeneration processes depends principally on the efficient implementation of the techniques for placing these cells in a host tissue. For this reason, the design of biomaterials and cellular scaffolds has gained importance in recent years because the topographical characteristics of the selected scaffold must ensure adhesion, proliferation and differentiation into the desired cell lineage in the microenvironment of the injured tissue. This option for the delivery of MSCs from human amniotic membrane (AM-hMSCs) employs bovine bone matrix as a cellular scaffold and is an efficient culture technique because the cells respond to the topographic characteristics of the bovine bone matrix Nukbone (NKB), i.e., spreading on the surface, macroporous covering and colonizing the depth of the biomaterial, after the cell isolation process. We present the procedure for isolating and culturing MSCs on a bovine matrix.

Mesenchymal stem cells (MSCs) have a differentiation potential towards osteoblastic lineage when they are stimulated with soluble factors or specific biomaterials. This work presents a novel option for the delivery of MSCs from human amniotic membrane (AM-hMSCs) that employs the bovine bone matrix Nukbone (NKB) as a scaffold.

Mesenchymal stem cells (MSCs) have a differentiation potential towards osteoblastic lineage when they are stimulated with soluble factors or specific ...

An Enzymatic Method to Rescue Mesenchymal Stem Cells from Clotted Bone Marrow Samples

Mesenchymal stem cells (MSCs) - usually obtained from bone marrow - often require expansion culture. Our protocol uses clinical grade urokinase to degrade clots in the bone marrow and release MSCs for further use. This protocol provides a rapid and inexpensive alternative to bone marrow resampling. Bone marrow is a major source of MSCs, which are interesting for tissue engineering and autologous stem cell therapies. Upon withdrawal bone marrow may clot, as it comprises all of the hematopoietic system. The resulting clots contain also MSCs that are lost for expansion culture or direct stem cell therapy. We experienced that 74% of canine bone marrow samples contained clots and yielded less than half of the stem cell number expected from unclotted samples. Thus, we developed a protocol for enzymatic digestion of those clots to avoid labor-intense and costly bone marrow resampling. Urokinase - a clinically approved and readily available thrombolytic drug &#8211; clears away the bone marrow clots almost completely. As a consequence, treated bone marrow aspirates yield similar numbers of MSCs as unclotted samples. Also, after urokinase treatment the cells kept their metabolic activity and the ability to differentiate into chondrogenic, osteogenic and adipogenic lineages. Our protocol salvages clotted blood and bone marrow samples without affecting the quality of the cells. This obsoletes resampling, considerably reduces sampling costs and enables the use of clotted samples for research or therapy.

Mesenchymal stem cells are usually obtained from bone marrow and require expansion culture. When samples clot before processing, a protocol using the (enzymatic) thrombolytic drug urokinase can be applied to degrade the clot. Thus, cells are released and available for expansion culture. This protocol provides a rapid and inexpensive alternative to resampling.

Mesenchymal stem cells (MSCs) - usually obtained from bone marrow - often require expansion culture. Our protocol uses clinical grade urokinase to degrade ...

Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

Stem cell transplantation protocols are finding their way into clinical practice1,2,3. Getting better results, making the protocols more robust, and finding new sources for implantable cells are the focus of recent research4,5. Investigating the effectiveness of cell therapies is not an easy task and new tools are needed to investigate the mechanisms involved in the treatment process6. We designed an experimental protocol of ischemia/reperfusion in order to allow the observation of cellular connections and even subcellular mechanisms during ischemia/reperfusion injury and after stem cell transplantation and to evaluate the efficacy of cell therapy. H9c2 cardiomyoblast cells were placed onto cell culture plates7,8. Ischemia was simulated with 150 minutes in a glucose free medium with oxygen level below 0.5%. Then, normal media and oxygen levels were reintroduced to simulate reperfusion. After oxygen glucose deprivation, the damaged cells were treated with transplantation of labeled human bone marrow derived mesenchymal stem cells by adding them to the culture. Mesenchymal stem cells are preferred in clinical trials because they are easily accessible with minimal invasive surgery, easily expandable and autologous. After 24 hours of co-cultivation, cells were stained with calcein and ethidium-homodimer to differentiate between live and dead cells. This setup allowed us to investigate the intercellular connections using confocal fluorescent microscopy and to quantify the survival rate of postischemic cells by flow cytometry. Confocal microscopy showed the interactions of the two cell populations such as cell fusion and formation of intercellular nanotubes. Flow cytometry analysis revealed 3 clusters of damaged cells which can be plotted on a graph and analyzed statistically. These populations can be investigated separately and conclusions can be drawn on these data on the effectiveness of the simulated therapeutical approach.

Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

We demonstrate how to set up an in vitro ischemia/reperfusion model and how to evaluate the effect of stem cell therapy on postischemic cardiac cells.

Stem cell transplantation protocols are finding their way into clinical practice1,2,3. Getting better results, making the protocols more robust, and ...

Medicine

Isolating Mesangiogenic Progenitor Cells (MPCs) from Human Bone Marrow

In a research study aimed to isolate human bone marrow (hBM)-derived Mesenchymal Stromal Cells (MSCs) for clinical applications, we identified a novel cell population specifically selected for growth in human serum supplemented medium. These cells are characterized by morphological, phenotypic, and molecular features distinct from MSCs and we named them Mesodermal Progenitor Cells (MPCs). MPCs are round, with a thick highly refringent core region; they show strong, trypsin resistant adherence to plastic. Failure to expand MPCs directly revealed that they are slow in cycling. This is as also suggested by Ki-67 negativity. On the other hand, culturing MPCs in standard medium designed for MSC expansion, gave rise to a population of exponentially growing MSC-like cells. Besides showing mesenchymal differentiation capacity MPCs retained angiogenic potential, confirming their multiple lineage progenitor nature. Here we describe an optimized highly reproducible protocol to isolate and characterize hBM-MPCs by flow cytometry (CD73, CD90, CD31, and CD45), nestin expression, and F-actin organization. Protocols for mesengenic and angiogenic differentiation of MPCs are also provided. Here we also suggest a more appropriate nomenclature for these cells, which has been re-named as "Mesangiogenic Progenitor Cells".

Isolating Mesangiogenic Progenitor Cells MPCs from Human Bone Marrow

Here we describe an optimized, highly reproducible protocol to isolate Mesodermal Progenitor Cells (MPCs) from human bone marrow (hBM). MPCs were characterized by flow cytometry and nestin expression. They showed the ability to give rise to exponentially growing MSC-like cell cultures while retaining their angiogenic potential.

In a research study aimed to isolate human bone marrow (hBM)-derived Mesenchymal Stromal Cells (MSCs) for clinical applications, we identified a novel ...

Treatment of Osteochondral Defects in the Rabbit's Knee Joint by Implantation of Allogeneic Mesenchymal Stem Cells in Fibrin Clots

The treatment of osteochondral articular defects has been challenging physicians for many years. The better understanding of interactions of articular cartilage and subchondral bone in recent years led to increased attention to restoration of the entire osteochondral unit. In comparison to chondral lesions the regeneration of osteochondral defects is much more complex and a far greater surgical and therapeutic challenge. The damaged tissue does not only include the superficial cartilage layer but also the subchondral bone. For deep, osteochondral damage, as it occurs for example with osteochondrosis dissecans, the full thickness of the defect needs to be replaced to restore the joint surface 1. Eligible therapeutic procedures have to consider these two different tissues with their different intrinsic healing potential 2. In the last decades, several surgical treatment options have emerged and have already been clinically established 3-6.
Autologous or allogeneic osteochondral transplants consist of articular cartilage and subchondral bone and allow the replacement of the entire osteochondral unit. The defects are filled with cylindrical osteochondral grafts that aim to provide a congruent hyaline cartilage covered surface 3,7,8. Disadvantages are the limited amount of available grafts, donor site morbidity (for autologous transplants) and the incongruence of the surface; thereby the application of this method is especially limited for large defects.
New approaches in the field of tissue engineering opened up promising possibilities for regenerative osteochondral therapy. The implantation of autologous chondrocytes marked the first cell based biological approach for the treatment of full-thickness cartilage lesions and is now worldwide established with good clinical results even 10 to 20 years after implantation 9,10. However, to date, this technique is not suitable for the treatment of all types of lesions such as deep defects involving the subchondral bone 11.
The sandwich-technique combines bone grafting with current approaches in Tissue Engineering 5,6. This combination seems to be able to overcome the limitations seen in osteochondral grafts alone. After autologous bone grafting to the subchondral defect area, a membrane seeded with autologous chondrocytes is sutured above and facilitates to match the topology of the graft with the injured site. Of course, the previous bone reconstruction needs additional surgical time and often even an additional surgery. Moreover, to date, long-term data is missing 12.
Tissue Engineering without additional bone grafting aims to restore the complex structure and properties of native articular cartilage by chondrogenic and osteogenic potential of the transplanted cells. However, again, it is usually only the cartilage tissue that is more or less regenerated. Additional osteochondral damage needs a specific further treatment. In order to achieve a regeneration of the multilayered structure of osteochondral defects, three-dimensional tissue engineered products seeded with autologous/allogeneic cells might provide a good regeneration capacity 11.
Beside autologous chondrocytes, mesenchymal stem cells (MSC) seem to be an attractive alternative for the development of a full-thickness cartilage tissue. In numerous preclinical in vitro and in vivo studies, mesenchymal stem cells have displayed excellent tissue regeneration potential 13,14. The important advantage of mesenchymal stem cells especially for the treatment of osteochondral defects is that they have the capacity to differentiate in osteocytes as well as chondrocytes. Therefore, they potentially allow a multilayered regeneration of the defect.
In recent years, several scaffolds with osteochondral regenerative potential have therefore been developed and evaluated with promising preliminary results 1,15-18. Furthermore, fibrin glue as a cell carrier became one of the preferred techniques in experimental cartilage repair and has already successfully been used in several animal studies 19-21 and even first human trials 22.
The following protocol will demonstrate an experimental technique for isolating mesenchymal stem cells from a rabbit's bone marrow, for subsequent proliferation in cell culture and for preparing a standardized in vitro-model for fibrin-cell-clots. Finally, a technique for the implantation of pre-established fibrin-cell-clots into artificial osteochondral defects of the rabbit's knee joint will be described.

An experimental technique for the treatment of osteochondral defects in the rabbit's knee joint is described. The implantation of allogeneic mesenchymal stem cells into osteochondral defects provides a promising development in the field of tissue engineering. The preparation of fibrin-cell-clots in vitro offers a standardized method for implantation.

The treatment of osteochondral articular defects has been challenging physicians for many years. The better understanding of interactions of articular ...

Mesenchymal Stromal Cell Culture and Delivery in Autologous Conditions: A Smart Approach for Orthopedic Applications

Human Mesenchymal Stromal Cells (hMSCs) are cultured in vitro with different media. Limits on their use in clinical settings, however, mainly depend on potential biohazard and inflammation risks exerted by xenogeneic nutrients for their culture. Human derivatives or recombinant materials are the first choice candidates to reduce these reactions. Therefore, culture supplements and materials of autologous origin represent the best nutrients and the safest products.
Here, we describe a new protocol for the isolation and culture of bone marrow hMSCs in autologous conditions &#8212;&#160;namely,&#160;patient-derived serum as a supplement for the culture medium and fibrin as a scaffold for hMSC administration. Indeed, hMSC/fibrin clot constructs could be extremely useful for several clinical applications. In particular, we focus on their use in orthopedic surgery, where the fibrin clot derived from the donor&#39;s own blood allowed effective cell delivery and nutrient/waste exchanges. To ensure optimal safety conditions, it is of the utmost importance to avoid the risks of hMSC transformation and tissue overgrowth. For these reasons, the approach described in this paper also indicates a minimally ex vivo hMSC expansion, to reduce cell senescence and morphologic changes, and short-term osteo-differentiation before implantation, to induce osteogenic lineage specification, thus decreasing the risk of subsequent uncontrolled proliferation.

Culturing human Mesenchymal Stromal Cells (hMSCs) with autologous serum, reduces the risks of rejection by xenogeneic material and other negative effects. It also allows for the recovery of a subset of mesodermal progenitors, which can deliver fresh hMSCs. Embedding hMSCs in an autologous fibrin clot enables easy handling and effective surgical implantation.

Human Mesenchymal Stromal Cells (hMSCs) are cultured in vitro with different media. Limits on their use in clinical settings, however, mainly depend on ...

Bioengineering

Recombinant Collagen I Peptide Microcarriers for Cell Expansion and Their Potential Use As Cell Delivery System in a Bioreactor Model

Tissue engineering is a promising field, focused on developing solutions for the increasing demand on tissues and organs regarding transplantation purposes. The process to generate such tissues is complex, and includes an appropriate combination of specific cell types, scaffolds, and physical or biochemical stimuli to guide cell growth and differentiation. Microcarriers represent an appealing tool to expand cells in a three-dimensional (3D) microenvironment, since they provide higher surface-to volume ratios and mimic more closely the in vivo situation compared to traditional two-dimensional methods. The vascular system, supplying oxygen and nutrients to the cells and ensuring waste removal, constitutes an important building block when generating engineered tissues. In fact, most constructs fail after being implanted due to lacking vascular support. In this study, we present a protocol for endothelial cell expansion on recombinant collagen-based microcarriers under dynamic conditions in spinner flask and bioreactors, and we explain how to determine in this setting cell viability and functionality. In addition, we propose a method for cell delivery for vascularization purposes without additional detachment steps necessary. Furthermore, we provide a strategy to evaluate the cell vascularization potential in a perfusion bioreactor on a decellularized&#160;biological matrix. We believe that the use of the presented methods could lead to the development of new cell-based therapies for a large range of tissue engineering applications in the clinical practice.

We propose a cell expansion protocol on macroporous microcarriers and their use as delivery system in a perfusion bioreactor to seed a decellularized tissue matrix. We also include different techniques to determine cell proliferation and viability of cells cultured on microcarriers. Furthermore, we demonstrate functionality of cells after bioreactor cultures.

Tissue engineering is a promising field, focused on developing solutions for the increasing demand on tissues and organs regarding transplantation ...

Protocol for MicroRNA Transfer into Adult Bone Marrow-derived Hematopoietic Stem Cells to Enable Cell Engineering Combined with Magnetic Targeting

While CD133+ hematopoietic stem cells (SCs) have been proven to provide high potential in the field of regenerative medicine, their low retention rates after injection into injured tissues as well as the observed massive cell death rates lead to very restricted therapeutic effects. To overcome these limitations, we sought to establish a non-viral based protocol for suitable cell engineering prior to their administration. The modification of human CD133+ expressing SCs using microRNA (miR) loaded magnetic polyplexes was addressed with respect to uptake efficiency and safety as well as the targeting potential of the cells. Relying on our protocol, we can achieve high miR uptake rates of 80&#8211;90% while the CD133+ stem cell properties remain unaffected. Moreover, these modified cells offer the option of magnetic targeting. We describe here a safe and highly efficient procedure for the modification of CD133+ SCs. We expect this approach to provide a standard technology for optimization of therapeutic stem cell effects and for monitoring of the administered cell product via magnetic resonance imaging (MRI).

This protocol illustrates a safe and efficient procedure to modify CD133+ hematopoietic stem cells. The presented non-viral, magnetic polyplex-based approach may provide a basis for the optimization of therapeutic stem cell effects as well as for monitoring the administered cell product via magnetic resonance imaging.

While CD133+ hematopoietic stem cells (SCs) have been proven to provide high potential in the field of regenerative medicine, their low retention rates ...

Isolation and Identification of Porcine Bone Marrow Mesenchymal Stem Cells and their Derived Extracellular Vesicles

With the development of stem cell therapy in translational research and regenerative medicine, bone marrow mesenchymal stem cells (BM-MSCs), as a kind of pluripotent stem cells, are favored for their instant availability and proven safety. It has been reported that transplantation of BM-MSCs is of great benefit to repairing injured tissues in various diseases, which might be related to modulating the immune and inflammatory responses via paracrine mechanisms. Extracellular vesicles (EVs), featuring a double-layer lipid membrane structure, are considered to be the main mediators of the paracrine effects of stem cells. Recognized for their crucial roles in cell communication and epigenetic regulation, EVs have already been applied in vivo for immunotherapy. However, similar to its maternal cells, most of the studies on the efficacy of transplantation of EVs still remain at the level of small animals, which is not enough to provide essential evidence for clinical translation. Here, we use density-gradient centrifugation to isolate bone marrow cells (BMC) from porcine bone marrow at first, and get porcine BM-MSCs (pBM-MSCs) by cell culture subsequently, identified by the results of observation under the microscope, induced differentiation assay, and flow cytometry. Furthermore, we isolate EVs derived from pBM-MSCs in cell supernatant by ultracentrifugation, proved by the techniques of transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and western blotting successfully. Overall, pBM-MSCs and their derived EVs can be isolated and identified effectively by the following protocols, which might be widely used in pre-clinical studies on the transplantation efficacy of BM-MSCs and their derived EVs.

This article elaborates a method to isolate and identify porcine bone marrow mesenchymal stem cells (pBM-MSCs) and extracellular vesicles (EVs) derived from them, providing a methodological basis for the pre-clinical evaluation of transplantation efficacy of BM-MSCs and their derived EVs.

With the development of stem cell therapy in translational research and regenerative medicine, bone marrow mesenchymal stem cells (BM-MSCs), as a kind of ...

Isolation, Characterization, and Therapeutic Application of Extracellular Vesicles from Cultured Human Mesenchymal Stem Cells

Extracellular vesicles (EVs) are heterogeneous membrane nanoparticles released by most cell types, and they are increasingly recognized as physiological regulators of organismal homeostasis and important indicators of pathologies; in the meantime, their immense potential to establish accessible and controllable disease therapeutics is emerging. Mesenchymal stem cells (MSCs) can release large amounts of EVs in culture, which have shown promise to jumpstart effective tissue regeneration and facilitate extensive therapeutic applications with good scalability and reproducibility. There is a growing demand for simple and effective protocols for collecting and applying MSC-EVs. Here, a detailed protocol is provided based on differential centrifugation to isolate and characterize representative EVs from cultured human MSCs, exosomes, and microvesicles for further applications. The adaptability of this method is shown for a series of downstream approaches, such as labeling, local transplantation, and systemic injection. The implementation of this procedure will address the need for simple and reliable MSC-EVs collection and application in translational research.

The present protocol describes the differential centrifugation for isolating and characterizing representative EVs (exosomes and microvesicles) from cultured human MSCs. Further applications of these EVs are also explained in this article.

Extracellular vesicles (EVs) are heterogeneous membrane nanoparticles released by most cell types, and they are increasingly recognized as physiological ...

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

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