mesenchymal stem cells in tooth and oral cavity
the origin of mesenchymal stem cells and their types in the mouth and tooth
dental pulp DPSC, apical papilla SCAP, periodontal ligaments PDLSC, dental follicle DFSC, gingival fibroblast GFSC, and gingival epithelial stem cells GESC.
it is functions and usage in dentistry and dental treatments.
Braz Dent J 22(2) 2011
Mesenchymal stem cells in the dental tissues 91
The discovery of stem cells and recent advances
in cellular and molecular biology has led to the
development of novel therapeutic strategies that aim at
the regeneration of many tissues that were injured by
disease. Generally, stem cells have two major properties:
they are capable of self-renewal and, upon division, they
can give rise to cells that have the potential to differentiate
(1). Tissue engineering is a multidisciplinary field that
combines biology, engineering, and clinical science
with the goal of generating new tissues and organs. It is
a science based on fundamental principles that involves
the identification of appropriate cells, the development
of scaffolds and morphogenic signals required to induce
Mesenchymal Stem Cells in the Dental Tissues:
Perspectives for Tissue Regeneration
Ana Helena Gonçalves de ALENCAR1
Gregory Thomas KITTEN2
Eneida Franco VENCIO1
1Dental School, UFG – Federal University of Goiás, Goiânia, GO, Brazil
2Institute of Biological Sciences, UFMG – Federal University of Minas Gerais, Belo Horizonte, MG, Brazil
In recent years, stem cell research has grown exponentially owing to the recognition that stem cell-based therapies have the potential
to improve the life of patients with conditions that range from Alzheimer’s disease to cardiac ischemia and regenerative medicine, like
bone or tooth loss. Based on their ability to rescue and/or repair injured tissue and partially restore organ function, multiple types of
stem/progenitor cells have been speculated. Growing evidence demonstrates that stem cells are primarily found in niches and that certain
tissues contain more stem cells than others. Among these tissues, the dental tissues are considered a rich source of mesenchymal stem
cells that are suitable for tissue engineering applications. It is known that these stem cells have the potential to differentiate into several
cell types, including odontoblasts, neural progenitors, osteoblasts, chondrocytes, and adipocytes. In dentistry, stem cell biology and
tissue engineering are of great interest since may provide an innovative for generation of clinical material and/or tissue regeneration.
Mesenchymal stem cells were demonstrated in dental tissues, including dental pulp, periodontal ligament, dental papilla, and dental
follicle. These stem cells can be isolated and grown under defined tissue culture conditions, and are potential cells for use in tissue
engineering, including, dental tissue, nerves and bone regeneration. More recently, another source of stem cell has been successfully
generated from human somatic cells into a pluripotent stage, the induced pluripotent stem cells (iPS cells), allowing creation of patientand
disease-specific stem cells. Collectively, the multipotency, high proliferation rates, and accessibility make the dental stem cell an
attractive source of mesenchymal stem cells for tissue regeneration. This review describes new findings in the field of dental stem cell
research and on their potential use in the tissue regeneration.
Key Words: Endodontics, stem cell, dental stem cell, tissue engineering.
cells to regenerate a tissue or organ (2). Over the last
few years, medicine has begun to explore the possible
applications of stem cells and tissue engineering towards
the repair and regeneration body structures (3). It is
becoming ever more clear that this conceptual come
up to therapy, named regenerative medicine, will have
its place in clinical practice in the future. It has been
shown that stem cells will play an important role in future
medical treatments because they can be readily grown
and induced to differentiate into any cell type in culture.
Stem cells are cells that have the ability to renew
themselves through mitosis and can differentiate into
several specialized cells. The embryonic stem cells (ESC)
are pluripotent and have the ability to become almost any
kind of cell of the body (4). The local microenvironment
Correspondence: Profa. Dra. Elisandra Gava, Departamento de Ciências Estomatológicas, Universidade Federal de Goiás, Praça Universitária S/N,
Setor Universitário, 74605-220 Goiânia, GO, Brasil. Tel: +55-62-3209-6053. e-mail: [email protected]
Invited Review Article
Braz Dent J (2011) 22(2): 91-98 ISSN 0103-6440
Braz Dent J 22(2) 2011
92 C. Estrela et al.
represents an important compartment in maintaining the
stem cells status. The microenvironment regulates the
balance between self-renewal and differentiation. This
intercellular communication has been characterized
between embryonal carcinoma cells and stromal cells,
and indicates changes in the expression on both cellular
Scientists can induce these cells to replicate
themselves in an undifferentiated state. However, the
use of ESC is controversial and associated with ethical
and legal issues, thus conditioning their application for
the development of new therapies (4).
Another source of stem cells is the umbilical cord.
Blood from the umbilical cord contains stem cells that
are genetically identical to those of the newborn baby.
These cells are multipotent, and are able to differentiate
into certain cell types. Umbilical cord stem cells can
be stored cryogenically after birth for use in a future
medical therapy (2).
Mesenchymal stem cells (MSC) are multipotent
progenitor cells, originally isolated from adult bone
marrow and subsequently from other tissues in both
adult and fetal life. Adult stem cells normally generate
cell types of the tissue in which they reside. However,
studies have shown that stem cells from one tissue could
generate cell types of a completely different tissue (3).
Unlike ESC, adult stem cells have the potential
to be used for treatment of regenerative disease, cardiac
ischemia, and bone or tooth loss. Future applications for
stem cells include the treatment of Parkinson’s disease
and cancer (5). The use of adult stem cells in research
and medical applications is less controversial because
they can be harvested without destroying an embryo.
Postnatal stem cells have been found in almost all body
tissues, including dental tissues. Dental stem cells have
been identified as candidates for tissue engineering (6).
Because of their multipotent differentiation ability, they
provide an alternative for use in regenerative medicine
since they can be used for not only to dental tissue
regeneration, but also to facilitate repair of non-dental
tissues such as bone and nerves (6,7).
A new source of stem cell has been generated
from human somatic cells into a pluripotent stage, the
induced pluripotent stem cells (iPS cells) (8,9). iPS cells
resemble human ESC and can differentiate into advanced
derivates of all three primary germ layers. Unlike ESC,
iPS cell technology can derive patient-specific stem cells
allowing derivation of tissue-matched differentiation
donor cells for basic research, disease modeling, and
regenerative medicine (9). This technology might be
the new era of personalized medicine.
This review discusses the perspectives in the field
of stem cell-based regenerative medicine, addressing
sources of stem cells identified in dental tissues; and
new findings in the field of dental stem cell research
and their potential use in the dental tissue engineering.
Several cell populations with stem cells properties
have been isolated from different parts of the tooth.
Since the discovery of the existence of adult stem cells
from the dental pulp in 2000 (10), several other types
of dental stem cells have been successively isolated
from mature and immature teeth, including stem cells
derived from exfoliated deciduous teeth (11), stem
cells derived from the apical papilla (12), MSC from
tooth germs (13) and from human periodontal ligament
(PDL) (14). It is considered that these stem cells are
undifferentiated mesenchymal cells present in dental
tissues and characterized by their unlimited self-renewal,
colony forming capacity, and multipotent differentiation
(1). During the characterization of these newly identified
dental stem cells, certain aspects of their proprieties
have been compared with those of bone-marrow-derived
stromal stem cells (BMMSC). Dental stem cells display
multidifferentiation potencial, with the capacity to
give rise to distinct cell lineages, osteo/osteogenic,
adipogenic, and neurogenic. Therefore, these cells have
been used for tissue-engineering studies to assess their
potential in preclinical applications (6).
It is, however, important to consider that, although
different types of dental-tissue derived MSC share
several common characteristics and present significant
heterogeneity, expressed by multiple phenotypic
differences, which most probably reflect distinct
functional properties (1). There is already evidence
that there are significant variations, for example, in
the odontogenic potential of single colony-derived
populations isolated from the dental pulp, reflecting
differences in their genotypic and protein expression
patterns (15). In addition, this heterogeneity may be
significantly enhanced as a function of their tissue
microenvironment (16). This issue becomes more
complicated as researchers have used quite different
methods to isolate and culture dental MSC and evaluate
their differentiation potential.
DENTAL PULP STEM CELLS
The first stem cells isolated from adult human
Braz Dent J 22(2) 2011
Mesenchymal stem cells in the dental tissues 93
dental pulp were termed dental pulp stem cells (DPSC).
They were isolated from permanent third molars and
exhibited high proliferation and high frequency of colony
formation that produced calcified nodules (10). DPSC
cultures from impacted third molars at the stage of root
development were able to differentiate into odontoblastlike
cells with a very active migratory and mineralization
potential, leading to organized three-dimensional dentinlike
structures in vitro (17).
There are different cell densities of the colonies in
DPSC, suggesting that each cell clone may have different
grown rate (10). Different cell morphologies and sizes
can be observed in the same colony. The differentiation
of DPSC to a specific cell lineage is mainly determined
by the components of local microenvironment, such as,
growth factors, receptor molecules, signaling molecules,
transcription factors and extracellular matrix protein.
DPSC can be reprogrammed into multiple cell lineages
such as, odontoblast, osteoblast, chondrocyte, myocyte,
neurocyte, adipocyte, corneal epithelial cell, melanoma
cell, and even induced pluripotent stem cells (iPS cells)
(18,19). Almushayt et al. (20) demonstrated that dentin
matrix protein 1 (DMP1), a non-collagen extracellular
matrix protein extract from dentin, can significantly
promote the odontoblastic differentiation of DPSC
and formation of reparative dentin over the exposed
pulp tissue. Additionally, DPSC can be induced into
odontoblast lineage when treated with transforming
growth factor ß1 (TGFß1) alone or in combination with
fibroblast growth factor (FGF2) (21).
Histologically, dentin lies outside of dental pulp,
and they intimately link to each other. Functionally,
dental pulp cells can regenerate dentin and provide it
with oxygen, nutrition and innervation, whereas the
hard dentin can protect soft dental pulp tissue. Together,
they maintain the integrity of tooth shape and function.
Any physiological or pathological reaction occurring at
one part, such as trauma, caries, and cavity preparation,
will affect the other. Both of them act as a dentin-pulp
complex and simultaneously participate in various
biological activities of the tooth. Several studies have
shown that DPSC play a vital role in the dentin-pulp
tissue regeneration (10). In vivo transplantation into
immunocompromised mice DPSC demonstrated the
ability to generate functional dental tissue in the form
of dentin/pulp-like complexes (22). Transplanted
ex vivo expanted DPSC mixed with hydroxyapatite/
tricalcium phosphate form ectopic dentin/pulp-like
complexes in immunocompromised mice. These polls
of heterogeneous DPSC form vascularizad pulp like
tissue and are surrounded by a layer of odontoblast-like
cells expressing factors that produce dentin containing
tubules similar those found in natural dentin (22,23).
Huang et al. (24) reported that dentin-pulp-like complex
with well-established vascularity can be regenerated
de novo in emptied root canal space by DPSC. These
studies provide a novel advance for future pulp tissue
preservation and a new alternative for the biological
treatment for endodontic diseases.
In addition, DPSC can express neural markers
and differentiate into functionally active neurons,
suggesting their potential as cellular therapy for neuronal
disorders (7). In recent study, DPSC were transplanted
into the cerebrospinal fluid of rats in which cortical
lesion was induced. Those cells migrated as single cells
into a variety of brain regions and were detected in
the injured cortex expressing neuron specific markers.
This showed that DPSC-derived cells integrate into
the host brain may serve as useful sources of neuro and
gliogenesis in vivo, especially when the brain is injured
(25). The spontaneous differentiating potential of these
cells strongly suggests their possible applications in
STEM CELLS FROM HUMAN EXFOLIATED
Stem cells may be also isolated from the pulp
of human exfoliated deciduous teeth (SHED). These
cells have the capacity of inducing bone formation,
generate dentin and differentiate into other nondental
mesenchymal cell derivatives in vitro. SHED
exhibit higher proliferation rates, increased population
doublings, in addition to osteoinductive capacity in vivo
and an ability to form sphere-like clusters. However,
unlike DPCSs, they are unable to regenerate complete
dentin/pulp-like complexes in vivo (10). With the
osteoinductive potential, SHED can repair critical
sized calvarial defects in mice with substantial bone
formation (26). Given their ability to produce and
secrete neurotrophic factors, dental stem cells may also
be beneficial for the treatment of neurodegenerative
diseases and the repair of motor neurons following
injury. Indeed, dental stem cells from deciduous teeth
have been induced to express neural markers such as
nestin (27). The expression of neural markers in dental
stem cells stimulates the imagination for their potential
use in neural regeneration such as in the treatment of
Braz Dent J 22(2) 2011
94 C. Estrela et al.
Parkinson’s disease. The potential of dental stem cells in
non-dental regeneration continues to be further explored
STEM CELLS FROM APICAL PAPILLA
The physical and histological characteristics
of the dental papilla located at the apex of developing
human permanent teeth has been recently been described
and this tissue has been termed apical papilla. This
tissue is loosely attached to the apex of the developing
root and can be easily detached. A population of stem
cells isolated from human teeth was found at the
tooth root apex. These cells are called stem cells from
apical papilla (SCAP) and have been demonstrated to
differentiate exhibit higher rates of proliferation in vitro
than do DPSC. There is an apical cell-rich zone lying
between the apical papilla and the pulp. Importantly,
stem/progenitor cells were located in both dental pulp
and the apical papilla, but they have somewhat different
characteristics (12). The higher proliferative potential
of SCAP makes this population of cells suitable for
cell-based regeneration and preferentially for forming
roots. They are capable of forming odontoblast-like
cells and produce dentin in vivo and are likely to be
the cell source of primary odontoblasts for the root
dentin formation (12). The discovery of SCAP may
also explain a clinical phenomenon that was presented
in a number of recent clinical case reports showing that
apexogenesis can occur in infected immature permanent
teeth with apical periodontitis or abscess (28). It is likely
that SCAP residing in the apical papilla survived the
infection due to their proximity to the periapical tissues.
This tissue may be benefited by its collateral circulation,
which enables it to survive during the process of pulp
necrosis. Perhaps, after endodontic disinfection, these
cells give rise to primary odontoblasts to complete the
PERIODONTAL LIGAMENT STEM CELLS
Periodontal ligament (PDL) is a space interlying
the cementum and alveolar bone, a replacement of the
follicle region surrounding the developing tooth in cap
and bud stages of development. Fibers inserted into
the cementum layer may be of follicle origin (termed
Sharpey’s fibers) or cementoblast origin (in cellular
intrinsic fiber cementum). The PDL matures during
tooth eruption, preparing to support the functional
tooth for the occlusal forces. In the mature PDL, major
collagen bundles (principal fibers) occupy the entire
PDL, embedding in both cementum and alveolar bone.
Fibers are arranged in specific orientations to maximize
absorption of the forces to be placed on the tooth during
mastication. The PDL has long been recognized to
contain a population of progenitor cells and recently,
studies identified a population of stem cells from human
PDL capable of differentiating along mesenchymal
cell lineages to produce cementoblast-like cells,
adipocytes and connective tissue rich in collagen I (14).
PDL stem cells (PDLSC) display cell surface marker
characteristics and differentiation potential similar to
bone marrow stromal stem cells and DPSC (14). After
PDLSC were transplanted into immunocompromised
mice, cementum/PDL-like structures were formed.
Human PDLSC expanded ex vivo and seeded in threedimensional
scaffolds (fibrin sponge, bovine-derived
substitutes) were shown to generate bone (29). These
cells have also been shown to retain stem cell properties
and tissue regeneration capacity. These findings suggest
that this population of cells might be used to create a
biological root that could be used in a similar way as a
metal implant, by capping with an artificial dental crown.
DENTAL FOLLICLE PRECURSOR CELLS
The dental follicle is a loose connective tissue
that surrounds the developing tooth. The dental follicle
has long been considered a multipotent tissue, based on
its ability to generate cementum, bone and PDL from
the ectomesenchyme-derived fibrous tissue. Dental
follicle precursor cells (DFPC) can be isolated and
grown under defined tissue culture conditions, and
recent characterization of these stem cells has increased
their potential for use in tissue engineering applications,
including periodontal and bone regeneration (12,30).
DFPC form the PDL by differentiating into PDL
fibroblasts that secrete collagen and interact with fibers
on the surfaces of adjacent bone and cementum. Dental
follicle progenitor cells isolated from human third
molars are characterized by their rapid attachment in
culture, and ability to form compact calcified nodules
in vitro (30). DFPC, in common with SCAP, represent
cells from a developing tissue and might thus exhibit a
greater plasticity than other dental stem cells. However,
in the same way as for SCAP, further research needs to
be carried out on the properties and potential uses of
these cells (Table 1).
Braz Dent J 22(2) 2011
Mesenchymal stem cells in the dental tissues 95
DENTAL PULP STEM CELLS AND DENTAL
There are several areas of research for which
dental stem cells are presently considered to offer
potential for tissue regeneration. These include the
obvious uses of cells to repair damaged tooth tissues
such as dentin, PDL and dental pulp (6,24). Even the
use of dental stem cells as sources of cells to facilitate
repair of additional tissues as bone and nerves (6,7,26).
Efforts to induce tissue regeneration in the pulp space
have been a long search. In 1962, Ostby (31) proposed
inducing hemorrhage and blood clot formation in the
canal space of mature teeth in the hope of guiding the
tissue repair in the canal. However, the connective
tissue that grew into the canal space was limited and
the origin of this tissue remains unproved. Regenerative
Endodontics represents a new treatment modality that
focuses on reestablishment of pulp vitality and continued
root development. This clinical procedure relies on the
intracanal delivery of a blood clot (scaffold), growth
factors (possibly from platelets and dentin), and stem
cells (32). In a recent study, it was demonstrated that
mesenchymal stem cells are delivered into root canal
spaces during regenerative endodontic procedures in
immature teeth with open apices (32). These findings
provide the biological basis for the participation of stem
cells in the continued root development and regenerative
response that follow this clinically performed procedure.
As DPSC have the potent dentinogenic ability,
they could be used for the vital pulp therapy. When DPSC
are transplanted alone or in combination with BMP2
in the pulp cavity, these stem cells can significantly
promote the repair and reconstruction of dentin-pulplike
complex (31). Prescott et al. (34) placed the triad of
DPSC, a collagen scaffold, and DMP1 in the simulated
perforation sites in dentin slices, and then transplanted
the recombination subcutaneously into the nude mice.
After 6 weeks of incubation, well-organized pulplike
tissue could be detected in the perforation site.
Table 1. Stem cell types in dental pulps (6,7,10-15,17,18,20).
Properties DPSC SCAP SHED PDLSC DFPC
Apical papilla of
Dental follicle of
Proliferation rate Moderate High High High High
Heterogeneity Yes Yes Yes Yes Yes
melanoma cell, iPS
DPSC = dental pulp stem cells; SCAPs = stem cells from the apical papila; SHED = stem cells from the pulp of human exfoliated
deciduous teeth; PDLSC = periodontal ligament stem cells; DFPC = dental follicle precursor cells.
Braz Dent J 22(2) 2011
96 C. Estrela et al.
Cordeiro et al. (35) demonstrated that SHED/scaffold
recombination prepared within human tooth slices also
have the potential to form dental pulp-like structures.
Huang et al. (24) reported that dentin-pulp-like complex
with well-established vascularity can be regenerated
de novo in emptied root canal space by either DPSC
or SHED (24). One of the most challenging aspects
of developing a regenerative endodontic therapy is to
understand how the various procedures involved can
be optimized and integrated to produce the outcome
of a regenerated pulp-dentin complex. The future
development of regenerative endodontic procedures will
require a comprehensive research program directed at
each of these components and their application in the
Periodontitis is the most common cause for tooth
loss in adults due to irreversible waste of connective
tissue attachment and the supporting alveolar bone. The
challenge for cell-based replacement of a functional
periodontium is therefore to form new ligament and
bone, and to ensure that the appropriate connections
are made between these tissues, as well as between the
bone and tooth root. This is not a trivial undertaking,
as these are very different tissues that are formed in
an ordered manner (spatially and temporally) during
tooth development (36). In recent years, guided tissue
regeneration has become the gold-standard surgery for
periodontal tissue regeneration. This procedure involves
draping a biocompatible membrane over the periodontal
defect from the root surface to the adjacent alveolar
bone, often in combination with a bone graft (37). The
barrier membrane prevents unwanted epithelium and
gingival connective tissue from entering the healing
site, while promoting repopulation of the defect site by
cells migrating in from the PDL (29). The rather limited
success of this approach has led scientists to develop
methods to improve this therapy, through the addition
of exogenous growth factors and via stem cell therapy
(38). One goal of current research is to use different
populations of dental stem cells to replicate the key
events in periodontal development both temporally
and spatially, so that healing can occur in a sequential
manner to regenerate the periodontium (39).
Commonly used growth factors for PDL
regeneration therapies include bone morphogenetic
proteins, platelet derived growth factor, Emdogain and
recombinant amelogenin protein. The resultant improved
regenerative capability could be related to increased
recruitment of progenitor MSC, which subsequently
differentiate to form PDL tissue. Recently, PDLSC
transfected with expression vectors for platelet-derived
growth factor and bone morphogenetic protein were
investigated in periodontal tissue engineering models
(40). These studies revealed the regeneration of normal
periodontal tissues, containing organized cementum,
alveolar bone and the PDL attachment apparatus. The
possibility of constructing a root-periodontal tissue
complex was further successfully demonstrated using
a pelleted hydroxyapatite/tricalcium phosphate scaffold
containing SCAP, coated with PDLSC-seeded Gelfoam,
implanted and grown in minipig tooth socket (11,41).
The multipotent differentiation properties of PDLSC
for generating both hard and soft tissues were further
demonstrated by constructing multilayered cell sheets
supported by woven polyglycolic acid. Transplanted
cell seeded polyglycolic acid sheets regenerated new
bone, cementum and well-oriented collagen fibers when
introduced into root surfaces. In addition to PDL-derived
DSCs, bone marrow-derived MSC and adipose-derived
stem cells have been shown to promote periodontal tissue
In a recent study (43), three kinds of dental
tissue derived adult stem cells were obtained from the
extracted immature molars of dogs, and ex vivo expanded
PDLSC, DPSC, and periapical follicular stem cells
were transplanted into the apical involvement defect.
Autologous PDLSC showed the best regenerating
capacity of PDL, alveolar bone and cementum as well as
peripheral nerve and blood vessel which were evaluated
by conventional and immune histology.
Successful therapies for PDL tissue regeneration
will not only facilitate the treatment of periodontal
diseases, but may also be used to improve current dental
implant therapies. Numerous attempts to reconstruct
periodontal tissues around dental implants revealed the
challenge of avoiding fibrous tissue encapsulation and
the formation of functional cementum on the implant
There is still much to learn about the nature,
potentiality and behavior of dental stem/progenitor
cells. However, the opportunities for their exploitation
in dental tissue regeneration are immense and will lead
to significant benefits for the management of the effects
of dental disease.
Dental stem cells display multifactorial potential
Braz Dent J 22(2) 2011
Mesenchymal stem cells in the dental tissues 97
such as high proliferation rate, multi-differentiation
ability, easy accessibility, high viability and easy to be
induced to distinct cell lineages.
Therefore, these cells have been used for tissueengineering
studies in large animals to assess their
potential in preclinical applications. However, although
numerous breakthroughs in stem cell research have been
made thus far, their success and applicability in clinical
trials remains to be ascertained. Solid research into the
basic science and biology behind stem cells must be
performed before scientists leap into the clinical trials.
Technologies using MSC and iPS cells might be the new
era of personalized medicine. The heterogeneity among
patient factors and the biology of different stem cell types
reinforces the need for an individual-targeted approach
to stem cell therapy and other cell-based treatments.
Nos últimos anos, as pesquisas com células tronco têm aumentado
exponencialmente devido ao reconhecimento de que seu potencial
terapêutico pode melhorar a qualidade de vida de pacientes
com diversas doenças, como a doença de Alzheimer, isquemias
cardíacas e, até mesmo, nas pesquisas de medicina regenerativa
que visa uma possível substituição de órgão perdidos, como por
exemplo, os dentes. Baseado em habilidades de reparar tecidos
injuriados e restaurar parcialmente as funções de um órgão,
diversos tipos de células-tronco têm sido estudadas. Recentes
evidências demonstram que as células-tronco são primariamente
encontradas em nichos e que certos tecidos apresentam mais
células-tronco que outros. Entre estes, os tecidos dentais são
considerados como uma fonte rica de células-tronco mesenquimais
adequado para aplicações em engenharia tecidual. Sabe-se que
estas células têm o potencial de diferenciarem-se em diversos
tipos celulares, incluindo osteoblastos, células progenitoras de
neurônios, osteoblastos, condrócitos e adipósitos. Na odontologia,
a biologia celular e a engenharia tecidual são de grande interesse,
pois fornecem inovações na geração de novos materiais clínicos e
ou na regeneração tecidual. Estas podem ser isoladas e crescidas
em diversos meios de cultura apresentando grande potencial
para ser usada na engenharia tecidual, incluindo regeneração de
tecidos dentais, nervos e ossos. Recentemente, outra fonte de
células tronco tem sido geradas a partir de células somáticas de
humanos a um estágio de pluripotência, chamados de célulastronco
pluripotente induzida (iPS) levando à criação de célulastronco
específicas. Coletivamente, a multipotencialidade, altas
taxas de proliferação e acessibilidade, faz das células-tronco
dentárias uma fonte atrativa de células-tronco mesenquimais
para regeneração tecidual. Esta revisão descreve novos achados
no campo da pesquisa com células-tronco dentais e seu potencial
uso na regeneração tecidual.
This study was supported in part by grants from the National
Council for Scientific and Technological Development (FAPEGO
to E.G., and CNPq grants #302875/2008-5 and CNPq grants
#474642/2009 to C.E.).
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Accepted February 28, 2011
Concise Review: The Surface Markers and Identity
of Human Mesenchymal Stem Cells
FENG-JUAN LV,a,b,c ROCKY S. TUAN,d KENNETH M.C. CHEUNG,a,b,c VICTOR Y.L. LEUNG
Key Words. Mesenchymal stem cells • Surface epitopes • CD271 • CD146 • Markers • Pericytes •
Niche • Regenerative medicine
The concept of mesenchymal stem cells (MSCs) is becoming increasingly obscure due to the
recent findings of heterogeneous populations with different levels of stemness within MSCs isolated
by traditional plastic adherence. MSCs were originally identified in bone marrow and later
detected in many other tissues. Currently, no cloning based on single surface marker is capable
of isolating cells that satisfy the minimal criteria of MSCs from various tissue environments.
Markers that associate with the stemness of MSCs await to be elucidated. A number of candidate
MSC surface markers or markers possibly related to their stemness have been brought forward
so far, including Stro-1, SSEA-4, CD271, and CD146, yet there is a large difference in their
expression in various sources of MSCs. The exact identity of MSCs in vivo is not yet clear,
although reports have suggested they may have a fibroblastic or pericytic origin. In this review,
we revisit the reported expression of surface molecules in MSCs from various sources, aiming
to assess their potential as MSC markers and define the critical panel for future investigation.
We also discuss the relationship of MSCs to fibroblasts and pericytes in an attempt to shed light
on their identity in vivo. STEM CELLS 2014;32:1408–1419
Mesenchymal stem cells (MSCs) were first identified
from bone marrow mononuclear cells
(BM-MNCs) as fibroblastic colony-forming units
(CFU-Fs). Due to their multipotency and paracrine
effect [1, 2], MSCs are ideal candidates for
regenerative medicine [3, 4]. Currently, there is
no consensus on a single surface molecule to
identify MSCs from various sources. The minimum
criteria of MSCs  include: (a) remain
plastic-adherent under standard culture conditions;
(b) express CD105, CD73, and CD90, and
lack expression of CD45, CD34, CD14 or CD11b,
CD79a or CD19, and HLA-DR; (c) differentiate
into osteoblasts, adipocytes, and chondrocytes
in vitro. Other surface antigens generally
expressed by MSCs include CD13, CD29, CD44,
and CD10 [6, 7]. Although bone marrow (BM) is
the most widely recognized source of MSCs,
recent research has identified alternative sources
of MSC-like cells, including adipose tissue
(AT) , placenta , dental pulp , synovial
membrane , peripheral blood , periodontal
ligament , endometrium ,
umbilical cord (UC) , and umbilical cord
blood (UCB) [16, 17]. In fact, evidence has suggested
that MSCs may be present virtually in
any vascularized tissues throughout the whole
Genuine MSCs are expected to possess
both clonogenicity and tripotency. However,
only a fraction of CFU-Fs from plastic adherence
isolated MSCs (PA-MSCs) exhibited multipotency
, indicating that PA-MSCs
comprised a heterogeneous population of cells
with different lineage commitment , which
may relate to their in vivo environment. This is
reflected in the differences in the protein
expression profile, cytokine profile, or differentiation
potency of various sources of MSCs
(reviewed in ). For example, the percentage
of BM CFU-Fs with osteogenic potency
was higher than that with adipogenic potency
. Similarly, ectopic transplantation of BMMSCs
resulted in heterotopic bone tissue formation,
while dental pulp-derived MSCs generated
reparative dentin-like tissue . It is
now widely accepted that in the MSCs population,
only a proportion of cells satisfy the
“MSCs” criteria at single cell level, while the
other cells are more committed. There is also
a more immature population in MSCs which
are embryonic stem cell (ESC)-like and express
Oct-4 and Sox2 .
So far, the markers proposed for MSCs fall
into two categories: sole markers and stemness
markers. A sole marker is an alternative
MSC selection tool to plastic adherence, which
alone is sufficient to identify or purify MSCaDepartment
and Traumatology, Li Ka
Shing Faculty of Medicine,
The University of Hong Kong,
Hong Kong, People’s Republic
of China; bStem Cell &
Consortium and cCenter for
and Growth, Li Ka Shing
Faculty of Medicine, The
University of Hong Kong,
Hong Kong SAR, People’s
Republic of China; dCenter
for Cellular and Molecular
Engineering, Department of
University of Pittsburgh
School of Medicine,
Pittsburgh, Pennyslvania, USA
Correspondence: Victor Y.L.
Leung, Ph.D., 9/F, Lab Block,
Department of Orthopaedics and
Traumatology, Li Ka Shing Faculty
of Medicine, The University of
Hong Kong, 21 Sassoon Road,
Hong Kong SAR, People’s
Republic of China. Telephone:
1852-2819-9589; Fax: 1852-
2818-5210; e-mail: [email protected]
hk or Kenneth M.C. Cheung,
M.D., Department of Orthopaedics
and Traumatology, The University
of Hong Kong Medical
Centre, Queen Mary Hospital,
Pokfulam Road, Pokfulam, Hong
Kong SAR, People’s Republic of
China. Telephone: 1852-2255-
4254; Fax: 1852-2817-4392;
e-mail: [email protected]
Received September 16, 2013;
accepted for publication
February 9, 2014; first published
online in STEM CELLS EXPRESS
February 28, 2014.
VC AlphaMed Press
Stem Cells 2014;32:1408–1419 www.StemCells.com VC AlphaMed Press 2014
TISSUE-SPECIFIC STEM CELLS
like cells from their in vivo environment . A “stemness”
marker is able to identify a subset of MSCs with high CFU-Fs
and trilineage potential or even identify ESC-like population.
Ideally, such stemness marker may facilitate selection and
therefore enrichment of subpopulation that exhibit superior
CFU-Fs and multipotency. Based on the nature of the two different
types of markers, the sole markers are normally highly
expressed, while the stemness markers may be moderately
The majority of MSC markers are identified for BM-MSCs.
To date, however, whether these markers can be applied to
other sources of MSCs is not very clear. Moreover, the exact
in situ identity of MSCs is not entirely clear, although reports
have suggested they may have a fibroblastic or pericytic origin.
This review attempt to address these issues through a
comprehensive analysis of the current findings on MSC isolated
from various tissues via the use of single surface
markers. Moreover, the capacity of stemness markers representing
the subset of more primitive MSCs is revisited and
the origin of MSCs is discussed.
SOLE AND STEMNESS MARKERS OF MSCS
A number of molecules have been suggested as MSC markers,
as shown in Table 1. Among them, Stro-1, CD271, stagespecific
embryonic antigen-4 (SSEA-4), and CD146 are the
ones that have received the most attention and adopted in
studies as markers to sort MSCs. The expression of these four
molecules in various sources of MSCs is listed in Table 2.
Stro-1 is one of the most well-known markers for MSCs. Stro-
1 is a cell membrane single pass type I protein that translocates
from the endoplasmic reticulum to the cell membrane
in response to the depletion of intracellular calcium . By
combining negative selection against glycophorin-A, Stro-1
enriched CFU-Fs from BM with multipotency . However, it
did not enrich CFU-Fs from human endometrial stroma .
The degree of homogeneity of the Stro-1-selected MSCs was
further enhanced 1000-fold by positive selection for CD106
compared to PA-MSCs . Injection of human Stro-1(1) but
not Stro-1(2) BM-MNCs into rat myocardium led to arteriogenesis
and functional cardiac recovery . Further in vivo
research demonstrated that Stro-1(2) MSCs supported higher
hematopoietic stem cells (HSC) engraftment in nonobese diabetic/
severe combined immunodeficiency (NOD/SCID) mice
while no support was detected by Stro-1(1) MSCs. However,
Stro-1(1) MSCs exhibited greater capability in homing to
spleen, BM, and kidney . Conditioned medium from Stro-
1(1) MSCs could induce a greater degree of cardiac vascular
repair than PA-MSCs . These suggest that Stro-1 may be
involved in clonogenicity and play a role in homing and angiogenesis
However, Stro-1 is not universally expressed in all
reported types of MSCs. Stro-1 is expressed in dental- ,
synovial membrane- , decidua parietalis-derived MSCs 
and multipotent dermal fibroblasts . AT- , UCB- ,
UC- , peripheral blood-derived MSCs  are negative/low
for Stro-1 expression. It is reported that placenta-derived
MSCs gradually lose Stro-1 expression in culture . In contrast,
however, the expression of Stro-1 in BM-MSCs increases
with culture time .
The potential of Stro-1 as an MSC marker is limited in several
ways. It is unclear whether Stro-1 expression correlates
with multipotency. Stro-1 is also unsuitable as a sole marker
to separate MSCs from its harboring tissue, at least not from
BM, as greater than 95% of Stro-1(1) cells in the human BM
were glycophorin A expressing nucleated erythroid cells .
Moreover, Stro-1 expression appears not universal for various
CD271 (also named as low-affinity nerve growth factor receptor)
is a receptor for neurotrophins, which stimulate neuronal
cells to survive and differentiate. CD271 has been used to
select CFU-Fs from BM-MNCs. The percentage of
CD90(1)CD105(1)CD45(2)CD34(2)CD79(2) cells in BMMNCs
coincided with the amount of CD271(1) cell subset
(0.54%) . CFU-Fs could only be generated from CD271(1)
subsets of CD45(2)glycophorin-A(2) human BM-MNCs while
the CD271(2) fraction showed no residual CFU-F activity [7,
29]. CD271(1) BM MSCs were shown to have enhanced capability
in promoting HSC engraftment compared to PA-MSCs
 and also induced superior chondral repair than the
CD271(2) BM MSCs . These together suggest a role of
CD271 in maintaining clonogenicity and function of MSCs.
However, the majority of the CD271(1) cells were found not
to coexpress CD90 and CD73, the two general markers of
MSCs. The percentage of CD90 and CD73 positive cells was
found to be very low (<10%) in CD271(1) cells from BM 
and from AT (10%–20%) . Moreover, nearly 50%–99% of
the CD271(1) cells in BM  and synovium  coexpressed
CD34, which disqualifies CD271 as a sole marker to isolate
MSCs from various tissues .
Similar to Stro-1, CD271 is not universally expressed in
various MSCs. CD271 shows high levels of expression in BM
and AT MSCs [6, 55] and is also expressed in periodontal ligament
MSCs . However, it is expressed at low levels in
placenta-derived MSCs [31, 63] and not expressed in synovial
membrane- [47, 65, 66], peripheral blood- , UC-, and
UCB-MSCs [48, 49]. Although Watson et al.  reported
detection of CD271 in UCB-MSCs, CD271 failed to enrich CFUFs
and multipotency. The potency of CD271(1) cells as a
stemness marker was further challenged by the finding of
lower trilineage differentiation potential in CD105(1)/
CD271(1) expanded BM-MNCs subsets compared to unsorted
BM-MNCs . Therefore, CD271 may not be considered as a
MSC stemness marker.
SSEA-4 is an embryonic stem cell marker. It has been documented
to isolate genuine MSCs from BM . SSEA-4(1)
BM cells can expand extensively, while SSEA-4(2) subsets fail
to grow. SSEA-4(1) cells also show tripotency . SSEA-4
expression gradually increases in BM culture over time .
Besides in BM-MSCs, SSEA-4 expression was also detected in
placenta- , periodontal ligament- , dental pulp- ,
and synovial membrane -derived MSCs. On the contrary,
AT-, UC-, or UCB-derived MSCs [48, 51] do not express SSEA-
4. A more important question that whether the clonogenicity
and multipotency of SSEA-4(1) cells is superior to the
Lv, Tuan, Cheung et al. 1409
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Table 1. Potential MSC sole markers and their expression in unsorted MSC population
Markers Marker potential % positive MSC source References
Stro-1 Enrich CFU-Fs from whole BM 10 Human BM-MNCs 
11.2 Human BM-MNCs 
6 Human BM-MSCs after 1 week in culture 
2.1 Human endometrial stroma cells 
1.29 Human amnion MSCs at passage 0–2 
8 Human dermal fibroblasts at passage 3 
28.96 Inflamed periodontal ligament MSCs 
37.84 Healthy periodontal ligament MSCs
CD271 Enrich CFU-Fs from CD45/A-glycophorin
A depleted BM-MNCs
2.3 Human BM-MNCs [7, 29]
Higher differentiation-related gene
expression after induction compared
to MNC-derived MSCs
Negligible Human placenta MSCs 
SSEA-4 Enrich CFU-Fs from whole BM 1–2 Mouse BM-MSCs at day 2 in culture 
71 Mouse BM-MSCs after 100 days in culture
2–4 Human BM cells
37.82 Human amnion MSC at passage 0–2 
CD146 Enrich CFU-Fs from BM-MSCs 9.4 Human endometrial stroma 
Enrich cells with multipotency from
1.5 CD146(1)PDGF-Rb(1) Human endometrial stroma 
Downregulated in differentiated cells 0.1 Human BM-MSCs [34, 35]
1.2 Human CD45 depleted BM-MNCs 
42.7 Human BM-MSCs 
17.2–37.9 Human UC-MSCs
11.55 Inflamed periodontal ligament MSCs 
21.85 Healthy periodontal ligament MSCs
9.4 Fresh isolated human endometrial stromal cells 
CD49f Enrich clonogenicity and differentiation
88.1 human BM-MSCs at passage 1 
Knocking down results in differentiation
15 Human BM-MSCs 
55 Human UCB-MSCs 
CD349 Enrich CFU-Fs from whole placenta cells 0.2 Total human plancenta cells 
GD2 High specificity for isolating MSCs from
95 Human CD45(2)CD105(1)CD73(1) BM-MNCs 
65 Human BM-MSCs 
3 Human UCB-MSCs 
3G5 Enrich CFU-Fs Decidua parietalis 
63 Dental pulp CFU-Fs 
14 BM CFU-Fs
SSEA-3 Enrich cells with clonogenicity and
ectodermal, endodermal, and mesodermal
1 BM-MSCs 
SUSD2 Enrich CFU-Fs and tri-potency 4.2 Human endometrial stromal cells 
Stro-4 Enrich CFU-Fs <5 Human and ovine BM-MSCs 
MSCA-1 MSCA-1 positivity enrich CFU-Fs by 90-
fold from BM-MNCs; MSCA-1 and
CD56 double positivity enrich CFU-Fs
by 180-fold from BM-MNCs; MSCA-
1(1)CD56(2) selects for better adipogenesis
in BM-MSCs; MSCA-
1(1)CD56(1) selects for better chondrogenesis
0.5–5.5 coexpressed CD271bright BM cells 
CD200 Enrich CFU-Fs from BM-MNCs; downregulated
in differentiated cells
0.15 Human BM-MNCs 
PODXL PODXL decreases in high-density
90 Human BM-MSCs at passage 2 
13 Human BM-MSCs 
25 Human UCB-MSCs 
Sox11 Downregulated during culture. Knockdown
affects proliferation and osteogenesis
Not tested Human BM-MSCs 
TM4SF1 Enriched in MSCs compared with their
source tissue or fibroblasts
Not specified BM, AT, and UCB 
AT, adipose tissue; BM, bone marrow. BM-MNCs: bone marrow mononuclear cells; CFU-Fs, fibroblastic colony-forming units; MSC, mesenchymal
stem cell; UCB, umbilical cord blood.
1410 Markers and Identity of MSCs
VC AlphaMed Press 2014 STEM CELLS
traditional PA-MSCs or MSCs sorted by other molecules is
unanswered. Notably, the expression of SSEA-4 in UCB-HSCs
was suggested to be an artificial induction in the in vitro culture
as fetal calf serum (FCS) contained globoseries glycosphingolipids
which can be recognized by a SSEA-4 antibody,
and in vitro FCS exposure may induce SSEA-4 expression .
In fact, some other studies reported no detection of SSEA-4
expressing cells in unsorted BM [51, 52, 76]. These findings
raise the issue of the physiological relevance and reliability of
SSEA-4 as the marker for MSCs.
CD146 is a key cell adhesion protein in vascular endothelial
cell activity and angiogenesis. Notably, CD146 emerged as an
attractive candidate for identifying genuine MSCs. In human
endometrial stroma population, CD146(1)PDGF-Rb(1) cells
show higher CFU-F enrichment compared with CD146(2)
PDGF-Rb(2) cells (7.761.7% vs. 0.760.2%) . CD146 has
a greater CFU-Fs enrichment capacity than CD90, Stro-1, or
CD133 . CD146 expression also defines MSCs with higher
multipotency. Russell’s group reported that the expression
level of CD146 in the tripotent clones is twofold of that in
the unipotent clones . Additionally, CD146 also identifies
MSCs with higher supporting capacity for hematopoiesis, as in
vitro, CD146(1) MSCs show more than 100-fold increase in
the long-term culture colony output by 8 weeks compared to
unsorted BM-MNCs , and in vivo, when transplanted into
mice, CD146(1) BM stroma subendothelial cells exhibit the
capacity to reorganize the hematopoietic microenvironment
to heterotopic sites .
Importantly, the expression of CD146 was found not only
in BM-MSCs  but also in almost all the other sources of
MSCs, including MSCs derived from AT , UC [15, 17], synovial
membrane , UCB , placenta , dermis ,
periodontal ligament , and intervertebral disc . In fact,
CD146-expressing MSC clones from multiple organs were
found to exhibit trilineage potency .
CD49f (a6-integrin) regulates signaling pathways in a variety of
cellular activities. Oct-4 and Sox-2 directly regulates the expression
of CD49f, and that the knockdown of CD49f in ESCs results
in differentiation into three germ layers, indicating CD49f is
involved in the maintenance of pluripotency and is an ESC
marker . CD49f has also been identified as a specific HSC
marker and shown to enrich cells capable of generating longterm
multilineage grafts . To date, CD49f expression has
been detected in BM-MSCs , fetal urinary bladder-derived
MSCs , and UCB-MSCs . It is possible that the expression
of CD49f may implicate the stemness of MSC culture. In fact,
study has shown that CD49f is associated with high clonogenicity
and multipotency in less confluent MSC culture . Condition
that induces MSC sphere formation can enrich CD49f(1)
population compared with MSCs in monolayer . Moreover,
higher expression level of CD49f, such as in UCB-MSCs, is functionally
linked with a higher lung clearance rate in systemic infusion
. Nonetheless, CD49f may not necessarily be of value
as a single specific marker of MSCs since it is also widely
expressed in epithelial cells as well as endothelial cells, monocytes,
platelets, and thymocytes .
CD349 (frizzled-9) is a transmembrane-spanning receptor that is
activated by Wnt ligands. It has been proposed to enrich CFU-Fs
from placenta cells . Additionally, CD349 expression has
been reported in periodontal ligament-derived MSCs . However,
whether CD349 being essential to the enrichment of clonogenicity
has been questioned by other reports. For example,
while CFU-F could be enriched by 60-fold in the
CD349(1)CD10(1)CD26(1) fraction, the CD349(1)CD10(2)
CD26(2) subsets did not show CFU-F capacity, implying that
CD349 alone is not sufficient for CFU-F enrichment. In fact,
CD349(2) subset has been shown to proliferate at a higher rate
than CD349(1) subset in periodontal ligament MSCs .
Moreover, CD349(2), rather than CD349(1) placenta MSCs,
show a function in recovering blood flow following vascular
occlusion . These suggest CD349 might not be a critical MSC
marker or essential in enriching MSC function.
GD2, the neural ganglioside, was found by Martinez et al. 
as a single surface marker sufficient to isolate MSCs from BM
Table 2. Detection of Stro-1, CD271, SSEA-4 and CD146 in various MSC sources
Stro-1 CD271 SSEA-4 CD146
Sources of MSCs Presence References Presence References Presence References Presence References
Bone marrow 1  1 [6, 7, 29, 47, 48] 1 [32, 43, 48] 1 [17, 19, 34, 35, 49, 50]
2  2 [51–53]
Adipose tissue 2  1  2  1 
Umbilical cord 2  2  2 [16, 48] 1 
Umbilical cord blood 2  2 [48, 49] 22/1 [48, 51] 1 [17, 49]
Peripheral blood 2  2 
Dental pulp 1 [10, 40] 2/low  1  1 
Placenta 2  2/low  1  1 
Synovial membrane 1  2 [47, 65, 66] 1  1 
Periodontal ligament 1  1  1 
Dermis 1  1  1  1 
Endometrium 1  1 [14, 33]
Decidua parietalis 1  1 
MSCs, mesenchymal stem cells; 1, expression detected; 2, expression not detected; 2/low, expression detected but very low.
Lv, Tuan, Cheung et al. 1411
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as GD2 is highly expressed in CD45(2)CD73(1) MSCs (>90%)
but not in CD45(2) BM cells. It is also expressed in AT-MSCs
and UC-MSCs , but not on foreskin fibroblasts. However, a
portion of CD34(1) or CD19(1) BM cells also express GD2
, suggesting GD2 expression is not limited within BMMSCs.
3G5 is a pericyte marker. Khan et al.  reported that
plastic-adherence isolated BM-MSCs were negative for CD271,
CD56, and Stro-1 but positive for 3G5. To date, 3G5 expression
is detected on BM-MSCs, dental pulp- , and decidua
parietalis-derived MSCs . Shi and Gronthos  showed
that a minor population of BM-MSCs positive for Stro-1
expression is also positive for 3G5. 3G5 positivity accounts for
14% of BM CFU-Fs and 63% of dental pulp CFU-Fs . However,
a large proportion (54%) of hematopoietic BM cells
express 3G5, eliminating its potential as a sole marker to isolate
MSCs from human bone marrow .
Stage-specific embryonic antigen-3 (SSEA-3) is a pluripotent
stem cell marker. Recently, evidence showed that a minor
subset of SSEA-3(1)CD105(1) cells in MSCs, namely
multilineage-differentiating stress enduring (MUSE) cells, are
able to differentiate into ectodermal, endodermal, and mesodermal
lineage cells in vivo . Induced pluripotent stem
cells (iPSCs) were only found to be derived from the MUSE
cell subset in fibroblasts but not the non-MUSE subset ,
suggesting that SSEA-3 and CD105 expressing MSCs (1%) as
progenitor cells reminiscent of, but not identical to,
pluripotent-like ESCs. MUSE cells coexpressed some other pluripotency
markers including Nanog, Oct3/4, PAR-4, Sox2 .
Since MSCs are strongly positive for CD105, MUSE cells can
be represented as the SSEA-3(1) subset of MSCs. MUSE cells
are not tumorigenic and can differentiate in vivo without
prior genetic manipulation or growth receptor induction ,
hence they may have practical advantages for regenerative
Type 1 integral membrane protein Sushi domain containing 2
(SUSD2) has been recently reported to enrich for CFU-Fs and
tripotency from endometrium- , and BM-derived MSCs
. SUSD2 can be detected by the W5C5 antibody .
SUSD2 is not expressed in hematopoietic cells. In the endometrium,
it is predominantly expressed in perivascular
regions. W5C5(1) cells are also capable of producing endometrial
stromal-like tissue in vivo . Whether SUSD2 being a
common MSC marker remains to be consolidated.
There are also some other MSC sole or stemness markers proposed
by some researchers which are well-investigated. Stro-
4, MSCA-1, CD56, CD200, and PODXL have been proposed as
MSC markers by their CFU-Fs enrichment capacity. The antibody
Stro-4 identified the beta isoform of heat shock protein-
90. It was found expressed in BM-, dental pulp-, periodontal
ligament-, and AT-derived MSCs, and enriched CFU-Fs from
both human and ovine BM by 16- and 8-fold compared to
BM-MNCs . MSCA-1 (mesenchymal stem cell antigen-1) is
identical to tissue nonspecific alkaline phosphatase . Compared
to unsorted BM-MNCs, MSCA-1 selection resulted in a
90-fold increase in enrichment of CFU-Fs and a 180-fold
increase when coselected for CD56 . Another surface molecule
CD200 was reported  to enrich CFU-Fs from BMMNCs
to 333-fold. PODXL, a sialomucin in the CD34 family,
was also reported to decrease in high-density cultures which
have lower clonogenicity and differentiation potency compared
to less confluent cultures .
Unlike the above molecules, neuron-glial antigen 2 (NG2),
Sox11, and TM4SF1 were proposed largely based on their
expression. NG2 is first observed on the surface of neural progenitors
and is a pericyte marker whose expression is also
shared by BM-MSCs [18, 86, 87]. Sox11, a transcription factor
previously identified in neural progenitor cells, was found to
significantly decrease during MSC passages and knockdown of
Sox11 with siRNA decreased the proliferation and osteogenic
differentiation potential of MSCs . TM4SF1 is another surface
protein highly expressed in BM-, UCB-, and AT-MSCs
which is not detected in mononuclear cells and fibroblasts,
suggesting it may be a potential marker for MSC selection
COMPARISON OF MSCS ISOLATED BY DIFFERENT MARKERS
With the array of potential markers identified in MSCs, it is
still unclear whether these markers define different or overlapping
subpopulations of MSCs. One of the reasons is that
the phenotypic or functional differences among the MSC subpopulations
selected by different markers are still poorly
understood. Here we aim to review studies that were
designed to compare various MSC populations sorted in parallel
and directly from single sources with respect to their coexpression
of MSC markers, CFU enrichment capacity or
CFU-Fs Enrichment Capacity
Delorme et al.  reported that, among nine molecules,
CD73, CD130, CD146, CD200, and integrin aV/b5 were able to
enrich CFU-Fs from CD235a(2)/CD45(2)/CD11b(2) BMMNCs,
while CD49b, CD90, and CD105 showed less enrichment.
Among various methods of MSC isolation from BMMNCs,
including plastic adherence, RosetteSep-isolation, and
CD105(1) and CD271(1) selection , CD271(1) fraction
showed the highest number of CFU-Fs colonies. Double selection
for CD56 or MSCA-1 enriched CFU-Fs to 3- or 2-fold,
respectively, in CD271 (bright) BM-MNCs . Schwab et al.
 found that CD146 but not Stro-1 or CD133 selection
enriched CFU-Fs from human endometrial stromal cells. Sorting
of CD34(2)CD45(2)  or CD45(2)  human BMMNCs
with CD271 and CD146 revealed that CFU-Fs units
remained exclusively in CD271(1) population regardless of
CD146 expression, with a tendency toward more CFU-Fs in
CD271(1)CD146(1) cells relative to CD271(1)CD146(2) cells.
CD146(1) subsets accounted for 96% of CFU-Fs in unfractionated
human dental pulp cells, while Stro-1(1) and 3G5(1)
subsets accounted for around 80% and 60%, respectively .
In addition, the cloning efficiency of W5C5(1)CD146(1) cells
was found significantly higher than CD140b(1)CD146(1) cells.
W5C5hi cells had a high clonal capacity equivalent to
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VC AlphaMed Press 2014 STEM CELLS
W5C5(1)CD146(1) cells . Taken together, these findings
imply that CD146 and CD271 positivity indicates superior
CFU-F capacity in MSCs. These findings are summarized in
Battula et al.  reported that chondrocytes and pancreaticlike
islets are predominantly induced from MSCA-
1(1)CD56(1) BM-MNCs whereas adipocytes emerge exclusively
from MSCA-1(1)CD56(2) subsets, indicating that CD56
is involved in differentiation tendency. Jarocha et al. 
reported that CD271(1) or CD105(1) MSCs have lower lineage
marker expression than PA-MSCs after osteogenic, chondrogenic,
and adipogenic induction. Arufe et al.  reported
that when comparing CD73, CD106, or CD271 positive human
synovial membrane cells, CD271(1) cells are highly chondrogenic,
whereas the CD73(1) cells are less chondrogenic and
the CD106(1) cells mostly undifferentiated after induction.
Vaculik et al.  reported that CD271(1) but not SSEA-4(1)
dermal cells exhibit osteogenic and chondrogenic differentiation
potential. Dermal SSEA-4(1) cells, in contrast, are only
responsive to adipogenic induction. In adult human BMMNCs,
CD271(1)CD146(2/low) and CD271(1)CD146(1) subsets
 show a similar capacity to differentiate and to support
hematopoiesis, but the two subsets have been found at
different sites; CD271(1)CD146(2/low) cells are bone-lining,
while CD271(1)CD146(1) cells have a perivascular localization,
suggesting that the two subsets play different roles in
HSC niche. The function of surface markers in multipotency
enrichment is summarized in Table 4.
Surface Marker Coexpression
Relevant studies on the degree of coexpression of surface
markers on MSCs is summarized in Table 5. The expression of
CD106 and CD146 was found to be restricted to the MSCA-
1(1)CD56(2) MSCs and CD166 to MSCA-1(1)CD56(1/2)
MSCs . Vaculik et al.  reported that in human dermis,
the expression pattern of SSEA-4 is almost analogous to
CD271. Both were found only weakly expressed and coexpressed
with CD45. Van Landuyt and Quirici also reported the
detection of CD34 expression on CD271(1) subpopulation of
human synovial and BM-MSCs [29, 47]. In human dermis,
CD73 and CD105 are coexpressed . A minor population of
the human dermis CD73(1) cells is CD90(2). Dermis
CD271(1) cells were CD73(1) and CD105(1), whereas the
majority of CD271(1) cells are CD90(2) . Similarly, in two
other reports, only a minor subset of the CD271(1) cells
express CD90, CD73 (<10% in cultured CD271(1) cells from
BM , 10%–20% in freshly purified CD271(1) cells from
adipose tissue ). Maijenburg further reported that the distribution
of CD271(1)CD146(2) and CD271(1)CD146(1) subsets
correlates with donor age. The main subset in pediatric
and fetal BM was reported to be CD271(1)CD146(1),
whereas CD271(1)CD146(2) population was dominant in
adult marrow . In endometrial MSCs, 28% of W5C5(1)
cells are CD146(1), while 60% of W5C5(1) cells are Stro-
1(1). A small population of W5C5(1) cells also express other
Table 3. Comparison of MSC sorting protocols for CFU-Fs enrichment
Cell subsets analyzed Whole cell population Result References
Stro-1(1), CD133(1), CD90(1),
Human endometrial stromal cells Only CD146 showed CFU-Fs
CD49b(1), CD90(1), CD105(1),
CD73(1), CD130(1), CD146(1),
Human BM-MNCs CD49b, CD105, and CD90 showed low
CFU-Fs enrichment. CD73, CD130,
CD146, CD200, and integrin aV/ß5
showed higher CFU-Fs enrichment
MSCA-1(1), CD271(1), CD56(1) Human BM-MNCs CD271(1)CD56(1) fraction enriched
CFU-Fs to threefold compared to
CD271(1)CD56(2) fraction. MSCA-
1(1)CD56(1) fraction gave rise to
two fold higher CFU-Fs than MSCA-
PA-MSCs, RosetteSep-, CD105(1) or
Human BM-MNCs CFU-Fs was most enriched in
CD271(1),CD146(1) CD34(2)CD45(2) human BM-MNCs CFU-Fs remained exclusively in
CD271(1)CD146(1) cells had more
CFU-Fs relative to
CD271(1),CD146(1) Human BM-MNCs CFU-Fs was not observed in CD271(-)
cell fraction. CD146 positivity further
enhanced CFU-Fs to 2.1 times in
Stro-1(1), CD146(1), 3G5(1) Human dental pulp cells No colony formation could be detected
in STRO-1bright/CD146(2) human
bone marrow. CD146(1) subsets
accounted for 96% of CFU-Fs in unfractured
dental pulp cells, while
Stro-1(1) and 3G5(1) subsets
accounted for around 80% and 60%,
BM-MNCs, bone marrow mononuclear cells; CFU-Fs, fibroblastic colony-forming units; MSC, mesenchymal stem cell; PA-MSCs, plastic adherence
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lineage markers, like CD24 (11.6%), CD31 (5 %), CD45 (4.7 %),
and epithelial cell adhesion molecule .
MSC IDENTITY IN VIVO: FIBROBLASTS OR PERIVASCULAR CELLS?
Adult stem cells are found in specialized niches that store and
maintain stem cells and mediate the balanced response of
stem cells to the needs of organisms. The definition of MSCs
has been based on their ability to self-renew and to differentiate
into certain mature cell types in vitro. Their identity in
vivo, however, remains unclear. Unlike the well-established
niche of BM for HSCs , the true identity of MSCs and their
niche in vivo is still under debate. Currently, it has been
raised that MSCs may derive from fibroblasts or pericytes.
Fibroblasts are a type of cells synthesizing collagen, the
major structural framework for animal tissues, and in the
human body they are found in virtually every organ and tissue.
MSCs have a close resemblance to fibroblasts . Fibroblasts
and MSCs are both plastic adherent and share similar
cell morphology. Human dermal fibroblasts express many cell
Table 4. Comparison of multipotency of sorted MSCs
Cell subsets analyzed Whole cell population Capacity compared Result References
Chondrogenesis Chondrogenic potential: CD271
MSCA-1(1), CD56(1) Human BM-MNCs Chondrogenesis, adipogenesis MSCA-1(1)CD56(1): Chondrogenic
and pancreatic differentiation
Pancreatic differentiation MSCA-1(1)CD56(2): Adipogenic
potential, no chondrogenic
and pancreatic differentiation
Human dermis MSCs Chondrogenesis, adipogenesis,
CD271(1) cells had tri-lineage
potential. Dermal SSEA-4(1)
cells could only go for adipogenesis.
showed a significantly higher
capacity than CD90(1) cells.
CD105(1) or CD271(1)
Human BM-MNCs Osteogenesis, chondrogenesis,
CD271(1) or CD105(1) MSCs
showed lower differentiation
related marker expression
than PA-MSCs after osteogenic,
chondrogenic and adipogenic
CD271(1), CD146(1) Human BM-MNCs Osteogenesis, chondrogenesis,
showed a similar differentiation
BM-MNCs, bone marrow mononuclear cells; MSCs, mesenchymal stem cells; SSEA-4, surface-specific embryonic antigen.
Table 5. Comparison of coexpressed markers in sorted MSCs
Cell subsets analyzed Whole cell population Result References
MSCA-1(1), CD271(1), CD56(1) Human BM-MNCs CD271 (1)CD56(2) cells expressed CD106
and CD146. CD271(1)CD56(1) cells
exclusively expressed CD166.
CD271(1)CD56(1) double positivity
enriched SSEA-4 expression.
CD271(1)CD56(1) double positivity
enriched MSCA-1 expression.
CD271(1), SSEA-4(1) Human dermis cells Expression pattern of SSEA-4 in dermis was
analogous to CD271. CD271 and SSEA-4
both coexpressed with CD45(1) cells.
CD73 and CD105 were coexpressed. A
minor population of the CD73(1) cells
was CD90(2). CD271(1) cells were
CD73(1) and CD105(1), whereas the
majority of CD271(1) cells were
W5C5(1) Human endometrial cells W5C5(1) cells were 28% CD146(1), 60%
Stro-1(1), 11.6% CD24(1), 5.3%
CD31(1), 4.7% CD45(1).
BM-MNCs, bone marrow mononuclear cells; MSCs, mesenchymal stem cells; SSEA, surface specific embryonic antigen.
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VC AlphaMed Press 2014 STEM CELLS
surface proteins similar to MSCs, including the general
markers used for MSC characterization . Human dermal
fibroblasts also have tripotency [91, 92], although contradictory
finding has been reported which suggests a lack of multipotency
. In addition, human dermal fibroblasts show
immunoregulatory functions similar to MSCs [93, 94].
Another hypothesis is that MSCs reside throughout the
body as pericytes or perivascular cells and that the perivascular
zone is the in vivo niche of MSCs . Pericytes are a relatively
elusive cell type recognized by virtue of their
anatomical location of their residence, that is on the abluminal
surface of endothelial cells in the microvasculature, rather
than by a precisely defined phenotype. As pericytes, MSCs
may be readily released from their niche and secrete immunoregulatory
and trophic bioactive factors upon tissue damage.
As such, MSCs may function as a source of stem cells for
A perivascular niche of MSCs is supported by the observation
that in the majority of solid tissues where MSCs have
been found, blood vessels may be the only common anatomical
structure. Consistent with the observation, the mesenchyme
acts as a “space filler” before the development of a
vascular system in early embryonic limb development .
Similar to MSCs, pericytes or perivascular cells are able to differentiate
into osteoblasts, chondrocytes, adipocytes, fibroblasts,
myofibroblasts, and smooth muscle cells in vitro .
In fact, CD146(1) perivascular cells from multiple organs
expressed general MSC surface antigens , as well as 3G5
 and NG2 . Observations in vivo also support the association
of pericytes with MSCs. For instance, multipotential
stem cells were identified in the mural cell population of the
vasculature . In rat malignant glioma, intratumoral injection
of MSCs  resulted in the engraftment of MSCs into
tumor vessel walls and the expression of several pericyte
Several studies have further compared the associations of
MSCs with fibroblasts and pericytes. Blasi et al.  reported
that AT-MSCs cannot be distinguished from human dermal
fibroblasts in vitro by phenotype or multipotency. However,
AT-MSCs, but not dermal fibroblasts, displayed strong angiogenic
and anti-inflammatory activity. Sacchetti et al. 
found that only CD146(1) MSCs, but not muscle or skin fibroblasts,
are capable of reconstructing BM and conferring a
hematopoietic microenvironment in immunocompromized
mice. Additionally, several transcripts were found differentially
expressed between HS68 fibroblasts and MSCs, whereas several
inhibitors of the Wnt pathway (DKK1, DKK3, SFRP1), an
important pathway in regulation of MSCs, were highly
expressed in fibroblasts, suggesting that MSCs and fibroblasts
have distinct gene expression profiles. Gene and microRNA
expression comparison of human MSCs and dermal fibroblasts
revealed a panel of MSC-specific molecular signature, which
mainly encode transmembrane proteins or associate with
tumors . In a comprehensive study by Covas et al. ,
the cell morphology and the phenotypes were found to be
comparable among 12 types of MSCs, 2 origins of pericytes,
and 4 sources of fibroblasts. However, different from MSCs
and pericytes, fibroblasts were reported to be weak for
CD146 expression and high for the expression of fibroblastspecific
protein-1 (FSP-1, also named as S100A4), a specific
fibroblast marker. Furthermore, serial analysis of gene expression
revealed a consistent grouping of MSCs with pericytes
and hepatic stellate cells, while fibroblasts differentially clustered
with smooth muscle cells and myofibroblasts rather
than MSCs .
The close relationship of MSCs with perivascular cells is
also reflected by the physical distribution of the MSC specific
markers in vivo. As summarized in Table 6, the general MSC
antigens, such as CD73, CD90, and CD105 have a vascular and
perivascular expression pattern [18, 68], although their
Table 6. Physical expression of MSC markers in vivo
Markers Expression site References
CD73, CD90, and CD105 Dermis: Vascular and perivascular expression 
Skeletal muscle, placenta, and white adipose tissue: Perivascular expression 
CD90 Endometrium: Expressed on all the stroma of the human endometrium, including the fibroblasts,
perivascular and endothelial cells
Stro-1 BM: Expressed on blood vessel walls 
Dental pulp: Expressed on blood vessels and around perineurium surrounding nerve bundles 
Endometrium: On endothelial cells and on the stroma around blood vessels 
Placenta: Expressed around the vessels 
NG2 Skeletal muscle, pancreas, placenta, white adipose tissue, fetal heart, fetal skin, lung, brain, eye, gut,
bone marrow, and umbilical cord: Only expressed in periphery of capillaries and microvessels in
almost all tissues
CD146 Skeletal muscle, pancreas, placenta, white adipose tissue, fetal heart, fetal skin, lung, brain, eye, gut,
bone marrow, and umbilical cord: Expressed on perivascular cells surrounding capillaries, arterioles
and venules, and on endothelium in capillaries, but not on microvessel endothelial cells
BM and dental pulp: Blood vessel wall expression 
Endometrium: Expressed on perivascular and endothelial cells 
Placenta: expressed around the vessels 
3G5 Placenta: expressed on scattered cells around the vessels. 
CD271 Dermis: presented on cutaneous nerve fibers, Schwann cells, dermal single cells, and, faintly, on clusters
of basal keratinocytes
BM: CD271(1)CD146(2/low) cells were bone-lining, while CD271(1)CD146(1) had a perivascular
SSEA-4 Dermis: Presented on cutaneous nerve fibers, Schwann cells, dermal single cells, and, faintly, on clusters
of basal keratinocytes
SUSD2/W5C5 Endometrial tissue: Perivascular location. 
BM, bone marrow; MSC, mesenchymal stem cells; SSEA, surface specific embryonic antigen.
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expression can also be found in fibroblasts . For the MSC
specific markers, Stro-1, NG2, CD146, and 3G5 expression was
mainly found in perivascular area of capillaries, microvessels,
and/or venules in many tissues [18, 40], despite additional
expression was found in certain endothelial cells for CD146
 and in endothelial cells and stroma for Stro-1 . This
further supports the identity of MSCs as perivascular cells in
vivo, and that MSCs may bear stronger resemblance to pericytes
and perivascular cells rather than to fibroblasts. However,
this “perivascular niche” theory cannot explain why
MSC-like cells are also detected in avascular tissues, such as
in articular cartilage  and nucleus pulposus . A further
postulation is that MSCs may have more than one origin
than the perivascular niche, as disclosed in the dual origin of
odontoblasts in the teeth by genetic lineage tracing .
This postulation of a nonpericytic origin of MSCs is also supported
by the fact that MUSE cells, a subset of MSCs with
higher stemness, do not express CD146 .
CONCLUSIONS AND PERSPECTIVES
A large number of markers have been brought forward to
facilitate the isolation of MSCs from their surrounding environment
or the selection of MSCs with high stemness. It
should be noted that the marker expression of MSCs is not in
a stable level. Culture conditions have potential influence on
the phenotype of MSCs and that such influence may contribute
to the contradictory reports on marker expression. Particularly,
some antigens may be artificially induced by in vitro
manipulation and culturing, such as the induction of SSEA-4
by FCS . Culture confluence can also induce certain
markers, such as CD49d, CD200, or CD106, or diminish them,
such as CD49f and PODXL . Certain growth factors and
cytokines, such as fibroblast growth factor and interferon-Ç,
or disease conditions such as inflammation, may also
modulate the phenotype of MSCs (Table 7). This therefore
emphasizes the importance of a standard operation procedure
for in vitro MSC expansion and validation of the markers
As shown in the above analysis, there is no sole marker
that is truly MSC-specific. Among the known MSC markers,
CD146 may be the most appropriate stemness marker, as it is
universally detected in the MSC population isolated from various
tissues, and enriches cells with clonogenicity and multipotency.
On the other hand, SSEA-3 may be a more immature
stemness marker which represents an ESCs-like phenotype.
This is consistent with the proposed in vivo identity of MSCs
as pericytes, as CD146 is also a pericyte marker. Interestingly,
CD146 is highly expressed in both MSCs and pericytes, but
not in dermal fibroblasts , while FSP-1, a fibroblast marker,
is lowly expressed in MSCs and pericytes , lending support
to the closer association of MSCs with pericytes. However,
this theory cannot explain the non-pericytic origin of MSCs
suggested in a number of reports. Further investigation to
Table 7. Effect of in vitro or in vivo conditions on MSC phenotype
Regulatory factors Markers investigated Findings References
Inflammation Stro-1 There was no significant difference in proliferation, differentiation or Stro-1
positivity between MSCs isolated from normal and inflamed dental pulps.
Stro-1, CD90, CD105, CD146 Inflammed dental pulps expressed higher levels of MSC markers STRO-1,
CD90, CD105, and CD146 compared with normal dental pulps.
Stro-1, SSEA-4 More Stro-1 and SSEA-4 positive cells were found in healthy than in
inflammed gingival tissues.
CD49d, CD49f, CD200,
Culture confluency was shown positively correlated with the expression of
CD49d, CD200 and CD106, and negatively correlated with CD49f and
Serum SSEA-4 FCS contained globoseries glycolipids which could be recognized by a SSEA-
4 antibody, and exposure to FCS induced the cell-surface expression of
SSEA-3 in cord-blood-derived HSCs
Interferon HLA class II HLA class II expression in MSCs was induced by IFN-c. [109, 110]
HLA-DR HLA–DR positivity upon addition of IFN remained unchanged. 
NG2 Addition of IFN-c repressed the transcription of NG2 in MSCs after neural
Growth factors CD105, CD73, CD90, CD29,
FGF induced expression of HLA-DR, and lowered the expression of CD146
and CD49a, as well as the expression of CD49c. Expression of the MSC
surface antigens HLA–A/B/C, CD105, CD73, CD90, CD29, and CD44 was
Stemness markers (Oct4A,
Notch 1, Hes5), neural
markers (Nestin, Pax6,
EGF1bFGF pretreatment downregulated the expression of stemness
markers Oct4A, Notch1 and Hes5, whereas neural/neuronal molecules
Nestin, Pax6, Ngn2 and the neurotrophin receptor tyrosine kinase 1 and
3 were upregulated.
CD44, CD90, CD146, CD105 FGF-2 resulted in reduced expression of CD146 and alkaline phosphatase,
which was partially reversed upon removal of the supplement. There was
no alteration in CD44 and CD90 with culture conditions, whereas the
CD146, CD105, and ALP expression profile was regulated by supplementation
with FGF-2, EGF, and PDGF-BB.
AA CD44, CD90, CD146, CD105 There was no alteration in CD44 and CD90 with culture conditions, whereas
the CD146, CD105, and ALP expression profile was regulated by supplementation
AA, ascorbic acid; ALP, alkaline phosphatase; EGF, epidermal growth factor; FCS, fetal calf serum; FGF, fibroblast growth factor; HLA-DR, human
leukocyte antigen DR; HSCs, hematopoietic stem cells; IFN, interferon; MSCs, mesenchymal stem cells; PDGF-BB, platelet-derived growth factor
1416 Markers and Identity of MSCs
VC AlphaMed Press 2014 STEM CELLS
understand the niche components of MSCs in vivo is therefore
demanded to validate the theory.
Accompanied by this dilemma is an emerging theory proposing
a neuroectodermal origin for MSCs, represented by
the expression of nestin . We have not included nestin,
Sox2, or Oct4, in the analysis, since these are intracellular
proteins but not surface markers. However, we noticed that
nestin(1)/CD271(2)/Stro-1(2) MSCs derived from human
ESCs were reported to differentiate into representative derivatives
of all three embryonic germ layers . Therefore,
compared with CD271 and Stro-1, nestin positivity may represent
a more primitive phenotype of MSCs. An interesting
hypothesis is that CD146(1) MSCs may be a lineage of
nestin(1) MSCs, since pericytes within several tissues were
reported to be derived from neural crest derivatives .
While nestin is a intracellular protein which may complicate
the isolation of nestin(1) MSCs, a recent paper suggests that
nestin (1) MSCs can be isolated by PDGFRalpha and CD51
double positivity , which may facilitate the future investigation
of the properties of nestin(1) MSCs.
This work was supported by Small Project Funding of the University
of Hong Kong (201209176179), General Funding from
National Science Foundation of China (NSFC, No. 81371993), and
by the Commonwealth of Pennsylvania, Department of Health.
F.-J.L.: conception and design, collection and/or assembly of
data, interpretation and analysis of data, manuscript writing,
and final approval of manuscript; R.S.T.: interpretation and analysis
of data, manuscript writing, and final approval of manuscript;
K.M.C.C. and V.Y.L.L.: financial support, administrative
support, manuscript writing, and final approval of manuscript.
DISCLOSURE OF POTENTIAL CONFLICT OF INTEREST
The authors indicate no potential conflicts of interest.
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