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 language | English |
|---|---|
| Title of host publication | Methods in Molecular Biology |
| Pages | 281-294 |
| Number of pages | 14 |
| Volume | 482 |
| DOIs | |
| Publication status | Published - 2009 |
Publication series
| Name | Methods in Molecular Biology |
|---|---|
| Volume | 482 |
| ISSN (Print) | 10643745 |
Fingerprint
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 proceeding › Chapter
}
TY - CHAP
T1 - Bone marrow-derived mesenchymal stem cells
T2 - Isolation, expansion, characterization, viral transduction, and production of conditioned medium
AU - Gnecchi, Massimiliano
AU - Melo, Luis G.
PY - 2009
Y1 - 2009
N2 - 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.
AB - 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.
KW - Cell surface markers
KW - Conditioned medium
KW - Drug discovery
KW - Flow cytometry
KW - Gene transfer
KW - Mesenchymal stem cells
KW - Paracrine effect
KW - Soluble factors
UR - http://www.scopus.com/inward/record.url?scp=59749100262&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=59749100262&partnerID=8YFLogxK
U2 - 10.1007/978-1-59745-060-7_18
DO - 10.1007/978-1-59745-060-7_18
M3 - Chapter
C2 - 19089363
AN - SCOPUS:59749100262
SN - 9781588297976
VL - 482
T3 - Methods in Molecular Biology
SP - 281
EP - 294
BT - Methods in Molecular Biology
ER -