J. Cell. Mol. Med. Vol 8, No 3, 2004 pp. 301-316 [626776]

J. Cell. Mol. Med. Vol 8, No 3, 2004 pp. 301-316
Adult mesenchymal stem cells: characterization,
differentiation, and application in cell and gene therapy
D. Baksh‡, L. Song‡, R. S. Tuan*
Cartilage Biology and Orthopaedics Branch,
National Institute of Arthritis, Musculoskeletal and Skin Diseases, National Institutes of Health,
Department of Health and Human Services, Bethesda, MD, USA
Received: September 14, 2004; Accepted: September 24, 2004
Abstract
Aconsiderable amount of retrospective data is available that describes putative mesenchymal stem cells (MSCs).
However, there is still very little knowledge available that documents the properties of a MSC in its native environ-
ment. Although the precise identity of MSCs remains a challenge, further understanding of their biological proper-
ties will be greatly advanced by analyzing the mechanisms that govern their self-renewal and differentiation poten-
tial. This review begins with the current state of knowledge on the biology of MSCs, specifically with respect to their
existence in the adult organism and postulation of their biological niche. While MSCs are considered suitable candi-
dates for cell-based strategies owing to their intrinsic capacity to self-renew and differentiate, there is currently little
information available regarding the molecular mechanisms that govern their stem cell potential. We propose here a
model for the regulation of MSC differentiation, and recent findings regarding the regulation of MSC differentiation
are discussed. Current research efforts focused on elucidating the mechanisms regulating MSC differentiation should
facilitate the design of optimal in vitroculture conditions to enhance their clinical utility cell and gene therapy.
Keywords :mesenchymal stem cells•stem cell niche•differentiation•Wnt•gene therapy
* Correspondence to: Rocky S. TUAN
Cartilage Biology and Orthopaedics Branch, National Institute
of Arthritis, and Musculoskeletal and Skin Diseases
50 South Dr., Room 1503, MSC 8022 National Institutes of Health, Bethesda, MD 20892-8022, USA.
Tel.: 301-451-6854, Fax: 301-435-8017
E-mail: [anonimizat]
‡These authors contributed equally.Stem Cell Review Series
• Introduction
• Existence of mesenchymal stem cells
• The mesenchymal stem cell niche
• Key characteristics of MSCs phenotype
– Self-renewal potential– Multilineage differentiation potential
• Regulation of differentiation
• Application of MSCs in cell
and gene therapy
• Conclusions

Introduction
Mesenchymal stem cells (MSCs) have generated a
great deal of excitement and promise as a potential
source of cells for cell-based therapeutic strategies,
primarily owing to their intrinsic ability to self-
renew and differentiate into functional cell types
that constitute the tissue in which they exist. MSCs
are considered a readily accepted source of stem
cells because such cells have already demonstrated
efficacy in multiple types of cellular therapeutic
strategies, including applications in treating chil-
dren with osteogenesis imperfecta[1], hematopoi-
etic recovery [2], and bone tissue regeneration
strategies [3]. More importantly, these cells may be
directly obtained from individual patients, thereby
eliminating the complications associated with
immune rejection of allogenic tissue. Despite
diverse and growing information concerning MSCs
and their use in cell-based strategies, the mecha-
nisms that govern MSC self-renewal and multilin-
eage differentiation are not well understood and
remain an active area of investigation. Therefore,
research efforts focused on identifying factors that
regulate and control MSC cell fate decisions are
crucial to promote a greater understanding of the
molecular, biological and physiological characteris-
tics of this potentially highly useful stem cell type.
Existence of mesenchymal stem cells
To date, there is no unequivocal evidence indicating
that MSCs existin vivo. Nevertheless, conventional
wisdom promotes the existence of such a cell type,
as connective tissue formation, the functional end-
point of MSC lineage development, occurs in an
organism during development and throughout post-
natal growth, repair and regeneration. Further sup-
port of their putative existence is derived from the
important role of subpopulations of stromal cells in
providing appropriate environmental cues essential
for normal adult hematopoiesis [4, 5].
Due to the lack of a single definitive marker and
knowledge regarding the anatomical location and
distribution of MSCs in vivo, the demonstration of
their existence has relied primarily on retrospective
assays. The gold standard assay utilized to identify
MSCs is the colony forming unit-fibroblast (CFU-F) assay which, at minimum, identifies adherent,
spindle-shaped cells that proliferate to form
colonies [6]. Some of the earliest experimental evi-
dence supporting the existence of MSCs originated
from the pioneering work of Friedenstein et al.,
who first demonstrated that bone marrow derived-
cells were capable of osteogenesis [7]. Accordingly,
this assay has been used as an in vitrocorrelate for
MSC potential. One of the most important caveats
of this assay involves its assumption that putative
MSCs can only be identified by their inherent abil-
ity to adhere, proliferate and develop on a static sur-
face. Therefore, the primary question introduced by
this system is whether these adhesion-derived cells
definitively correlate to an in vivopopulation of
MSCs.
Since the early work of Castro-Malaspina et al.
[8], many researchers have employed different
methods to isolate MSCs, in both serum and serum-
deprived conditions, and have developed novel
approaches to isolate purified populations of MSCs.
These advances have furthered our understanding
of MSC biology but have also created differences in
terminology and read-out measures (i.e., based on
morphology, phenotype, gene expression, and com-
binations thereof) for describing the adherent-capa-
ble cells derived from many adult tissue sources
displaying fibroblast-like morphology (Table 1).
Although none of these terms can accurately
account for both the developmental origin and dif-
ferentiation capacity of these cells, the term ‘mes-
enchymal stem cell’(MSC) is currently most often
employed. However, both this and the other named
cell types depend, for their definition, on the adher-
ence of a population of harvested cells to a tissue
culture substrate, and therefore none can represent
the actual progenitors existant in adult human mar-
row. Despite considerable amount of retrospective
data available that describe the putative MSCs, the
existence of a single MSC in vivoremains to be
determined.
The mesenchymal stem cell niche
There is much research interest in determining what
defines and constitutes the mesenchymal stem cell
niche. It is clearly described that distinct niches
exist within the bone marrow that support
302

hematopoietic stem cell (HSC) survival and
growth, by providing the requisite factors and adhe-
sive properties to maintain their viability, while
facilitating an appropriate balanced output of
mature progeny for the lifetime of an organism [9].
It has also been determined that these niches are
formed by stromal precursor cells, specifically
osteoblasts [5]. The stroma, and stromal cells,
together, provide a physical support for maturing
precursors of blood cells, and serve as a repository
of a broad range of cell-derived cues and signals
driving the commitment, differentiation and matu-
ration of hematopoietic cells [10-12]. Specifically,
endothelial cells, adipocytes, macrophages, reticu-
lar cells, fibroblasts, osteoprogenitors, HSCs and
their progeny are the primary cellular components
of the marrow stroma [13, 14]. It is within this
dynamic and cellular microenvironment where
MSCs are presumed to exist. The question, howev-er, is: Do MSCs reside in their own unique stem
niche amidst hematopoietic stem cells or do they
share the same niche with hematopoietic cells? It
may be argued that these two cell compartments
occupy the same niche, given the close physical
proximity to one another of both hematopoietic and
mesenchymal cells in the bone marrow. However,
the extracellular and/or intercellular signals that are
required to maintain both the hematopoietic and
mesenchymal stem cell developmental program in
the bone marrow microenvironment are likely to be
vastly different. Acomplete characterization of the
cellular, biochemical, and molecular interactions of
MSCs within their niche is needed in order to
understand how these cells can be optimally regu-
lated in vitro.
Despite the fact that bone marrow is considered
a well-accepted source of MSCs, MSCs have been
isolated from other tissue sources, including trabec-
303J. Cell. Mol. Med. Vol 8, No 3, 2004
Term Cell type(s) identified Animal Source/Reference(s)
Precursors of non-hematopoietic
tissueAdherent cells of bone marrow that include
fibroblast-like cells, endothelial cells, and
monocytes/macrophageGuinea pig [6] Mouse [88]
Colony forming unit-fibroblast
(CFU-F)Colonies of fibroblastic cells, with the occa-
sional monocyte/macrophage presentHuman [8] Mouse [89, 90]
Rabbit [91]
Mesenchymal stem cells
(MSCs)Cells defined by their selective attachment to a
solid surfaceHuman [92]
Marrow stromal cells Adherent cells of bone marrow that include
and/or adherent fibroblast-like cells, endothelial
cells and colonies monocytes/macrophage Mouse [39, 93, 94]
Bone marrow stromal [stem]
cells [BMSSCs] and/or Stromal
precursors cells (SPCs)Non-hematopoietic cells of mesenchymal ori-
gin, displaying fibroblastic morphologyMouse [95] Human [86, 96]
RS-1, RS-2, mMSCs (RS:
Recycling stem cell) (m:
mature) RS-1: thin, spindle-shaped cells RS-2: moder-
ately thin, spindle-shaped cells mMSCs: wider,
spindle-shaped cellsHuman [27, 97]
Multipotent adult progenitor
cells (MAPCs)Culture-derived bone marrow-derived progeni-
tor cellsHumans [98] Murine [25] Rat
[25]Table 1 Representative examples of terms given to mesenchymal stem cells.

ular bone [15], adipose tissue, synovium, skeletal
muscle, lung, deciduous teeth (reviewed in Tuan et
al. [16]), and human umbilical cord perivascular
cells derived from the Wharton’s Jelly [17], sug-
gesting that the MSC niche may not be restricted to
just bone marrow. These findings reveal that MSCs
are diversely distributedin vivo, and as a result may
occupy a ubiquitous stem cell niche.Key characteristics of MSCs
phenotype
Considerable progress has been made towards char-
acterizing the cell surface antigenic profile of
human bone marrow-derived MSC populations
using fluorescence activated cell sorting (FACS)
and magnetic bead sorting techniques. To date,
304Table 2 Examples of human MSC frequency and phenotypic properties calculated from representative studies.
Study Cell fraction iso-
latedFrequency Major cell properties
Castro-Malaspina
et al., [8]1.07 g/ml 68 – 10 in 5 x 106• Adherent fibroblastic-like cells
Lazarus et al., [99]70% Percoll
(1.03 g/ml)1 in 1 x 105 • Adherent fibroblastic-like cells
• CD45–, CD14
Pittenger et al.,
[19]70% Percoll
(1.073 g/ml)1 in 1 x 105 • Adherent fibroblastic-like cells
• SH2+, SH3+, CD29+, CD44+, CD71+, CD90+,
CD106+, CD120a+, CD124+
Koç et al., [2] Percoll (1.073 g/ml)
23.4 – 5.9 ml BM1.4 – 0.7 in 1 x
105(a)• Adherent fusiform fibroblastic-like cells
• SH2+, SH3+, SH4+, CD45–, CD14, CD34–
Kuznetsov et al.,
[100]BM aspirates 34.2 – 6 in 1 x 105• Adherent colonies of fibroblastic-like cells
Reyes et al, [101]Ficoll-Paque
(1.077 g/ml)1 in 1 x 106 • Clusters of small adherent cells
• CD34–, CD44low, CD45–, CD117–, class I-HLA–,
class 2-HLA-DR CD45–GlyAcells
Quirici et al., [102]NGFR+ cells 1,584 in 1 x 106 NGFR+cells
• Isolated fraction consists of small round cells
that rapidly adhere to plastic
• NGFR+cells express CD34+(44.1 – 45.8%),
CD113+(49.4 – 29.9%)
• Minority of cells expressed SH2, CD90, TE7
Gronthos et al.,
[47]STRO-1+ VCAM+1 in 3 STRO1+
VCAM+ cells• Adherent fibroblastic-like cells (> 50 cells) with
occasional cluster of cells (>10–50 cells)
• 0.02% STRO-1+VCAM+cells in BM MNC
population
• >90% of cells stained for collagen type 1
• CD45–
• Quiescent in vivo
• No detection of mature mesenchymal cell markers
(i.e. osteopontin, parathyroid hormone receptor,
Cbfa1/Runx2, osterix).
Suva et al., [103] Ficoll-Paque
(1.077 g/ml)1 in 13,000 • CD45–, CD14–, CD34–, CD11b–, CD90+,
HLA–ABC+
(a) Amean of 1.4 – 0.7 x 105MSCs are recovered at the first passage from 1 x 106input BM MNC.

however, a single marker that definitively delin-
eates the in vivo MSCs has yet to be identified, due
to the lack of consensus from diverse documenta-
tions of the MSC phenotype [18-21] (Table 2).
However, analyses using a combination of mono-
clonal antibodies raised against surface markers of
in vitro-derived MSCs (e.g., STRO-1, SH2, SH3,
SH4) [18, 22] have shown some promise toward
immuno-phenotyping these cells. On the other
hand, the fact that MSCs share common features
with endothelial, epithelial and muscle cells
(reviewed in Minguell et al. [20]) and present a
highly variable profile of cell surface antigens [23-
25] makes it a daunting task to identify a universal
single marker for MSCs. Despite this controversy
of what defines a ‘mesenchymal stem cell’, there is
general agreement that MSCs lack typical
hematopoietic antigens, namely, CD45, CD34 and
CD14 [19].
Self-renewal potential
One of the defining characteristics of stem cells is
their self-renewal potential, the ability to generate
identical copies of themselves through mitotic
division over extended time periods (even the
entire lifetime of an organism). The absolute self-
renewal potential of MSCs remains an open ques-
tion, due in large part to the different methods
employed to derive populations of MSCs and the
varying approaches used to evaluate their self-
renewal capacity. As a population, bone marrow
derived MSCs have been demonstrated to have a
significant but highly variable self-renewal poten-
tial during in vitro serial propagation [26, 27].
Continuous labeling of fresh bone marrow cell har-
vests with tritiated thymidine reveals that CFU-Fs
are not cyclingin vivo[28], and their entry into cell
cycle and subsequent development into colonies
depend on serum growth factors [8]. In fact, high-
er population doublings (i.e. >50 PDs) have been
achieved as a consequence of the addition of spe-
cific growth factors [e.g., fibroblast growth factor-
2 (FGF-2)], to the basal culture medium [29]. Cell
seeding density also plays a role in the expansion
capacity of MSCs. For example, Colter et al [27]
demonstrated that higher expansion profiles of
MSCs can be attained when plated at low density
(1.5-3 cells/cm2) but not at high density (12cells/cm2), resulting in a dramatic increase in the
fold expansion of total cells (2,000-fold vs. 60-fold
expansion, respectively). This work and other sim-
ilarly reported work (reviewed in Bianco et al.
[30]) strongly suggest that MSCs and isolated
MSC clones are heterogeneous with respect to their
self- renewal capacity.
Multilineage differentiation potential
The multilineage differentiation potential of MSC
populations derived from a variety of different
species has been extensively studied in vitrosince
their first discovery in 1960s [31]. These studies
demonstrate that populations of bone marrow-
derived MSCs from human, canine, rabbit, rat, and
mouse have the capacity to develop into terminally
differentiated mesenchymal phenotypes both in
vitroand in vivo, including bone [26, 32], cartilage
[33], tendon [34, 35], muscle [36, 37], adipose tis-
sue [38, 39], and hematopoietic-supporting stroma
[39] (Fig. 1A). The ability of MSCs to differentiate
into a variety of connective tissue cell types has
rendered them an ideal candidate cell source for
clinical tissue regeneration strategies, including the
augmentation and local repair and regeneration of
bone [33, 40], cartilage [41] and tendon [34].
Individual colonies derived from single MSC
precursors have also been reported to be heteroge-
neous in terms of their multilineage differentiation
potential. For instance, Pittenger et al. [19] report-
ed that only one-third of the initial adherent bone
marrow-derived MSC clones are pluripotent
(osteo/chondro/adipo). Furthermore, nonimmortal-
ized cell clones examined by Muraglia et al. [42]
demonstrated that 30% of thein vitroderived MSC
clones exhibited a tri-lineage (osteo/chondro/adipo)
differentiation potential, while the remainder dis-
played a bi-lineage (osteo/chondro) or uni-lineage
potential (osteo). These observations are consistent
with otherin vitrostudies using conditionally
immortalized clones [43-45]. Additionally,
Kuznetsov et al. [46] demonstrated that only 58.8%
of the single colony-derived clones had the ability
to form bone within hydroxyapatite-tricalcium
phosphate ceramic scaffolds after implantation in
immunodeficient mice. Similar results were report-
ed by using purer populations of MSCs maintained
in vitro[47]. Taken together, these results suggest
305J. Cell. Mol. Med. Vol 8, No 3, 2004

that clonally-derived MSCs are heterogeneous with
respect to their developmental potential.
The heterogeneity of adult MSCs, demonstrated
in bothin vivoand in vitrostudies, with respect to
their self-renewal and differentiation potential,
could be explained by the notion that in bone mar-
row, the MSC pool comprises not only putative
“mesenchymal stem cells” but also subpopulations
at different states of differentiation (Fig. 1B). In this
model, MSCs in the bone marrow constitute a prim-
itive stem cell population (multipotent MSCs), sim-
ilar to the hematopoietic stem cell system that is
capable of extensive self-renewal and formation of
all the differentiated connective tissues, as well as
MSCs with different multilineage potential (e.g.,
quadra-, tri-, bi-, and uni-potential MSCs). These
various multi-potential MSCs have limited self-
renewal capacity and give rise to specific cell types
with terminally differentiated phenotype. The
multi-potent MSCs are eventually depleted from
the MSC pool during long-term culture, due to theirlow frequency in relation to more differentiated
MSC phenotypes, present at higher frequency in the
primary tissue source. The question, therefore, is
how can these highly multipotent MSCs be main-
tained during in vitroculture expansion.
Several strategies have been employed to
enhance and maintain the multilineage potential of
MSCs, such as culturing cells with specific growth
factors, enriching cells prior to initial plating,
and/or culturing cells in a non-contact suspension
culture configuration. However, the general
approach to the culture of MSCs involves isolating
the mononucleated cells containing MSCs from
bone marrow aspirates and seeding these cells on
tissue culture plates at a standard plating density in
a minimal essential medium base containing fetal
bovine serum (FBS). Within 24-48 hours, nonad-
herent hematopoietic cells are removed, and the
adherent cells are cultured and passaged to expand
the MSC population [26, 48]. Under this condition,
cells can be expanded typically to 40 PDs until their
306
Fig. 1 Models of mesenchymal stem cell differentiation. (A) In this theoretical model, a mesenchymal stem cell
(MSC) has the capacity to differentiate into all connective tissue cell types, including bone, cartilage, tendon, mus-
cle, marrow, fat and dermis. Furthermore, MSCs have the potential for self-renewal and proliferation and, under
defined environmental cues, can commit to a particular differentiation pathway. The lineage-committed cell progress-
es through several stages of maturation prior to the onset of terminal differentiation, which is marked by the cessa-
tion of proliferative capacity and shift toward synthesis of tissue-specific markers, including components of the extra-
cellular matrix. (B) An alternative model illustrating thatin vivo, MSCs comprise a cell population that consists of
mesenchymal cells, which have different differentiation potentials (i.e., quadra-, tri-, bi and uni-potential). During in
vitro culture, all or a subset of these mesenchymal cells are isolated. During differentiation, the proliferative poten-
tial of these different mesenchymal cells decreases and, depending on the initial state of differentiation, both their pro-
liferative and multilineage potential become limited.

growth rate is significantly reduced. Furthermore,
addition of specific growth factors in the MSC cul-
tures has resulted in selective enrichment of differ-
ent subsets of MSCs [25, 29, 49]. For example, sup-
plementation of FGF-2 in the presence of 10% FBS
prolongs the lifespan of bone marrow-derived
MSCs to more than 70 PDs and maintains their dif-
ferentiation potential until 50 PDs [29]. These
results suggest that FGF-2 preferentially selects for
the survival of a particular subset of MSCs with a
higher self-renewal potential. Enrichment of a more
homogeneous MSC starting population, particular-
ly those that have a multilineage differentiation
potential (i.e., quadra- vs. bipotent cells) could also
prolong the life-span of MSCs during in vitroexpansion. Anumber of techniques have been
developed to fulfill this purpose, such as cell size-
based physical enrichment, plating property-based
selection (low vs. high plating densities) [27, 50],
and cell surface marker selection [22, 47, 51]. Since
these approaches usually generate diverse results
with respect to the expansion potential of the isolat-
ed cells, it is apparent that a clearly established,
efficient, and reproducible method to the isolation,
culture and expansion of putative MSCs has yet to
be developed. An optimal culturing strategy would
involve recapitulating the in vivo environment of
MSCs. It has been reported that non-hematopoietic
cells that display fibroblastic cell morphology,
under CFU-F assay conditions, can be isolated from
307J. Cell. Mol. Med. Vol 8, No 3, 2004
Fig. 2 Schematic model depicting adult stem cell differentiation. Uncommitted MSCs undergo two stages, occur-
ring in the stem cell compartment and the committed cell compartment, prior to acquiring specific phenotypes. In the
stem cell compartment, multipotent MSCs give rise to a less potent cell population via asymmetric cell division (A),
which then generate more precursor cells with less self-renewal capacity and more restricted differentiation potential
via symmetric division (S). In the committed cell compartment, these tri-or bi-potent precursor cells continue to
divide symmetrically and generate bi- or unipotent progenitor cells with pre-determined cell fate, which eventually
give rise to fully differentiated cells. Recent studies also suggest that the fully committed cells are able to dedifferen-
tiate into more potent cells, and acquire a different phenotype under inductive cues (open arrows).

biological fluids, including adult peripheral blood
and fetal blood [52, 53]. These cells show charac-
teristics of adherent-derived MSCs in that they
share a similar phenotypic profile (CD45-, CD42+,
SH2+, SH3+, SH4+) and have the capacity to differ-
entiate into a variety of mesenchymal tissues (i.e.,
bone, cartilage and adipose) both in vitroand in
vivo[52-54]. These results suggest that in vitro
derived MSCs might be able to survive and prolif-
erate in a non-adherent environment, such as that
already demonstrated in a stirred suspension culture
system [55]. The suspension cells grown under
these non-contact conditions maintain their ability
to form functional connective tissue types.
Importantly, this approach provides an alternative
strategy to expand adult bone marrow-derived non-
hematopoietic progenitor cell numbers in a scalable
and controllable bioprocess and also provides new
insight into, and possibilities to explore, mesenchy-
mal stem/progenitor cell biology.
Regulation of differentiation
As state above, an important feature about MSCs is
their multilineage differentiation potential. Under
defined inductive conditions, MSCs are able to
acquire characteristics of cells derived from embry-
onic mesoderm, such as osteoblasts, chondrocytes,
adipocytes, tendon cells, as well as cells possessing
ectodermal and neuronal properties. However, the
molecular mechanisms that govern MSC differenti-
ation are incompletely understood. Based on the
genetic and genomic information provided by vari-
ous studies, we propose a model for the regulation
of adult stem cell differentiation, which incorpo-rates two continuous yet distinct compartments
(Fig. 2). In the first compartment, MSCs undergo
transcriptional modification, generating precursor
cells without apparent changes in phenotype and
self-renewal capacity. Similar to MSCs residing in
adult bone marrow, the majority of MSCs cultured
in vitroremain quiescent and growth arrested in
G0/G1, until stimulated, for example, by the sup-
plementation of growth factors. Upon stimulation,
multipotent, uncommitted MSCs undergo asym-
metric division, giving rise to two daughter cells,
one being the exact replica of the mother cell and
maintaining multilineage potential, and the other
daughter cell becoming a precursor cell, with a
more restricted developmental program. In this
model, the precursor cell continues to divide sym-
metrically, generating more tripotent and bipotent
precursor cells. These tripotent and bipotent precur-
sor cells are morphologically similar to the multipo-
tent MSCs, but differ in their gene transcription
repertoire, and therefore, still reside in the stem cell
compartment. The progression of MSCs to precur-
sor cells is considered the first step in stem cell
commitment. The transition or exit from the ‘stem
cell compartment’to the ‘commitment compart-
ment’occurs when precursor cells continue to
divide symmetrically to generate unipotent progen-
itor cells, simultaneous with the acquisition of lin-
eage specific properties, rendering them fully com-
mitted mature cells with distinguishable pheno-
types. At present, what is not fully understood is the
mechanism that governs the transit of uncommitted
stem cells to partially committed precursor or pro-
genitor cells, and then to fully differentiated cells.
To better understand this phenomenon, a number of
questions need to be answered. For example, is
there a common regulatory pathway that functions
308
Fig. 3 V enn diagram showing
the number of candidate genes
that are upregulated during MSC
commitment into osteoblasts
(osteogenesis), adipocytes (adipo-
genesis), and chondrocytes (chon-
drogenesis), and those that are
common to two or all three lin-
eages (see text for details).

as a master ‘switch’that can be manipulated to turn
on stem cell differentiation? How do precursor and
progenitor cells selectively differentiate into one
specific phenotype but not the other? Can pre-deter-
mined progenitor cells change their commitment
and phenotype? Do fully differentiated cells retain
multipotentiality?
The commitment and differentiation of MSCs to
specific mature cell types is a tightly and temporal-
ly controlled process, involving the activities of
various transcription factors, cytokines, growth fac-
tors, and extracellular matrix molecules. Global
gene expression profiling using DNAmicroarray
technology is a useful tool to identify genes
involved in stem cell commitment and differentia-
tion as a function of different inductive microenvi-
ronments. In fact, this approach has already been
used successfully to identify genes that regulate
osteogenic, adipogenic, and chondrogenic differen-
tiation of MSCs [56, 57], which has greatly facili-
tated our effort to elucidate the mechanism control-
ling adult stem cell differentiation. However,
although studies focused on individual lineage(s)
could identify the genes essential for specific lin-
eage (s), they often failed to identify genes that
might be involved in more than one differentiation
lineages, i.e., the master control genes. To deter-
mine if such master control genes exist, we have
compared the transcriptome profiles associated
with three mesenchymal lineages derived from
human MSCs, namely, osteoblasts, chondrocytes,
and adipocytes, to that of uncommitted MSCs using
Affymetrix human genome U133 array set (Song
and Tuan, manuscript in preparation). Genes that
showed 1.5-fold or higher levels of increased
expression during differentiation were selected and
categorized into three subclasses, depending on
their upregulation in only one lineage, in two lin-
eages, or in all three lineages. Among 39,000 tran-
scripts analyzed for osteogenesis, adipogenesis, and
chondrogenesis, respectively, 914, 947, and 52
genes increased their expression in one mesenchy-
mal lineage, while 235, 3, and 10 genes shared
upregulated expression between two lineages (Fig.
3). Most interestingly, there are 8 genes whose
expression are increased during all three mesenchy-
mal lineage differentiation, suggesting that they
might function in all three lineages, and thus may
represent the putative master control genes. These
genes are identified as period homolog1 (PER1),nebulette (NEBL), neuronal cell adhesion molecule
(NRCAM), FK506 binding protein 5 (FKBP5),
interleukin 1 type II receptor (IL1R2), zinc finger
protein 145 (ZNF145), tissue inhibitor of metallo-
proteinase 4 (TIMP4), and serum amyloid A2. The
function of these genes cover a broad range of cel-
lular processes, including cell adhesion, protein
folding, organization of actin cytoskeleton, as well
as inflammatory response, implying that the initia-
tion and commitment of adult stem cells is a com-
plex process requiring the coordination of multiple
molecules and signaling pathways. Functional anal-
ysis of these genes is necessary to determine if and
how they are involved in the progression of stem
cells from one differentiation stage to the next. The
fact that osteoblasts and adipocytes shared more
upregulated genes during their phenotypic acquisi-
tion (235 genes), compared to 3 genes shared
between osteoblasts and chondrocytes, and 10 genes
shared between chondrocytes and adipocytes, also
implies that osteoblasts and adipocytes might share a
common precursor, while chondrocytes are derived
from a different precursor. Further analysis of shared
genes among different lineages should advance our
understanding of the hierarchical sequence of stem
cell commitment during development.
The conventional view of linear hierarchical
progression of stem cells from one differentiation
stage to the next during their phenotypic determina-
tion (Fig. 1A) has been challenged by the recent
findings that adult stem cells can give rise to cells
other than their residing tissues upon in vivotrans-
plantation [58-60]. Using an in vitrodifferentiation
strategy, we recently showed that MSC-derived,
fully differentiated osteoblasts, adipocytes, and
chondrocytes can switch their phenotypes to other
mesenchymal lineages in response to specific extra-
cellular stimuli [61]. During the transdifferentiation
process, extensive cell proliferation is observed and
committed cells lose their lineage-specific pheno-
type before resuming a cell state similar to primi-
tive stem cells, both in morphology and function.
Furthermore, upon induction, these dedifferentiated
cells are able to acquire a new differentiated pheno-
type, that is, undergo redifferentiation (Fig. 4).
Taken together, it is reasonable to conclude that
both pre-committed progenitor cells and fully dif-
ferentiated cells retain the multipotentiality, and
that their plasticity during ‘phenotypic switching’
can be preserved during differentiation and be
309J. Cell. Mol. Med. Vol 8, No 3, 2004

reaquired under defined, appropriate microenviron-
mental circumstances, such as tissue repair and
regeneration.
Studies using transgenic and knockout mice
and human musculoskeletal disorders have pro-
vided valuable information on how MSC differen-
tiate into multiple lineages during embryonic
development and adult homeostasis [62]. On the
other hand, analyses of in vitrodifferentiation of
MSCs under appropriate conditions that recapitu-
late the in vivoprocess have led to the identifica-
tion of various factors essential for stem cell com-
mitment. Among them, secreted molecules and
their receptors (e.g., transforming growth factor-
!), extracellular matrix molecules (e.g., collagens
and proteoglycans), actin cytoskeleton, and intra-
cellular transcription factors (e.g., Cbfa1/Runx2,
PPAR", Sox9, and MEF2) play important roles in
driving the commitment of multipotent stem cells
into specific lineages, and maintain their differen-
tiated phenotypes [63-66]. For example, osteoge-
nesis of MSCs, both in vitroand in vivo, is a well-
orchestrated sequence of events, involving multi-
ple steps and expression of various regulatory fac-
tors. During osteogenesis, multipotent MSCs
undergo asymmetric division and generate osteo-precursors, which then progress to form osteopro-
genitors, preosteoblasts, functional osteoblasts,
and eventually osteocytes [61]. This progression
from one differentiation stage to the next is
accompanied by the activation and subsequent
inactivation of transcription factors, i.e.,
Cbfa1/Runx2, Msx2, Dlx5, Osx, and expression
of bone-related marker genes, i.e., osteopontin,
collagen type I, alkaline phosphatase, bone sialo-
protein, and osteocalcin [66, 67]. Disruption of
the timely sequential expression of these genes
results in the delay of the cell’s progression to the
osteoblast phenotype and the subsequent failure
to form functional osteoblasts.
Members of the Wnt family have recently
shown to impact MSC osteogenesis [68, 69]. Wnts
are a family of secreted cysteine-rich glycopro-
teins that have been implicated in the regulation of
stem cell maintenance, proliferation, and differen-
tiation during embryonic development. Canonical
Wnt signaling increases the stability of cytoplas-
mic !-catenin by receptor-mediated inactivation
of GSK-3 kinase activity and promotes !-catenin
translocation into the nucleus. The active !-
catenin/TCF/LEF complex then regulates the tran-
scription of genes involved in cell proliferation
310
Fig. 4 Model of mesenchymal
stem cell plasticity . Experimental
evidence has demonstrated the abili-
ty of MSCs to transdifferentiate and
dedifferentiate as a function of spe-
cific culture conditions. MSCs have
the potential to differentiate into
osteoblasts, chondrocytes and
adipocytes (solid black arrows), and
may also transdifferentiate directly
into other mature connective tissue
cell types (solid red arrows).
However, these differentiated cells
from MSCs are also able to re-enter a
proliferation stage and resume the
characteristics of undifferentiated
MSCs through genomic reprogram-
ming (dashed orange lines). At this
stage, these cells can become a new
connective tissue cell type. Factors or
signals involved in maintaining the
MSC biological properties (question
marks) require further investigation.

and differentiation. In humans, mutations in the
Wnt co-receptor, LRP5, lead to defective bone for-
mation. Gain of function mutation results in high
bone mass, whereas loss of function causes an
overall loss of bone mass and strength, indicating
that Wnt signaling is positively involved in
embryonic osteogenesis. Canonical Wnt signaling
pathway also functions as a stem cell mitogen, via
the stabilization of intracellular !-catenin and acti-
vation of the !-catenin/TCF/LEF transcription
complex, resulting in activated expression of cell
cycle regulatory genes, such as Myc, cyclin D1,
and Msx1 [70]. When MSCs are exposed to
Wnt3a, a prototypic canonical Wnt signal, under
standard growth medium conditions, they show
markedly increased cell proliferation and a
decrease in apoptosis [69], consistent with the
mitogenic role of Wnts in hematopoietic stem cells
[71]. However, exposure of MSCs to Wnt3a con-
ditioned medium or overexpression of ectopic
Wnt3a during osteogenic differentiation inhibits
osteogenesis in vitrothrough !-catenin mediated
down-regulation of TCF activity [69]. The expres-
sion of several osteoblast specific genes, e.g.,
alkaline phosphatase, bone sialoprotein, and
osteocalcin, is dramatically reduced, while the
expression of Cbfa1/Runx2, an early osteo-induc-
tive transcription factor was not altered, implying
that Wnt3a-mediated canonical signaling pathway
is necessary, but not sufficient, to completely block
MSC osteogenesis. These results raise the question
of whether there are other signaling pathways
involved in triggering osteogenic commitment. On
the other hand, Wnt5a, a typical non-canonical Wnt
member, has been shown to promote osteogenesis
in vitro[69]. Since Wnt3a promotes MSC prolifer-
ation during early osteogenesis, it is very likely that
canonical Wnt signaling functions in the initiation
of early osteogenic commitment by increasing the
number of osteoprecursors in the stem cell compart-
ment, while non-canonical Wnt drives the progres-
sion of osteoprecursors to mature functional
osteoblasts. Interestingly, several osteoblast marker
genes, e.g., alkaline phosphatase, osteocalcin,
appear to contain putative TCF/LEF binding sites.
It will be of interest to determine whether the
inhibitory effect of Wnt3a on osteogenesis is the
direct result of suppression of osteogenic gene
expression, or the secondary effect of increasing
cell proliferation.Application of MSCs in cell and gene
therapy
Adult MSCs have shown great promise in cell and
gene therapy applications, because of their multipo-
tentiality and capacity for extensive self-renewal. In
a large number of animal transplantation studies,
MSCs expanded ex vivowere able to differentiate
into cells of the residing tissue, repair the damaged
tissue due to trauma or disease, and partially restore
its normal function. They not only regenerate tis-
sues of mesenchymal lineages, such as interverte-
bral disc cartilage [72], bone [73, 74], cardiomy-
ocytes [75], and articular cartilage at knee joints
[76], but also differentiate into cells derived from
other embryonic layers, including neurons [77] and
epithelia in skin, lung, liver, intestine, kidney, and
spleen [78-80]. These applications demonstrate the
plasticity of these adult stem cells and their useful-
ness in multiple tissue repair and regeneration and
in cell therapy applications. It is also noteworthy
that neither autologous nor allogeneic MSCs induce
any immunoreactivity in the host upon local trans-
plantation or systemic administration [74, 75, 79,
81], thus rendering MSCs an ideal carrier to deliver
genes into the tissues of interest for gene therapy
applications.
Several approaches have been examined and
used to introduce exogenous DNAinto MSCs to
render them useful in tissue regeneration therapies.
Viral transduction, particularly using adenovirus-
mediated gene transfer, can generate stable cell
clones with high efficiency and low cell mortality,
thus making it a popular option in gene therapy. For
example, MSCs infected with an adenovirus vector
containing dominant-negative mutant collagen type
I gene have been used successfully to repair the
bone in individuals with the brittle bone disorder,
osteogenesis imperfecta [73]. However, the safety
concerns associated with viral transduction have
prompted us to look for alternative non-viral gene
delivery approaches. Traditional transfection meth-
ods, such as calcium phosphate precipitation, lipo-
fection, and electroporation, have shown little suc-
cess in delivering plasmid DNAinto primary MSCs,
usually resulting in less than 1% transfection effi-
ciency and high cell mortality [82]; therefore, these
methods are not suitable for producing sufficient
amount of transfected cells for gene delivery and
transplantation. Recently, two new methods have
311J. Cell. Mol. Med. Vol 8, No 3, 2004

been developed to transfect primary MSCs, namely
NucleofectionTMand vibration-based transfection
using SymphonizerTM. NucleofectionTM (Amaxa
Biosystems), combining electroporation and a pro-
prietary transfection solution, has been shown to
successfully introduce a GFPreporter plasmid into
primary MSCs with up to 80% transfection efficien-
cy and 50% cell viability [83]. Approximately 10%
of the transfected cells retain GFPexpression after 3
weeks, suggesting that the plasmid is transiently
incorporated into the cell nucleus. There was no
apparent adverse effects on normal cellular function
as transfected cells were able to differentiate into
chondrocytes at similar efficiency as untransfected
cells upon induction. Song and Tuan [61] have
recently demonstrated that MSCs transfected using
NucleofectionTM with a lineage-specific promoter
reporter, i.e., an osteocalcin promoter driven GFP
plasmid, acquired osteoblast phenotype as a func-
tion of induction time and maintained their multilin-
eage transdifferentiation capacity. Taken together,
these results strongly suggest the utility of this
method in delivering functional genes into MSCs
used for transplantation to either promote repair and
regeneration of diseased or damaged tissue or rescue
defective genes.
Another recently developed method of nonviral-
ly transfecting cells is based on electric field-
induced molecular vibration using a newly intro-
duced machine, Gene SymphonizerTM(Mollennium
Inc., Japan). This non-invasive method can intro-
duce foreign DNAinto both established cell lines,
such as murine C3H10T1/2 cells, and primary cells,
including chondrocytes, embryonic mesenchymal
cells, and MSCs, at high transfection efficiency (20-
80%) with low cell mortality [82]. This approach
also does not interfere with the normal cellular dif-
ferentiation activities of human and chick mes-
enchymal progenitors. Another unique and impor-
tant feature about this method is its ability to also
deliver exogenous DNAinto multilayered tissue,
such as sternum cartilage and skeletal muscle. As
such, this method could be applied to deliver foreign
DNAdirectly into target tissue/organs in vivo, an
ideal option for gene therapy.
Despite their enormous potential, one of the
major bottlenecks in the use of MSCs has been their
limited numbers, given that a variety of clinical
applications require significant cell numbers to
achieve a clinically successful result (e.g., bonemarrow transplantations and regeneration of large
segmental bone defects). The yield of MSCs from
the primary tissue source is insufficient for such
clinical applications. Unlike embryonic stem cells,
adult MSCs, which lack telomerase activity [84],
show defined ex vivoproliferation capability, reach-
ing senescence and losing multilineage differentia-
tion potential after 34-50 population doublings in
culture. Thus, it is necessary and critical to develop
new strategies to prolong the replicative capacity of
MSCs without impairing their multipotentiality.
Several studies have shown that forced ectopic
expression of human telomerase reverse transcrip-
tase (hTERT) in postnatal MSCs could extend their
life span to more than 260 population doublings,
while maintaining their osteogenic, chondrogenic,
adipogenic, neurogenic, and stromal differentiation
potential [85, 86] . Importantly, these hTERT-trans-
duced, immortalized MSCs have normal karyotype
and do not cause tumor formation in xenogenic
transplants, thus making them an attractive candi-
date source of cells for tissue repair and regenera-
tion. However, caution must be exercised in using
these immortalized MSCs since they express high-
er levels of osteogenic lineage specific genes, such
as Cbfa1/Runx2, osterix, and osteocalcin, com-
pared to non-transduced MSCs [87], which could
potentially compromise their ability to commit to
other cell lineages.
Conclusions
Agrowing body of research evidence has defini-
tively demonstrated that MSCs exist in the adult tis-
sue/organs. Despite the lack of knowledge of the
origin of the putative MSCs, they have been suc-
cessfully isolated from various tissue sources,
mostly prominently, from bone marrow. These cells
have already shown great regenerative potential.
However, to continue to take advantage of these
cells for cell and gene therapy applications will
require a complete understanding of how the main-
tenance and differentiation of MSCs are regulated
both in vivoand in vitro. Knowledge gained in these
areas will facilitate the design of optimalin vitro
conditions that incorporate regimes targeted
towards generating highly functional MSCs for
cell-based clinical applications.
312

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