Isolation and characterization of microvesicles from mesenchymal stem cells

Methods. 2020 May 1:177:50-57. doi: 10.1016/j.ymeth.2019.10.010. Epub 2019 Oct 25.

Abstract

Mesenchymal stem or stromal cells are currently under clinical investigation for multiple diseases. While their mechanism of action is still not fully elucidated, vesicles secreted by MSCs are believed to recapitulate their therapeutic potentials to some extent. Microvesicles (MVs), also called as microparticles or ectosome, are among secreted vesicles that could transfer cytoplasmic cargo, including RNA and proteins, from emitting (source) cells to recipient cells. Given the importance of MVs, we here attempted to establish a method to isolate and characterize MVs secreted from unmodified human bone marrow derived MSCs (referred to as native MSCs, and their microvesicles as Native-MVs) and IFNγ stimulated MSCs (referred to as IFNγ-MSCs, and their microvesicles as IFNγ-MVs). We first describe an ultracentrifugation technique to isolate MVs from the conditioned cell culture media of MSCs. Next, we describe characterization and quality control steps to analyze the protein and RNA content of MVs. Finally, we examined the potential of MVs to exert immunomodulatory effects through induction of regulatory T cells (Tregs). Secretory vesicles from MSCs are promising alternatives for cell therapy with applications in drug delivery, regenerative medicine, and immunotherapy.

Keywords: Cell-free therapy; Drug delivery; Extracellular vesicles (EVs); Mesenchymal Stem Cells (MSCs); Microvesicles (MVs).

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Bone Marrow Cells / chemistry
  • Bone Marrow Cells / cytology
  • Bone Marrow Cells / drug effects
  • Bone Marrow Cells / immunology
  • Cell Separation / methods
  • Cell-Derived Microparticles / chemistry*
  • Cell-Derived Microparticles / immunology
  • Culture Media, Conditioned / chemistry
  • Drug Delivery Systems / methods*
  • Humans
  • Immunotherapy / methods
  • Interferon-gamma / pharmacology
  • Mesenchymal Stem Cells / chemistry*
  • Mesenchymal Stem Cells / cytology
  • Mesenchymal Stem Cells / drug effects
  • Mesenchymal Stem Cells / immunology
  • Proteins / classification
  • Proteins / isolation & purification
  • Proteomics / methods*
  • RNA / classification
  • RNA / isolation & purification
  • Regenerative Medicine / methods*
  • T-Lymphocytes, Regulatory / drug effects
  • T-Lymphocytes, Regulatory / immunology

Substances

  • Culture Media, Conditioned
  • Proteins
  • RNA
  • Interferon-gamma