mesenchymal stem cells in tooth and oral cavity<\/p>\n
Order Description
\nthe origin of mesenchymal stem cells and their types in the mouth and tooth
\ne.g.
\ndental pulp DPSC, apical papilla SCAP, periodontal ligaments PDLSC, dental follicle DFSC, gingival fibroblast GFSC, and gingival epithelial stem cells GESC.<\/p>\n
it is functions and usage in dentistry and dental treatments.<\/p>\n
Braz Dent J 22(2) 2011
\nMesenchymal stem cells in the dental tissues 91
\nINTRODUCTION
\nThe discovery of stem cells and recent advances
\nin cellular and molecular biology has led to the
\ndevelopment of novel therapeutic strategies that aim at
\nthe regeneration of many tissues that were injured by
\ndisease. Generally, stem cells have two major properties:
\nthey are capable of self-renewal and, upon division, they
\ncan give rise to cells that have the potential to differentiate
\n(1). Tissue engineering is a multidisciplinary field that
\ncombines biology, engineering, and clinical science
\nwith the goal of generating new tissues and organs. It is
\na science based on fundamental principles that involves
\nthe identification of appropriate cells, the development
\nof scaffolds and morphogenic signals required to induce
\nMesenchymal Stem Cells in the Dental Tissues:
\nPerspectives for Tissue Regeneration
\nCarlos ESTRELA1
\nAna Helena Gon\u00e7alves de ALENCAR1
\nGregory Thomas KITTEN2
\nEneida Franco VENCIO1
\nElisandra GAVA1,2
\n1Dental School, UFG – Federal University of Goi\u00e1s, Goi\u00e2nia, GO, Brazil
\n2Institute of Biological Sciences, UFMG – Federal University of Minas Gerais, Belo Horizonte, MG, Brazil
\nIn recent years, stem cell research has grown exponentially owing to the recognition that stem cell-based therapies have the potential
\nto improve the life of patients with conditions that range from Alzheimer\u2019s disease to cardiac ischemia and regenerative medicine, like
\nbone or tooth loss. Based on their ability to rescue and\/or repair injured tissue and partially restore organ function, multiple types of
\nstem\/progenitor cells have been speculated. Growing evidence demonstrates that stem cells are primarily found in niches and that certain
\ntissues contain more stem cells than others. Among these tissues, the dental tissues are considered a rich source of mesenchymal stem
\ncells that are suitable for tissue engineering applications. It is known that these stem cells have the potential to differentiate into several
\ncell types, including odontoblasts, neural progenitors, osteoblasts, chondrocytes, and adipocytes. In dentistry, stem cell biology and
\ntissue engineering are of great interest since may provide an innovative for generation of clinical material and\/or tissue regeneration.
\nMesenchymal stem cells were demonstrated in dental tissues, including dental pulp, periodontal ligament, dental papilla, and dental
\nfollicle. These stem cells can be isolated and grown under defined tissue culture conditions, and are potential cells for use in tissue
\nengineering, including, dental tissue, nerves and bone regeneration. More recently, another source of stem cell has been successfully
\ngenerated from human somatic cells into a pluripotent stage, the induced pluripotent stem cells (iPS cells), allowing creation of patientand
\ndisease-specific stem cells. Collectively, the multipotency, high proliferation rates, and accessibility make the dental stem cell an
\nattractive source of mesenchymal stem cells for tissue regeneration. This review describes new findings in the field of dental stem cell
\nresearch and on their potential use in the tissue regeneration.
\nKey Words: Endodontics, stem cell, dental stem cell, tissue engineering.
\ncells to regenerate a tissue or organ (2). Over the last
\nfew years, medicine has begun to explore the possible
\napplications of stem cells and tissue engineering towards
\nthe repair and regeneration body structures (3). It is
\nbecoming ever more clear that this conceptual come
\nup to therapy, named regenerative medicine, will have
\nits place in clinical practice in the future. It has been
\nshown that stem cells will play an important role in future
\nmedical treatments because they can be readily grown
\nand induced to differentiate into any cell type in culture.
\nStem cells are cells that have the ability to renew
\nthemselves through mitosis and can differentiate into
\nseveral specialized cells. The embryonic stem cells (ESC)
\nare pluripotent and have the ability to become almost any
\nkind of cell of the body (4). The local microenvironment
\nCorrespondence: Profa. Dra. Elisandra Gava, Departamento de Ci\u00eancias Estomatol\u00f3gicas, Universidade Federal de Goi\u00e1s, Pra\u00e7a Universit\u00e1ria S\/N,
\nSetor Universit\u00e1rio, 74605-220 Goi\u00e2nia, GO, Brasil. Tel: +55-62-3209-6053. e-mail: elisandragava@yahoo.com.br
\nInvited Review Article
\nBraz Dent J (2011) 22(2): 91-98 ISSN 0103-6440
\nBraz Dent J 22(2) 2011
\n92 C. Estrela et al.
\nrepresents an important compartment in maintaining the
\nstem cells status. The microenvironment regulates the
\nbalance between self-renewal and differentiation. This
\nintercellular communication has been characterized
\nbetween embryonal carcinoma cells and stromal cells,
\nand indicates changes in the expression on both cellular
\ncompartments (5).
\nScientists can induce these cells to replicate
\nthemselves in an undifferentiated state. However, the
\nuse of ESC is controversial and associated with ethical
\nand legal issues, thus conditioning their application for
\nthe development of new therapies (4).
\nAnother source of stem cells is the umbilical cord.
\nBlood from the umbilical cord contains stem cells that
\nare genetically identical to those of the newborn baby.
\nThese cells are multipotent, and are able to differentiate
\ninto certain cell types. Umbilical cord stem cells can
\nbe stored cryogenically after birth for use in a future
\nmedical therapy (2).
\nMesenchymal stem cells (MSC) are multipotent
\nprogenitor cells, originally isolated from adult bone
\nmarrow and subsequently from other tissues in both
\nadult and fetal life. Adult stem cells normally generate
\ncell types of the tissue in which they reside. However,
\nstudies have shown that stem cells from one tissue could
\ngenerate cell types of a completely different tissue (3).
\nUnlike ESC, adult stem cells have the potential
\nto be used for treatment of regenerative disease, cardiac
\nischemia, and bone or tooth loss. Future applications for
\nstem cells include the treatment of Parkinson\u2019s disease
\nand cancer (5). The use of adult stem cells in research
\nand medical applications is less controversial because
\nthey can be harvested without destroying an embryo.
\nPostnatal stem cells have been found in almost all body
\ntissues, including dental tissues. Dental stem cells have
\nbeen identified as candidates for tissue engineering (6).
\nBecause of their multipotent differentiation ability, they
\nprovide an alternative for use in regenerative medicine
\nsince they can be used for not only to dental tissue
\nregeneration, but also to facilitate repair of non-dental
\ntissues such as bone and nerves (6,7).
\nA new source of stem cell has been generated
\nfrom human somatic cells into a pluripotent stage, the
\ninduced pluripotent stem cells (iPS cells) (8,9). iPS cells
\nresemble human ESC and can differentiate into advanced
\nderivates of all three primary germ layers. Unlike ESC,
\niPS cell technology can derive patient-specific stem cells
\nallowing derivation of tissue-matched differentiation
\ndonor cells for basic research, disease modeling, and
\nregenerative medicine (9). This technology might be
\nthe new era of personalized medicine.
\nThis review discusses the perspectives in the field
\nof stem cell-based regenerative medicine, addressing
\nsources of stem cells identified in dental tissues; and
\nnew findings in the field of dental stem cell research
\nand their potential use in the dental tissue engineering.
\nSeveral cell populations with stem cells properties
\nhave been isolated from different parts of the tooth.
\nSince the discovery of the existence of adult stem cells
\nfrom the dental pulp in 2000 (10), several other types
\nof dental stem cells have been successively isolated
\nfrom mature and immature teeth, including stem cells
\nderived from exfoliated deciduous teeth (11), stem
\ncells derived from the apical papilla (12), MSC from
\ntooth germs (13) and from human periodontal ligament
\n(PDL) (14). It is considered that these stem cells are
\nundifferentiated mesenchymal cells present in dental
\ntissues and characterized by their unlimited self-renewal,
\ncolony forming capacity, and multipotent differentiation
\n(1). During the characterization of these newly identified
\ndental stem cells, certain aspects of their proprieties
\nhave been compared with those of bone-marrow-derived
\nstromal stem cells (BMMSC). Dental stem cells display
\nmultidifferentiation potencial, with the capacity to
\ngive rise to distinct cell lineages, osteo\/osteogenic,
\nadipogenic, and neurogenic. Therefore, these cells have
\nbeen used for tissue-engineering studies to assess their
\npotential in preclinical applications (6).
\nIt is, however, important to consider that, although
\ndifferent types of dental-tissue derived MSC share
\nseveral common characteristics and present significant
\nheterogeneity, expressed by multiple phenotypic
\ndifferences, which most probably reflect distinct
\nfunctional properties (1). There is already evidence
\nthat there are significant variations, for example, in
\nthe odontogenic potential of single colony-derived
\npopulations isolated from the dental pulp, reflecting
\ndifferences in their genotypic and protein expression
\npatterns (15). In addition, this heterogeneity may be
\nsignificantly enhanced as a function of their tissue
\nmicroenvironment (16). This issue becomes more
\ncomplicated as researchers have used quite different
\nmethods to isolate and culture dental MSC and evaluate
\ntheir differentiation potential.
\nDENTAL PULP STEM CELLS
\nThe first stem cells isolated from adult human
\nBraz Dent J 22(2) 2011
\nMesenchymal stem cells in the dental tissues 93
\ndental pulp were termed dental pulp stem cells (DPSC).
\nThey were isolated from permanent third molars and
\nexhibited high proliferation and high frequency of colony
\nformation that produced calcified nodules (10). DPSC
\ncultures from impacted third molars at the stage of root
\ndevelopment were able to differentiate into odontoblastlike
\ncells with a very active migratory and mineralization
\npotential, leading to organized three-dimensional dentinlike
\nstructures in vitro (17).
\nThere are different cell densities of the colonies in
\nDPSC, suggesting that each cell clone may have different
\ngrown rate (10). Different cell morphologies and sizes
\ncan be observed in the same colony. The differentiation
\nof DPSC to a specific cell lineage is mainly determined
\nby the components of local microenvironment, such as,
\ngrowth factors, receptor molecules, signaling molecules,
\ntranscription factors and extracellular matrix protein.
\nDPSC can be reprogrammed into multiple cell lineages
\nsuch as, odontoblast, osteoblast, chondrocyte, myocyte,
\nneurocyte, adipocyte, corneal epithelial cell, melanoma
\ncell, and even induced pluripotent stem cells (iPS cells)
\n(18,19). Almushayt et al. (20) demonstrated that dentin
\nmatrix protein 1 (DMP1), a non-collagen extracellular
\nmatrix protein extract from dentin, can significantly
\npromote the odontoblastic differentiation of DPSC
\nand formation of reparative dentin over the exposed
\npulp tissue. Additionally, DPSC can be induced into
\nodontoblast lineage when treated with transforming
\ngrowth factor \u00df1 (TGF\u00df1) alone or in combination with
\nfibroblast growth factor (FGF2) (21).
\nHistologically, dentin lies outside of dental pulp,
\nand they intimately link to each other. Functionally,
\ndental pulp cells can regenerate dentin and provide it
\nwith oxygen, nutrition and innervation, whereas the
\nhard dentin can protect soft dental pulp tissue. Together,
\nthey maintain the integrity of tooth shape and function.
\nAny physiological or pathological reaction occurring at
\none part, such as trauma, caries, and cavity preparation,
\nwill affect the other. Both of them act as a dentin-pulp
\ncomplex and simultaneously participate in various
\nbiological activities of the tooth. Several studies have
\nshown that DPSC play a vital role in the dentin-pulp
\ntissue regeneration (10). In vivo transplantation into
\nimmunocompromised mice DPSC demonstrated the
\nability to generate functional dental tissue in the form
\nof dentin\/pulp-like complexes (22). Transplanted
\nex vivo expanted DPSC mixed with hydroxyapatite\/
\ntricalcium phosphate form ectopic (sample nursing essay examples by the best nursing assignment writing service) dentin\/pulp-like
\ncomplexes in immunocompromised mice. These polls
\nof heterogeneous DPSC form vascularizad pulp like
\ntissue and are surrounded by a layer of odontoblast-like
\ncells expressing factors that produce dentin containing
\ntubules similar those found in natural dentin (22,23).
\nHuang et al. (24) reported that dentin-pulp-like complex
\nwith well-established vascularity can be regenerated
\nde novo in emptied root canal space by DPSC. These
\nstudies provide a novel advance for future pulp tissue
\npreservation and a new alternative for the biological
\ntreatment for endodontic diseases.
\nIn addition, DPSC can express neural markers
\nand differentiate into functionally active neurons,
\nsuggesting their potential as cellular therapy for neuronal
\ndisorders (7). In recent study, DPSC were transplanted
\ninto the cerebrospinal fluid of rats in which cortical
\nlesion was induced. Those cells migrated as single cells
\ninto a variety of brain regions and were detected in
\nthe injured cortex expressing neuron specific markers.
\nThis showed that DPSC-derived cells integrate into
\nthe host brain may serve as useful sources of neuro and
\ngliogenesis in vivo, especially when the brain is injured
\n(25). The spontaneous differentiating potential of these
\ncells strongly suggests their possible applications in
\nregenerative medicine.
\nSTEM CELLS FROM HUMAN EXFOLIATED
\nDECIDUOUS TEETH
\nStem cells may be also isolated from the pulp
\nof human exfoliated deciduous teeth (SHED). These
\ncells have the capacity of inducing bone formation,
\ngenerate dentin and differentiate into other nondental
\nmesenchymal cell derivatives in vitro. SHED
\nexhibit higher proliferation rates, increased population
\ndoublings, in addition to osteoinductive capacity in vivo
\nand an ability to form sphere-like clusters. However,
\nunlike DPCSs, they are unable to regenerate complete
\ndentin\/pulp-like complexes in vivo (10). With the
\nosteoinductive potential, SHED can repair critical
\nsized calvarial defects in mice with substantial bone
\nformation (26). Given their ability to produce and
\nsecrete neurotrophic factors, dental stem cells may also
\nbe beneficial for the treatment of neurodegenerative
\ndiseases and the repair of motor neurons following
\ninjury. Indeed, dental stem cells from deciduous teeth
\nhave been induced to express neural markers such as
\nnestin (27). The expression of neural markers in dental
\nstem cells stimulates the imagination for their potential
\nuse in neural regeneration such as in the treatment of
\nBraz Dent J 22(2) 2011
\n94 C. Estrela et al.
\nParkinson\u2019s disease. The potential of dental stem cells in
\nnon-dental regeneration continues to be further explored
\nby researchers.
\nSTEM CELLS FROM APICAL PAPILLA
\nThe physical and histological characteristics
\nof the dental papilla located at the apex of developing
\nhuman permanent teeth has been recently been described
\nand this tissue has been termed apical papilla. This
\ntissue is loosely attached to the apex of the developing
\nroot and can be easily detached. A population of stem
\ncells isolated from human teeth was found at the
\ntooth root apex. These cells are called stem cells from
\napical papilla (SCAP) and have been demonstrated to
\ndifferentiate exhibit higher rates of proliferation in vitro
\nthan do DPSC. There is an apical cell-rich zone lying
\nbetween the apical papilla and the pulp. Importantly,
\nstem\/progenitor cells were located in both dental pulp
\nand the apical papilla, but they have somewhat different
\ncharacteristics (12). The higher proliferative potential
\nof SCAP makes this population of cells suitable for
\ncell-based regeneration and preferentially for forming
\nroots. They are capable of forming odontoblast-like
\ncells and produce dentin in vivo and are likely to be
\nthe cell source of primary odontoblasts for the root
\ndentin formation (12). The discovery of SCAP may
\nalso explain a clinical phenomenon that was presented
\nin a number of recent clinical case reports showing that
\napexogenesis can occur in infected immature permanent
\nteeth with apical periodontitis or abscess (28). It is likely
\nthat SCAP residing in the apical papilla survived the
\ninfection due to their proximity to the periapical tissues.
\nThis tissue may be benefited by its collateral circulation,
\nwhich enables it to survive during the process of pulp
\nnecrosis. Perhaps, after endodontic disinfection, these
\ncells give rise to primary odontoblasts to complete the
\nroot formation.
\nPERIODONTAL LIGAMENT STEM CELLS
\nPeriodontal ligament (PDL) is a space interlying
\nthe cementum and alveolar bone, a replacement of the
\nfollicle region surrounding the developing tooth in cap
\nand bud stages of development. Fibers inserted into
\nthe cementum layer may be of follicle origin (termed
\nSharpey\u2019s fibers) or cementoblast origin (in cellular
\nintrinsic fiber cementum). The PDL matures during
\ntooth eruption, preparing to support the functional
\ntooth for the occlusal forces. In the mature PDL, major
\ncollagen bundles (principal fibers) occupy the entire
\nPDL, embedding in both cementum and alveolar bone.
\nFibers are arranged in specific orientations to maximize
\nabsorption of the forces to be placed on the tooth during
\nmastication. The PDL has long been recognized to
\ncontain a population of progenitor cells and recently,
\nstudies identified a population of stem cells from human
\nPDL capable of differentiating along mesenchymal
\ncell lineages to produce cementoblast-like cells,
\nadipocytes and connective tissue rich in collagen I (14).
\nPDL stem cells (PDLSC) display cell surface marker
\ncharacteristics and differentiation potential similar to
\nbone marrow stromal stem cells and DPSC (14). After
\nPDLSC were transplanted into immunocompromised
\nmice, cementum\/PDL-like structures were formed.
\nHuman PDLSC expanded ex vivo and seeded in threedimensional
\nscaffolds (fibrin sponge, bovine-derived
\nsubstitutes) were shown to generate bone (29). These
\ncells have also been shown to retain stem cell properties
\nand tissue regeneration capacity. These findings suggest
\nthat this population of cells might be used to create a
\nbiological root that could be used in a similar way as a
\nmetal implant, by capping with an artificial dental crown.
\nDENTAL FOLLICLE PRECURSOR CELLS
\nThe dental follicle is a loose connective tissue
\nthat surrounds the developing tooth. The dental follicle
\nhas long been considered a multipotent tissue, based on
\nits ability to generate cementum, bone and PDL from
\nthe ectomesenchyme-derived fibrous tissue. Dental
\nfollicle precursor cells (DFPC) can be isolated and
\ngrown under defined tissue culture conditions, and
\nrecent characterization of these stem cells has increased
\ntheir potential for use in tissue engineering applications,
\nincluding periodontal and bone regeneration (12,30).
\nDFPC form the PDL by differentiating into PDL
\nfibroblasts that secrete collagen and interact with fibers
\non the surfaces of adjacent bone and cementum. Dental
\nfollicle progenitor cells isolated from human third
\nmolars are characterized by their rapid attachment in
\nculture, and ability to form compact calcified nodules
\nin vitro (30). DFPC, in common with SCAP, represent
\ncells from a developing tissue and might thus exhibit a
\ngreater plasticity than other dental stem cells. However,
\nin the same way as for SCAP, further research needs to
\nbe carried out on the properties and potential uses of
\nthese cells (Table 1).
\nBraz Dent J 22(2) 2011
\nMesenchymal stem cells in the dental tissues 95
\nDENTAL PULP STEM CELLS AND DENTAL
\nTISSUE ENGINEERING
\nThere are several areas of research for which
\ndental stem cells are presently considered to offer
\npotential for tissue regeneration. These include the
\nobvious uses of cells to repair damaged tooth tissues
\nsuch as dentin, PDL and dental pulp (6,24). Even the
\nuse of dental stem cells as sources of cells to facilitate
\nrepair of additional tissues as bone and nerves (6,7,26).
\nEfforts to induce tissue regeneration in the pulp space
\nhave been a long search. In 1962, Ostby (31) proposed
\ninducing hemorrhage and blood clot formation in the
\ncanal space of mature teeth in the hope of guiding the
\ntissue repair in the canal. However, the connective
\ntissue that grew into the canal space was limited and
\nthe origin of this tissue remains unproved. Regenerative
\nEndodontics represents a new treatment modality that
\nfocuses on reestablishment of pulp vitality and continued
\nroot development. This clinical procedure relies on the
\nintracanal delivery of a blood clot (scaffold), growth
\nfactors (possibly from platelets and dentin), and stem
\ncells (32). In a recent study, it was demonstrated that
\nmesenchymal stem cells are delivered into root canal
\nspaces during regenerative endodontic procedures in
\nimmature teeth with open apices (32). These findings
\nprovide the biological basis for the participation of stem
\ncells in the continued root development and regenerative
\nresponse that follow this clinically performed procedure.
\nAs DPSC have the potent dentinogenic ability,
\nthey could be used for the vital pulp therapy. When DPSC
\nare transplanted alone or in combination with BMP2
\nin the pulp cavity, these stem cells can significantly
\npromote the repair and reconstruction of dentin-pulplike
\ncomplex (31). Prescott et al. (34) placed the triad of
\nDPSC, a collagen scaffold, and DMP1 in the simulated
\nperforation sites in dentin slices, and then transplanted
\nthe recombination subcutaneously into the nude mice.
\nAfter 6 weeks of incubation, well-organized pulplike
\ntissue could be detected in the perforation site.
\nTable 1. Stem cell types in dental pulps (6,7,10-15,17,18,20).
\nProperties DPSC SCAP SHED PDLSC DFPC
\nLocation Permanent
\ntooth pulp
\nApical papilla of
\ndeveloping root
\nExfoliated
\ndeciduous tooth
\npulp
\nPeriodontal
\nligament
\nDental follicle of
\ndeveloping tooth
\nProliferation rate Moderate High High High High
\nHeterogeneity Yes Yes Yes Yes Yes
\nMultipotentialy
\nOdontoblast,
\nosteoblast,
\nchondrocyte,
\nmyocyte, neurocyte,
\nadipocyte, corneal
\nepithelial cell,
\nmelanoma cell, iPS
\nOdontoblast,
\nosteoblast,
\nneurocyte,
\nadipocyte, iPS
\nOdontoblast,
\nosteoblast,
\nchondrocyte,
\nmyocyte,
\nneurocyte,
\nadipocyte, iPS
\nOdontoblast,
\nosteoblast,
\nchondrocyte,
\ncementoblast,
\nneurocyte
\nOdontoblast,
\nosteoblast,
\nneurocyte
\nTissue repair
\nBone regeneration,
\nneuroregeneration,
\nmyogenic
\nregeneration, dentinpulp
\nregeneration
\nBone regeneration,
\nneuroregeneration,
\ndentin-pulp
\nregeneration,
\nroot formation
\nBone regeneration,
\nneuroregeneration,
\ntubular dentin
\nBone regeneration,
\nroot formation,
\nperiodontal
\nregeneration
\nBone
\nregeneration,
\nperiodontal
\nregeneration
\nDPSC = dental pulp stem cells; SCAPs = stem cells from the apical papila; SHED = stem cells from the pulp of human exfoliated
\ndeciduous teeth; PDLSC = periodontal ligament stem cells; DFPC = dental follicle precursor cells.
\nBraz Dent J 22(2) 2011
\n96 C. Estrela et al.
\nCordeiro et al. (35) demonstrated that SHED\/scaffold
\nrecombination prepared within human tooth slices also
\nhave the potential to form dental pulp-like structures.
\nHuang et al. (24) reported that dentin-pulp-like complex
\nwith well-established vascularity can be regenerated
\nde novo in emptied root canal space by either DPSC
\nor SHED (24). One of the most challenging aspects
\nof developing a regenerative endodontic therapy is to
\nunderstand how the various procedures involved can
\nbe optimized and integrated to produce the outcome
\nof a regenerated pulp-dentin complex. The future
\ndevelopment of regenerative endodontic procedures will
\nrequire a comprehensive research program directed at
\neach of these components and their application in the
\nclinical practice.
\nPeriodontitis is the most common cause for tooth
\nloss in adults due to irreversible waste of connective
\ntissue attachment and the supporting alveolar bone. The
\nchallenge for cell-based replacement of a functional
\nperiodontium is therefore to form new ligament and
\nbone, and to ensure that the appropriate connections
\nare made between these tissues, as well as between the
\nbone and tooth root. This is not a trivial undertaking,
\nas these are very different tissues that are formed in
\nan ordered manner (spatially and temporally) during
\ntooth development (36). In recent years, guided tissue
\nregeneration has become the gold-standard surgery for
\nperiodontal tissue regeneration. This procedure involves
\ndraping a biocompatible membrane over the periodontal
\ndefect from the root surface to the adjacent alveolar
\nbone, often in combination with a bone graft (37). The
\nbarrier membrane prevents unwanted epithelium and
\ngingival connective tissue from entering the healing
\nsite, while promoting repopulation of the defect site by
\ncells migrating in from the PDL (29). The rather limited
\nsuccess of this approach has led scientists to develop
\nmethods to improve this therapy, through the addition
\nof exogenous growth factors and via stem cell therapy
\n(38). One goal of current research is to use different
\npopulations of dental stem cells to replicate the key
\nevents in periodontal development both temporally
\nand spatially, so that healing can occur in a sequential
\nmanner to regenerate the periodontium (39).
\nCommonly used growth factors for PDL
\nregeneration therapies include bone morphogenetic
\nproteins, platelet derived growth factor, Emdogain and
\nrecombinant amelogenin protein. The resultant improved
\nregenerative capability could be related to increased
\nrecruitment of progenitor MSC, which subsequently
\ndifferentiate to form PDL tissue. Recently, PDLSC
\ntransfected with expression vectors for platelet-derived
\ngrowth factor and bone morphogenetic protein were
\ninvestigated in periodontal tissue engineering models
\n(40). These studies revealed the regeneration of normal
\nperiodontal tissues, containing organized cementum,
\nalveolar bone and the PDL attachment apparatus. The
\npossibility of constructing a root-periodontal tissue
\ncomplex was further successfully demonstrated using
\na pelleted hydroxyapatite\/tricalcium phosphate scaffold
\ncontaining SCAP, coated with PDLSC-seeded Gelfoam,
\nimplanted and grown in minipig tooth socket (11,41).
\nThe multipotent differentiation properties of PDLSC
\nfor generating both hard and soft tissues were further
\ndemonstrated by constructing multilayered cell sheets
\nsupported by woven polyglycolic acid. Transplanted
\ncell seeded polyglycolic acid sheets regenerated new
\nbone, cementum and well-oriented collagen fibers when
\nintroduced into root surfaces. In addition to PDL-derived
\nDSCs, bone marrow-derived MSC and adipose-derived
\nstem cells have been shown to promote periodontal tissue
\nregeneration (42).
\nIn a recent study (43), three kinds of dental
\ntissue derived adult stem cells were obtained from the
\nextracted immature molars of dogs, and ex vivo expanded
\nPDLSC, DPSC, and periapical follicular stem cells
\nwere transplanted into the apical involvement defect.
\nAutologous PDLSC showed the best regenerating
\ncapacity of PDL, alveolar bone and cementum as well as
\nperipheral nerve and blood vessel which were evaluated
\nby conventional and immune histology.
\nSuccessful therapies for PDL tissue regeneration
\nwill not only facilitate the treatment of periodontal
\ndiseases, but may also be used to improve current dental
\nimplant therapies. Numerous attempts to reconstruct
\nperiodontal tissues around dental implants revealed the
\nchallenge of avoiding fibrous tissue encapsulation and
\nthe formation of functional cementum on the implant
\nsurface (44).
\nCONCLUDING REMARKS
\nThere is still much to learn about the nature,
\npotentiality and behavior of dental stem\/progenitor
\ncells. However, the opportunities for their exploitation
\nin dental tissue regeneration are immense and will lead
\nto significant benefits for the management of the effects
\nof dental disease.
\nDental stem cells display multifactorial potential
\nBraz Dent J 22(2) 2011
\nMesenchymal stem cells in the dental tissues 97
\nsuch as high proliferation rate, multi-differentiation
\nability, easy accessibility, high viability and easy to be
\ninduced to distinct cell lineages.
\nTherefore, these cells have been used for tissueengineering
\nstudies in large animals to assess their
\npotential in preclinical applications. However, although
\nnumerous breakthroughs in stem cell research have been
\nmade thus far, their success and applicability in clinical
\ntrials remains to be ascertained. Solid research into the
\nbasic science and biology behind stem cells must be
\nperformed before scientists leap into the clinical trials.
\nTechnologies using MSC and iPS cells might be the new
\nera of personalized medicine. The heterogeneity among
\npatient factors and the biology of different stem cell types
\nreinforces the need for an individual-targeted approach
\nto stem cell therapy and other cell-based treatments.
\nRESUMO
\nNos \u00faltimos anos, as pesquisas com c\u00e9lulas tronco t\u00eam aumentado
\nexponencialmente devido ao reconhecimento de que seu potencial
\nterap\u00eautico pode melhorar a qualidade de vida de pacientes
\ncom diversas doen\u00e7as, como a doen\u00e7a de Alzheimer, isquemias
\ncard\u00edacas e, at\u00e9 mesmo, nas pesquisas de medicina regenerativa
\nque visa uma poss\u00edvel substitui\u00e7\u00e3o de \u00f3rg\u00e3o perdidos, como por
\nexemplo, os dentes. Baseado em habilidades de reparar tecidos
\ninjuriados e restaurar parcialmente as fun\u00e7\u00f5es de um \u00f3rg\u00e3o,
\ndiversos tipos de c\u00e9lulas-tronco t\u00eam sido estudadas. Recentes
\nevid\u00eancias demonstram que as c\u00e9lulas-tronco s\u00e3o primariamente
\nencontradas em nichos e que certos tecidos apresentam mais
\nc\u00e9lulas-tronco que outros. Entre estes, os tecidos dentais s\u00e3o
\nconsiderados como uma fonte rica de c\u00e9lulas-tronco mesenquimais
\nadequado para aplica\u00e7\u00f5es em engenharia tecidual. Sabe-se que
\nestas c\u00e9lulas t\u00eam o potencial de diferenciarem-se em diversos
\ntipos celulares, incluindo osteoblastos, c\u00e9lulas progenitoras de
\nneur\u00f4nios, osteoblastos, condr\u00f3citos e adip\u00f3sitos. Na odontologia,
\na biologia celular e a engenharia tecidual s\u00e3o de grande interesse,
\npois fornecem inova\u00e7\u00f5es na gera\u00e7\u00e3o de novos materiais cl\u00ednicos e
\nou na regenera\u00e7\u00e3o tecidual. Estas podem ser isoladas e crescidas
\nem diversos meios de cultura apresentando grande potencial
\npara ser usada na engenharia tecidual, incluindo regenera\u00e7\u00e3o de
\ntecidos dentais, nervos e ossos. Recentemente, outra fonte de
\nc\u00e9lulas tronco tem sido geradas a partir de c\u00e9lulas som\u00e1ticas de
\nhumanos a um est\u00e1gio de pluripot\u00eancia, chamados de c\u00e9lulastronco
\npluripotente induzida (iPS) levando \u00e0 cria\u00e7\u00e3o de c\u00e9lulastronco
\nespec\u00edficas. Coletivamente, a multipotencialidade, altas
\ntaxas de prolifera\u00e7\u00e3o e acessibilidade, faz das c\u00e9lulas-tronco
\ndent\u00e1rias uma fonte atrativa de c\u00e9lulas-tronco mesenquimais
\npara regenera\u00e7\u00e3o tecidual. Esta revis\u00e3o descreve novos achados
\nno campo da pesquisa com c\u00e9lulas-tronco dentais e seu potencial
\nuso na regenera\u00e7\u00e3o tecidual.
\nACKNOWLEDGEMENTS
\nThis study was supported in part by grants from the National
\nCouncil for Scientific and Technological Development (FAPEGO
\nto E.G., and CNPq grants #302875\/2008-5 and CNPq grants
\n#474642\/2009 to C.E.).
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\nAccepted February 28, 2011<\/p>\n
Concise Review: The Surface Markers and Identity
\nof Human Mesenchymal Stem Cells
\nFENG-JUAN LV,a,b,c ROCKY S. TUAN,d KENNETH M.C. CHEUNG,a,b,c VICTOR Y.L. LEUNG
\na,b,c
\nKey Words. Mesenchymal stem cells \u2022 Surface epitopes \u2022 CD271 \u2022 CD146 \u2022 Markers \u2022 Pericytes \u2022
\nNiche \u2022 Regenerative medicine
\nABSTRACT
\nThe concept of mesenchymal stem cells (MSCs) is becoming increasingly obscure due to the
\nrecent findings of heterogeneous populations with different levels of stemness within MSCs isolated
\nby traditional plastic adherence. MSCs were originally identified in bone marrow and later
\ndetected in many other tissues. Currently, no cloning based on single surface marker is capable
\nof isolating cells that satisfy the minimal criteria of MSCs from various tissue environments.
\nMarkers that associate with the stemness of MSCs await to be elucidated. A number of candidate
\nMSC surface markers or markers possibly related to their stemness have been brought forward
\nso far, including Stro-1, SSEA-4, CD271, and CD146, yet there is a large difference in their
\nexpression in various sources of MSCs. The exact identity of MSCs in vivo is not yet clear,
\nalthough reports have suggested they may have a fibroblastic or pericytic origin. In this review,
\nwe revisit the reported expression of surface molecules in MSCs from various sources, aiming
\nto assess their potential as MSC markers and define the critical panel for future investigation.
\nWe also discuss the relationship of MSCs to fibroblasts and pericytes in an attempt to shed light
\non their identity in vivo. STEM CELLS 2014;32:1408\u20131419
\nINTRODUCTION
\nMesenchymal stem cells (MSCs) were first identified
\nfrom bone marrow mononuclear cells
\n(BM-MNCs) as fibroblastic colony-forming units
\n(CFU-Fs). Due to their multipotency and paracrine
\neffect [1, 2], MSCs are ideal candidates for
\nregenerative medicine [3, 4]. Currently, there is
\nno consensus on a single surface molecule to
\nidentify MSCs from various sources. The minimum
\ncriteria of MSCs [5] include: (a) remain
\nplastic-adherent under standard culture conditions;
\n(b) express CD105, CD73, and CD90, and
\nlack expression of CD45, CD34, CD14 or CD11b,
\nCD79a or CD19, and HLA-DR; (c) differentiate
\ninto osteoblasts, adipocytes, and chondrocytes
\nin vitro. Other surface antigens generally
\nexpressed by MSCs include CD13, CD29, CD44,
\nand CD10 [6, 7]. Although bone marrow (BM) is
\nthe most widely recognized source of MSCs,
\nrecent research has identified alternative sources
\nof MSC-like cells, including adipose tissue
\n(AT) [8], placenta [9], dental pulp [10], synovial
\nmembrane [11], peripheral blood [12], periodontal
\nligament [13], endometrium [14],
\numbilical cord (UC) [15], and umbilical cord
\nblood (UCB) [16, 17]. In fact, evidence has suggested
\nthat MSCs may be present virtually in
\nany vascularized tissues throughout the whole
\nbody [18].
\nGenuine MSCs are expected to possess
\nboth clonogenicity and tripotency. However,
\nonly a fraction of CFU-Fs from plastic adherence
\nisolated MSCs (PA-MSCs) exhibited multipotency
\n[19], indicating that PA-MSCs
\ncomprised a heterogeneous population of cells
\nwith different lineage commitment [19], which
\nmay relate to their in vivo environment. This is
\nreflected in the differences in the protein
\nexpression profile, cytokine profile, or differentiation
\npotency of various sources of MSCs
\n(reviewed in [20]). For example, the percentage
\nof BM CFU-Fs with osteogenic potency
\nwas higher than that with adipogenic potency
\n[21]. Similarly, ectopic (sample nursing essay examples by the best nursing assignment writing service) transplantation of BMMSCs
\nresulted in heterotopic (sample nursing essay examples by the best nursing assignment writing service) bone tissue formation,
\nwhile dental pulp-derived MSCs generated
\nreparative dentin-like tissue [22]. It is
\nnow widely accepted that in the MSCs population,
\nonly a proportion of cells satisfy the
\n\u201cMSCs\u201d criteria at single cell level, while the
\nother cells are more committed. There is also
\na more immature population in MSCs which
\nare embryonic stem cell (ESC)-like and express
\nOct-4 and Sox2 [23].
\nSo far, the markers proposed for MSCs fall
\ninto two categories: sole markers and stemness
\nmarkers. A sole marker is an alternative
\nMSC selection tool to plastic adherence, which
\nalone is sufficient to identify or purify MSCaDepartment
\nof Orthopaedics
\nand Traumatology, Li Ka
\nShing Faculty of Medicine,
\nThe University of Hong Kong,
\nHong Kong, People\u2019s Republic
\nof China; bStem Cell &
\nRegenerative Medicine
\nConsortium and cCenter for
\nReproduction, Development
\nand Growth, Li Ka Shing
\nFaculty of Medicine, The
\nUniversity of Hong Kong,
\nHong Kong SAR, People\u2019s
\nRepublic of China; dCenter
\nfor Cellular and Molecular
\nEngineering, Department of
\nOrthopaedic Surgery,
\nUniversity of Pittsburgh
\nSchool of Medicine,
\nPittsburgh, Pennyslvania, USA
\nCorrespondence: Victor Y.L.
\nLeung, Ph.D., 9\/F, Lab Block,
\nDepartment of Orthopaedics and
\nTraumatology, Li Ka Shing Faculty
\nof Medicine, The University of
\nHong Kong, 21 Sassoon Road,
\nHong Kong SAR, People\u2019s
\nRepublic of China. Telephone:
\n1852-2819-9589; Fax: 1852-
\n2818-5210; e-mail: vicleung@hku.
\nhk or Kenneth M.C. Cheung,
\nM.D., Department of Orthopaedics
\nand Traumatology, The University
\nof Hong Kong Medical
\nCentre, Queen Mary Hospital,
\nPokfulam Road, Pokfulam, Hong
\nKong SAR, People\u2019s Republic of
\nChina. Telephone: 1852-2255-
\n4254; Fax: 1852-2817-4392;
\ne-mail: cheungmc@hku.hk
\nReceived September 16, 2013;
\naccepted for publication
\nFebruary 9, 2014; first published
\nonline in STEM CELLS EXPRESS
\nFebruary 28, 2014.
\nVC AlphaMed Press
\n1066-5099\/2014\/$30.00\/0
\nhttp:\/\/dx.doi.org\/
\n10.1002\/stem.1681
\nStem Cells 2014;32:1408\u20131419 www.StemCells.com VC AlphaMed Press 2014
\nTISSUE-SPECIFIC STEM CELLS
\nlike cells from their in vivo environment [5]. A \u201cstemness\u201d
\nmarker is able to identify a subset of MSCs with high CFU-Fs
\nand trilineage potential or even identify ESC-like population.
\nIdeally, such stemness marker may facilitate selection and
\ntherefore enrichment of subpopulation that exhibit superior
\nCFU-Fs and multipotency. Based on the nature of the two different
\ntypes of markers, the sole markers are normally highly
\nexpressed, while the stemness markers may be moderately
\ndetected.
\nThe majority of MSC markers are identified for BM-MSCs.
\nTo date, however, whether these markers can be applied to
\nother sources of MSCs is not very clear. Moreover, the exact
\nin situ identity of MSCs is not entirely clear, although reports
\nhave suggested they may have a fibroblastic or pericytic origin.
\nThis review attempt to address these issues through a
\ncomprehensive analysis of the current findings on MSC isolated
\nfrom various tissues via the use of single surface
\nmarkers. Moreover, the capacity of stemness markers representing
\nthe subset of more primitive MSCs is revisited and
\nthe origin of MSCs is discussed.
\nSOLE AND STEMNESS MARKERS OF MSCS
\nA number of molecules have been suggested as MSC markers,
\nas shown in Table 1. Among them, Stro-1, CD271, stagespecific
\nembryonic antigen-4 (SSEA-4), and CD146 are the
\nones that have received the most attention and adopted in
\nstudies as markers to sort MSCs. The expression of these four
\nmolecules in various sources of MSCs is listed in Table 2.
\nStro-1
\nStro-1 is one of the most well-known markers for MSCs. Stro-
\n1 is a cell membrane single pass type I protein that translocates
\nfrom the endoplasmic reticulum to the cell membrane
\nin response to the depletion of intracellular calcium [69]. By
\ncombining negative selection against glycophorin-A, Stro-1
\nenriched CFU-Fs from BM with multipotency [24]. However, it
\ndid not enrich CFU-Fs from human endometrial stroma [14].
\nThe degree of homogeneity of the Stro-1-selected MSCs was
\nfurther enhanced 1000-fold by positive selection for CD106
\ncompared to PA-MSCs [25]. Injection of human Stro-1(1) but
\nnot Stro-1(2) BM-MNCs into rat myocardium led to arteriogenesis
\nand functional cardiac recovery [70]. Further in vivo
\nresearch demonstrated that Stro-1(2) MSCs supported higher
\nhematopoietic stem cells (HSC) engraftment in nonobese diabetic\/
\nsevere combined immunodeficiency (NOD\/SCID) mice
\nwhile no support was detected by Stro-1(1) MSCs. However,
\nStro-1(1) MSCs exhibited greater capability in homing to
\nspleen, BM, and kidney [26]. Conditioned medium from Stro-
\n1(1) MSCs could induce a greater degree of cardiac vascular
\nrepair than PA-MSCs [25]. These suggest that Stro-1 may be
\ninvolved in clonogenicity and play a role in homing and angiogenesis
\nof MSCs.
\nHowever, Stro-1 is not universally expressed in all
\nreported types of MSCs. Stro-1 is expressed in dental- [10],
\nsynovial membrane- [11], decidua parietalis-derived MSCs [39]
\nand multipotent dermal fibroblasts [28]. AT- [54], UCB- [57],
\nUC- [16], peripheral blood-derived MSCs [59] are negative\/low
\nfor Stro-1 expression. It is reported that placenta-derived
\nMSCs gradually lose Stro-1 expression in culture [62]. In contrast,
\nhowever, the expression of Stro-1 in BM-MSCs increases
\nwith culture time [36].
\nThe potential of Stro-1 as an MSC marker is limited in several
\nways. It is unclear whether Stro-1 expression correlates
\nwith multipotency. Stro-1 is also unsuitable as a sole marker
\nto separate MSCs from its harboring tissue, at least not from
\nBM, as greater than 95% of Stro-1(1) cells in the human BM
\nwere glycophorin A expressing nucleated erythroid cells [24].
\nMoreover, Stro-1 expression appears not universal for various
\nMSC types.
\nCD271
\nCD271 (also named as low-affinity nerve growth factor receptor)
\nis a receptor for neurotrophins, which stimulate neuronal
\ncells to survive and differentiate. CD271 has been used to
\nselect CFU-Fs from BM-MNCs. The percentage of
\nCD90(1)CD105(1)CD45(2)CD34(2)CD79(2) cells in BMMNCs
\ncoincided with the amount of CD271(1) cell subset
\n(0.54%) [71]. CFU-Fs could only be generated from CD271(1)
\nsubsets of CD45(2)glycophorin-A(2) human BM-MNCs while
\nthe CD271(2) fraction showed no residual CFU-F activity [7,
\n29]. CD271(1) BM MSCs were shown to have enhanced capability
\nin promoting HSC engraftment compared to PA-MSCs
\n[72] and also induced superior chondral repair than the
\nCD271(2) BM MSCs [73]. These together suggest a role of
\nCD271 in maintaining clonogenicity and function of MSCs.
\nHowever, the majority of the CD271(1) cells were found not
\nto coexpress CD90 and CD73, the two general markers of
\nMSCs. The percentage of CD90 and CD73 positive cells was
\nfound to be very low (<10%) in CD271(1) cells from BM [29]
\nand from AT (10%\u201320%) [72]. Moreover, nearly 50%\u201399% of
\nthe CD271(1) cells in BM [29] and synovium [47] coexpressed
\nCD34, which disqualifies CD271 as a sole marker to isolate
\nMSCs from various tissues [5].
\nSimilar to Stro-1, CD271 is not universally expressed in
\nvarious MSCs. CD271 shows high levels of expression in BM
\nand AT MSCs [6, 55] and is also expressed in periodontal ligament
\nMSCs [13]. However, it is expressed at low levels in
\nplacenta-derived MSCs [31, 63] and not expressed in synovial
\nmembrane- [47, 65, 66], peripheral blood- [60], UC-, and
\nUCB-MSCs [48, 49]. Although Watson et al. [58] reported
\ndetection of CD271 in UCB-MSCs, CD271 failed to enrich CFUFs
\nand multipotency. The potency of CD271(1) cells as a
\nstemness marker was further challenged by the finding of
\nlower trilineage differentiation potential in CD105(1)\/
\nCD271(1) expanded BM-MNCs subsets compared to unsorted
\nBM-MNCs [30]. Therefore, CD271 may not be considered as a
\nMSC stemness marker.
\nSSEA-4
\nSSEA-4 is an embryonic stem cell marker. It has been documented
\nto isolate genuine MSCs from BM [32]. SSEA-4(1)
\nBM cells can expand extensively, while SSEA-4(2) subsets fail
\nto grow. SSEA-4(1) cells also show tripotency [57]. SSEA-4
\nexpression gradually increases in BM culture over time [57].
\nBesides in BM-MSCs, SSEA-4 expression was also detected in
\nplacenta- [64], periodontal ligament- [74], dental pulp- [61],
\nand synovial membrane [65]-derived MSCs. On the contrary,
\nAT-, UC-, or UCB-derived MSCs [48, 51] do not express SSEA-
\n4. A more important question that whether the clonogenicity
\nand multipotency of SSEA-4(1) cells is superior to the
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\nTable 1. Potential MSC sole markers and their expression in unsorted MSC population
\nMarkers Marker potential % positive MSC source References
\nStro-1 Enrich CFU-Fs from whole BM 10 Human BM-MNCs [24]
\n11.2 Human BM-MNCs [25]
\n6 Human BM-MSCs after 1 week in culture [26]
\n2.1 Human endometrial stroma cells [14]
\n1.29 Human amnion MSCs at passage 0\u20132 [27]
\n8 Human dermal fibroblasts at passage 3 [28]
\n28.96 Inflamed periodontal ligament MSCs [13]
\n37.84 Healthy periodontal ligament MSCs
\nCD271 Enrich CFU-Fs from CD45\/A-glycophorin
\nA depleted BM-MNCs
\n2.3 Human BM-MNCs [7, 29]
\nHigher differentiation-related gene
\nexpression after induction compared
\nto MNC-derived MSCs
\n[30]
\nNegligible Human placenta MSCs [31]
\nSSEA-4 Enrich CFU-Fs from whole BM 1\u20132 Mouse BM-MSCs at day 2 in culture [32]
\n71 Mouse BM-MSCs after 100 days in culture
\n2\u20134 Human BM cells
\n37.82 Human amnion MSC at passage 0\u20132 [27]
\nCD146 Enrich CFU-Fs from BM-MSCs 9.4 Human endometrial stroma [14]
\nEnrich cells with multipotency from
\nBM-MSCs
\n1.5 CD146(1)PDGF-Rb(1) Human endometrial stroma [33]
\nDownregulated in differentiated cells 0.1 Human BM-MSCs [34, 35]
\n1.2 Human CD45 depleted BM-MNCs [35]
\n42.7 Human BM-MSCs [17]
\n17.2\u201337.9 Human UC-MSCs
\n11.55 Inflamed periodontal ligament MSCs [13]
\n21.85 Healthy periodontal ligament MSCs
\n9.4 Fresh isolated human endometrial stromal cells [14]
\nCD49f Enrich clonogenicity and differentiation
\npotency
\n88.1 human BM-MSCs at passage 1 [36]
\nKnocking down results in differentiation
\nof HSCs
\n15 Human BM-MSCs [37]
\n55 Human UCB-MSCs [37]
\nCD349 Enrich CFU-Fs from whole placenta cells 0.2 Total human plancenta cells [31]
\nGD2 High specificity for isolating MSCs from
\nBM
\n95 Human CD45(2)CD105(1)CD73(1) BM-MNCs [38]
\n65 Human BM-MSCs [37]
\n3 Human UCB-MSCs [37]
\n3G5 Enrich CFU-Fs Decidua parietalis [39]
\n63 Dental pulp CFU-Fs [40]
\n14 BM CFU-Fs
\nSSEA-3 Enrich cells with clonogenicity and
\nectodermal, endodermal, and mesodermal
\ndifferentiation potency
\n1 BM-MSCs [23]
\nSUSD2 Enrich CFU-Fs and tri-potency 4.2 Human endometrial stromal cells [41]
\nStro-4 Enrich CFU-Fs <5 Human and ovine BM-MSCs [42]
\nMSCA-1 MSCA-1 positivity enrich CFU-Fs by 90-
\nfold from BM-MNCs; MSCA-1 and
\nCD56 double positivity enrich CFU-Fs
\nby 180-fold from BM-MNCs; MSCA-
\n1(1)CD56(2) selects for better adipogenesis
\nin BM-MSCs; MSCA-
\n1(1)CD56(1) selects for better chondrogenesis
\nin BM-MSCs
\n0.5\u20135.5 coexpressed CD271bright BM cells [43]
\nCD200 Enrich CFU-Fs from BM-MNCs; downregulated
\nin differentiated cells
\n0.15 Human BM-MNCs [44]
\nPODXL PODXL decreases in high-density
\ncultures
\n90 Human BM-MSCs at passage 2 [36]
\n13 Human BM-MSCs [37]
\n25 Human UCB-MSCs [37]
\nSox11 Downregulated during culture. Knockdown
\naffects proliferation and osteogenesis
\npotential
\nNot tested Human BM-MSCs [45]
\nTM4SF1 Enriched in MSCs compared with their
\nsource tissue or fibroblasts
\nNot specified BM, AT, and UCB [46]
\nAT, adipose tissue; BM, bone marrow. BM-MNCs: bone marrow mononuclear cells; CFU-Fs, fibroblastic colony-forming units; MSC, mesenchymal
\nstem cell; UCB, umbilical cord blood.
\n1410 Markers and Identity of MSCs
\nVC AlphaMed Press 2014 STEM CELLS
\ntraditional PA-MSCs or MSCs sorted by other molecules is
\nunanswered. Notably, the expression of SSEA-4 in UCB-HSCs
\nwas suggested to be an artificial induction in the in vitro culture
\nas fetal calf serum (FCS) contained globoseries glycosphingolipids
\nwhich can be recognized by a SSEA-4 antibody,
\nand in vitro FCS exposure may induce SSEA-4 expression [75].
\nIn fact, some other studies reported no detection of SSEA-4
\nexpressing cells in unsorted BM [51, 52, 76]. These findings
\nraise the issue of the physiological relevance and reliability of
\nSSEA-4 as the marker for MSCs.
\nCD146
\nCD146 is a key cell adhesion protein in vascular endothelial
\ncell activity and angiogenesis. Notably, CD146 emerged as an
\nattractive candidate for identifying genuine MSCs. In human
\nendometrial stroma population, CD146(1)PDGF-Rb(1) cells
\nshow higher CFU-F enrichment compared with CD146(2)
\nPDGF-Rb(2) cells (7.761.7% vs. 0.760.2%) [33]. CD146 has
\na greater CFU-Fs enrichment capacity than CD90, Stro-1, or
\nCD133 [14]. CD146 expression also defines MSCs with higher
\nmultipotency. Russell\u2019s group reported that the expression
\nlevel of CD146 in the tripotent clones is twofold of that in
\nthe unipotent clones [19]. Additionally, CD146 also identifies
\nMSCs with higher supporting capacity for hematopoiesis, as in
\nvitro, CD146(1) MSCs show more than 100-fold increase in
\nthe long-term culture colony output by 8 weeks compared to
\nunsorted BM-MNCs [34], and in vivo, when transplanted into
\nmice, CD146(1) BM stroma subendothelial cells exhibit the
\ncapacity to reorganize the hematopoietic microenvironment
\nto heterotopic (sample nursing essay examples by the best nursing assignment writing service) sites [35].
\nImportantly, the expression of CD146 was found not only
\nin BM-MSCs [50] but also in almost all the other sources of
\nMSCs, including MSCs derived from AT [56], UC [15, 17], synovial
\nmembrane [47], UCB [49], placenta [63], dermis [68],
\nperiodontal ligament [13], and intervertebral disc [77]. In fact,
\nCD146-expressing MSC clones from multiple organs were
\nfound to exhibit trilineage potency [18].
\nCD49f
\nCD49f (a6-integrin) regulates signaling pathways in a variety of
\ncellular activities. Oct-4 and Sox-2 directly regulates the expression
\nof CD49f, and that the knockdown of CD49f in ESCs results
\nin differentiation into three germ layers, indicating CD49f is
\ninvolved in the maintenance of pluripotency and is an ESC
\nmarker [78]. CD49f has also been identified as a specific HSC
\nmarker and shown to enrich cells capable of generating longterm
\nmultilineage grafts [79]. To date, CD49f expression has
\nbeen detected in BM-MSCs [36], fetal urinary bladder-derived
\nMSCs [80], and UCB-MSCs [37]. It is possible that the expression
\nof CD49f may implicate the stemness of MSC culture. In fact,
\nstudy has shown that CD49f is associated with high clonogenicity
\nand multipotency in less confluent MSC culture [36]. Condition
\nthat induces MSC sphere formation can enrich CD49f(1)
\npopulation compared with MSCs in monolayer [78]. Moreover,
\nhigher expression level of CD49f, such as in UCB-MSCs, is functionally
\nlinked with a higher lung clearance rate in systemic infusion
\n[37]. Nonetheless, CD49f may not necessarily be of value
\nas a single specific marker of MSCs since it is also widely
\nexpressed in epithelial cells as well as endothelial cells, monocytes,
\nplatelets, and thymocytes [79].
\nCD349
\nCD349 (frizzled-9) is a transmembrane-spanning receptor that is
\nactivated by Wnt ligands. It has been proposed to enrich CFU-Fs
\nfrom placenta cells [31]. Additionally, CD349 expression has
\nbeen reported in periodontal ligament-derived MSCs [74]. However,
\nwhether CD349 being essential to the enrichment of clonogenicity
\nhas been questioned by other reports. For example,
\nwhile CFU-F could be enriched by 60-fold in the
\nCD349(1)CD10(1)CD26(1) fraction, the CD349(1)CD10(2)
\nCD26(2) subsets did not show CFU-F capacity, implying that
\nCD349 alone is not sufficient for CFU-F enrichment. In fact,
\nCD349(2) subset has been shown to proliferate at a higher rate
\nthan CD349(1) subset in periodontal ligament MSCs [74].
\nMoreover, CD349(2), rather than CD349(1) placenta MSCs,
\nshow a function in recovering blood flow following vascular
\nocclusion [81]. These suggest CD349 might not be a critical MSC
\nmarker or essential in enriching MSC function.
\nGD2
\nGD2, the neural ganglioside, was found by Martinez et al. [38]
\nas a single surface marker sufficient to isolate MSCs from BM
\nTable 2. Detection of Stro-1, CD271, SSEA-4 and CD146 in various MSC sources
\nStro-1 CD271 SSEA-4 CD146
\nSources of MSCs Presence References Presence References Presence References Presence References
\nBone marrow 1 [24] 1 [6, 7, 29, 47, 48] 1 [32, 43, 48] 1 [17, 19, 34, 35, 49, 50]
\n2 [49] 2 [51\u201353]
\nAdipose tissue 2 [54] 1 [55] 2 [51] 1 [56]
\n2 [49]
\nUmbilical cord 2 [16] 2 [48] 2 [16, 48] 1 [17]
\nUmbilical cord blood 2 [57] 2 [48, 49] 22\/1 [48, 51] 1 [17, 49]
\n1 [58]
\nPeripheral blood 2 [59] 2 [60]
\nDental pulp 1 [10, 40] 2\/low [31] 1 [61] 1 [40]
\nPlacenta 2 [62] 2\/low [63] 1 [64] 1 [63]
\nSynovial membrane 1 [11] 2 [47, 65, 66] 1 [66] 1 [47]
\n1 [67]
\nPeriodontal ligament 1 [13] 1 [64] 1 [13]
\nDermis 1 [28] 1 [68] 1 [68] 1 [68]
\nEndometrium 1 [14] 1 [14, 33]
\nDecidua parietalis 1 [39] 1 [39]
\nMSCs, mesenchymal stem cells; 1, expression detected; 2, expression not detected; 2\/low, expression detected but very low.
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\nas GD2 is highly expressed in CD45(2)CD73(1) MSCs (>90%)
\nbut not in CD45(2) BM cells. It is also expressed in AT-MSCs
\nand UC-MSCs [82], but not on foreskin fibroblasts. However, a
\nportion of CD34(1) or CD19(1) BM cells also express GD2
\n[38], suggesting GD2 expression is not limited within BMMSCs.
\n3G5
\n3G5 is a pericyte marker. Khan et al. [83] reported that
\nplastic-adherence isolated BM-MSCs were negative for CD271,
\nCD56, and Stro-1 but positive for 3G5. To date, 3G5 expression
\nis detected on BM-MSCs, dental pulp- [40], and decidua
\nparietalis-derived MSCs [39]. Shi and Gronthos [40] showed
\nthat a minor population of BM-MSCs positive for Stro-1
\nexpression is also positive for 3G5. 3G5 positivity accounts for
\n14% of BM CFU-Fs and 63% of dental pulp CFU-Fs [40]. However,
\na large proportion (54%) of hematopoietic BM cells
\nexpress 3G5, eliminating its potential as a sole marker to isolate
\nMSCs from human bone marrow [40].
\nSSEA-3
\nStage-specific embryonic antigen-3 (SSEA-3) is a pluripotent
\nstem cell marker. Recently, evidence showed that a minor
\nsubset of SSEA-3(1)CD105(1) cells in MSCs, namely
\nmultilineage-differentiating stress enduring (MUSE) cells, are
\nable to differentiate into ectodermal, endodermal, and mesodermal
\nlineage cells in vivo [23]. Induced pluripotent stem
\ncells (iPSCs) were only found to be derived from the MUSE
\ncell subset in fibroblasts but not the non-MUSE subset [84],
\nsuggesting that SSEA-3 and CD105 expressing MSCs (1%) as
\nprogenitor cells reminiscent of, but not identical to,
\npluripotent-like ESCs. MUSE cells coexpressed some other pluripotency
\nmarkers including Nanog, Oct3\/4, PAR-4, Sox2 [23].
\nSince MSCs are strongly positive for CD105, MUSE cells can
\nbe represented as the SSEA-3(1) subset of MSCs. MUSE cells
\nare not tumorigenic and can differentiate in vivo without
\nprior genetic manipulation or growth receptor induction [23],
\nhence they may have practical advantages for regenerative
\nmedicine.
\nSUSD2 (W5C5)
\nType 1 integral membrane protein Sushi domain containing 2
\n(SUSD2) has been recently reported to enrich for CFU-Fs and
\ntripotency from endometrium- [41], and BM-derived MSCs
\n[85]. SUSD2 can be detected by the W5C5 antibody [85].
\nSUSD2 is not expressed in hematopoietic cells. In the endometrium,
\nit is predominantly expressed in perivascular
\nregions. W5C5(1) cells are also capable of producing endometrial
\nstromal-like tissue in vivo [41]. Whether SUSD2 being a
\ncommon MSC marker remains to be consolidated.
\nOthers
\nThere are also some other MSC sole or stemness markers proposed
\nby some researchers which are well-investigated. Stro-
\n4, MSCA-1, CD56, CD200, and PODXL have been proposed as
\nMSC markers by their CFU-Fs enrichment capacity. The antibody
\nStro-4 identified the beta isoform of heat shock protein-
\n90. It was found expressed in BM-, dental pulp-, periodontal
\nligament-, and AT-derived MSCs, and enriched CFU-Fs from
\nboth human and ovine BM by 16- and 8-fold compared to
\nBM-MNCs [42]. MSCA-1 (mesenchymal stem cell antigen-1) is
\nidentical to tissue nonspecific alkaline phosphatase [52]. Compared
\nto unsorted BM-MNCs, MSCA-1 selection resulted in a
\n90-fold increase in enrichment of CFU-Fs and a 180-fold
\nincrease when coselected for CD56 [43]. Another surface molecule
\nCD200 was reported [44] to enrich CFU-Fs from BMMNCs
\nto 333-fold. PODXL, a sialomucin in the CD34 family,
\nwas also reported to decrease in high-density cultures which
\nhave lower clonogenicity and differentiation potency compared
\nto less confluent cultures [36].
\nUnlike the above molecules, neuron-glial antigen 2 (NG2),
\nSox11, and TM4SF1 were proposed largely based on their
\nexpression. NG2 is first observed on the surface of neural progenitors
\nand is a pericyte marker whose expression is also
\nshared by BM-MSCs [18, 86, 87]. Sox11, a transcription factor
\npreviously identified in neural progenitor cells, was found to
\nsignificantly decrease during MSC passages and knockdown of
\nSox11 with siRNA decreased the proliferation and osteogenic
\ndifferentiation potential of MSCs [45]. TM4SF1 is another surface
\nprotein highly expressed in BM-, UCB-, and AT-MSCs
\nwhich is not detected in mononuclear cells and fibroblasts,
\nsuggesting it may be a potential marker for MSC selection
\n[46].
\nCOMPARISON OF MSCS ISOLATED BY DIFFERENT MARKERS
\nWith the array of potential markers identified in MSCs, it is
\nstill unclear whether these markers define different or overlapping
\nsubpopulations of MSCs. One of the reasons is that
\nthe phenotypic or functional differences among the MSC subpopulations
\nselected by different markers are still poorly
\nunderstood. Here we aim to review studies that were
\ndesigned to compare various MSC populations sorted in parallel
\nand directly from single sources with respect to their coexpression
\nof MSC markers, CFU enrichment capacity or
\ndifferentiation potential.
\nCFU-Fs Enrichment Capacity
\nDelorme et al. [44] reported that, among nine molecules,
\nCD73, CD130, CD146, CD200, and integrin aV\/b5 were able to
\nenrich CFU-Fs from CD235a(2)\/CD45(2)\/CD11b(2) BMMNCs,
\nwhile CD49b, CD90, and CD105 showed less enrichment.
\nAmong various methods of MSC isolation from BMMNCs,
\nincluding plastic adherence, RosetteSep-isolation, and
\nCD105(1) and CD271(1) selection [30], CD271(1) fraction
\nshowed the highest number of CFU-Fs colonies. Double selection
\nfor CD56 or MSCA-1 enriched CFU-Fs to 3- or 2-fold,
\nrespectively, in CD271 (bright) BM-MNCs [43]. Schwab et al.
\n[14] found that CD146 but not Stro-1 or CD133 selection
\nenriched CFU-Fs from human endometrial stromal cells. Sorting
\nof CD34(2)CD45(2) [88] or CD45(2) [76] human BMMNCs
\nwith CD271 and CD146 revealed that CFU-Fs units
\nremained exclusively in CD271(1) population regardless of
\nCD146 expression, with a tendency toward more CFU-Fs in
\nCD271(1)CD146(1) cells relative to CD271(1)CD146(2) cells.
\nCD146(1) subsets accounted for 96% of CFU-Fs in unfractionated
\nhuman dental pulp cells, while Stro-1(1) and 3G5(1)
\nsubsets accounted for around 80% and 60%, respectively [40].
\nIn addition, the cloning efficiency of W5C5(1)CD146(1) cells
\nwas found significantly higher than CD140b(1)CD146(1) cells.
\nW5C5hi cells had a high clonal capacity equivalent to
\n1412 Markers and Identity of MSCs
\nVC AlphaMed Press 2014 STEM CELLS
\nW5C5(1)CD146(1) cells [41]. Taken together, these findings
\nimply that CD146 and CD271 positivity indicates superior
\nCFU-F capacity in MSCs. These findings are summarized in
\nTable 3.
\nDifferentiation Potential
\nBattula et al. [43] reported that chondrocytes and pancreaticlike
\nislets are predominantly induced from MSCA-
\n1(1)CD56(1) BM-MNCs whereas adipocytes emerge exclusively
\nfrom MSCA-1(1)CD56(2) subsets, indicating that CD56
\nis involved in differentiation tendency. Jarocha et al. [30]
\nreported that CD271(1) or CD105(1) MSCs have lower lineage
\nmarker expression than PA-MSCs after osteogenic, chondrogenic,
\nand adipogenic induction. Arufe et al. [67] reported
\nthat when comparing CD73, CD106, or CD271 positive human
\nsynovial membrane cells, CD271(1) cells are highly chondrogenic,
\nwhereas the CD73(1) cells are less chondrogenic and
\nthe CD106(1) cells mostly undifferentiated after induction.
\nVaculik et al. [68] reported that CD271(1) but not SSEA-4(1)
\ndermal cells exhibit osteogenic and chondrogenic differentiation
\npotential. Dermal SSEA-4(1) cells, in contrast, are only
\nresponsive to adipogenic induction. In adult human BMMNCs,
\nCD271(1)CD146(2\/low) and CD271(1)CD146(1) subsets
\n[76] show a similar capacity to differentiate and to support
\nhematopoiesis, but the two subsets have been found at
\ndifferent sites; CD271(1)CD146(2\/low) cells are bone-lining,
\nwhile CD271(1)CD146(1) cells have a perivascular localization,
\nsuggesting that the two subsets play different roles in
\nHSC niche. The function of surface markers in multipotency
\nenrichment is summarized in Table 4.
\nSurface Marker Coexpression
\nRelevant studies on the degree of coexpression of surface
\nmarkers on MSCs is summarized in Table 5. The expression of
\nCD106 and CD146 was found to be restricted to the MSCA-
\n1(1)CD56(2) MSCs and CD166 to MSCA-1(1)CD56(1\/2)
\nMSCs [43]. Vaculik et al. [68] reported that in human dermis,
\nthe expression pattern of SSEA-4 is almost analogous to
\nCD271. Both were found only weakly expressed and coexpressed
\nwith CD45. Van Landuyt and Quirici also reported the
\ndetection of CD34 expression on CD271(1) subpopulation of
\nhuman synovial and BM-MSCs [29, 47]. In human dermis,
\nCD73 and CD105 are coexpressed [68]. A minor population of
\nthe human dermis CD73(1) cells is CD90(2). Dermis
\nCD271(1) cells were CD73(1) and CD105(1), whereas the
\nmajority of CD271(1) cells are CD90(2) [68]. Similarly, in two
\nother reports, only a minor subset of the CD271(1) cells
\nexpress CD90, CD73 (<10% in cultured CD271(1) cells from
\nBM [29], 10%\u201320% in freshly purified CD271(1) cells from
\nadipose tissue [72]). Maijenburg further reported that the distribution
\nof CD271(1)CD146(2) and CD271(1)CD146(1) subsets
\ncorrelates with donor age. The main subset in pediatric
\nand fetal BM was reported to be CD271(1)CD146(1),
\nwhereas CD271(1)CD146(2) population was dominant in
\nadult marrow [88]. In endometrial MSCs, 28% of W5C5(1)
\ncells are CD146(1), while 60% of W5C5(1) cells are Stro-
\n1(1). A small population of W5C5(1) cells also express other
\nTable 3. Comparison of MSC sorting protocols for CFU-Fs enrichment
\nCell subsets analyzed Whole cell population Result References
\nStro-1(1), CD133(1), CD90(1),
\nCD146(1)
\nHuman endometrial stromal cells Only CD146 showed CFU-Fs
\nenrichment
\n[14]
\nCD49b(1), CD90(1), CD105(1),
\nCD73(1), CD130(1), CD146(1),
\nCD200(1), aV\/b5(1)
\nHuman BM-MNCs CD49b, CD105, and CD90 showed low
\nCFU-Fs enrichment. CD73, CD130,
\nCD146, CD200, and integrin aV\/\u00df5
\nshowed higher CFU-Fs enrichment
\n[44]
\nMSCA-1(1), CD271(1), CD56(1) Human BM-MNCs CD271(1)CD56(1) fraction enriched
\nCFU-Fs to threefold compared to
\nCD271(1)CD56(2) fraction. MSCA-
\n1(1)CD56(1) fraction gave rise to
\ntwo fold higher CFU-Fs than MSCA-
\n1(1)CD56(\u2013) cells.
\n[43]
\nPA-MSCs, RosetteSep-, CD105(1) or
\nCD271(1) sorted
\nHuman BM-MNCs CFU-Fs was most enriched in
\nCD271(1) fraction.
\n[30]
\nCD271(1),CD146(1) CD34(2)CD45(2) human BM-MNCs CFU-Fs remained exclusively in
\nCD271(1) population;
\nCD271(1)CD146(1) cells had more
\nCFU-Fs relative to
\nCD271(1)CD146(2) cells
\n[87]
\nCD271(1),CD146(1) Human BM-MNCs CFU-Fs was not observed in CD271(-)
\ncell fraction. CD146 positivity further
\nenhanced CFU-Fs to 2.1 times in
\nCD271(1)CD45(2) fraction.
\n[76]
\nStro-1(1), CD146(1), 3G5(1) Human dental pulp cells No colony formation could be detected
\nin STRO-1bright\/CD146(2) human
\nbone marrow. CD146(1) subsets
\naccounted for 96% of CFU-Fs in unfractured
\ndental pulp cells, while
\nStro-1(1) and 3G5(1) subsets
\naccounted for around 80% and 60%,
\nrespectively.
\n[40]
\nBM-MNCs, bone marrow mononuclear cells; CFU-Fs, fibroblastic colony-forming units; MSC, mesenchymal stem cell; PA-MSCs, plastic adherence
\nisolated MSCs.
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\nlineage markers, like CD24 (11.6%), CD31 (5 %), CD45 (4.7 %),
\nand epithelial cell adhesion molecule [41].
\nMSC IDENTITY IN VIVO: FIBROBLASTS OR PERIVASCULAR CELLS
\nAdult stem cells are found in specialized niches that store and
\nmaintain stem cells and mediate the balanced response of
\nstem cells to the needs of organisms. The definition of MSCs
\nhas been based on their ability to self-renew and to differentiate
\ninto certain mature cell types in vitro. Their identity in
\nvivo, however, remains unclear. Unlike the well-established
\nniche of BM for HSCs [89], the true identity of MSCs and their
\nniche in vivo is still under debate. Currently, it has been
\nraised that MSCs may derive from fibroblasts or pericytes.
\nFibroblasts are a type of cells synthesizing collagen, the
\nmajor structural framework for animal tissues, and in the
\nhuman body they are found in virtually every organ and tissue.
\nMSCs have a close resemblance to fibroblasts [90]. Fibroblasts
\nand MSCs are both plastic adherent and share similar
\ncell morphology. Human dermal fibroblasts express many cell
\nTable 4. Comparison of multipotency of sorted MSCs
\nCell subsets analyzed Whole cell population Capacity compared Result References
\nCD73(1), CD106(1),
\nCD271(1)
\nHuman synovial
\nmembrane cells
\nChondrogenesis Chondrogenic potential: CD271
\n(1)>CD73(1)>CD106 (1)
\n[67]
\nMSCA-1(1), CD56(1) Human BM-MNCs Chondrogenesis, adipogenesis MSCA-1(1)CD56(1): Chondrogenic
\nand pancreatic differentiation
\npotential, no
\nadipogenic potential.
\n[43]
\nPancreatic differentiation MSCA-1(1)CD56(2): Adipogenic
\npotential, no chondrogenic
\nand pancreatic differentiation
\npotential.
\nCD271(1), SSEA-4(1),
\nCD73(1), CD90(1)
\nHuman dermis MSCs Chondrogenesis, adipogenesis,
\nosteogenesis
\nCD271(1) cells had tri-lineage
\npotential. Dermal SSEA-4(1)
\ncells could only go for adipogenesis.
\nCD73(1) cells
\nshowed a significantly higher
\nadipogenic differentiation
\ncapacity than CD90(1) cells.
\n[68]
\nPA-MSCs, RosetteSep-,
\nCD105(1) or CD271(1)
\nsorted
\nHuman BM-MNCs Osteogenesis, chondrogenesis,
\nadipogenesis
\nCD271(1) or CD105(1) MSCs
\nshowed lower differentiation
\nrelated marker expression
\nthan PA-MSCs after osteogenic,
\nchondrogenic and adipogenic
\ninduction.
\n[30]
\nCD271(1), CD146(1) Human BM-MNCs Osteogenesis, chondrogenesis,
\nadipogenesis
\nCD271(1)CD146(2\/low) and
\nCD271(1)CD146(1) subsets
\nshowed a similar differentiation
\npotential
\n[76]
\nBM-MNCs, bone marrow mononuclear cells; MSCs, mesenchymal stem cells; SSEA-4, surface-specific embryonic antigen.
\nTable 5. Comparison of coexpressed markers in sorted MSCs
\nCell subsets analyzed Whole cell population Result References
\nMSCA-1(1), CD271(1), CD56(1) Human BM-MNCs CD271 (1)CD56(2) cells expressed CD106
\nand CD146. CD271(1)CD56(1) cells
\nexclusively expressed CD166.
\nCD271(1)CD56(1) double positivity
\nenriched SSEA-4 expression.
\nCD271(1)CD56(1) double positivity
\nenriched MSCA-1 expression.
\n[43]
\nCD271(1), SSEA-4(1) Human dermis cells Expression pattern of SSEA-4 in dermis was
\nanalogous to CD271. CD271 and SSEA-4
\nboth coexpressed with CD45(1) cells.
\nCD73 and CD105 were coexpressed. A
\nminor population of the CD73(1) cells
\nwas CD90(2). CD271(1) cells were
\nCD73(1) and CD105(1), whereas the
\nmajority of CD271(1) cells were
\nCD90(2).
\n[68]
\nW5C5(1) Human endometrial cells W5C5(1) cells were 28% CD146(1), 60%
\nStro-1(1), 11.6% CD24(1), 5.3%
\nCD31(1), 4.7% CD45(1).
\n[41]
\nBM-MNCs, bone marrow mononuclear cells; MSCs, mesenchymal stem cells; SSEA, surface specific embryonic antigen.
\n1414 Markers and Identity of MSCs
\nVC AlphaMed Press 2014 STEM CELLS
\nsurface proteins similar to MSCs, including the general
\nmarkers used for MSC characterization [91]. Human dermal
\nfibroblasts also have tripotency [91, 92], although contradictory
\nfinding has been reported which suggests a lack of multipotency
\n[51]. In addition, human dermal fibroblasts show
\nimmunoregulatory functions similar to MSCs [93, 94].
\nAnother hypothesis is that MSCs reside throughout the
\nbody as pericytes or perivascular cells and that the perivascular
\nzone is the in vivo niche of MSCs [95]. Pericytes are a relatively
\nelusive cell type recognized by virtue of their
\nanatomical location of their residence, that is on the abluminal
\nsurface of endothelial cells in the microvasculature, rather
\nthan by a precisely defined phenotype. As pericytes, MSCs
\nmay be readily released from their niche and secrete immunoregulatory
\nand trophic bioactive factors upon tissue damage.
\nAs such, MSCs may function as a source of stem cells for
\nphysiological turnover.
\nA perivascular niche of MSCs is supported by the observation
\nthat in the majority of solid tissues where MSCs have
\nbeen found, blood vessels may be the only common anatomical
\nstructure. Consistent with the observation, the mesenchyme
\nacts as a \u201cspace filler\u201d before the development of a
\nvascular system in early embryonic limb development [96].
\nSimilar to MSCs, pericytes or perivascular cells are able to differentiate
\ninto osteoblasts, chondrocytes, adipocytes, fibroblasts,
\nmyofibroblasts, and smooth muscle cells in vitro [97].
\nIn fact, CD146(1) perivascular cells from multiple organs
\nexpressed general MSC surface antigens [18], as well as 3G5
\n[98] and NG2 [87]. Observations in vivo also support the association
\nof pericytes with MSCs. For instance, multipotential
\nstem cells were identified in the mural cell population of the
\nvasculature [99]. In rat malignant glioma, intratumoral injection
\nof MSCs [100] resulted in the engraftment of MSCs into
\ntumor vessel walls and the expression of several pericyte
\nmarkers.
\nSeveral studies have further compared the associations of
\nMSCs with fibroblasts and pericytes. Blasi et al. [101] reported
\nthat AT-MSCs cannot be distinguished from human dermal
\nfibroblasts in vitro by phenotype or multipotency. However,
\nAT-MSCs, but not dermal fibroblasts, displayed strong angiogenic
\nand anti-inflammatory activity. Sacchetti et al. [35]
\nfound that only CD146(1) MSCs, but not muscle or skin fibroblasts,
\nare capable of reconstructing BM and conferring a
\nhematopoietic microenvironment in immunocompromized
\nmice. Additionally, several transcripts were found differentially
\nexpressed between HS68 fibroblasts and MSCs, whereas several
\ninhibitors of the Wnt pathway (DKK1, DKK3, SFRP1), an
\nimportant pathway in regulation of MSCs, were highly
\nexpressed in fibroblasts, suggesting that MSCs and fibroblasts
\nhave distinct gene expression profiles. Gene and microRNA
\nexpression comparison of human MSCs and dermal fibroblasts
\nrevealed a panel of MSC-specific molecular signature, which
\nmainly encode transmembrane proteins or associate with
\ntumors [102]. In a comprehensive study by Covas et al. [86],
\nthe cell morphology and the phenotypes were found to be
\ncomparable among 12 types of MSCs, 2 origins of pericytes,
\nand 4 sources of fibroblasts. However, different from MSCs
\nand pericytes, fibroblasts were reported to be weak for
\nCD146 expression and high for the expression of fibroblastspecific
\nprotein-1 (FSP-1, also named as S100A4), a specific
\nfibroblast marker. Furthermore, serial analysis of gene expression
\nrevealed a consistent grouping of MSCs with pericytes
\nand hepatic stellate cells, while fibroblasts differentially clustered
\nwith smooth muscle cells and myofibroblasts rather
\nthan MSCs [86].
\nThe close relationship of MSCs with perivascular cells is
\nalso reflected by the physical distribution of the MSC specific
\nmarkers in vivo. As summarized in Table 6, the general MSC
\nantigens, such as CD73, CD90, and CD105 have a vascular and
\nperivascular expression pattern [18, 68], although their
\nTable 6. Physical expression of MSC markers in vivo
\nMarkers Expression site References
\nCD73, CD90, and CD105 Dermis: Vascular and perivascular expression [68]
\nSkeletal muscle, placenta, and white adipose tissue: Perivascular expression [18]
\nCD90 Endometrium: Expressed on all the stroma of the human endometrium, including the fibroblasts,
\nperivascular and endothelial cells
\n[14]
\nStro-1 BM: Expressed on blood vessel walls [40]
\nDental pulp: Expressed on blood vessels and around perineurium surrounding nerve bundles [40]
\nEndometrium: On endothelial cells and on the stroma around blood vessels [14]
\nPlacenta: Expressed around the vessels [105]
\nNG2 Skeletal muscle, pancreas, placenta, white adipose tissue, fetal heart, fetal skin, lung, brain, eye, gut,
\nbone marrow, and umbilical cord: Only expressed in periphery of capillaries and microvessels in
\nalmost all tissues
\n[18]
\nCD146 Skeletal muscle, pancreas, placenta, white adipose tissue, fetal heart, fetal skin, lung, brain, eye, gut,
\nbone marrow, and umbilical cord: Expressed on perivascular cells surrounding capillaries, arterioles
\nand venules, and on endothelium in capillaries, but not on microvessel endothelial cells
\n[18]
\nBM and dental pulp: Blood vessel wall expression [40]
\nEndometrium: Expressed on perivascular and endothelial cells [14]
\nPlacenta: expressed around the vessels [105]
\n3G5 Placenta: expressed on scattered cells around the vessels. [105]
\nCD271 Dermis: presented on cutaneous nerve fibers, Schwann cells, dermal single cells, and, faintly, on clusters
\nof basal keratinocytes
\n[68]
\nBM: CD271(1)CD146(2\/low) cells were bone-lining, while CD271(1)CD146(1) had a perivascular
\nlocalization
\n[76]
\nSSEA-4 Dermis: Presented on cutaneous nerve fibers, Schwann cells, dermal single cells, and, faintly, on clusters
\nof basal keratinocytes
\n[68]
\nSUSD2\/W5C5 Endometrial tissue: Perivascular location. [41]
\nBM, bone marrow; MSC, mesenchymal stem cells; SSEA, surface specific embryonic antigen.
\nLv, Tuan, Cheung et al. 1415
\nwww.StemCells.com VC AlphaMed Press 2014
\nexpression can also be found in fibroblasts [14]. For the MSC
\nspecific markers, Stro-1, NG2, CD146, and 3G5 expression was
\nmainly found in perivascular area of capillaries, microvessels,
\nand\/or venules in many tissues [18, 40], despite additional
\nexpression was found in certain endothelial cells for CD146
\n[14] and in endothelial cells and stroma for Stro-1 [14]. This
\nfurther supports the identity of MSCs as perivascular cells in
\nvivo, and that MSCs may bear stronger resemblance to pericytes
\nand perivascular cells rather than to fibroblasts. However,
\nthis \u201cperivascular niche\u201d theory cannot explain why
\nMSC-like cells are also detected in avascular tissues, such as
\nin articular cartilage [103] and nucleus pulposus [77]. A further
\npostulation is that MSCs may have more than one origin
\nthan the perivascular niche, as disclosed in the dual origin of
\nodontoblasts in the teeth by genetic lineage tracing [104].
\nThis postulation of a nonpericytic origin of MSCs is also supported
\nby the fact that MUSE cells, a subset of MSCs with
\nhigher stemness, do not express CD146 [84].
\nCONCLUSIONS AND PERSPECTIVES
\nA large number of markers have been brought forward to
\nfacilitate the isolation of MSCs from their surrounding environment
\nor the selection of MSCs with high stemness. It
\nshould be noted that the marker expression of MSCs is not in
\na stable level. Culture conditions have potential influence on
\nthe phenotype of MSCs and that such influence may contribute
\nto the contradictory reports on marker expression. Particularly,
\nsome antigens may be artificially induced by in vitro
\nmanipulation and culturing, such as the induction of SSEA-4
\nby FCS [75]. Culture confluence can also induce certain
\nmarkers, such as CD49d, CD200, or CD106, or diminish them,
\nsuch as CD49f and PODXL [36]. Certain growth factors and
\ncytokines, such as fibroblast growth factor and interferon-\u00c7,
\nor disease conditions such as inflammation, may also
\nmodulate the phenotype of MSCs (Table 7). This therefore
\nemphasizes the importance of a standard operation procedure
\nfor in vitro MSC expansion and validation of the markers
\nin vivo.
\nAs shown in the above analysis, there is no sole marker
\nthat is truly MSC-specific. Among the known MSC markers,
\nCD146 may be the most appropriate stemness marker, as it is
\nuniversally detected in the MSC population isolated from various
\ntissues, and enriches cells with clonogenicity and multipotency.
\nOn the other hand, SSEA-3 may be a more immature
\nstemness marker which represents an ESCs-like phenotype.
\nThis is consistent with the proposed in vivo identity of MSCs
\nas pericytes, as CD146 is also a pericyte marker. Interestingly,
\nCD146 is highly expressed in both MSCs and pericytes, but
\nnot in dermal fibroblasts [35], while FSP-1, a fibroblast marker,
\nis lowly expressed in MSCs and pericytes [86], lending support
\nto the closer association of MSCs with pericytes. However,
\nthis theory cannot explain the non-pericytic origin of MSCs
\nsuggested in a number of reports. Further investigation to
\nTable 7. Effect of in vitro or in vivo conditions on MSC phenotype
\nRegulatory factors Markers investigated Findings References
\nInflammation Stro-1 There was no significant difference in proliferation, differentiation or Stro-1
\npositivity between MSCs isolated from normal and inflamed dental pulps.
\n[106]
\nStro-1, CD90, CD105, CD146 Inflammed dental pulps expressed higher levels of MSC markers STRO-1,
\nCD90, CD105, and CD146 compared with normal dental pulps.
\n[107]
\nStro-1, SSEA-4 More Stro-1 and SSEA-4 positive cells were found in healthy than in
\ninflammed gingival tissues.
\n[108]
\nCulture
\nconfluency
\nCD49d, CD49f, CD200,
\nCD106, PODXL
\nCulture confluency was shown positively correlated with the expression of
\nCD49d, CD200 and CD106, and negatively correlated with CD49f and
\nPODXL.
\n[36]
\nSerum SSEA-4 FCS contained globoseries glycolipids which could be recognized by a SSEA-
\n4 antibody, and exposure to FCS induced the cell-surface expression of
\nSSEA-3 in cord-blood-derived HSCs
\n[75]
\nInterferon HLA class II HLA class II expression in MSCs was induced by IFN-c. [109, 110]
\nHLA-DR HLA\u2013DR positivity upon addition of IFN remained unchanged. [111]
\nNG2 Addition of IFN-c repressed the transcription of NG2 in MSCs after neural
\ninduction procedures.
\n[112]
\nGrowth factors CD105, CD73, CD90, CD29,
\nCD44, CD146
\nFGF induced expression of HLA-DR, and lowered the expression of CD146
\nand CD49a, as well as the expression of CD49c. Expression of the MSC
\nsurface antigens HLA\u2013A\/B\/C, CD105, CD73, CD90, CD29, and CD44 was
\nnot affected.
\n[111]
\nStemness markers (Oct4A,
\nNotch 1, Hes5), neural
\nmarkers (Nestin, Pax6,
\nNgn2)
\nEGF1bFGF pretreatment downregulated the expression of stemness
\nmarkers Oct4A, Notch1 and Hes5, whereas neural\/neuronal molecules
\nNestin, Pax6, Ngn2 and the neurotrophin receptor tyrosine kinase 1 and
\n3 were upregulated.
\n[113]
\nCD44, CD90, CD146, CD105 FGF-2 resulted in reduced expression of CD146 and alkaline phosphatase,
\nwhich was partially reversed upon removal of the supplement. There was
\nno alteration in CD44 and CD90 with culture conditions, whereas the
\nCD146, CD105, and ALP expression profile was regulated by supplementation
\nwith FGF-2, EGF, and PDGF-BB.
\n[114]
\nAA CD44, CD90, CD146, CD105 There was no alteration in CD44 and CD90 with culture conditions, whereas
\nthe CD146, CD105, and ALP expression profile was regulated by supplementation
\nwith AA.
\n[114]
\nAA, ascorbic acid; ALP, alkaline phosphatase; EGF, epidermal growth factor; FCS, fetal calf serum; FGF, fibroblast growth factor; HLA-DR, human
\nleukocyte antigen DR; HSCs, hematopoietic stem cells; IFN, interferon; MSCs, mesenchymal stem cells; PDGF-BB, platelet-derived growth factor
\nBB.
\n1416 Markers and Identity of MSCs
\nVC AlphaMed Press 2014 STEM CELLS
\nunderstand the niche components of MSCs in vivo is therefore
\ndemanded to validate the theory.
\nAccompanied by this dilemma is an emerging theory proposing
\na neuroectodermal origin for MSCs, represented by
\nthe expression of nestin [115]. We have not included nestin,
\nSox2, or Oct4, in the analysis, since these are intracellular
\nproteins but not surface markers. However, we noticed that
\nnestin(1)\/CD271(2)\/Stro-1(2) MSCs derived from human
\nESCs were reported to differentiate into representative derivatives
\nof all three embryonic germ layers [116]. Therefore,
\ncompared with CD271 and Stro-1, nestin positivity may represent
\na more primitive phenotype of MSCs. An interesting
\nhypothesis is that CD146(1) MSCs may be a lineage of
\nnestin(1) MSCs, since pericytes within several tissues were
\nreported to be derived from neural crest derivatives [117].
\nWhile nestin is a intracellular protein which may complicate
\nthe isolation of nestin(1) MSCs, a recent paper suggests that
\nnestin (1) MSCs can be isolated by PDGFRalpha and CD51
\ndouble positivity [118], which may facilitate the future investigation
\nof the properties of nestin(1) MSCs.
\nACKNOWLEDGEMENTS
\nThis work was supported by Small Project Funding of the University
\nof Hong Kong (201209176179), General Funding from
\nNational Science Foundation of China (NSFC, No. 81371993), and
\nby the Commonwealth of Pennsylvania, Department of Health.
\nAUTHOR CONTRIBUTIONS
\nF.-J.L.: conception and design, collection and\/or assembly of
\ndata, interpretation and analysis of data, manuscript writing,
\nand final approval of manuscript; R.S.T.: interpretation and analysis
\nof data, manuscript writing, and final approval of manuscript;
\nK.M.C.C. and V.Y.L.L.: financial support, administrative
\nsupport, manuscript writing, and final approval of manuscript.
\nDISCLOSURE OF POTENTIAL CONFLICT OF INTEREST
\nThe authors indicate no potential conflicts of interest.
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