Bone marrow-derived mesenchymal stem cells: Isolation, expansion, characterization, viral transduction, and production of conditioned medium

Massimiliano Gnecchi, Luis G. Melo

Research output: Chapter in Book/Report/Conference proceedingChapter

173 Citations (Scopus)

Abstract

Mesenchymal stem cells (MSCs) are defined as self-renewing and multipotent cells capable of differentiating into multiple cell types, including osteocytes, chondrocytes, adipocytes, hepatocytes, myocytes, neurons, and cardiomyocytes. MSCs were originally isolated from the bone marrow stroma but they have recently been identified also in other tissues, such as fat, epidermis, and cord blood. Several methods have been used for MSC isolation. The most common method is based on the ability of the MSCs to selectively adhere to plastic surfaces. Phenotypic characterization of MSCs is usually carried out using immunocytochemical detection or fluorescence-activated cell sorting (FACS) analysis of cell surface molecule expression. However, the lack of specific markers renders the characterization of MSCs difficult and sometimes ambiguous. MSCs posses remarkable expansion potential in culture and are highly amenable to genetic modification with various viral vectors rendering them optimal vehicles for cell-based gene therapy. Most importantly, MSC plasticity and the possibility to use them as autologous cells render MSCs suitable for cell therapy and tissue engineering. Furthermore, it is known that MSCs produce and secrete a great variety of cytokines and chemokines that play beneficial paracrine actions when MSCs are used for tissue repair. In this chapter, we describe methods for isolation, ex vivo expansion, phenotypic characterization, and viral infection of MSCs from mouse bone marrow. We also describe a method for preparation of conditioned and concentrated conditioned medium from MSCs. The conditioned medium can be easily tested both in vitro and in vivo when a particular paracrine effect (i.e., cytoprotection) is hypothesized to be an important mechanism of action of the MSCs and/or screened to identify a target paracrine/autocrine mediator.

Original languageEnglish
Title of host publicationMethods in Molecular Biology
Pages281-294
Number of pages14
Volume482
DOIs
Publication statusPublished - 2009

Publication series

NameMethods in Molecular Biology
Volume482
ISSN (Print)10643745

Fingerprint

Cell Separation
Conditioned Culture Medium
Mesenchymal Stromal Cells
Bone Marrow
Cell Engineering
Osteocytes
Cytoprotection
Virus Diseases
Tissue Engineering
Chondrocytes
Cell- and Tissue-Based Therapy
Fetal Blood
Chemokines
Adipocytes
Cardiac Myocytes
Epidermis
Genetic Therapy
Muscle Cells
Plastics
Hepatocytes

Keywords

  • Cell surface markers
  • Conditioned medium
  • Drug discovery
  • Flow cytometry
  • Gene transfer
  • Mesenchymal stem cells
  • Paracrine effect
  • Soluble factors

ASJC Scopus subject areas

  • Genetics
  • Molecular Biology

Cite this

Bone marrow-derived mesenchymal stem cells : Isolation, expansion, characterization, viral transduction, and production of conditioned medium. / Gnecchi, Massimiliano; Melo, Luis G.

Methods in Molecular Biology. Vol. 482 2009. p. 281-294 (Methods in Molecular Biology; Vol. 482).

Research output: Chapter in Book/Report/Conference proceedingChapter

Gnecchi, Massimiliano ; Melo, Luis G. / Bone marrow-derived mesenchymal stem cells : Isolation, expansion, characterization, viral transduction, and production of conditioned medium. Methods in Molecular Biology. Vol. 482 2009. pp. 281-294 (Methods in Molecular Biology).
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