HEP (2006) 174:249–282

© Springer-Verlag Berlin Heidelberg 2006

Mesenchymal Stem Cells: Isolation, In Vitro Expansion

and Characterization
N. Beyer Nardi (u) · L. da Silva Meirelles
Genetics Department, Universidade Federal do Rio Grande do Sul, Av Bento Gonçalves
9500, Porto Alegre RS, CEP 91540–000, Brazil
nardi@ufrgs.br

1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 250

2 The Identity of the Mesenchymal Stem Cell . . . . . . . . . . . . . . . . . . . 251

2.1 The Colony-Forming Unit-Fibroblast . . . . . . . . . . . . . . . . . . . . . . . 251
2.2 The Bone Marrow Stroma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 252
2.3 The Mesenchymal Stem Cell . . . . . . . . . . . . . . . . . . . . . . . . . . . 253
2.4 Other Cell Populations Related to the Mesenchymal Stem Cell . . . . . . . . . 254

3 Distribution of the Mesenchymal Stem Cell . . . . . . . . . . . . . . . . . . . 255

4 Isolation and Culture of Mesenchymal Stem Cells . . . . . . . . . . . . . . . . 256

5 Homing and Engraftment of Transplanted Mesenchymal Stem Cells . . . . . 259

6 Characterization of Mesenchymal Stem Cells . . . . . . . . . . . . . . . . . . 260

7 Differentiation of Mesenchymal Stem Cells . . . . . . . . . . . . . . . . . . . 262

8 Applications of Mesenchymal Stem Cells in Cell and Gene Therapy . . . . . . 263

8.1 Study of Cancer Biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 264
8.2 Cell Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 264
8.2.1 Fibrosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 265
8.2.2 Cardiovasculogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 265
8.2.3 Arteriogenic Effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 266
8.2.4 Immunosuppressive Effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267
8.3 Mesenchymal Stem Cells and Tissue Engineering . . . . . . . . . . . . . . . . 267
8.4 Genetic Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 268
8.4.1 Correction of Genetic Disorders . . . . . . . . . . . . . . . . . . . . . . . . . 269
8.4.2 Cancer Suppression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 270

9 Pharmacologic Aspects of Mesenchymal Stem Cell Biology . . . . . . . . . . 271

10 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 272

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 274
250 N. Beyer Nardi · L. da Silva Meirelles

Abstract Mesenchymal stem cells (MSC), one type of adult stem cell, are easy to isolate,
culture, and manipulate in ex vivo culture. These cells have great plasticity and the potential
for therapeutic applications, but their properties are poorly understood. MSCs can be
found in bone marrow and in many other tissues, and these cells are generally identified
through a combination of poorly defined physical, phenotypic, and functional properties;
consequently, multiple names have been given to these cell populations. Murine MSCs have
been directly applied to a wide range of murine models of diseases, where they can act as
therapeutic agents per se, or as vehicles for the delivery of therapeutic genes. In addition
to their systemic engraftment capabilities, MSCs show great potential for the replacement
of damaged tissues such as bone, cartilage, tendon, and ligament. Their pharmacological
importance is related to four points: MSCs secrete biologically important molecules, express
specific receptors, can be genetically manipulated, and are susceptible to molecules that
modify their natural behavior. Due to their low frequency and the lack of knowledge on
cell surface markers and their location of origin, most information concerning MSCs is
derived from in vitro studies. The search for the identity of the mesenchymal stem cell has
depended mainly on three culture systems: the CFU-F assay, the analysis of bone marrow
stroma, and the cultivation of mesenchymal stem cell lines. Other cell populations, more
or less related to the MSC, have also been described. Isolation and culture conditions
used to expand these cells rely on the ability of MSCs, although variable, to adhere to plastic
surfaces. Whether these conditions selectively favor the expansion of different bone marrow
precursors or cause similar cell populations to acquire different phenotypes is not clear.
The cell populations could also represent different points of a hierarchy or a continuum of
differentiation. These issues reinforce the urgent need for a more comprehensive view of
the mesenchymal stem cell identity and characteristics.

Keywords Mesenchymal stem cell · Bone marrow stroma · Differentiation ·

Stem cell niche · Cell therapy · Genetic therapy

1
Introduction

Stem cells present in early embryonic stages are pluripotent and can generate
all of the cell types found in adult organisms, whereas, adult stem cells exhibit
a continuum of plasticity or multipotency. In adult humans, the first and
one of the best-known stem cells to be described is the hematopoietic stem
cell (HSC). A great variety of other stem/precursor cell types have also been
described, but much less is known about their origin and maintenance in vivo
as organ-specific stem cell pools (Nardi 2005).
The mesenchymal stem cell (MSC) is one of the most interesting of the adult
stem cell types. These cells are easily isolated, cultured, and manipulated ex
vivo. MSCs exhibit great plasticity and harbor the potential for therapeutic
applications, but these cells are poorly defined. This has led to a heterogeneity
of names and phenotypes ascribed by different groups to this cell population.
MSCs are present in the bone marrow and in many other tissues, and these
cells are presently identified through a combination of poorly defined physi-
cal, phenotypic, and functional properties. A number of recent reviews have
adequately described the nature of MSCs (Short et al. 2003; Zipori 2004; Barry