Localization of mesenchymal stem cells populations in the adult human dental pulp

Localization of mesenchymal stem cells populations in the adult human dental pulp | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Localization of mesenchymal stem cells populations in the adult human dental pulp Heba Alzer, Firas Alsoleihat, Mohammed Al-Shayyab, Nisreen Abu Shahin, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3067290/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background : Despite comprehensive studies and reports about the properties of dental pulp stem cells (DPSCs) in vitro, we still lack the knowledge of whether these in vitro characteristics coincide with the original nature of DPSCs in situ. The histological location of DPSCs populations in the dental pulp has been rarely investigated. Methods : In this study, well-recognized mesenchymal stem cell markers MCAM, CD73, STRO1, Thy1, and CD105 are being investigated by immunohistochemistry to localize the precise location of these populations of DPSCs within the human adult dental pulp. Results . All previously mentioned markers were expressed in the dental pulp, and their intensity and location of expression were reported. Conclusion : The observations that multiple mesenchymal stem cell niches exist may have further implications for their recruitment in situ for multiple regenerative therapies. Biological sciences/Developmental biology Biological sciences/Stem cells MCAM CD73 STRO1 Thy1 CD105 Osteocalcin human dental pulp stem cells mesenchymal stem cells marker Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction The dental pulp is a loose connective tissue surrounded by specialized dentin-forming cells called odontoblasts. Dental pulp stem cells (DPSCs) originate from the cranial neural crest [1,2] and the mesoderm [3], and both populations are heterogeneous [4–16]. DPSCs from mesenchymal origin have phenotypic characteristics similar to bone marrow mesenchymal stem cells [17]. They are suggested to migrate from the intra-pulpal blood vessels toward the pulp tissue when regeneration is anticipated [3,18]. MCAM, CD73, STRO1, Thy1, and CD105 are among the most recognized mesenchymal stem cell (MSCs) markers [18–22]. To our knowledge, previous studies to investigate the distribution of these markers of MSCs in the human adult dental pulp by immunohistochemistry do not exist. Accordingly, this study tries to illuminate the distribution and intensity of DPSCs that express these MSCs. The difference between these DPSCs populations' percentage in situ and in vitro could be further studied to elucidate the effect of culturing methods on the enrichment or extinction of these populations, as it was suggested earlier that culture conditions are responsible for such effects [12,23]. Methods Sample collection Wisdom teeth from healthy patients aged 17-19 years old were collected, and teeth were extracted for dental reasons. The Institutional Review Board and Ethics Committee approved the study in accordance with The Declaration of Helsinki, and the decision number is 2017/158. Informed consent was obtained to acquire the teeth for analysis, and data privacy was preserved. Sample processing Following extraction, the teeth were fixed in 10% neutral buffered formalin and transformed to the laboratory. The teeth were immersed in the decalcification solution (1:1 volume of 8% formic acid + 8% HCL) and then incubated in a water bath at 50°C for 3h and left until they were soft enough to be sectioned. The teeth were fixed in formalin for 24h and then inserted into an auto processor where they were subjected to fixation in formal saline, dehydration with ascending concentrations of ethanol, clearing with xylene, and finally, infiltration and embedding in paraffin blocks. With the microtome, serial sections of 3-micrometer thickness were utilized and transferred onto Immunohistochemistry slides. Immunohistochemical staining Slides were dried in the dryer for 15min and then incubated overnight at room temperature. Subsequently, they were deparaffinized with xylene for 15 min, rehydrated with graded ethanol (70% - 100%), and then washed in distilled water. Heat-mediated antigen retrieval was performed using Tris/EDTA buffer pH9 or sodium citrate pH 6. Table 1 shows the data of the primary antibodies, dilutions, incubation conditions, and antigen retrieving methods utilized. The rest of the procedure was performed exactly as stated in the EXPOSE Mouse and Rabbit Specific HRP/DAB Detection IHC Kit (ab80436) (Abcam/UK). Bound antibodies were visualized with a microscope (Nikon Eclipse Ts2). The location and intensity of the signal were graded as explained in the results in Table 2. Results All five stem cell markers and the osteogenic differentiation marker osteocalcin were identified in the dental pulp. Figures 1-5 and Table 2 summarize each marker’s localization and intensity of expression. MCAM was expressed strongly in the nuclei, cell membrane, and cytoplasm of most of the dental pulp cells (Figure 1) and radicular odontoblasts (Figure 1, A), some of the positive pulp cells showed large nuclei and a kind of clustering (Figure 1, C). Further, MCAM was expressed moderately in the walls of blood vessels (Figure 1, C) and intensively in cell sheets in the coronal pulp cell-rich zone (Figure 1, D). CD73 expression was detected as a few big and small clusters scattered in the dental pulp (Figure 2, A, C). Moreover, a multinucleated lymphocyte expressed it (Figure 2, B). STRO1 was moderately expressed in dental pulp blood vessels and nerve bundles (Figure 3, A). CD105 showed strong nuclear staining in most dental pulp cells (Figure 3, B). Thy1 showed an intense expression in the nuclei and cytoplasm of the blood vessel walls cells, nerve bundles, and the nuclei of pulp core fibroblasts (Figure 4, A, B, D). Moreover, accessory canals were intensively positive, and cells with strong nuclear Thy1 expression seemed to be entering the dental pulp from the accessory canals (Figure 4, C). Osteocalcin expression was intense and uniform in the nuclei and cytoplasm of dental pulp cells throughout the dental pulp and odontoblasts (Figure 5). Antibody Clone Source Dilution/ Incubation conditions Antigen retrieval method MCAM Mouse monoclonal [P1H12] Abcam, UK 1:500 overnight 4°C Tris/ EDTA pH 9 CD73 Mouse monoclonal [7G2] Abcam, UK 1:200 overnight 4°C Tris/ EDTA pH 9 STRO1 Mouse monoclonal [STRO1] Abcam, UK 1:200 1 hour 25°C Sodium citrate pH 6 Thy1 Rabbit Monoclonal [EPR3133] Abcam, UK 1:500 overnight 4°C Sodium citrate pH 6 CD105 Mouse monoclonal [SN6] Abcam, UK 1:500 overnight 4°C Tris/ EDTA pH 9 Osteocalcin Rabbit polyclonal [AB10911] Millipore, USA 1:500 overnight 4°C Sodium citrate pH 6 Table 1: Primary antibodies utilized for immunohistochemistry. Their clone, source, dilutions, incubation conditions, and antigen retrieval methods. Antibody Locations of marker expression Intensity MCAM Dental pulp core cells Cell clusters scattered in the dental pulp Blood vessels of the dental pulp Sheets in the coronal pulp cell-rich zone Odontoblastic layer - root Odontoblastic layer- the crown and cervical part of the root ++ § + +++ ++ - CD73 Cell clusters scattered in the dental pulp – large Cell clusters scattered in the dental pulp – small Multinucleated lymphocyte +++ + ++ STRO1 Blood vessels of the dental pulp Nerve bundles + + Thy1 Dental pulp core cells Blood vessels of the dental pulp Nerve bundles Accessory canals +++ +++ +++ +++ CD105 Dental pulp core cells ++ Osteocalcin Dental pulp core cells Odontoblasts +++ +++ Table 2: The distribution and intensity of expression of MCAM, CD73, STRO1, Thy1, CD105, and osteocalcin in the adult human dental pulp, the intensity of expression is defined in all locations where the antibodies were detected as follows: +++intense, ++ strong, + moderate, § weak, - negative. Discussion DPSCs reside in a quiescent state within their niche; they migrate and proliferate in response to certain stimuli to regenerate damaged tissues. Exploring their niche may provide valuable information helping scale up proper culturing and enrichment of DPSCs [24]. In 2006, the International Society for Cellular Therapy (ISCT) proposed CD73, CD90, and CD105 as MSCs markers [19,20]. STRO1 CD90, CD73, and MCAM are reported repeatedly to be expressed in DPSCs [18,21,22]. Using immunohistochemistry, we have investigated the localization of these mesenchymal stem cell markers in the pulp tissue of third molars of young adult human males and females. Our results identified multiple DPSCs niches located in the dental pulp cell-rich zone, walls of blood vessels, the odontoblastic layer, nerve bundles, accessory canals, subodontoblastic zone, and the radicular pulp. Markers' expression and intensity showed a gradual decrease from the apex toward the coronal part of the pulp, which coincides with a previous study that investigated the proliferation capacity of dental pulp cells [25]. Earlier studies reported similar DPSCs’ niche locations to the ones reported here [25,26]. However, these studies experimented with a combination of stem cell markers different from those described here or with deciduous teeth [26]. MCAM or CD146 is a cell adhesion molecule and integral membrane glycoprotein that exhibits many functions in development, signaling transduction, cell migration, mesenchymal stem cell differentiation, angiogenesis, and immune response [27–29]. MCAM is considered a stem cell and endothelial marker co-expressed with STRO1 on the walls of blood vessels and is reported to reside in the perivascular niche of dental pulp [18]. CD146+ DPSCs showed higher mineralization and adipogenicity than CD146- cells [30]. MCAM expression was strong in the radicular odontoblasts (Figure 1, A) and most dental pulp cells' nuclei, cell membranes, and cytoplasm (Figure 1). Some positive pulp cells showed large nuclei and a kind of clustering (Figure 1, C). Further, MCAM was expressed moderately in the walls of blood vessels (Figure 1, C) and intensively in cell sheets in the coronal pulp cell-rich zone (Figure 1 and Table 2). This observation contradicts a previous report by Shi and Gronthos 2003 who reported that MCAM was only expressed in the walls of blood vessels of the dental pulp but coincided with Alongi et al. 2010 and Martens et al. 2012 who mentioned MCAM was expressed in multiple locations in the dental pulp without specification [18,24,31]. This contradiction in reports could be attributed to differences in the donor’s age in each study, as age is previously suggested to play a role in DPSCs’ nature in vitro [23]. Shi and Gronthos 2003 presumed that the co-localization of STRO1 and CD146 on the walls of large blood vessels in dental pulp tissue implied that most DPSCs arise from microvasculature [18]. We respectfully disagree as MCAM’s strong expression was observed in multiple niches other than the perivascular niche. Moreover, they suggested that during dentin regeneration, pre-odontoblasts may originate from perivascular cells migrating out of the capillary walls into the surrounding pulp tissue [18]. Here, we demonstrated that both odontoblasts and capillary walls expressed MCAM, which could support this suggestion, but further investigation is recommended. MCAM showed nuclear expression in some cells despite it being a membrane glycoprotein. The phenomena could be explained by the accumulated evidence that considers the shuttling of adhesion transmembrane proteins between the plasma membrane and nucleus to be common, where adhesion transmembrane proteins interact with transcription factors to perform certain functions [32]. CD73 is a cell surface protein that hydrolyzes extracellular nucleotides into permeable membrane nucleosides and is expressed in MSCs [33–35]. CD73 expression was observed in multinucleated lymphocytes or as large and small clusters rarely seen scattered in the dental pulp (Figure 2 and Table 2). STRO1 is a cell surface antigen, like MCAM, expressed by MSCs in the bone marrow and dental pulp blood vessels. It is considered a marker of pre-osteogenic populations and an early marker of different MSCs populations in the perivascular niche [17,18]. Here, STRO1 expression was restricted to blood vessel walls and the nerve bundles (Figure 3 and Table 2), which coincides with the previous reports[18,26]. CD105 showed strong nuclear staining in most dental pulp cell-rich zone (Figure 4 and Table 2), as reported previously [24,31]. Thy1, also known as CD90, is expressed on fibroblasts, neurons, endothelial cells, and thymocytes. It has been identified as an MSCs marker and has a crucial role in MSCs fate decisions [36]. Thy1 showed an intensive expression in the nuclei and cytoplasm of the blood vessel walls (arterioles and venules), nerve bundles, and cells of the pulp core. Moreover, accessory canals were intensively positive, and cells with strong positive nucleareThy1 expression seemed to be entering the dental pulp from the accessory canals (Figure 4 and Table 2). Osteocalcin is the main non-collagenous hydroxyapatite-binding protein synthesized by osteoblasts, odontoblasts, and hypertrophic chondrocytes. It regulates mineralization and is considered a marker of bone cell metabolism [37]. Osteocalcin expression was intense and uniform in the nuclei and cytoplasm of dental pulp cells throughout the dental pulp and odontoblasts. The radicular pulp has the most intense expression of osteocalcin (Figure 5 and Table 2). This antibody was investigated to illuminate the differentiation status of the dental pulp, which is highly differentiated, as osteocalcin was strongly expressed throughout the dental pulp. To our knowledge, no previous reports explored the expression of CD73, Thy1, or osteocalcin in dental pulp tissue by immunohistochemistry. Conclusion Information concerning DPSCs proliferation and differentiation is essential to understanding tooth response to regenerative stimuli. Since there is little data concerning DPSCs in vivo, we have investigated the localization of DPSCs populations of third molars of young adult humans using immunohistochemistry. The results demonstrated the presence of multiple DPSCs niches, which are observed in the dental pulp cell-rich zone, walls of blood vessels, the odontoblastic layer, nerve bundles, accessory canals, subodontoblastic zone, and the radicular pulp. The observations that multiple DPSCs niches exist may have further implications for their recruitment in situ for multiple regenerative therapies. However, these stem cells' exact location, developmental potential, and ontogeny are still largely unknown. In the present study, identifying mesenchymal DPSCs niches may help elucidate the fundamental conditions necessary to selectively maintain and expand DPSCs in vitro and direct their potentials in vivo . Abbreviations DPSCs: Dental Pulp Stem Cells MSCs: Mesenchymal Stem Cells Declarations Ethics approval and consent to participate The Institutional Review Board and Ethics Committee at the University of Jordan approved the study ‘localization of the different populations of stem cells existing in the dental pulp of adult human teeth’ in accordance with The Declaration of Helsinki, and the decision was given the number 2017/158 in 2017. Informed consent was obtained to acquire the teeth for analysis, and data privacy was preserved. Data Availability All data used to publish these findings are available upon request from Dr. Heba Alzer. Disclaimer Statement The authors have no conflict of interest to announce Funding Statement This study was funded bythe Deanship of Scientific Research at the University of Jordan [47/2015/2016]. The funding body played no role in the design of the study and collection, analysis, and interpretation of data nor in writing the manuscript. Acknowledgment The authors thank the Deanship of Scientific Research at the University of Jordan for funding this project and the Maxillofacial Surgery Department at the University of Jordan Hospital for their cooperation in sample collection. Author contribution HA: contributed to conception, design, data acquisition and interpretation, drafted and revised the manuscript. NAS: contributed to conception and design and critically revised the manuscript. MA: contributed to sample collection, patient selection, acquiring consent, surgical extraction of the samples, and revised the manuscript. HK: contributed to the conception and design and revised the manuscript. FA: contributed to conception, design, data acquisition and interpretation, and critically revised the manuscript. Consent for Publication All authors gave their final approval and agreed to be accountable for all aspects of the work. References Chai Y, Jiang X, Ito Y, et al. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3067290","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":374228892,"identity":"41510baa-a28a-43f8-a58f-d970d61f820c","order_by":0,"name":"Heba Alzer","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA30lEQVRIiWNgGAWjYDACdjB5gMGAgfkAA2MDMVqY4VrYEkjWwmNAnBb+ZvaHjysY7siZs5/5JvFzh40cA/vhoxvwaZE4zJBseIbhmbFlT+42yd4zacYMPGlpN/Bac5jhmGQDw+HEDQdyt0nwth1ObJDgMcOrRf4wY/tPoJb6DeffPJP8S4wWg8PMbEBPH04wuJHDJk2ULYaH2ZhBDjPcOeOZsbVsW5oxGyG/yB1vf/gRqEXenD/54c23bTZy/OyHj+H3Pggw/gNTLBIgko2gciTA/IEU1aNgFIyCUTByAABCc0tKZt94GwAAAABJRU5ErkJggg==","orcid":"","institution":"University of Jordan","correspondingAuthor":true,"prefix":"","firstName":"Heba","middleName":"","lastName":"Alzer","suffix":""},{"id":374228893,"identity":"b5ed256c-2068-4692-b170-45f7fb5c74c6","order_by":1,"name":"Firas Alsoleihat","email":"","orcid":"","institution":"University of Jordan","correspondingAuthor":false,"prefix":"","firstName":"Firas","middleName":"","lastName":"Alsoleihat","suffix":""},{"id":374228894,"identity":"23c83af3-3915-44da-b7ef-9c6772b552ab","order_by":2,"name":"Mohammed Al-Shayyab","email":"","orcid":"","institution":"University of Jordan","correspondingAuthor":false,"prefix":"","firstName":"Mohammed","middleName":"","lastName":"Al-Shayyab","suffix":""},{"id":374228895,"identity":"58c1e1f0-f254-4ba6-9399-281257a1f2c0","order_by":3,"name":"Nisreen Abu Shahin","email":"","orcid":"","institution":"University of Jordan","correspondingAuthor":false,"prefix":"","firstName":"Nisreen","middleName":"Abu","lastName":"Shahin","suffix":""},{"id":374228896,"identity":"2f423e7e-b65c-4f08-94b8-b8eaab2d5611","order_by":4,"name":"Heba Kalbouneh","email":"","orcid":"","institution":"University of Jordan","correspondingAuthor":false,"prefix":"","firstName":"Heba","middleName":"","lastName":"Kalbouneh","suffix":""}],"badges":[],"createdAt":"2023-06-15 10:29:50","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3067290/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3067290/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":68347425,"identity":"0da14d5e-5680-4a57-bca4-4ba5f8fcf69a","added_by":"auto","created_at":"2024-11-06 09:54:47","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":943213,"visible":true,"origin":"","legend":"\u003cp\u003eThe distribution of expression of MCAM in adult human dental pulp. A) The odontoblastic layer in the radicular area shows a strong positive signal (black arrow), 10x. B) Dental pulp core cells expressed MCAM (black arrows) and showed some clustering (white arrows), 20x. C) Some dental pulp core cells that expressed MCAM featured large nuclei (black arrows) and showed clustering (circle). Endothelial cells of blood vessels (white arrows) expressed MCAM, 20x. D) MCAM was expressed strongly in cell sheets found in the coronal pulp cell-rich zone (black arrows). The odontoblasts in this cervical part of the root are negative (white arrow) compared with those observed in A, 20x. Scale bar 50 um\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-3067290/v1/5021a9f73d7b34d45c11a996.png"},{"id":68347282,"identity":"c8ff2276-5550-4de1-ab8e-3bcd6c9670e9","added_by":"auto","created_at":"2024-11-06 09:54:40","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":479002,"visible":true,"origin":"","legend":"\u003cp\u003eThe distribution of expression of CD73 in adult human dental pulp. A) and C) CD73 expression was detected as a few big and small clusters scattered in the dental pulp (black arrows), both 10x. B) A multinucleated lymphocyte expressing CD73 (black arrow), 20x. Scale bar 50 um\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-3067290/v1/820c46dab4c3b4d9bc95fbfc.png"},{"id":68347517,"identity":"e35c3ab2-0d3b-456a-8a7d-3ecfd2a4835d","added_by":"auto","created_at":"2024-11-06 09:54:49","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":780493,"visible":true,"origin":"","legend":"\u003cp\u003eThe distribution of expression of STRO1 and CD105 in adult human dental pulp. A) STRO1 was expressed in the dental pulp blood vessels and nerve bundles (black arrows), 10x. B) CD105 showed strong nuclear staining in most dental pulp cells (black arrows), 10x. Scale bar 50 um\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-3067290/v1/89ed645158a572aa49fec146.png"},{"id":68347266,"identity":"39335847-b864-42fc-af0d-0c1e406bcfae","added_by":"auto","created_at":"2024-11-06 09:54:38","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":849748,"visible":true,"origin":"","legend":"\u003cp\u003eThe distribution of expression of Thy1 in adult human dental pulp. A), B), and D) Thy1 showed an intense expression in the nuclei and cytoplasm of the blood vessel walls and nerve bundles (black arrows), all are 20x, B) and D) Thy1 showed an intense expression in the nuclei of pulp core fibroblasts. C) Accessory canals were intensively positive, and cells with strong nuclear Thy1 expression seemed to be entering the dental pulp from the accessory canals (black rectangle showing positive cells entering from the canal), 10x. Scale bar 50 um\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-3067290/v1/18a51ed8b67d467cc16ac6a8.png"},{"id":68347281,"identity":"f59edb5c-4d3f-4348-8c2b-bdd94f440f67","added_by":"auto","created_at":"2024-11-06 09:54:39","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":551734,"visible":true,"origin":"","legend":"\u003cp\u003eThe distribution of osteocalcin expression in the adult human dental pulp. A) The odontoblastic layer showed a strong positive signal, 10x. B) Osteocalcin expression was intense and uniform in the nuclei and cytoplasm of dental pulp cells throughout the dental pulp, 20x. Scale bar 50 um\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-3067290/v1/89612fbe4c64d5dc714c012e.png"},{"id":68350392,"identity":"085b1e87-1c24-4e8f-ad18-bf92e70fd97b","added_by":"auto","created_at":"2024-11-06 10:27:17","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4590739,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3067290/v1/312fe2fa-67c3-4717-b2e1-8a32975ce151.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Localization of mesenchymal stem cells populations in the adult human dental pulp","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe dental pulp is a loose connective tissue surrounded by specialized dentin-forming cells called odontoblasts.\u0026nbsp;Dental pulp stem cells (DPSCs) originate from the cranial neural crest [1,2] and the mesoderm [3], and both populations are heterogeneous [4\u0026ndash;16].\u0026nbsp;DPSCs from mesenchymal origin have phenotypic characteristics similar to bone marrow mesenchymal stem cells [17]. They are suggested to migrate from the intra-pulpal blood vessels toward the pulp tissue when regeneration is anticipated [3,18]. MCAM, CD73, STRO1, Thy1, and CD105 are among the most recognized mesenchymal stem cell (MSCs) markers [18\u0026ndash;22]. To our knowledge, previous studies to investigate the distribution of these markers of MSCs in the human adult dental pulp by immunohistochemistry do not exist. Accordingly, this study tries to illuminate the distribution and intensity of DPSCs that express these MSCs.\u003c/p\u003e\n\u003cp\u003eThe difference between these DPSCs populations\u0026apos; percentage \u003cem\u003ein situ\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e could be further studied to elucidate the effect of culturing methods on the enrichment or extinction of these populations, as it was suggested earlier that culture conditions are responsible for such effects [12,23].\u0026nbsp;\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eSample collection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWisdom teeth from healthy patients aged 17-19 years old were collected, and teeth were extracted for dental reasons. The Institutional Review Board and Ethics Committee approved the study in accordance with The Declaration of Helsinki, and the decision number is 2017/158. Informed consent was obtained to acquire the teeth for analysis, and data privacy was preserved.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSample processing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFollowing extraction, the teeth were fixed in 10% neutral buffered formalin and transformed to the laboratory. The teeth were immersed\u0026nbsp;in\u0026nbsp;the decalcification solution (1:1\u0026nbsp;volume\u0026nbsp;of 8% formic acid + 8% HCL)\u0026nbsp;and then incubated\u0026nbsp;in a water bath at 50\u0026deg;C for 3h\u0026nbsp;and left\u0026nbsp;until they were soft enough to be sectioned. The teeth were fixed in\u0026nbsp;formalin for 24h and then inserted into an auto processor where they were subjected to\u0026nbsp;fixation in formal saline, dehydration with ascending concentrations of ethanol, clearing with xylene, and finally, infiltration and embedding in paraffin blocks. With the microtome, serial\u0026nbsp;sections of 3-micrometer thickness were utilized and\u0026nbsp;transferred onto Immunohistochemistry slides.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunohistochemical staining\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSlides were dried in the dryer for 15min and then incubated overnight at room temperature. Subsequently, they were deparaffinized with xylene for 15 min, rehydrated with graded ethanol (70% - 100%), and then washed in distilled water. Heat-mediated antigen retrieval was performed using Tris/EDTA buffer pH9 or sodium citrate pH 6. Table 1 shows the data of the primary antibodies, dilutions, incubation conditions, and antigen retrieving methods utilized. The rest of the procedure was performed exactly as stated in the EXPOSE Mouse and Rabbit Specific HRP/DAB Detection IHC Kit (ab80436) (Abcam/UK). Bound antibodies were visualized with a microscope (Nikon Eclipse Ts2). The location and intensity of the signal were graded as explained in the results in Table 2.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eAll five stem cell markers and the osteogenic differentiation marker osteocalcin were identified in the dental pulp. Figures 1-5 and Table 2 summarize each marker\u0026rsquo;s localization and intensity of expression.\u003c/p\u003e\n\u003cp\u003eMCAM was expressed strongly in the nuclei, cell membrane, and cytoplasm of most of the dental pulp cells (Figure 1) and radicular odontoblasts (Figure 1, A), some of the positive pulp cells showed large nuclei and a kind of clustering (Figure 1, C). Further, MCAM was expressed moderately in the walls of blood vessels (Figure 1, C) and intensively in cell sheets in the coronal pulp cell-rich zone (Figure 1, D). CD73 expression was detected as a few big and small clusters scattered in the dental pulp (Figure 2, A, C). Moreover, a multinucleated lymphocyte expressed it (Figure 2, B). STRO1 was moderately expressed in dental pulp blood vessels and nerve bundles (Figure 3, A). CD105 showed strong nuclear staining in most dental pulp cells (Figure 3, B). Thy1 showed an intense expression in the nuclei and cytoplasm of the blood vessel walls cells, nerve bundles, and the nuclei of pulp core fibroblasts (Figure 4, A, B, D). Moreover, accessory canals were intensively positive, and cells with strong nuclear Thy1 expression seemed to be entering the dental pulp from the accessory canals (Figure 4, C). Osteocalcin expression was intense and uniform in the nuclei and cytoplasm of dental pulp cells throughout the dental pulp and odontoblasts (Figure 5).\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"595\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 15.8249%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eAntibody\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25.4209%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eClone\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14.3098%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eSource\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19.0236%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eDilution/ Incubation conditions\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25.4209%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAntigen retrieval method\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 15.8249%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eMCAM\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25.4209%;\"\u003e\n \u003cp\u003e\u0026nbsp; Mouse monoclonal\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e[P1H12]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14.3098%;\"\u003e\n \u003cp\u003eAbcam, UK\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19.0236%;\"\u003e\n \u003cp\u003e1:500\u003c/p\u003e\n \u003cp\u003eovernight 4\u0026deg;C\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25.4209%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eTris/ EDTA pH 9\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 15.8249%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eCD73 \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25.4209%;\"\u003e\n \u003cp\u003eMouse monoclonal [7G2]\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14.3098%;\"\u003e\n \u003cp\u003eAbcam, UK\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19.0236%;\"\u003e\n \u003cp\u003e1:200\u003c/p\u003e\n \u003cp\u003eovernight 4\u0026deg;C\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25.4209%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eTris/ EDTA pH 9\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 15.8249%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eSTRO1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25.4209%;\"\u003e\n \u003cp\u003eMouse monoclonal [STRO1]\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14.3098%;\"\u003e\n \u003cp\u003eAbcam, UK\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19.0236%;\"\u003e\n \u003cp\u003e1:200\u003c/p\u003e\n \u003cp\u003e1 hour 25\u0026deg;C\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25.4209%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eSodium citrate pH 6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 15.8249%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eThy1\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25.4209%;\"\u003e\n \u003cp\u003eRabbit Monoclonal [EPR3133]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14.3098%;\"\u003e\n \u003cp\u003eAbcam, UK\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19.0236%;\"\u003e\n \u003cp\u003e1:500\u003c/p\u003e\n \u003cp\u003eovernight 4\u0026deg;C\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25.4209%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eSodium citrate pH 6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 15.8249%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eCD105\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25.4209%;\"\u003e\n \u003cp\u003eMouse monoclonal [SN6] \u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14.3098%;\"\u003e\n \u003cp\u003eAbcam, UK\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19.0236%;\"\u003e\n \u003cp\u003e1:500\u003c/p\u003e\n \u003cp\u003eovernight 4\u0026deg;C\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25.4209%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eTris/ EDTA pH 9\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 15.8249%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eOsteocalcin\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25.4209%;\"\u003e\n \u003cp\u003eRabbit polyclonal\u003c/p\u003e\n \u003cp\u003e[AB10911]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14.3098%;\"\u003e\n \u003cp\u003eMillipore, USA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19.0236%;\"\u003e\n \u003cp\u003e1:500\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eovernight 4\u0026deg;C\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25.4209%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eSodium citrate pH 6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1:\u0026nbsp;\u003c/strong\u003ePrimary antibodies utilized for immunohistochemistry. Their clone, source, dilutions, incubation conditions, and antigen retrieval methods.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 16.5785%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAntibody\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 70.0176%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eLocations of marker expression\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.4039%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eIntensity\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 16.5785%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eMCAM\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 70.0176%;\"\u003e\n \u003cp\u003eDental pulp core cells\u003c/p\u003e\n \u003cp\u003eCell clusters scattered in the dental pulp\u003c/p\u003e\n \u003cp\u003eBlood vessels of the dental pulp\u003c/p\u003e\n \u003cp\u003eSheets in the coronal pulp cell-rich zone\u003c/p\u003e\n \u003cp\u003eOdontoblastic layer - root\u003c/p\u003e\n \u003cp\u003eOdontoblastic layer- the crown and cervical part of the root\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.4039%;\"\u003e\n \u003cp\u003e++\u003c/p\u003e\n \u003cp\u003e\u0026sect;\u003c/p\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003cp\u003e+++\u003c/p\u003e\n \u003cp\u003e++\u003c/p\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 16.5785%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eCD73 \u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 70.0176%;\"\u003e\n \u003cp\u003eCell clusters scattered in the dental pulp \u0026ndash; large\u003c/p\u003e\n \u003cp\u003eCell clusters scattered in the dental pulp \u0026ndash; small\u003c/p\u003e\n \u003cp\u003eMultinucleated lymphocyte\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.4039%;\"\u003e\n \u003cp\u003e+++\u003c/p\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003cp\u003e++\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 16.5785%;\"\u003e\n \u003cp\u003eSTRO1\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 70.0176%;\"\u003e\n \u003cp\u003eBlood vessels of the dental pulp\u003c/p\u003e\n \u003cp\u003eNerve bundles\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.4039%;\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 16.5785%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eThy1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 70.0176%;\"\u003e\n \u003cp\u003eDental pulp core cells\u003c/p\u003e\n \u003cp\u003eBlood vessels of the dental pulp\u003c/p\u003e\n \u003cp\u003eNerve bundles\u003c/p\u003e\n \u003cp\u003eAccessory canals\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.4039%;\"\u003e\n \u003cp\u003e+++\u003c/p\u003e\n \u003cp\u003e+++\u003c/p\u003e\n \u003cp\u003e+++\u003c/p\u003e\n \u003cp\u003e+++\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 16.5785%;\"\u003e\n \u003cp\u003eCD105\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 70.0176%;\"\u003e\n \u003cp\u003eDental pulp core cells\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.4039%;\"\u003e\n \u003cp\u003e++\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 16.5785%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eOsteocalcin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 70.0176%;\"\u003e\n \u003cp\u003eDental pulp core cells\u003c/p\u003e\n \u003cp\u003eOdontoblasts\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.4039%;\"\u003e\n \u003cp\u003e+++\u003c/p\u003e\n \u003cp\u003e+++\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2: \u0026nbsp;\u0026nbsp;\u003c/strong\u003eThe distribution and intensity of expression of MCAM, CD73, STRO1, Thy1, CD105, and osteocalcin in the adult human dental pulp, the intensity of expression is defined in all locations where the antibodies were detected as follows: +++intense, ++ strong, + moderate, \u0026sect; weak, - negative.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eDPSCs reside in a\u0026nbsp;quiescent state within their niche; they migrate and proliferate in response to certain stimuli to regenerate damaged tissues. Exploring their niche may provide valuable information helping scale up proper culturing and enrichment of DPSCs [24].\u003c/p\u003e\n\u003cp\u003eIn 2006, the International Society for Cellular Therapy (ISCT) proposed CD73, CD90, and CD105 as MSCs markers [19,20]. STRO1 CD90, CD73, and MCAM are reported repeatedly to be expressed in DPSCs [18,21,22]. Using immunohistochemistry, we have investigated the localization of these mesenchymal stem cell markers in the pulp tissue of third molars of young adult human males and females.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOur results identified multiple DPSCs niches located in the dental pulp cell-rich zone, walls of blood vessels, the odontoblastic layer, nerve bundles, accessory canals, subodontoblastic zone, and the radicular pulp. Markers' expression and intensity showed a gradual decrease from the apex toward the coronal part of the pulp, which coincides with a previous study that investigated the proliferation capacity of dental pulp cells\u0026nbsp;[25]. Earlier studies reported similar DPSCs’ niche locations to the ones reported here\u0026nbsp;[25,26]. However, these studies experimented with a combination of stem cell markers different from those described here or with deciduous teeth\u0026nbsp;[26].\u003c/p\u003e\n\u003cp\u003eMCAM or CD146 is a cell adhesion molecule and integral membrane glycoprotein that exhibits many functions in development, signaling transduction, cell migration, mesenchymal stem cell differentiation, angiogenesis, and immune response [27–29]. MCAM is considered a stem cell and endothelial marker co-expressed with STRO1 on the walls of blood vessels\u0026nbsp;and is reported to reside in the perivascular niche of dental pulp [18]. CD146+ DPSCs showed higher mineralization and adipogenicity than CD146- cells [30].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMCAM expression was strong in the radicular odontoblasts (Figure 1, A) and most dental pulp cells' nuclei, cell membranes, and cytoplasm (Figure 1). Some positive pulp cells showed large nuclei and a kind of clustering (Figure 1, C). Further, MCAM was expressed moderately in the walls of blood vessels (Figure 1, C) and intensively in cell sheets in the coronal pulp cell-rich zone (Figure 1 and Table 2). This observation contradicts a previous report by Shi and\u0026nbsp;Gronthos 2003\u0026nbsp;who reported that MCAM was only expressed in the walls of blood vessels of the dental pulp but coincided with Alongi et al. 2010 and Martens et al. 2012 who mentioned\u0026nbsp;MCAM was expressed in multiple locations in the dental pulp without specification\u0026nbsp;[18,24,31]. This contradiction in reports could be attributed to differences in the donor’s age in each study, as age is previously suggested to play a role in DPSCs’ nature \u003cem\u003ein vitro\u0026nbsp;\u003c/em\u003e[23].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eShi and\u0026nbsp;Gronthos 2003\u0026nbsp;presumed that the co-localization of STRO1 and CD146 on the walls of large blood vessels in dental pulp tissue implied that most DPSCs arise from microvasculature\u0026nbsp;[18]. We\u0026nbsp;respectfully disagree as MCAM’s\u0026nbsp;strong expression was observed in multiple niches other than the perivascular niche. Moreover, they suggested that during dentin regeneration, pre-odontoblasts may originate from perivascular cells migrating out of the capillary walls into the surrounding pulp tissue [18]. Here, we demonstrated that both odontoblasts and capillary walls expressed MCAM, which could support this suggestion, but further investigation is recommended.\u0026nbsp;MCAM showed nuclear expression in some cells despite it being a membrane glycoprotein. The phenomena could be explained by the accumulated evidence that considers the shuttling of adhesion transmembrane proteins between the plasma membrane and nucleus to be common, where adhesion transmembrane proteins interact with transcription factors to perform certain functions [32].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCD73 is a cell surface protein that hydrolyzes extracellular nucleotides into permeable membrane nucleosides and is expressed in MSCs [33–35]. CD73 expression was observed in multinucleated lymphocytes or as large and small clusters\u0026nbsp;rarely seen\u0026nbsp;scattered in the dental pulp (Figure 2 and Table 2). STRO1 is a cell surface antigen, like\u0026nbsp;MCAM, expressed by MSCs in the bone marrow and dental pulp blood vessels. It is considered a marker of pre-osteogenic populations and an early marker of different MSCs populations in the perivascular niche\u0026nbsp;[17,18].\u0026nbsp;Here, STRO1 expression was restricted to blood vessel walls and the nerve bundles (Figure 3 and Table 2), which coincides with the previous reports[18,26].\u0026nbsp;CD105 showed strong nuclear staining in most dental pulp cell-rich zone (Figure 4 and Table 2), as reported previously [24,31].\u003c/p\u003e\n\u003cp\u003eThy1,\u0026nbsp;also known as CD90,\u0026nbsp;is expressed on fibroblasts, neurons, endothelial cells, and thymocytes. It has been identified as an MSCs marker and has a crucial role in MSCs fate decisions\u0026nbsp;[36].\u0026nbsp;Thy1 showed an intensive expression in the nuclei and cytoplasm of the blood vessel walls (arterioles and venules), nerve bundles, and cells of the pulp core. Moreover, accessory canals were intensively positive, and cells with strong positive nucleareThy1 expression seemed to be entering the dental pulp from the accessory canals (Figure 4 and Table 2).\u0026nbsp;Osteocalcin is the main non-collagenous hydroxyapatite-binding protein synthesized by osteoblasts, odontoblasts, and hypertrophic chondrocytes.\u0026nbsp;It regulates mineralization and is considered a marker of bone cell metabolism [37]. Osteocalcin\u0026nbsp;expression was intense and uniform in the nuclei and cytoplasm of dental pulp cells throughout the dental pulp and odontoblasts. The radicular pulp has the most intense expression of osteocalcin (Figure 5 and Table 2). This antibody was investigated to illuminate the differentiation status of the dental pulp, which is highly differentiated, as osteocalcin was strongly expressed throughout the dental pulp. To our knowledge, no previous reports explored the expression of CD73, Thy1, or osteocalcin in dental pulp tissue by immunohistochemistry.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eInformation concerning DPSCs proliferation and differentiation is essential to understanding tooth response to regenerative stimuli. Since there is little data concerning DPSCs in vivo, we have investigated the localization of DPSCs populations of third molars of young adult humans using immunohistochemistry. The results demonstrated the presence of multiple DPSCs niches, which are observed in the dental pulp cell-rich zone, walls of blood vessels, the odontoblastic layer, nerve bundles, accessory canals, subodontoblastic zone, and the radicular pulp. The observations that multiple DPSCs niches exist may have further implications for their recruitment in situ for multiple regenerative therapies. However, these stem cells\u0026apos; exact location, developmental potential, and ontogeny are still largely unknown. In the present study, identifying mesenchymal DPSCs niches may help elucidate the fundamental conditions necessary to selectively maintain and expand DPSCs \u003cem\u003ein\u003c/em\u003e\u003cem\u003evitro\u003c/em\u003e and direct their potentials \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eDPSCs: Dental Pulp Stem Cells\u003c/p\u003e\n\u003cp\u003eMSCs: Mesenchymal Stem Cells\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Institutional Review Board and Ethics Committee at the University of Jordan approved the study ‘localization of the different populations of stem cells existing in the dental pulp of adult human teeth’ in accordance with The Declaration of Helsinki, and the decision was given the number 2017/158 in 2017. Informed consent was obtained to acquire the teeth for analysis, and data privacy was preserved.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data used to publish these findings are available upon request from Dr. Heba Alzer.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDisclaimer Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no conflict of interest to announce\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding Statement\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was funded bythe Deanship of Scientific Research at the University of Jordan [47/2015/2016]. The funding body played no role in the design of the study and collection, analysis, and interpretation of data nor in writing the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thank the Deanship of Scientific Research at the University of Jordan for funding this project and the Maxillofacial Surgery Department at the University of Jordan Hospital for their cooperation in sample collection.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHA: contributed to conception, design, data acquisition and interpretation, drafted and revised the manuscript.\u003c/p\u003e\n\u003cp\u003eNAS: contributed to conception and design and critically revised the manuscript.\u003c/p\u003e\n\u003cp\u003eMA: contributed to sample collection, patient selection, acquiring consent, surgical extraction of the samples, and revised the manuscript.\u003c/p\u003e\n\u003cp\u003eHK: contributed to the conception and design and revised the manuscript.\u003c/p\u003e\n\u003cp\u003eFA: contributed to conception, design, data acquisition and interpretation, and critically revised the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for Publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors gave their final approval and agreed to be accountable for all aspects of the work.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eChai Y, Jiang X, Ito Y, \u003cem\u003eet al.\u003c/em\u003e Fate of the mammalian cranial neural crest during tooth and mandibular morphogenesis. \u003cem\u003eDevelopment\u003c/em\u003e [Internet]. 127(8), 1671\u0026ndash;1679 (2000). 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Available from: https://doi.org/10.1007/s12020-014-0401-0.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"MCAM, CD73, STRO1, Thy1, CD105, Osteocalcin, human dental pulp, stem cells, mesenchymal stem cells marker","lastPublishedDoi":"10.21203/rs.3.rs-3067290/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3067290/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e: Despite comprehensive studies and reports about the properties of dental pulp stem cells (DPSCs)\u0026nbsp;in vitro, we still lack the knowledge of whether these\u0026nbsp;in vitro\u0026nbsp;characteristics coincide with the original nature of DPSCs in situ. The histological location of DPSCs populations in the dental pulp has been rarely investigated.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e: In this study, well-recognized mesenchymal stem cell markers\u0026nbsp;MCAM, CD73, STRO1, Thy1, and CD105\u0026nbsp;are being investigated by immunohistochemistry to localize the precise location of these populations of DPSCs within the human adult dental pulp.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e. All previously mentioned markers were expressed in the dental pulp, and their intensity and location of expression were reported.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e: The observations that multiple mesenchymal stem cell niches exist may have further implications for their recruitment in situ for multiple regenerative therapies.\u003c/p\u003e","manuscriptTitle":"Localization of mesenchymal stem cells populations in the adult human dental pulp","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-06 09:53:17","doi":"10.21203/rs.3.rs-3067290/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"2ef4a18d-ace2-4244-928d-e6a05246cdd1","owner":[],"postedDate":"November 6th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":39830155,"name":"Biological sciences/Developmental biology"},{"id":39830156,"name":"Biological sciences/Stem cells"}],"tags":[],"updatedAt":"2024-12-19T13:54:04+00:00","versionOfRecord":[],"versionCreatedAt":"2024-11-06 09:53:17","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3067290","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3067290","identity":"rs-3067290","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}