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

Summary

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.

Abstract

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'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 µm 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.

Introduction

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 applications^1,2,3. 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'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 diseases^{5,6,7,8,9,10,11}. 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 "off-the-shelf," 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-µm 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.

Protocol

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

1. To ensure sterility, turn on the UV light of a tissue culture hood for 30 min before use.
2. 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.
3. 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.
4. 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 µm pores.
5. Reassemble the unit by screwing the cap tightly against the bottom. Make sure that the membrane is not dislodged from the center.
6. 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.

2. Generation of Plasma Membrane Vesicles (PMVs)

1. 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 °C.
2. To propagate the cells, remove the culture medium and rinse the well with 0.5 mL of calcium-free PBS.
3. Add 0.5 mL of 0.25% trypsin-EDTA and incubate the cells at 37 °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.
4. 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.
5. 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.
6. 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.
7. 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.
8. Load 150 µL 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.

3. Examination of the Content within the PMVs Using Confocal Microscopy

1. To perform transfection in the BMSCs, dilute 2 µg of supercoiled plasmid DNA and 6 µL of cationic transfection reagent (e.g., PolyJet) in 50 µL of serum-free DMEM. Vortex to mix well and spin briefly to collect all liquid from the sides of the tube.
2. 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.
3. 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.
4. Replace with 2 mL of fresh medium after 12 h of transfection.
5. 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.
6. To trace the mitochondria, stain the cells with mitochondrial dye (1 µM, MitoTracker) in fresh culture medium for 30 min at 37 °C.
7. To detect the membrane potential of the mitochondria, stain the cells with the cyanine dye JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimi-dazolylcarbocyanine iodide) (10 µg/mL) in fresh culture medium for 30 min at 37 °C.
   1. 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.
   2. Load 150 µL 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.
8. 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.

4. Preparation of Concentrated PMVs by Centrifugation

1. Harvest the cells by trypsin digestion and generate PMVs, as described above, in 0.5 mL of culture medium.
2. 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 µL of culture medium with gentle swirling.
3. Load 50 µL 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.
4. 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 µg/mL was chosen since no sign of toxicity was detected in BMSCs.
5. Harvest the cells by trypsin digestion and add PEI at 2 µg/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.

5. Injection of PMVs into the Gastrocnemius Muscle

1. To stain the membrane, add CM-DiI (chloromethyl dialkyl-indocarbocyanine) (10 μM) dye in 1 mL of fresh culture medium to the BMSCs for 30 min.
2. Harvest the cells (about 5 x 10⁵) by trypsin digestion and resuspend them in 0.5 mL of culture medium. Add PEI (2 µg/mL) for 1 h. Generate and concentrate the PMVs in 100 µL of culture medium, as described above.
3. Anaesthetize a BALB/c mouse by injecting sodium barbital (10 mg/kg) after 12 h of fasting.
4. Clean the outside of the gastrocnemius muscle with 75% ethanol. Slowly inject the PMVs (100 µL) into the gastrocnemius muscle using a 30 G insulin syringe.
5. 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.
6. To harvest the gastrocnemius muscle 12 h post-operation, kill the mouse by cervical dislocation.
7. Sprinkle 75% ethanol over the gastrocnemius muscle, open the skin with scissors, and cut out the entire gastrocnemius muscle at both ends.
8. Rinse the muscle with PBS and examine it under a fluorescence microscope using the 20X objective lens.
9. 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.
10. Embed each cube in optimal cutting temperature medium (OCT), submerge it in liquid nitrogen for 5 min, and store it in a -20 °C freezer until use.
11. Cut the frozen sections into pieces 20 µm thick using a cryostat and adhere each section to a glass slide. Rinse the section once with PBS.
12. Draw a circle around the section with a stain circle pen and add 100 µL of DAPI (1 µg/mL). Aspirate the DAPI solution after 10 min of incubation at room temperature in the dark and wash three times with PBS.
13. 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.
14. 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.

Results

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 µm, that wo...

Discussion

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...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
IsoporeTM membranes	Millipore	TSTP04700	3 mm pore
Disposable filter unit	Xinya, Shanghai, China	25 mm	Medical grade polypropylene
Insulin syringe	BD	328446	1 mL
pN1-EGFP	Clontech	6085-1
MitoTracker	Molecular Probes	M7514	Green FM, 1 μM
JC-1	Beyotime, Haimen, China	C2006	10 mg/mL
CM-DiI	Beyotime, Haimen, China	C1036	10 mM
PEI	Sigma	P3143	Mn = 75,000
Fluorescence Microscope	Nikon	Eclipse TE 2000	With CCD camera
Confocol Microscope	Carl Zeiss	LSM 510 Meta
PolyJet	SigaGen	SL100688	For cell transfection