mesenchymal stem cells in tooth and oral cavity

mesenchymal stem cells in tooth and oral cavity Order Description the origin of mesenchymal stem cells and their types in the mouth and tooth e.g. 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 INTRODUCTION 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 Carlos ESTRELA1 Ana Helena Gonçalves de ALENCAR1 Gregory Thomas KITTEN2 Eneida Franco VENCIO1 Elisandra GAVA1,2 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: elisandragava@yahoo.com.br 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 compartments (5). 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 regenerative medicine. STEM CELLS FROM HUMAN EXFOLIATED DECIDUOUS TEETH 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 by researchers. 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 root formation. 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 TISSUE ENGINEERING 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 Location Permanent tooth pulp Apical papilla of developing root Exfoliated deciduous tooth pulp Periodontal ligament Dental follicle of developing tooth Proliferation rate Moderate High High High High Heterogeneity Yes Yes Yes Yes Yes Multipotentialy Odontoblast, osteoblast, chondrocyte, myocyte, neurocyte, adipocyte, corneal epithelial cell, melanoma cell, iPS Odontoblast, osteoblast, neurocyte, adipocyte, iPS Odontoblast, osteoblast, chondrocyte, myocyte, neurocyte, adipocyte, iPS Odontoblast, osteoblast, chondrocyte, cementoblast, neurocyte Odontoblast, osteoblast, neurocyte Tissue repair Bone regeneration, neuroregeneration, myogenic regeneration, dentinpulp regeneration Bone regeneration, neuroregeneration, dentin-pulp regeneration, root formation Bone regeneration, neuroregeneration, tubular dentin Bone regeneration, root formation, periodontal regeneration Bone regeneration, periodontal regeneration 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 clinical practice. 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 regeneration (42). 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 surface (44). CONCLUDING REMARKS 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. RESUMO 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. ACKNOWLEDGEMENTS 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.). REFERENCES 1. Bianco P, Robey PG, Simmons PJ. Mesenchymal stem cells: revisiting history, concepts, and assays. Cell Stem Cell 2008;2:313-319. 2. Arien-Zakay H, Lazarovici P, Nagler A. Tissue regeneration potential in human umbilical cord blood. Best Pract Res Clin Haematol 2010;23:291-303. 3. Meirelles Lda S, Nardi NB: Methodology, biology and clinical applications of mesenchymal stem cells. Front Biosci 2009;14:4281-4298. 4. 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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 a,b,c Key Words. Mesenchymal stem cells • Surface epitopes • CD271 • CD146 • Markers • Pericytes • Niche • Regenerative medicine ABSTRACT 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 INTRODUCTION 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 [5] 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) [8], placenta [9], dental pulp [10], synovial membrane [11], peripheral blood [12], periodontal ligament [13], endometrium [14], umbilical cord (UC) [15], 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 body [18]. 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 [19], indicating that PA-MSCs comprised a heterogeneous population of cells with different lineage commitment [19], 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 [20]). For example, the percentage of BM CFU-Fs with osteogenic potency was higher than that with adipogenic potency [21]. Similarly, ectopic transplantation of BMMSCs resulted in heterotopic bone tissue formation, while dental pulp-derived MSCs generated reparative dentin-like tissue [22]. 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 [23]. 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 of Orthopaedics and Traumatology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, People’s Republic of China; bStem Cell & Regenerative Medicine Consortium and cCenter for Reproduction, Development 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 Orthopaedic Surgery, 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: vicleung@hku. 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: cheungmc@hku.hk Received September 16, 2013; accepted for publication February 9, 2014; first published online in STEM CELLS EXPRESS February 28, 2014. VC AlphaMed Press 1066-5099/2014/$30.00/0 http://dx.doi.org/ 10.1002/stem.1681 Stem Cells 2014;32:1408–1419 www.StemCells.com VC AlphaMed Press 2014 TISSUE-SPECIFIC STEM CELLS like cells from their in vivo environment [5]. 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 detected. 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 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 [69]. By combining negative selection against glycophorin-A, Stro-1 enriched CFU-Fs from BM with multipotency [24]. However, it did not enrich CFU-Fs from human endometrial stroma [14]. The degree of homogeneity of the Stro-1-selected MSCs was further enhanced 1000-fold by positive selection for CD106 compared to PA-MSCs [25]. Injection of human Stro-1(1) but not Stro-1(2) BM-MNCs into rat myocardium led to arteriogenesis and functional cardiac recovery [70]. 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 [26]. Conditioned medium from Stro- 1(1) MSCs could induce a greater degree of cardiac vascular repair than PA-MSCs [25]. These suggest that Stro-1 may be involved in clonogenicity and play a role in homing and angiogenesis of MSCs. However, Stro-1 is not universally expressed in all reported types of MSCs. Stro-1 is expressed in dental- [10], synovial membrane- [11], decidua parietalis-derived MSCs [39] and multipotent dermal fibroblasts [28]. AT- [54], UCB- [57], UC- [16], peripheral blood-derived MSCs [59] are negative/low for Stro-1 expression. It is reported that placenta-derived MSCs gradually lose Stro-1 expression in culture [62]. In contrast, however, the expression of Stro-1 in BM-MSCs increases with culture time [36]. 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 [24]. Moreover, Stro-1 expression appears not universal for various MSC types. CD271 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%) [71]. 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 [72] and also induced superior chondral repair than the CD271(2) BM MSCs [73]. 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 [29] and from AT (10%–20%) [72]. Moreover, nearly 50%–99% of the CD271(1) cells in BM [29] and synovium [47] coexpressed CD34, which disqualifies CD271 as a sole marker to isolate MSCs from various tissues [5]. 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 [13]. However, it is expressed at low levels in placenta-derived MSCs [31, 63] and not expressed in synovial membrane- [47, 65, 66], peripheral blood- [60], UC-, and UCB-MSCs [48, 49]. Although Watson et al. [58] 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 [30]. Therefore, CD271 may not be considered as a MSC stemness marker. SSEA-4 SSEA-4 is an embryonic stem cell marker. It has been documented to isolate genuine MSCs from BM [32]. SSEA-4(1) BM cells can expand extensively, while SSEA-4(2) subsets fail to grow. SSEA-4(1) cells also show tripotency [57]. SSEA-4 expression gradually increases in BM culture over time [57]. Besides in BM-MSCs, SSEA-4 expression was also detected in placenta- [64], periodontal ligament- [74], dental pulp- [61], and synovial membrane [65]-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 www.StemCells.com VC AlphaMed Press 2014 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 [24] 11.2 Human BM-MNCs [25] 6 Human BM-MSCs after 1 week in culture [26] 2.1 Human endometrial stroma cells [14] 1.29 Human amnion MSCs at passage 0–2 [27] 8 Human dermal fibroblasts at passage 3 [28] 28.96 Inflamed periodontal ligament MSCs [13] 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 [30] Negligible Human placenta MSCs [31] SSEA-4 Enrich CFU-Fs from whole BM 1–2 Mouse BM-MSCs at day 2 in culture [32] 71 Mouse BM-MSCs after 100 days in culture 2–4 Human BM cells 37.82 Human amnion MSC at passage 0–2 [27] CD146 Enrich CFU-Fs from BM-MSCs 9.4 Human endometrial stroma [14] Enrich cells with multipotency from BM-MSCs 1.5 CD146(1)PDGF-Rb(1) Human endometrial stroma [33] Downregulated in differentiated cells 0.1 Human BM-MSCs [34, 35] 1.2 Human CD45 depleted BM-MNCs [35] 42.7 Human BM-MSCs [17] 17.2–37.9 Human UC-MSCs 11.55 Inflamed periodontal ligament MSCs [13] 21.85 Healthy periodontal ligament MSCs 9.4 Fresh isolated human endometrial stromal cells [14] CD49f Enrich clonogenicity and differentiation potency 88.1 human BM-MSCs at passage 1 [36] Knocking down results in differentiation of HSCs 15 Human BM-MSCs [37] 55 Human UCB-MSCs [37] CD349 Enrich CFU-Fs from whole placenta cells 0.2 Total human plancenta cells [31] GD2 High specificity for isolating MSCs from BM 95 Human CD45(2)CD105(1)CD73(1) BM-MNCs [38] 65 Human BM-MSCs [37] 3 Human UCB-MSCs [37] 3G5 Enrich CFU-Fs Decidua parietalis [39] 63 Dental pulp CFU-Fs [40] 14 BM CFU-Fs SSEA-3 Enrich cells with clonogenicity and ectodermal, endodermal, and mesodermal differentiation potency 1 BM-MSCs [23] SUSD2 Enrich CFU-Fs and tri-potency 4.2 Human endometrial stromal cells [41] Stro-4 Enrich CFU-Fs <5 Human and ovine BM-MSCs [42] 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 in BM-MSCs 0.5–5.5 coexpressed CD271bright BM cells [43] CD200 Enrich CFU-Fs from BM-MNCs; downregulated in differentiated cells 0.15 Human BM-MNCs [44] PODXL PODXL decreases in high-density cultures 90 Human BM-MSCs at passage 2 [36] 13 Human BM-MSCs [37] 25 Human UCB-MSCs [37] Sox11 Downregulated during culture. Knockdown affects proliferation and osteogenesis potential Not tested Human BM-MSCs [45] TM4SF1 Enriched in MSCs compared with their source tissue or fibroblasts Not specified BM, AT, and UCB [46] 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 [75]. 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 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%) [33]. CD146 has a greater CFU-Fs enrichment capacity than CD90, Stro-1, or CD133 [14]. 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 [19]. 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 [34], and in vivo, when transplanted into mice, CD146(1) BM stroma subendothelial cells exhibit the capacity to reorganize the hematopoietic microenvironment to heterotopic sites [35]. Importantly, the expression of CD146 was found not only in BM-MSCs [50] but also in almost all the other sources of MSCs, including MSCs derived from AT [56], UC [15, 17], synovial membrane [47], UCB [49], placenta [63], dermis [68], periodontal ligament [13], and intervertebral disc [77]. In fact, CD146-expressing MSC clones from multiple organs were found to exhibit trilineage potency [18]. CD49f 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 [78]. CD49f has also been identified as a specific HSC marker and shown to enrich cells capable of generating longterm multilineage grafts [79]. To date, CD49f expression has been detected in BM-MSCs [36], fetal urinary bladder-derived MSCs [80], and UCB-MSCs [37]. 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 [36]. Condition that induces MSC sphere formation can enrich CD49f(1) population compared with MSCs in monolayer [78]. Moreover, higher expression level of CD49f, such as in UCB-MSCs, is functionally linked with a higher lung clearance rate in systemic infusion [37]. 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 [79]. CD349 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 [31]. Additionally, CD349 expression has been reported in periodontal ligament-derived MSCs [74]. 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 [74]. Moreover, CD349(2), rather than CD349(1) placenta MSCs, show a function in recovering blood flow following vascular occlusion [81]. These suggest CD349 might not be a critical MSC marker or essential in enriching MSC function. GD2 GD2, the neural ganglioside, was found by Martinez et al. [38] 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 [24] 1 [6, 7, 29, 47, 48] 1 [32, 43, 48] 1 [17, 19, 34, 35, 49, 50] 2 [49] 2 [51–53] Adipose tissue 2 [54] 1 [55] 2 [51] 1 [56] 2 [49] Umbilical cord 2 [16] 2 [48] 2 [16, 48] 1 [17] Umbilical cord blood 2 [57] 2 [48, 49] 22/1 [48, 51] 1 [17, 49] 1 [58] Peripheral blood 2 [59] 2 [60] Dental pulp 1 [10, 40] 2/low [31] 1 [61] 1 [40] Placenta 2 [62] 2/low [63] 1 [64] 1 [63] Synovial membrane 1 [11] 2 [47, 65, 66] 1 [66] 1 [47] 1 [67] Periodontal ligament 1 [13] 1 [64] 1 [13] Dermis 1 [28] 1 [68] 1 [68] 1 [68] Endometrium 1 [14] 1 [14, 33] Decidua parietalis 1 [39] 1 [39] MSCs, mesenchymal stem cells; 1, expression detected; 2, expression not detected; 2/low, expression detected but very low. Lv, Tuan, Cheung et al. 1411 www.StemCells.com VC AlphaMed Press 2014 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 [82], but not on foreskin fibroblasts. However, a portion of CD34(1) or CD19(1) BM cells also express GD2 [38], suggesting GD2 expression is not limited within BMMSCs. 3G5 3G5 is a pericyte marker. Khan et al. [83] 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- [40], and decidua parietalis-derived MSCs [39]. Shi and Gronthos [40] 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 [40]. 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 [40]. SSEA-3 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 [23]. Induced pluripotent stem cells (iPSCs) were only found to be derived from the MUSE cell subset in fibroblasts but not the non-MUSE subset [84], 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 [23]. 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 [23], hence they may have practical advantages for regenerative medicine. SUSD2 (W5C5) Type 1 integral membrane protein Sushi domain containing 2 (SUSD2) has been recently reported to enrich for CFU-Fs and tripotency from endometrium- [41], and BM-derived MSCs [85]. SUSD2 can be detected by the W5C5 antibody [85]. 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 [41]. Whether SUSD2 being a common MSC marker remains to be consolidated. Others 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 [42]. MSCA-1 (mesenchymal stem cell antigen-1) is identical to tissue nonspecific alkaline phosphatase [52]. 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 [43]. Another surface molecule CD200 was reported [44] 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 [36]. 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 [45]. 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 [46]. 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 differentiation potential. CFU-Fs Enrichment Capacity Delorme et al. [44] 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 [30], 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 [43]. Schwab et al. [14] found that CD146 but not Stro-1 or CD133 selection enriched CFU-Fs from human endometrial stromal cells. Sorting of CD34(2)CD45(2) [88] or CD45(2) [76] 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 [40]. 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 1412 Markers and Identity of MSCs VC AlphaMed Press 2014 STEM CELLS W5C5(1)CD146(1) cells [41]. Taken together, these findings imply that CD146 and CD271 positivity indicates superior CFU-F capacity in MSCs. These findings are summarized in Table 3. Differentiation Potential Battula et al. [43] 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. [30] 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. [67] 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. [68] 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 [76] 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 [43]. Vaculik et al. [68] 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 [68]. 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) [68]. 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 [29], 10%–20% in freshly purified CD271(1) cells from adipose tissue [72]). 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 [88]. 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), CD146(1) Human endometrial stromal cells Only CD146 showed CFU-Fs enrichment [14] CD49b(1), CD90(1), CD105(1), CD73(1), CD130(1), CD146(1), CD200(1), aV/b5(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 [44] 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- 1(1)CD56(–) cells. [43] PA-MSCs, RosetteSep-, CD105(1) or CD271(1) sorted Human BM-MNCs CFU-Fs was most enriched in CD271(1) fraction. [30] CD271(1),CD146(1) CD34(2)CD45(2) human BM-MNCs CFU-Fs remained exclusively in CD271(1) population; CD271(1)CD146(1) cells had more CFU-Fs relative to CD271(1)CD146(2) cells [87] 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 CD271(1)CD45(2) fraction. [76] 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%, respectively. [40] BM-MNCs, bone marrow mononuclear cells; CFU-Fs, fibroblastic colony-forming units; MSC, mesenchymal stem cell; PA-MSCs, plastic adherence isolated MSCs. Lv, Tuan, Cheung et al. 1413 www.StemCells.com VC AlphaMed Press 2014 lineage markers, like CD24 (11.6%), CD31 (5 %), CD45 (4.7 %), and epithelial cell adhesion molecule [41]. 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 [89], 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 [90]. 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 CD73(1), CD106(1), CD271(1) Human synovial membrane cells Chondrogenesis Chondrogenic potential: CD271 (1)>CD73(1)>CD106 (1) [67] MSCA-1(1), CD56(1) Human BM-MNCs Chondrogenesis, adipogenesis MSCA-1(1)CD56(1): Chondrogenic and pancreatic differentiation potential, no adipogenic potential. [43] Pancreatic differentiation MSCA-1(1)CD56(2): Adipogenic potential, no chondrogenic and pancreatic differentiation potential. CD271(1), SSEA-4(1), CD73(1), CD90(1) Human dermis MSCs Chondrogenesis, adipogenesis, osteogenesis CD271(1) cells had tri-lineage potential. Dermal SSEA-4(1) cells could only go for adipogenesis. CD73(1) cells showed a significantly higher adipogenic differentiation capacity than CD90(1) cells. [68] PA-MSCs, RosetteSep-, CD105(1) or CD271(1) sorted Human BM-MNCs Osteogenesis, chondrogenesis, adipogenesis CD271(1) or CD105(1) MSCs showed lower differentiation related marker expression than PA-MSCs after osteogenic, chondrogenic and adipogenic induction. [30] CD271(1), CD146(1) Human BM-MNCs Osteogenesis, chondrogenesis, adipogenesis CD271(1)CD146(2/low) and CD271(1)CD146(1) subsets showed a similar differentiation potential [76] 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. [43] 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 CD90(2). [68] 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). [41] BM-MNCs, bone marrow mononuclear cells; MSCs, mesenchymal stem cells; SSEA, surface specific embryonic antigen. 1414 Markers and Identity of MSCs VC AlphaMed Press 2014 STEM CELLS surface proteins similar to MSCs, including the general markers used for MSC characterization [91]. Human dermal fibroblasts also have tripotency [91, 92], although contradictory finding has been reported which suggests a lack of multipotency [51]. 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 [95]. 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 physiological turnover. 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 [96]. Similar to MSCs, pericytes or perivascular cells are able to differentiate into osteoblasts, chondrocytes, adipocytes, fibroblasts, myofibroblasts, and smooth muscle cells in vitro [97]. In fact, CD146(1) perivascular cells from multiple organs expressed general MSC surface antigens [18], as well as 3G5 [98] and NG2 [87]. 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 [99]. In rat malignant glioma, intratumoral injection of MSCs [100] resulted in the engraftment of MSCs into tumor vessel walls and the expression of several pericyte markers. Several studies have further compared the associations of MSCs with fibroblasts and pericytes. Blasi et al. [101] 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. [35] 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 [102]. In a comprehensive study by Covas et al. [86], 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 [86]. 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 [68] Skeletal muscle, placenta, and white adipose tissue: Perivascular expression [18] CD90 Endometrium: Expressed on all the stroma of the human endometrium, including the fibroblasts, perivascular and endothelial cells [14] Stro-1 BM: Expressed on blood vessel walls [40] Dental pulp: Expressed on blood vessels and around perineurium surrounding nerve bundles [40] Endometrium: On endothelial cells and on the stroma around blood vessels [14] Placenta: Expressed around the vessels [105] 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 [18] 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 [18] BM and dental pulp: Blood vessel wall expression [40] Endometrium: Expressed on perivascular and endothelial cells [14] Placenta: expressed around the vessels [105] 3G5 Placenta: expressed on scattered cells around the vessels. [105] CD271 Dermis: presented on cutaneous nerve fibers, Schwann cells, dermal single cells, and, faintly, on clusters of basal keratinocytes [68] BM: CD271(1)CD146(2/low) cells were bone-lining, while CD271(1)CD146(1) had a perivascular localization [76] SSEA-4 Dermis: Presented on cutaneous nerve fibers, Schwann cells, dermal single cells, and, faintly, on clusters of basal keratinocytes [68] SUSD2/W5C5 Endometrial tissue: Perivascular location. [41] BM, bone marrow; MSC, mesenchymal stem cells; SSEA, surface specific embryonic antigen. Lv, Tuan, Cheung et al. 1415 www.StemCells.com VC AlphaMed Press 2014 expression can also be found in fibroblasts [14]. 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 [14] and in endothelial cells and stroma for Stro-1 [14]. 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 [103] and nucleus pulposus [77]. 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 [104]. 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 [84]. 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 [75]. Culture confluence can also induce certain markers, such as CD49d, CD200, or CD106, or diminish them, such as CD49f and PODXL [36]. 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 in vivo. 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 [35], while FSP-1, a fibroblast marker, is lowly expressed in MSCs and pericytes [86], 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. [106] 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. [107] Stro-1, SSEA-4 More Stro-1 and SSEA-4 positive cells were found in healthy than in inflammed gingival tissues. [108] Culture confluency CD49d, CD49f, CD200, CD106, PODXL Culture confluency was shown positively correlated with the expression of CD49d, CD200 and CD106, and negatively correlated with CD49f and PODXL. [36] 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 [75] 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. [111] NG2 Addition of IFN-c repressed the transcription of NG2 in MSCs after neural induction procedures. [112] Growth factors CD105, CD73, CD90, CD29, CD44, CD146 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 not affected. [111] Stemness markers (Oct4A, Notch 1, Hes5), neural markers (Nestin, Pax6, Ngn2) 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. [113] 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. [114] 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 with AA. [114] 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 BB. 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 [115]. 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 [116]. 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 [117]. 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 [118], which may facilitate the future investigation of the properties of nestin(1) MSCs. ACKNOWLEDGEMENTS 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. AUTHOR CONTRIBUTIONS 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. REFERENCES 1 Manuguerra-Gagne R, Boulos PR, Ammar A et al. Transplantation of mesenchymal stem cells promotes tissue regeneration in a glaucoma model through laser-induced paracrine factor secretion and progenitor cell recruitment. Stem Cells 2013;31:1136–1148. 2 Yang F, Leung VY, Luk KD et al. Mesenchymal stem cells arrest intervertebral disc degeneration through chondrocytic differentiation and stimulation of endogenous cells. Mol Ther 2009;17:1959–1966. 3 Noth U, Steinert AF, Tuan RS. 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