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Comparison of the differentiation potential of neural crest derived progenitor cells from apical papilla (dNC-PCs) and stem cells from exfoliated deciduous teeth (SHED) into mineralising cells
archives of oral biology 58 (2013) 699–706
Available online at www.sciencedirect.com
journal homepage: http://www.elsevier.com/locate/aob
Comparison of the differentiation potential of neural crest derived progenitor cells from apical papilla (dNC-PCs) and stem cells from exfoliated deciduous teeth (SHED) into mineralising cells Martin Gosau a,c,*, Werner Go¨tz b,c, Oliver Felthaus a,c, Tobias Ettl a, Andreas Ja¨ger b, Christian Morsczeck a a b
Department of Cranio- and Maxillofacial Surgery, University Hospital Regensburg, Germany Department of Orthodontics, Oral Biology Laboratory, Dental Clinic, University of Bonn, Germany
article info
abstract
Article history:
Objective: Recently, cells from the apical papilla of retained human third molars (dental
Accepted 7 November 2012
neural crest-derived progenitor cells, dNC-PCs) have been isolated and characterised as multipotent progenitor cells. Nonetheless, molecular processes during differentiation into
Keywords:
mineralising cells are still unknown. This study evaluated the osteogenic/odontogenic
Dental stem cells
differentiation of dNC-PCs under in vitro conditions and compared these cells with already
Odontogenic differentiation
known odontoblast precursor cells (dental stem cells from exfoliated human deciduous
Osteogenic differentiation
teeth, SHED).
Dental neural crest-derived
Methods: The differentiation of dNC-PCs and SHED under in vitro conditions was verified by
progenitor cells
Alizarin red staining (mineralisation), alkaline phosphatase activity and the expression of
SHED
osteogenic/odontogenic markers (RT-PCRs). The genome wide expression-profiles were
Microarray analysis
investigated with Affymetrix DNA-microarrays and the cell migration with a gel spot cell migration assay. Results: In our study dNC-PCs differentiated like SHED in mineralising cells. The expression of odontoblast markers suggested that dNC-PCs and SHED differentiated into different types of odontoblasts. This supposition was supported by genome wide gene expression profiles of dNC-PCs and SHED after cell differentiation. Typical biological processes of undifferentiated cells, for example ‘‘mitosis’’, were regulated in dNC-PCs. In SHED biological processes like ‘‘response to wounding’’ or ‘‘cell migration’’ were regulated, which are associated with replacement odontoblasts and their precursors. Moreover, a gel-spot assay revealed that SHED migrated faster than dNC-PCs. Conclusion: Our results suggest that dNC-PCs are precursors for primary odontoblasts, whereas SHED differentiate into replacement odontoblasts. These different odontogenic differentiation potentials of dNC-PCs and SHED have to be considered for cellular therapies and tissue engineering approaches in the future. # 2012 Elsevier Ltd. All rights reserved.
* Corresponding author at: Department of Cranio-Maxillofacial Surgery, University Hospital Regensburg, Franz-Josef-Strauss-Allee 11, D93053 Regensburg, Germany. Tel.: +49 944 941 6345; fax: +49 944 941 6342. E-mail addresses: [email protected], [email protected], [email protected] (M. Gosau). c These three authors contributed equally to this work. 0003–9969/$ – see front matter # 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.archoralbio.2012.11.004
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1.
archives of oral biology 58 (2013) 699–706
Introduction
Somatic stem cells (SC) or progenitor cells (PC) have been identified in nearly all tissues of the stomatognathic system.1,2 Stem cells in the dental pulp (DPSCs) are neural crest (NC)-derived and they have common characteristics with bone marrow derived mesenchymal SCs.3,4 These cells are multipotent, and they differentiate for example into odontoblasts. This characteristic makes DPSCs a valuable tool for regenerative dentistry. They are also of increasing interest for dental tissue engineering approaches.5,6 A subpopulation of human DPSCs was isolated by Miura et al. from exfoliated deciduous teeth (SHED).4 SHED have a higher proliferation rate than bone marrow derived mesenchymal SCs (BMSCs).4 SHED differentiate into odontoblasts under in vitro conditions and they form a typical dental pulp/dentine complex in the tooth slice/scaffold model.7,8 Recently, dental neural crest derived progenitor cells (dNCPCs) have been isolated from a region located apically from the developing root of impacted third molars, which is also known as the ‘‘apical papilla’’ or the ‘‘apical pad’’.9 These cells express neuroectodermal progenitor cell markers, but they do not express the mesenchymal stem cell marker STRO-1.10 This distinguishes them from another closely related type of stem cell from the apical papilla (SCAP), which are positive for STRO-1.11,12 SCAP and dNC-PCs are differ in their isolation method. Whereas SCAP are isolated as single cell suspension dNC-PCs are isolated as microexplants. Although dNC-PCs are multipotent,9 their mineralising potential has to be confirmed, especially regarding the expression of odontoblastic markers. In this study, we compared dNC-PCs with SHED after the differentiation into mineralising cells under in vitro conditions. Amongst other things we investigated their genome wide gene expression profiles using microarrays. Only a few studies have hitherto investigated gene expression profiles of dental stem cells after differentiation.13 In this study we focus on the differentiation of dNC-PCs and SHED into mineralising cells and analysed genome-wide gene expression profiles to reveal molecular mechanisms in these dental NC derived cells after differentiation.
2.
Materials and methods
2.1.
Cell culture
dNC-PCs were isolated from retained third molars of juvenile patients after informed consent. The isolation of dNC-PCs was previously described by Dr. M. Thie and colleagues.9 Briefly, the pad-like tissue from the apex that is located under the pulpal tissue was minced into small pieces and digested at 37 8C using a collagenase/dispase solution (Sigma, Munich, Germany) for 1 h. Subsequently, digested tissue was centrifuged at 350 g for 5 min and the pellet was resuspended in culture medium. Microexplants were further cultured until outgrowing cells reached subconfluency. SHED isolated from deciduous teeth of 7- to 8-year-old children were kindly provided by Prof. S. Shi (University of Southern California, Los Angeles, USA). All cells were cultivated in standard medium
comprising of DMEM supplemented with 10% foetal calf serum and a Pen/Strep solution (PAA, Pasching, Austria) at 37 8C in 5% CO2. Dental cell cultures were not pooled from individual patients.
2.2.
Flow cytometry analysis
SHED and dNC-PCs at passage 6 were analysed for stem cell associated markers by flow cytometry. The following antibodies were used: CD105-APC (Miltenyi Biotec; Bergisch Gladbach; Germany), CD44-FITC (Miltenyi Biotec), CD146-FITC (Miltenyi Biotec), CD133/2(293C3)–APC (Miltenyi Biotec), Antihuman nestin-PE Monoclonal Antibody (R&D Systems, Inc., Minneapolis, USA), Anti-human STRO-1 AlexaFluor1 647 (BioLegend, San Diego, CA, USA). Single cell suspensions of dental cells were incubated with monoclonal antibodies for 45 min at 4 8C, washed once in PBS with 2 nM EDTA and 0.5% BSA. For intracellular staining, cells were permeabilised with 0.2% saponin and 0.1% BSA for 15 min and washed once in PBS with 2 nM EDTA and 0.1% BSA before staining. The following antibodies were used: mouse IgG2b-APC isotype control antibody (Miltenyi Biotec), mouse IgG-FITC (Miltenyi Biotec), mouse IgG1 isotype Control-PE (R&D Systems, Inc.), Mouse IgM, k isotype Control, Alexa Fluor1 647 (BioLegend). Flow cytometry analyses were done using the FACS Canto II (Becton Dickinson, Heidelberg, Germany).
2.3.
Differentiation protocol
For differentiation, cells at passage 6 were cultivated in standard medium until sub-confluency. Subsequently, the medium was changed and cells were cultivated in an osteogenic differentiation medium (StemPro Osteogenesis Differentiation Kit, Invitrogen, Karlsruhe, Germany: ODM) for 7 days, 21 days, and 28 days, respectively. For control, cells were cultivated in standard DMEM medium. For quantitative evaluation of alkaline phosphatase (ALP) activity after 7 days of differentiation the JBS Phosphatase Assay kit (Jena Biosciences, Jena, Germany) was used. The ALP activity of the sample was normalised to the DNA content measured with the Quant-iTTM PicoGreen1 dsDNA Assay Kit (Invitrogen). Samples were measured in quintuplicates. After 28 days of cell differentiation, staining with alizarin red was performed for the evaluation of biomineralisation. Cell cultures were evaluated with phase contrast microscopy (Leica, Wetzlar, Germany). For quantitative measurement, alizarin was dissolved in 10% cetylpyridinium chloride monohydrate solution for 30 min. 50 ml samples were measured in a TECAN infinite F200 plate reader (TECAN, Crailsheim, Germany) at 540 nm. Samples were measured in quadruplicates.
2.4. PCR
Reverse transcription polymerase chain reaction (RT)-
For RNA isolation, cells from day 0, day 7, and day 21 of osteogenic differentiation were processed according to the manual of the RNA isolation kit RNeasy Mini Kit (Qiagen, Hilden, Germany). The QuantiTect Reverse Transcription Kit (Qiagen) was utilised for a reverse transcription of 500 ng total
archives of oral biology 58 (2013) 699–706
[(Fig._1)TD$IG]
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Fig. 1 – Flow cytometry analysis: characteristics of cultured dNC-PCs and SHED at passage 6. Typical flow cytometric analysis diagrams on the expression of antigens CD44, CD105, nestin, STRO-1 and CD 133. The relative levels of cell surface expression are shown by the shift of the grey histograms to the right compared to the fluorescence of the isotype-matched antibodies used as controls (light grey histograms). The percentage of positive cells is indicated. Histogram: peak of each marker expression. Please note positive expression of CD44, CD 105 and nestin in both cell types, no expression of STRO-1 and CD133.
RNA into cDNA. The HotStarTaq Kit (Qiagen) was used for PCRs. Primer sequences are as follows: GAPDH forward 50 CGTCTTCACCACCATGGAGA-30 ; GAPDH reverse 50 -CGGCCATCACGCCACAGTTT-30 ; DSPP forward 50 -AAAAGTCCAGGACAGTGGGC-30 ; DSPP reverse 50 -GCTTTGAGGAACTGGAATG GC-30 ; DMP1 forward 50 -AACACCACCCTTGGAGAGCAGTGA30 ; DMP1 reverse 50 -AGGCCCTCCTCTGACTGAGTGC-30 ; BSP forward 50 -GCATTTTGGGAATGGCCTGTGCT-30 ; BSP reverse 50 -CGTGGCCTGTACTTAAAGACCCCA-30 ; OCN forward 50 -TGAGAGCCCTCACACTCCTC-30 ; OCN reverse 50 -ACCTTTGCTGGAC TCTGCAC-30 . GAPDH was used as housekeeping gene. PCR products were separated with agarose gel electrophoresis. The methods for the real-time RT-PCR and Western blot analysis are described in Supplemental Materials.
2.5.
Microarray analysis
Total RNAs were quality-controlled using the RNA 6000 Nano LabChip (Agilent Technologies, Santa Clara, CA, USA). DNA microarray analyses were carried out with Affymetrix Human Gene 1.1 ST array according to the Affymetrix standard protocol. RNAs from dNC-PCs and SHED before differentiation (day 0) and after 7 days of cultivation in osteogenic differentiation medium were used for comparison of differential gene expression. Microarray hybridisations were carried out at the ‘‘Centre of Excellence for Fluorescent Bioanalytics’’ at the University of Regensburg (Germany). Data were analysed with the NetAffx Analysis Centre and the RMA algorithm.14 A change of more than twofold with a p-value of maximal 0.05 was considered as significant. The database for Annotation, Visualization, and Integrated Discovery (DAVID; http:// niaid.abcc.ncifcrf.gov/) was used for annotations of significant regulated transcripts after differentiation.15 Samples were measured in duplicates.
The Affymetrix Expression Console Software Version 1.0 was used to create summarised expression values (CHP-files) from Human Gene 1.1 ST array intensities (CEL-files). Microarray raw data are published online at the NCBI in Gene Expression Omnibus GSE37189.
2.6.
Cell migration assay
For RadiusTM 96-well cell migration assay (Cell Biolabs, San Diego, CA, USA), SHED and dNC-PCs were seeded in 96-well plates that are precoated with a ca. 0.68 mm diameter gel spot in the centre of each well on which the cells are unable to attach. After cells reached confluence, the gel spot was removed, allowing the cells to migrate into the cell free centre of the well. The remaining cell free area was measured after 8 h, 1 day, and 2 days, respectively. Experiments were done in triplicates. All statistics in this manuscript were done using student’s t-test (*p-value < 0.05; **p-value < 0.01; ***p-value < 0.001)
3.
Results
Flow cytometry analyses revealed that dNC-PCS and SHED at passage 6 express dental stem cell markers CD44, CD 105, and nestin, but they were negative for STRO-1 and CD 133 (Fig. 1). SHED and dNC-PCs showed similar specific ALP activities after 7 days. In both dental cell types the ALP-activity was increased after cultivation in ODM compared with cells which were cultivated in DMEM (Fig. 2A). The alizarin red staining showed a strong biomineralisation in cell cultures after 28 days of differentiation (Fig. 2B and C). The RT-PCR analysis of RNA extracted from SHED and dNC-PCs revealed that OCN expression was very weak and that DSPP, DMP1, and BSP were not expressed in undifferentiated cells. After 7 days of
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archives of oral biology 58 (2013) 699–706
[(Fig._2)TD$IG]
Fig. 2 – (A) ALP activity of SHED and dNC-PCs was similar after 7 days of differentiation. (B) Alizarin red staining was measured quantitatively after washing off with 10% cetylpyridinium chloride monohydrate solution. Abbreviations: ODM: osteogenic differentiation medium; DMEM: control medium without osteogenic inducer. (C) Microscopic pictures of undifferentiated and differentiated SHED and dNC-PCs. Magnification on the left is 25T, magnification on the right is 100T (bars equal 100 mm). (D) RT-PCR analysis: comparison of odontoblast marker genes DSPP, DMP1, BSP, and OCN in SHED and dNC-PCs (passage 6) at days 0, 7, and 21 of differentiation. The gene expression of GAPDH was used as a house-keeping gene.
differentiation, a considerable expression of OCN was observed in both cell types and DMP1 was expressed in dNC-PCs but not in SHED. At day 21 of differentiation DSPP was expressed in both cell types. While the expression of DSPP and OCN was similar in SHED and dNC-PCs, the odontoblast marker DMP1 was highly expressed in dNC-PCs (Fig. 2D). Interestingly, BSP was expressed in differentiated SHED, only. Western blot showed that the expression of osteopontin was considerably stronger after differentiation in both SHED and dNC-PCs (Fig. S1). Genome-wide gene expression profiles of SHED and dNCPCs were investigated after 7 days of differentiation. 960 genes were regulated in dNC-PCs and 374 genes in SHED. 204 of these genes were regulated in both SHED and dNC-PCs (Fig. 3), but only two genes (VLDLR, SLC1A3) were regulated contrarily. The reliability of the microarray results was confirmed by a realtime PCR with three selected genes (Fig. S2). The regulated genes are overrepresented in certain biological processes in dNC-PCs and SHED (Fig. 3). A gene ontology analysis using the Database for Annotation, Visualization, and Integrated Discovery (DAVID) revealed that in dNC-PCs biological processes such as ‘‘cell cycle’’, ‘‘regulation of mitotic cell cycle’’ or ‘‘cell
division’’ are regulated. In SHED biological processes that are involved in the regeneration of damaged tissues are regulated, which also is a feature of replacement odontoblasts or their progenitors. These processes comprise ‘‘response to wounding’’, ‘‘cell differentiation’’, ‘‘regulation of acute inflammatory response’’, and ‘‘cell migration’’. Moreover, SHED migrated faster than dNC-PCs in a gel spot assay (Fig. 4). Differentially expressed genes were also analysed with DAVID functional annotation clustering (DFAC) (Fig. 5A and B). The DFAC analysis tool annotated functional groups of differentially expressed genes, which represent biological processes such as skeletal system or bone development, ossification (SHED, dNC-PCs), and osteoblast differentiation (dNC-PCs). 25 genes of dNC-PCs (2.6%), and 18 genes of SHED (4.9%) could be clustered into these functional groups (Fig. 5A). Nine genes, which were significantly regulated in dNC-PCs and SHED, are related to biological processes in skeletal development (ALP, PTGS2, Col3A1, FOXC2, STC1, POSTN, NZBTB16, FRZB, IGFBP3; Fig. 5B). Whereas in both dental cell types PTGS2, POSTN, and IGFBP3 were down regulated ALP, Col3A1, FOXC2, STC1, ZBTB16 and FRZB were up regulated (Fig. 5B).
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[(Fig._3)TD$IG]
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Fig. 3 – (A and B) Gene expression profiles after 7 days; (A) Venn diagram of significant regulated genes. (B) Gene Ontology: overrepresented biological functions after 7 days of differentiation (DAVID).
4.
Discussion
The aim of this study was to show the odontogenic differentiation potential of dNC-PCs and to compare the gene expression profiles of dNC-PCs and SHED after differentiation.
[(Fig._4)TD$IG]
cell free area (%)
Migration
*** **
100 80
0h 8h 1day 2days
60 40 20 0 SHED
dNC-PC
Fig. 4 – Cell migration assay with SHED and dNC-PCs. Cells were cultivated under standard cell culture conditions and remaining cell free areas were determined at indicated time points.
The flow cytometry data of our study confirmed that both cell types are ectomesenchymal cells.10 While it is already known that SHED are odontoblast progenitors,4,8 the odontogenic differentiation potential of dNC-PCs was unknown before our study. As undifferentiated cells from the ‘‘apical pad’’, dNCPCs remain in an undifferentiated state until tooth development is accomplished.9,10 However, Thie and colleagues have already shown that dNC-PCs differentiate into mineralising cells under in vitro and in vivo conditions.9 In our study the differentiation potential of SHED and dNC-PCs into mineralising cells was similar. We determined the ALP activity at day 7 and the mineralisation (alizarin red staining) at day 28 of cell differentiation. Although the increase in ALP activity and the strong mineralisation of dNC-PCs and SHED after differentiation were nearly identical, the expression of odontoblastic markers revealed the differentiation of dNCPCs and SHED into two different types of cells of the odontoblast lineage. It is the first time that the expression of odontogenic markers was evaluated for dNC-PCs. The absence of DMP1 and the high expression for BSP in differentiated SHED indicated a differentiation into odontoblast-like cells or ‘‘replacement odontoblasts’’ of reparative dentine.16 In contrast, the expression of DMP1 and DSPP and the absence of BSP in differentiated dNC-PCs indicate a
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Fig. 5 – (A) Gene clusters with genes that are associated with osteogenesis and up-regulated in SHED and dNC-PCs. (B) Regulation of osteogenic genes in SHED and dNC-PCs after 7 days of differentiation (microarray analyses).
differentiation into primary odontoblasts.16 DMP-1 is an important inducer for the differentiation of dental pulp cells into odontoblasts, and it regulates dentine mineralisation.17 Moreover it can act as a morphogen on undifferentiated mesenchymal cells present in the dental pulp/dentinecomplex.18 Interestingly, Sonoyama et al. already suggested that cells of the apical papilla (apical pad) contains progenitors for primary odontoblasts.11 In the microarray study approximately three times more genes were regulated in dNC-PCs than in SHED after cell differentiation. This result may suggest that dNC-PCs have a more undifferentiated developmental stage than SHED. Gene ontology analyses support this assumption, because regulated biological processes in dNC-PCs are associated with proliferation, chromosome segregation, or cell division, which are typical characteristics of highly undifferentiated cells such as tooth germ cells.11 In contrast, biological processes, which are related to tissue regeneration, like ‘‘cell differentiation’’, ‘‘response to wounding’’ or ‘‘cell migration’’ were overrepresented in SHED. These processes characterise dNC-PCs and SHED as progenitors for primary odontoblasts and replacement odontoblasts, respectively. Similar results were identified in a recent microarray study with SHED and DPSCs of Nakamura et al.19 Here, a higher expression in SHED was observed for genes that participate in pathways related to cell
proliferation and extracellular matrix, including several cytokines such as fibroblast growth factor and tumour growth factor beta. Although little is known about molecular processes in dental stem cells, microarray analyses disclose regulated genes and correlated biological processes in these cells. Many genes were regulated in both dental cell types in our microarray analyses. However, only a small number of these regulated genes are associated with osteogenesis, odontogenesis or related processes of tooth development. This result is very similar to that of previous microarray studies with dental stem cells.13 However, the focus on these genes, which are regulated in dNC-PCs and SHED may shed new light on molecular processes in dental stem cells during odontogenic differentiation. Among well-known osteogenesis markers such as ALP and Col3A1 other interesting genes like FOXC2, stanniocalcin 1 (STC1), ZBTB16, and frizzledrelated protein (FRZB) were up-regulated after differentiation. FOXC2 is involved in a number of different developmental processes. It is important for the regulation of epithelial-mesenchymal transitions and promotes osteogenesis via the induction of integrin b1.20 STC1 is a 27.6 kDa homologue of the fish hormone stanniocalcin and has been linked to bone physiology. It regulates mineral homeostasis and has a strong influence on the transport of phosphates in
archives of oral biology 58 (2013) 699–706
a variety of mammalian cells.21 Moreover, it is also involved in the cranial intramembranous ossification.22 The induction of STC1 has been already shown in dental follicle cells after overexpression of the transcription factor DLX3, which induces the osteogenic differentiation in dental follicle cells.23 ZBTB16 promotes osteogenic differentiation of human MSCs and it is an upstream regulator of RUNX2 and regulates limb and axial skeletal patterning.24 This transcription factor is also induced in human dental follicle cells after cell differentiation.25 Frizzled-related proteins are involved in the regulation of Wnt and BMP signalling.26 The up-regulation of FRZB in dNC-PCs and SHED suggests a role of Wnt-signalling during the differentiation into mineralising cells. However, a previous study has shown that Wnt signalling inhibits the cementoblast differentiation and promotes proliferation of dental cells.27 Three osteogenesis related genes were down-regulated after cell differentiation in dNC-PCs and SHED. One gene encodes for the prostaglandin-endoperoxide synthase 2 (PTGS2), which is also known as COX-2. This protein is constitutively expressed in the skeleton and a key regulator of bone formation.28 Periostin is another down-regulated gene after the differentiation of dNC-PCs and SHED. Although periostin is expressed in cranial NC cells during tissue development29 its role during the differentiation of dental stem cells is still unknown. Finally. IGFBP-3 was down-regulated in dNC-PCs and SHED. This IGF binding protein is an important part of the IGF system and involved in many aspects of dental physiology, pathology and development.30 In conclusion, dNC-PCs and SHED seem to differ in their odontogenic differentiation potential. While differentiated dNC-PCs share more similarities with primary odontoblasts, differentiated SHED are more similar to replacement odontoblasts. These findings may be of interest for further studies about dNC-PCs or SHED in regenerative medicine and dental development.
Funding None.
Competing interests None declared.
Ethical approval Informed consent was obtained from all subjects.
Acknowledgements We thank Ms. Anja Reck for excellent technical support. We thank Prof. Songtao Shi (University of Southern California, Los Angeles, USA) for providing SHED. This study was supported by the German Society for Oral and Maxillofacial Medicine (DGZMK).
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Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.archoralbio.2012.11.004.
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Available online at www.sciencedirect.com
journal homepage: http://www.elsevier.com/locate/aob
Comparison of the differentiation potential of neural crest derived progenitor cells from apical papilla (dNC-PCs) and stem cells from exfoliated deciduous teeth (SHED) into mineralising cells Martin Gosau a,c,*, Werner Go¨tz b,c, Oliver Felthaus a,c, Tobias Ettl a, Andreas Ja¨ger b, Christian Morsczeck a a b
Department of Cranio- and Maxillofacial Surgery, University Hospital Regensburg, Germany Department of Orthodontics, Oral Biology Laboratory, Dental Clinic, University of Bonn, Germany
article info
abstract
Article history:
Objective: Recently, cells from the apical papilla of retained human third molars (dental
Accepted 7 November 2012
neural crest-derived progenitor cells, dNC-PCs) have been isolated and characterised as multipotent progenitor cells. Nonetheless, molecular processes during differentiation into
Keywords:
mineralising cells are still unknown. This study evaluated the osteogenic/odontogenic
Dental stem cells
differentiation of dNC-PCs under in vitro conditions and compared these cells with already
Odontogenic differentiation
known odontoblast precursor cells (dental stem cells from exfoliated human deciduous
Osteogenic differentiation
teeth, SHED).
Dental neural crest-derived
Methods: The differentiation of dNC-PCs and SHED under in vitro conditions was verified by
progenitor cells
Alizarin red staining (mineralisation), alkaline phosphatase activity and the expression of
SHED
osteogenic/odontogenic markers (RT-PCRs). The genome wide expression-profiles were
Microarray analysis
investigated with Affymetrix DNA-microarrays and the cell migration with a gel spot cell migration assay. Results: In our study dNC-PCs differentiated like SHED in mineralising cells. The expression of odontoblast markers suggested that dNC-PCs and SHED differentiated into different types of odontoblasts. This supposition was supported by genome wide gene expression profiles of dNC-PCs and SHED after cell differentiation. Typical biological processes of undifferentiated cells, for example ‘‘mitosis’’, were regulated in dNC-PCs. In SHED biological processes like ‘‘response to wounding’’ or ‘‘cell migration’’ were regulated, which are associated with replacement odontoblasts and their precursors. Moreover, a gel-spot assay revealed that SHED migrated faster than dNC-PCs. Conclusion: Our results suggest that dNC-PCs are precursors for primary odontoblasts, whereas SHED differentiate into replacement odontoblasts. These different odontogenic differentiation potentials of dNC-PCs and SHED have to be considered for cellular therapies and tissue engineering approaches in the future. # 2012 Elsevier Ltd. All rights reserved.
* Corresponding author at: Department of Cranio-Maxillofacial Surgery, University Hospital Regensburg, Franz-Josef-Strauss-Allee 11, D93053 Regensburg, Germany. Tel.: +49 944 941 6345; fax: +49 944 941 6342. E-mail addresses: [email protected], [email protected], [email protected] (M. Gosau). c These three authors contributed equally to this work. 0003–9969/$ – see front matter # 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.archoralbio.2012.11.004
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1.
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Introduction
Somatic stem cells (SC) or progenitor cells (PC) have been identified in nearly all tissues of the stomatognathic system.1,2 Stem cells in the dental pulp (DPSCs) are neural crest (NC)-derived and they have common characteristics with bone marrow derived mesenchymal SCs.3,4 These cells are multipotent, and they differentiate for example into odontoblasts. This characteristic makes DPSCs a valuable tool for regenerative dentistry. They are also of increasing interest for dental tissue engineering approaches.5,6 A subpopulation of human DPSCs was isolated by Miura et al. from exfoliated deciduous teeth (SHED).4 SHED have a higher proliferation rate than bone marrow derived mesenchymal SCs (BMSCs).4 SHED differentiate into odontoblasts under in vitro conditions and they form a typical dental pulp/dentine complex in the tooth slice/scaffold model.7,8 Recently, dental neural crest derived progenitor cells (dNCPCs) have been isolated from a region located apically from the developing root of impacted third molars, which is also known as the ‘‘apical papilla’’ or the ‘‘apical pad’’.9 These cells express neuroectodermal progenitor cell markers, but they do not express the mesenchymal stem cell marker STRO-1.10 This distinguishes them from another closely related type of stem cell from the apical papilla (SCAP), which are positive for STRO-1.11,12 SCAP and dNC-PCs are differ in their isolation method. Whereas SCAP are isolated as single cell suspension dNC-PCs are isolated as microexplants. Although dNC-PCs are multipotent,9 their mineralising potential has to be confirmed, especially regarding the expression of odontoblastic markers. In this study, we compared dNC-PCs with SHED after the differentiation into mineralising cells under in vitro conditions. Amongst other things we investigated their genome wide gene expression profiles using microarrays. Only a few studies have hitherto investigated gene expression profiles of dental stem cells after differentiation.13 In this study we focus on the differentiation of dNC-PCs and SHED into mineralising cells and analysed genome-wide gene expression profiles to reveal molecular mechanisms in these dental NC derived cells after differentiation.
2.
Materials and methods
2.1.
Cell culture
dNC-PCs were isolated from retained third molars of juvenile patients after informed consent. The isolation of dNC-PCs was previously described by Dr. M. Thie and colleagues.9 Briefly, the pad-like tissue from the apex that is located under the pulpal tissue was minced into small pieces and digested at 37 8C using a collagenase/dispase solution (Sigma, Munich, Germany) for 1 h. Subsequently, digested tissue was centrifuged at 350 g for 5 min and the pellet was resuspended in culture medium. Microexplants were further cultured until outgrowing cells reached subconfluency. SHED isolated from deciduous teeth of 7- to 8-year-old children were kindly provided by Prof. S. Shi (University of Southern California, Los Angeles, USA). All cells were cultivated in standard medium
comprising of DMEM supplemented with 10% foetal calf serum and a Pen/Strep solution (PAA, Pasching, Austria) at 37 8C in 5% CO2. Dental cell cultures were not pooled from individual patients.
2.2.
Flow cytometry analysis
SHED and dNC-PCs at passage 6 were analysed for stem cell associated markers by flow cytometry. The following antibodies were used: CD105-APC (Miltenyi Biotec; Bergisch Gladbach; Germany), CD44-FITC (Miltenyi Biotec), CD146-FITC (Miltenyi Biotec), CD133/2(293C3)–APC (Miltenyi Biotec), Antihuman nestin-PE Monoclonal Antibody (R&D Systems, Inc., Minneapolis, USA), Anti-human STRO-1 AlexaFluor1 647 (BioLegend, San Diego, CA, USA). Single cell suspensions of dental cells were incubated with monoclonal antibodies for 45 min at 4 8C, washed once in PBS with 2 nM EDTA and 0.5% BSA. For intracellular staining, cells were permeabilised with 0.2% saponin and 0.1% BSA for 15 min and washed once in PBS with 2 nM EDTA and 0.1% BSA before staining. The following antibodies were used: mouse IgG2b-APC isotype control antibody (Miltenyi Biotec), mouse IgG-FITC (Miltenyi Biotec), mouse IgG1 isotype Control-PE (R&D Systems, Inc.), Mouse IgM, k isotype Control, Alexa Fluor1 647 (BioLegend). Flow cytometry analyses were done using the FACS Canto II (Becton Dickinson, Heidelberg, Germany).
2.3.
Differentiation protocol
For differentiation, cells at passage 6 were cultivated in standard medium until sub-confluency. Subsequently, the medium was changed and cells were cultivated in an osteogenic differentiation medium (StemPro Osteogenesis Differentiation Kit, Invitrogen, Karlsruhe, Germany: ODM) for 7 days, 21 days, and 28 days, respectively. For control, cells were cultivated in standard DMEM medium. For quantitative evaluation of alkaline phosphatase (ALP) activity after 7 days of differentiation the JBS Phosphatase Assay kit (Jena Biosciences, Jena, Germany) was used. The ALP activity of the sample was normalised to the DNA content measured with the Quant-iTTM PicoGreen1 dsDNA Assay Kit (Invitrogen). Samples were measured in quintuplicates. After 28 days of cell differentiation, staining with alizarin red was performed for the evaluation of biomineralisation. Cell cultures were evaluated with phase contrast microscopy (Leica, Wetzlar, Germany). For quantitative measurement, alizarin was dissolved in 10% cetylpyridinium chloride monohydrate solution for 30 min. 50 ml samples were measured in a TECAN infinite F200 plate reader (TECAN, Crailsheim, Germany) at 540 nm. Samples were measured in quadruplicates.
2.4. PCR
Reverse transcription polymerase chain reaction (RT)-
For RNA isolation, cells from day 0, day 7, and day 21 of osteogenic differentiation were processed according to the manual of the RNA isolation kit RNeasy Mini Kit (Qiagen, Hilden, Germany). The QuantiTect Reverse Transcription Kit (Qiagen) was utilised for a reverse transcription of 500 ng total
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Fig. 1 – Flow cytometry analysis: characteristics of cultured dNC-PCs and SHED at passage 6. Typical flow cytometric analysis diagrams on the expression of antigens CD44, CD105, nestin, STRO-1 and CD 133. The relative levels of cell surface expression are shown by the shift of the grey histograms to the right compared to the fluorescence of the isotype-matched antibodies used as controls (light grey histograms). The percentage of positive cells is indicated. Histogram: peak of each marker expression. Please note positive expression of CD44, CD 105 and nestin in both cell types, no expression of STRO-1 and CD133.
RNA into cDNA. The HotStarTaq Kit (Qiagen) was used for PCRs. Primer sequences are as follows: GAPDH forward 50 CGTCTTCACCACCATGGAGA-30 ; GAPDH reverse 50 -CGGCCATCACGCCACAGTTT-30 ; DSPP forward 50 -AAAAGTCCAGGACAGTGGGC-30 ; DSPP reverse 50 -GCTTTGAGGAACTGGAATG GC-30 ; DMP1 forward 50 -AACACCACCCTTGGAGAGCAGTGA30 ; DMP1 reverse 50 -AGGCCCTCCTCTGACTGAGTGC-30 ; BSP forward 50 -GCATTTTGGGAATGGCCTGTGCT-30 ; BSP reverse 50 -CGTGGCCTGTACTTAAAGACCCCA-30 ; OCN forward 50 -TGAGAGCCCTCACACTCCTC-30 ; OCN reverse 50 -ACCTTTGCTGGAC TCTGCAC-30 . GAPDH was used as housekeeping gene. PCR products were separated with agarose gel electrophoresis. The methods for the real-time RT-PCR and Western blot analysis are described in Supplemental Materials.
2.5.
Microarray analysis
Total RNAs were quality-controlled using the RNA 6000 Nano LabChip (Agilent Technologies, Santa Clara, CA, USA). DNA microarray analyses were carried out with Affymetrix Human Gene 1.1 ST array according to the Affymetrix standard protocol. RNAs from dNC-PCs and SHED before differentiation (day 0) and after 7 days of cultivation in osteogenic differentiation medium were used for comparison of differential gene expression. Microarray hybridisations were carried out at the ‘‘Centre of Excellence for Fluorescent Bioanalytics’’ at the University of Regensburg (Germany). Data were analysed with the NetAffx Analysis Centre and the RMA algorithm.14 A change of more than twofold with a p-value of maximal 0.05 was considered as significant. The database for Annotation, Visualization, and Integrated Discovery (DAVID; http:// niaid.abcc.ncifcrf.gov/) was used for annotations of significant regulated transcripts after differentiation.15 Samples were measured in duplicates.
The Affymetrix Expression Console Software Version 1.0 was used to create summarised expression values (CHP-files) from Human Gene 1.1 ST array intensities (CEL-files). Microarray raw data are published online at the NCBI in Gene Expression Omnibus GSE37189.
2.6.
Cell migration assay
For RadiusTM 96-well cell migration assay (Cell Biolabs, San Diego, CA, USA), SHED and dNC-PCs were seeded in 96-well plates that are precoated with a ca. 0.68 mm diameter gel spot in the centre of each well on which the cells are unable to attach. After cells reached confluence, the gel spot was removed, allowing the cells to migrate into the cell free centre of the well. The remaining cell free area was measured after 8 h, 1 day, and 2 days, respectively. Experiments were done in triplicates. All statistics in this manuscript were done using student’s t-test (*p-value < 0.05; **p-value < 0.01; ***p-value < 0.001)
3.
Results
Flow cytometry analyses revealed that dNC-PCS and SHED at passage 6 express dental stem cell markers CD44, CD 105, and nestin, but they were negative for STRO-1 and CD 133 (Fig. 1). SHED and dNC-PCs showed similar specific ALP activities after 7 days. In both dental cell types the ALP-activity was increased after cultivation in ODM compared with cells which were cultivated in DMEM (Fig. 2A). The alizarin red staining showed a strong biomineralisation in cell cultures after 28 days of differentiation (Fig. 2B and C). The RT-PCR analysis of RNA extracted from SHED and dNC-PCs revealed that OCN expression was very weak and that DSPP, DMP1, and BSP were not expressed in undifferentiated cells. After 7 days of
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Fig. 2 – (A) ALP activity of SHED and dNC-PCs was similar after 7 days of differentiation. (B) Alizarin red staining was measured quantitatively after washing off with 10% cetylpyridinium chloride monohydrate solution. Abbreviations: ODM: osteogenic differentiation medium; DMEM: control medium without osteogenic inducer. (C) Microscopic pictures of undifferentiated and differentiated SHED and dNC-PCs. Magnification on the left is 25T, magnification on the right is 100T (bars equal 100 mm). (D) RT-PCR analysis: comparison of odontoblast marker genes DSPP, DMP1, BSP, and OCN in SHED and dNC-PCs (passage 6) at days 0, 7, and 21 of differentiation. The gene expression of GAPDH was used as a house-keeping gene.
differentiation, a considerable expression of OCN was observed in both cell types and DMP1 was expressed in dNC-PCs but not in SHED. At day 21 of differentiation DSPP was expressed in both cell types. While the expression of DSPP and OCN was similar in SHED and dNC-PCs, the odontoblast marker DMP1 was highly expressed in dNC-PCs (Fig. 2D). Interestingly, BSP was expressed in differentiated SHED, only. Western blot showed that the expression of osteopontin was considerably stronger after differentiation in both SHED and dNC-PCs (Fig. S1). Genome-wide gene expression profiles of SHED and dNCPCs were investigated after 7 days of differentiation. 960 genes were regulated in dNC-PCs and 374 genes in SHED. 204 of these genes were regulated in both SHED and dNC-PCs (Fig. 3), but only two genes (VLDLR, SLC1A3) were regulated contrarily. The reliability of the microarray results was confirmed by a realtime PCR with three selected genes (Fig. S2). The regulated genes are overrepresented in certain biological processes in dNC-PCs and SHED (Fig. 3). A gene ontology analysis using the Database for Annotation, Visualization, and Integrated Discovery (DAVID) revealed that in dNC-PCs biological processes such as ‘‘cell cycle’’, ‘‘regulation of mitotic cell cycle’’ or ‘‘cell
division’’ are regulated. In SHED biological processes that are involved in the regeneration of damaged tissues are regulated, which also is a feature of replacement odontoblasts or their progenitors. These processes comprise ‘‘response to wounding’’, ‘‘cell differentiation’’, ‘‘regulation of acute inflammatory response’’, and ‘‘cell migration’’. Moreover, SHED migrated faster than dNC-PCs in a gel spot assay (Fig. 4). Differentially expressed genes were also analysed with DAVID functional annotation clustering (DFAC) (Fig. 5A and B). The DFAC analysis tool annotated functional groups of differentially expressed genes, which represent biological processes such as skeletal system or bone development, ossification (SHED, dNC-PCs), and osteoblast differentiation (dNC-PCs). 25 genes of dNC-PCs (2.6%), and 18 genes of SHED (4.9%) could be clustered into these functional groups (Fig. 5A). Nine genes, which were significantly regulated in dNC-PCs and SHED, are related to biological processes in skeletal development (ALP, PTGS2, Col3A1, FOXC2, STC1, POSTN, NZBTB16, FRZB, IGFBP3; Fig. 5B). Whereas in both dental cell types PTGS2, POSTN, and IGFBP3 were down regulated ALP, Col3A1, FOXC2, STC1, ZBTB16 and FRZB were up regulated (Fig. 5B).
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Fig. 3 – (A and B) Gene expression profiles after 7 days; (A) Venn diagram of significant regulated genes. (B) Gene Ontology: overrepresented biological functions after 7 days of differentiation (DAVID).
4.
Discussion
The aim of this study was to show the odontogenic differentiation potential of dNC-PCs and to compare the gene expression profiles of dNC-PCs and SHED after differentiation.
[(Fig._4)TD$IG]
cell free area (%)
Migration
*** **
100 80
0h 8h 1day 2days
60 40 20 0 SHED
dNC-PC
Fig. 4 – Cell migration assay with SHED and dNC-PCs. Cells were cultivated under standard cell culture conditions and remaining cell free areas were determined at indicated time points.
The flow cytometry data of our study confirmed that both cell types are ectomesenchymal cells.10 While it is already known that SHED are odontoblast progenitors,4,8 the odontogenic differentiation potential of dNC-PCs was unknown before our study. As undifferentiated cells from the ‘‘apical pad’’, dNCPCs remain in an undifferentiated state until tooth development is accomplished.9,10 However, Thie and colleagues have already shown that dNC-PCs differentiate into mineralising cells under in vitro and in vivo conditions.9 In our study the differentiation potential of SHED and dNC-PCs into mineralising cells was similar. We determined the ALP activity at day 7 and the mineralisation (alizarin red staining) at day 28 of cell differentiation. Although the increase in ALP activity and the strong mineralisation of dNC-PCs and SHED after differentiation were nearly identical, the expression of odontoblastic markers revealed the differentiation of dNCPCs and SHED into two different types of cells of the odontoblast lineage. It is the first time that the expression of odontogenic markers was evaluated for dNC-PCs. The absence of DMP1 and the high expression for BSP in differentiated SHED indicated a differentiation into odontoblast-like cells or ‘‘replacement odontoblasts’’ of reparative dentine.16 In contrast, the expression of DMP1 and DSPP and the absence of BSP in differentiated dNC-PCs indicate a
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Fig. 5 – (A) Gene clusters with genes that are associated with osteogenesis and up-regulated in SHED and dNC-PCs. (B) Regulation of osteogenic genes in SHED and dNC-PCs after 7 days of differentiation (microarray analyses).
differentiation into primary odontoblasts.16 DMP-1 is an important inducer for the differentiation of dental pulp cells into odontoblasts, and it regulates dentine mineralisation.17 Moreover it can act as a morphogen on undifferentiated mesenchymal cells present in the dental pulp/dentinecomplex.18 Interestingly, Sonoyama et al. already suggested that cells of the apical papilla (apical pad) contains progenitors for primary odontoblasts.11 In the microarray study approximately three times more genes were regulated in dNC-PCs than in SHED after cell differentiation. This result may suggest that dNC-PCs have a more undifferentiated developmental stage than SHED. Gene ontology analyses support this assumption, because regulated biological processes in dNC-PCs are associated with proliferation, chromosome segregation, or cell division, which are typical characteristics of highly undifferentiated cells such as tooth germ cells.11 In contrast, biological processes, which are related to tissue regeneration, like ‘‘cell differentiation’’, ‘‘response to wounding’’ or ‘‘cell migration’’ were overrepresented in SHED. These processes characterise dNC-PCs and SHED as progenitors for primary odontoblasts and replacement odontoblasts, respectively. Similar results were identified in a recent microarray study with SHED and DPSCs of Nakamura et al.19 Here, a higher expression in SHED was observed for genes that participate in pathways related to cell
proliferation and extracellular matrix, including several cytokines such as fibroblast growth factor and tumour growth factor beta. Although little is known about molecular processes in dental stem cells, microarray analyses disclose regulated genes and correlated biological processes in these cells. Many genes were regulated in both dental cell types in our microarray analyses. However, only a small number of these regulated genes are associated with osteogenesis, odontogenesis or related processes of tooth development. This result is very similar to that of previous microarray studies with dental stem cells.13 However, the focus on these genes, which are regulated in dNC-PCs and SHED may shed new light on molecular processes in dental stem cells during odontogenic differentiation. Among well-known osteogenesis markers such as ALP and Col3A1 other interesting genes like FOXC2, stanniocalcin 1 (STC1), ZBTB16, and frizzledrelated protein (FRZB) were up-regulated after differentiation. FOXC2 is involved in a number of different developmental processes. It is important for the regulation of epithelial-mesenchymal transitions and promotes osteogenesis via the induction of integrin b1.20 STC1 is a 27.6 kDa homologue of the fish hormone stanniocalcin and has been linked to bone physiology. It regulates mineral homeostasis and has a strong influence on the transport of phosphates in
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a variety of mammalian cells.21 Moreover, it is also involved in the cranial intramembranous ossification.22 The induction of STC1 has been already shown in dental follicle cells after overexpression of the transcription factor DLX3, which induces the osteogenic differentiation in dental follicle cells.23 ZBTB16 promotes osteogenic differentiation of human MSCs and it is an upstream regulator of RUNX2 and regulates limb and axial skeletal patterning.24 This transcription factor is also induced in human dental follicle cells after cell differentiation.25 Frizzled-related proteins are involved in the regulation of Wnt and BMP signalling.26 The up-regulation of FRZB in dNC-PCs and SHED suggests a role of Wnt-signalling during the differentiation into mineralising cells. However, a previous study has shown that Wnt signalling inhibits the cementoblast differentiation and promotes proliferation of dental cells.27 Three osteogenesis related genes were down-regulated after cell differentiation in dNC-PCs and SHED. One gene encodes for the prostaglandin-endoperoxide synthase 2 (PTGS2), which is also known as COX-2. This protein is constitutively expressed in the skeleton and a key regulator of bone formation.28 Periostin is another down-regulated gene after the differentiation of dNC-PCs and SHED. Although periostin is expressed in cranial NC cells during tissue development29 its role during the differentiation of dental stem cells is still unknown. Finally. IGFBP-3 was down-regulated in dNC-PCs and SHED. This IGF binding protein is an important part of the IGF system and involved in many aspects of dental physiology, pathology and development.30 In conclusion, dNC-PCs and SHED seem to differ in their odontogenic differentiation potential. While differentiated dNC-PCs share more similarities with primary odontoblasts, differentiated SHED are more similar to replacement odontoblasts. These findings may be of interest for further studies about dNC-PCs or SHED in regenerative medicine and dental development.
Funding None.
Competing interests None declared.
Ethical approval Informed consent was obtained from all subjects.
Acknowledgements We thank Ms. Anja Reck for excellent technical support. We thank Prof. Songtao Shi (University of Southern California, Los Angeles, USA) for providing SHED. This study was supported by the German Society for Oral and Maxillofacial Medicine (DGZMK).
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Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.archoralbio.2012.11.004.
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