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-face....

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-....

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