Molecular differences between stromal cell populations from deciduous and permanent human teeth
© Kaukua et al.; licensee BioMed Central. 2015
Received: 15 May 2014
Accepted: 18 March 2015
Published: 18 April 2015
Deciduous and permanent human teeth represent an excellent model system to study aging of stromal populations. Aging is tightly connected to self-renewal and proliferation and thus, mapping potential molecular differences in these characteristics between populations constitutes an important task.
Using specifically designed microarray panels, Real-Time Quantitative Polymerase Chain Reaction (RT q-PCR), Western blot, immunohistochemistry and siRNA-mediated knock down experiments, we have detected a number of molecules that were differentially expressed in dental pulp from deciduous and permanent teeth extracted from young children and adults, respectively.
Among the differentially regulated genes, high-mobility group AT-hook 2 (HMGA2), a stem cell-associated marker, stood out as a remarkable example with a robust expression in deciduous pulp cells. siRNA-mediated knock down of HMGA2 expression in cultured deciduous pulp cells caused a down-regulated expression of the pluripotency marker NANOG. This finding indicates that HMGA2 is a pulpal stem cell regulatory factor. In addition to this, we discovered that several proliferation-related genes, including CDC2A and CDK4, were up-regulated in deciduous pulp cells, while matrix genes COL1A1, fibronectin and several signaling molecules, such as VEGF, FGFr-1 and IGFr-1 were up-regulated in the pulp cells from permanent teeth.
Taken together, our data suggest that deciduous pulp cells are more robust in self- renewal and proliferation, whereas adult dental pulp cells are more capable of signaling and matrix synthesis.
Senescence and aging are associated with the loss of self-renewing capacity of stem cells. This principle is valid for multiple locations in the body, including the nervous system, connective tissue and bone marrow, and plays a significant role in the regenerative potential of stem cells [1-3]. There are several extrinsic and intrinsic elements that contribute to the aging of stem cells. These include changes in the systemic environment through factors from the blood or modifications of the stem cell niche, and alterations of elements within the stem cells such as protein accumulation, damage to mitochondrial as well as nuclear DNA, telomere attrition and cell cycle inhibition that eventually leads to failure of function [2,4]. Identification of potent tissue-specific stem cells and their banking is crucial for regenerative medicine. Teeth host pulpal stromal cells with subpopulations that have mesenchymal stem cell characteristics [5-7] that are easily extracted and amenable for manipulation. In animal models, these pulp cells seem to have a beneficial effect on spinal cord regeneration  and other types of trauma and disease, although their precise function is not clear and there is no evidence of any transdifferentiation into other cell types [9,10]. For reasons that remain elusive and contradictory to their apparent in vivo quiescence , dental pulp cells undergo rapid proliferation in ex vivo culture, apparently more rapid than bone marrow mesenchymal stem cells (BMMSCs) [5,12]. Previous work on genetic profiles of dental pulp cells has yielded several important clues [13-17]. Comparisons of gene expression between fast growing and slow growing pulp cell populations showed robust expression of transcription factors, E2F2, PTTG1, TWIST-1, and transcriptional cofactor, LDB2, each with critical roles in cell growth and survival, in dental pulp cells, just as in the periodontal ligament and bone marrow .
Here, we address the issue of molecular differences between pulp cells from deciduous and permanent teeth to elucidate the mechanism that provides high stemness and self-renewal capacity. These two types of teeth offer an excellent model for studies of molecular differences between stromal cell populations, especially in relation to their age. We approached this task by comparing genetic profiles from cells isolated from different origins and generations of human teeth with subsequent functional studies. We found that the genetic profile of deciduous dental pulp cells differs from that of adult dental pulp cells, due to ontogenic discontinuity between deciduous and permanent teeth.
Deciduous teeth and permanent teeth extracted for medical-dental reasons after thorough diagnoses by clinicians were collected from patients following approval by The Institutional Review Board at the Columbia University Medical Center and a Regional Ethical Committee in Stockholm. Patients provided informed consent to participate in this study. When deciduous teeth were obtained from children, informed consent was given from the next of kin, caretakers, or guardians on behalf of the children enrolled in our study. Informed consent was recorded, with age and gender of the donor and parent/patient approval but without any personal identifiers.
The Regional Ethical Committee in Stockholm approved the isolation and establishment of human embryonic stem cell lines. The HS181 cell line used in this study was derived at Karolinska Institutet, Karolinska University Hospital, Huddinge, Sweden.
The procedures for the use of human tissues, adult and embryonic gingiva, for experimental studies were approved by the Regional Ethical Committee, Stockholm.
The deciduous teeth were collected from 3- to 12-year-old children (n = 8), and permanent teeth were collected from adults, 19 to 52 years of age (n = 8). Both deciduous and permanent teeth had full length roots with no visible root resorption and were extracted due to pericoronitis/periodontal issues, caries or orthodontic reasons. Cell isolation followed our previously described methods and approaches [9,18]. The tooth was mechanically separated into two halves to remove all soft tissue in the pulp chamber and the root canal. The pulp tissue was digested with 3 mg/ml collagenase I and 4 mg/ml dispase (Invitrogen, Carlsbad, CA, USA) for one hour at 37°C. The dissociated cells were passed through a 70-μm-cell strainer (BD Biosciences, San Jose, CA, USA) and plated in MEM-alpha medium (Invitrogen) containing 10% fetal bovine serum (Gibco, Grand Island, NY, USA) and penicillin-streptomycin (Atlanta Biologicals, Lawrenceville, GA, USA). The cells from each pulp were cultured separately and then passaged at 90% confluency in different 10 cm dishes (Nunc, Rochester, NY, USA) or several wells of the six-well plate model (BD Biosciences) for expansion, and utilized for further analysis. The analyses were performed on pulp cells from different individual pulps without pooling.
Deciduous pulp cells were transfected with Dharmafect transfection reagent nr.4 (Thermo Scientific, Lafayette, CO, USA) according to the manufacturer’s protocol, with a final concentration of 100 nM siRNA ON-TARGETplus Human HMGA2 and 100 nM siRNA ON-TARGETplus Human Control Pool (Thermo Scientific), for 48 hours. The cells were transfected after one passage, at 40% confluency in 10% fetal bovine serum MEM-alpha medium containing no antibiotics.
RNA and protein extraction
RNA and protein were extracted from passage one cells with the Qiagen RNeasy Mini Kit (Valencia, CA, USA). At 90% confluency, cells were washed with PBS (Lonza, Walkersville, MD, USA) and then placed in lysis buffer following the manufacture’s protocols. For siRNA transfection experiments, total RNA was extracted according to the Qiagen AllPrep DNA/RNA/Protein Mini Kit protocol with a Qiagen AllPrep DNA/RNA/ Protein Mini Kit (Qiagen, Hilden, Germany).
Microarray gene expression analysis
Samples were analyzed using a PIQOR Stem Cell Microarray chip (Miltenyi Biotec, Auburn, CA, USA). This consisted of 942 relevant genes for stem/progenitor cells and key genes for cell differentiation to identify gene sets that are differentially expressed between dental pulp cells in the deciduous and adult teeth. All microarray data have been deposited in a public repository, Gene Expression Omnibus (GEO), with accession number GSE58668.
The isolated RNA was subjected to microarray analyses. A total of 1 μg RNA for each sample was used for amplification and further analysis with the PIQOR stem cell microarray chip, followed by detection with a laser scanner (Agilent Technologies, Santa Clara, CA, USA). The dataset consisted of two microarrays, each containing 11 slides for a total of 16 samples. The Linear Model for Microarray Data package (Limma), within R/Bioconductor statistical framework, was used to pre-process the raw signal intensities, perform quality controls and estimate statistical significance . Expression intensities were background-corrected using a convolution of normal and exponential distributions with an offset of 50, which were then normalized within every slide using LOESS normalization. We analyzed the red and green channels separately using the mixed model method and different groups were quantile-normalized separately. The correlation was computed between the two channels for the same spot. Differentially expressed genes for the eight different comparisons considered were identified by computing the contrasts, fitting to a linear model, performing the hypothesis tests and correcting for multiple testing using the Benjamin-Hochberg correction (p ≤ 0.05). DAVID [20,21] was used to test over-represented functional groups, and the output was further analyzed with Enrichment Map  a plugin for Cytoscape . Additionally we used Ingenuity Pathway Analysis (IPA)(Ingenuity®Systems) to generate the network of connections between modulated genes, using the knowledge-based topology. We then isolated the HMGA2 to carry out the causal network analysis with IPA.
cDNA synthesis and real-time quantitative RT q-PCR
Primers for RT q-PCR
For siRNA transfection experiments, cDNA synthesis was performed using the High Capacity cDNA Reverse Transcription Kit (Invitrogen, Foster City, USA) protocol. Further on, the RT q-PCR procedure was performed according to an Applied Biosystems protocol with gene expression assays GAPDH (FAM, Hs02758991_g1), NANOG (FAM, Hs02387400_g1), OCT4 (Hs03005111_g1) and HMGA2 (FAM, Hs00971725_m1) (Applied Biosystems, Foster City, CA, USA). PCR reactions were applied on ABI 7500 Fast- Real Time PCR System. As above, the relative comparative method was used to analyze data.
The cells from the HS181 cell line were maintained in knockout Dulbecco’s modified Eagle’s medium supplemented with 20% knockout serum replacement, 2 mM glutamax, 0.5% penicillin–streptomycin, 1% non-essential amino acids, 0.5 mM 2-mercaptoethanol and 8 ng/ml basic fibroblast growth factor (bFGF) (all from Life Technologies, Invitrogen, Stockholm, Sweden).
BMMSCs (Life Technologies, Invitrogen) at passage 3 were cultured in MesenPRO RS basal medium, supplemented with low serum of 2% MesenPRO RS growth supplement and 1% penicillin and streptomycin (Life Technologies, Invitrogen).
Total RNA was purified according to the manufacturer’s instructions from deciduous pulp cells, HS181 and BMMSCs using a commercially available RNeasy Mini Kit (Qiagen, Germany). cDNA samples were prepared from 100 ng of total RNA with superscript III (Invitrogen) using a Veriti thermal cycler (Applied Biosystems, Stockholm, Sweden). RT q-PCR was processed on a 7500 Fast Real-Time PCR System (Applied Biosystems, USA) with Taqman Universal Master Mix and Taqman primer/probe for OCT4 (Hs03005111_g1), NANOG (Hs04260366_g1) and HMGA2 (Hs00971725_m1). The housekeeping gene, GAPDH (Hs02758991_g1) was used as an endogenous control. The expression level for each sample was normalized to GAPDH and quantification of expression was estimated using the relative comparative method and was presented as relative fold change.
SDS-PAGE and Western blot
Proteins were isolated with the Qiagen RNeasy Mini Kit followed by acetone precipitation (deciduous pulps: n = 3, permanent pulps: n = 3). The protein extract was dissolved in 1% SDS. Protein concentration was measured with a Biorad RC DC Protein Assay Kit and SmartSpec Plus Spectrophotometer. A total of 20 μg of protein was loaded into wells on NuPAGE® Novex 4% to 12% Bis-Tris Gels and then transferred onto polyvinylidene difluoride (PVDF) membranes (Biorad, Hercules, CA, USA) using electroblotting. The membranes were blocked with blocking buffer (Licor, Lincoln, NE, USA) and then probed with the antibodies for beta-actin (1:500, Santa Cruz Biotechnology, Santa Cruz, CA, USA), cdc2 (1:2000, Cell Signaling Technology, Danvers, MA, USA), Fatty Acid Binding Protein 5 (1 μg/ml, Abcam, Cambridge, MA, USA), HMGA2 (1:1000, Cell Signaling Technology) and PEDF (aka Serpinf1, 2 μg/ml, Abcam) overnight in 4°C on a shaker. Thereafter, the corresponding secondary antibodies goat anti-mouse immunoglobulin G (IgG)-AP and goat anti-rabbit IgG-AP (Santa Cruz Biotechnology) were added for one hour at room temperature on a shaker. Proteins were detected using the 1-Step NBT/BC1P system (Thermo Scientific, Rockford, IL, USA). Quantification of the intensity levels of the Western blot bands was performed with ImageJ software.
Deciduous teeth were collected from 3- to 11-year-old patients (n = 3) and permanent teeth were collected from adult patients (19 to 81 years of age, n = 3). Teeth were cracked open and pulps were collected and snap frozen in optimal cutting temperature (OCT) cryomount (Histolab, Gothenburg, Sweden) for subsequent 5 μm sectioning. The sections were fixed for 20 minutes in 4% paraformaldehyde (Merck, Darmstadt, Germany), rinsed and then incubated overnight at room temperature with primary antibodies against HMGA2 (1:500, Cell Signaling Technology) and vimentin (1:200, Abcam). Additional staining was performed the following day with 4',6-diamidino-2-phenylindole (DAPI) (Invitrogen, Burlington, Canada) at 300 nM applied for three minutes. Secondary antibodies used were Alexa Fluor® 555 Donkey Anti-Rabbit IgG (H + L) and Alexa Fluor® 488 Goat Anti-Chicken IgG (H + L) (Invitrogen, Eugene, OR, USA). Control sections were incubated where the primary antibody was omitted. This resulted in no detectable artifact. Additional negative control stainings were performed with isotype control for rabbit primary antibody (Invitrogen, Eugene, OR, USA), which resulted in no unspecific staining. Confocal microscopy was performed with Zeiss LSM700 CLSM and image processing was carried out with Imaris software.
Hematoxylin & eosin staining of deciduous and permanent tooth pulp
Slides with pulp sections were washed in distilled water for one minute. They were then immersed in modified Mayers solution hematoxylin (Histolab, Gothenburg, Sweden) for ten minutes, followed by rinsing in distilled water. Subsequently, slides were incubated in 0.2% eosin (Histolab) rinsed, dehydrated in graded steps of ethanol and xylene (VWR Chemicals, Fontenay-sous-Bois, France) and then mounted in Entellan (Merck). Light microscopy was performed with a Nikon Eclipse E600 microscope and images were processed in Adobe Photoshop CS5.
Immunohistochemistry of embryonic and adult gingiva
Human fetal lower jaw tissue (8.5 weeks after conception) was retrieved from elective routine abortions, with written consent from the pregnant women. The specimens (n = 2) were immersed in 4% paraformaldehyde (Merck) overnight and then rinsed in 30% sucrose for 24 hours. They were then snap frozen in OCT cryomount (Histolab) for subsequent sectioning. Biopsies of human healthy gingiva from patients 54- to 67-years old (n = 3) were obtained with written consent from the donors, after approval by the Regional Ethical Committee, Stockholm. These samples were fixed in paraformaldehyde, rinsed, dehydrated and embedded in paraffin according to standard methods. After sectioning and deparaffination, they were processed for immunohistochemistry. Immunohistochemical staining was performed as described above with HMGA2 antibody (1:500, Cell Signaling Technology), CD31 antibody (1:300, Dako, Glostrup, Denmark) and Vectashield mounting medium with DAPI (Vector Laboratories, Burlingame, CA, USA). Imaging was performed using a confocal microscope, Zeiss LSM710 CLSM, and image processing was carried out with Imaris software.
Microarray gene expression analysis reveals differences between deciduous and permanent pulp cells
Up-regulated genes in pulp cells from permanent teeth in microarray
Log 2 fold change
Regulation of nerve cell survival, embryonic axial development and bone growth
Inhibits angiogenesis, is a neurotrophic factor involved in neuronal differentiation in retinoblastoma cells
NM_002026,NM_212474, NM_212475, NM_212476,NM_212478, NM_212482
Cell adhesion, motility, opsonization, wound healing, and maintenance of cell shape
Found in most connective tissues
Inhibitors of the matrix metalloproteinases, which are involved in degradation of the extracellular matrix. They promote cell proliferation in a wide range of cell types.
NM_002211,NM_033666, NM_033667,NM_033668, NM_033669,NM_133376
Receptors for collagen, fibronectin, fibrinogen and laminin
Active in angiogenesis, vasculogenesis and endothelial cell growth
Binds insulin-like growth factor
Epithelial and neural tissue development
NM_015850,NM_023105, NM_023106,NM_023107 NM_023108,NM_023110
Interacts with fibroblast growth factors, influences mitogenesis and differentiation
Stem cell differentiation, negative regulation of cell proliferation
Down-regulated genes in pulp cells from permanent teeth in microarray
Log 2 fold change
Expressed during mammalian embryonic development. High expression in neural stem cells and decreases with age
Found in epidermal cells, may be involved in keratinocyte differentiation
Cell cycle key mediator of neuronal cell death in brain development and degeneration
Cell division and mitosis
Cell division and mitosis
Cytosolic enzyme, high in proliferating cells
Involved in the breakdown of extracellular matrix
Genes encoding extracellular matrix proteins, including collagen type I (COL1A1) and fibronectin were differentially expressed in favor of pulp cells from permanent teeth. In addition, genes associated with cell differentiation were robustly expressed by pulp cells of the permanent teeth, including INHBA, SERPINF1, PLXNA3 and JMJ. In comparison, genes promoting cell proliferation, mitosis and division, including MAD2L1, MMP23A-MMP23B, MMP6 and PTTG, were highly expressed in pulp cells from deciduous teeth. Remarkably, HMGA2, a gene strongly expressed by neural stem cells during embryogenesis, was highly expressed in pulp cells of deciduous but not permanent teeth (Figure 1B and Table 3). The differences in mRNA expression are significant with the moderated T-test, P ≤0.05.
IPA revealed that several genes are in association with HMGA2, in particular genes that are involved with stemness, tumorigenesis and cell regulation, such as SP1, BRCA1 and cyclin A (Additional files 2 and 3).
siRNA-mediated loss-of-function experiments show association between HMGA2 and NANOG in pulp cells
Immunohistochemistry reveals that HMGA2 expression in deciduous pulp tissue is unrelated to blood vessels
Immunohistochemistry reveals HMGA2 expression in embryonic gingiva
HMGA2 nuclear immunoreactivity was abundant in cells of embryonic gingiva, in epithelial as well as in the mesenchymal compartments of 24.76 ± 9.57% (Additional file 6). Adult gingival tissue did not express discernible HMGA2 protein immunostaining (Additional file 7).
Humans are diphyodont and have two generations of teeth, where the second permanent set slowly begins to replace the first deciduous set around the age of six. Studies of molecular differences in pulpal cells from teeth extracted from children and adults, respectively, provide unique opportunities to compare general age-related differences in human stromal populations. The regenerative capacity of human tissue is much greater during early childhood compared to later stages in life . We hypothesized that this would be reflected in age-related changes in molecular characteristics of human pulpal stromal cells.
For this purpose, we compared gene expression profiles of pulp cells from deciduous and permanent teeth using a microarray chip that is specifically designed for stem/progenitor cells. This was performed to evaluate their genetic signatures with regard to key differences related to self-renewal, proliferation and differentiation. Cells from deciduous teeth were obtained from children (3- to 12-years old), thus being younger than the permanent teeth cells we collected from adults (18- to 52-years old). For obvious reasons, there was no possibility to match deciduous and permanent teeth to the same donor, which is reflected in the microarray results (Figure 1A). However, with this taken into account, our results clearly show that there are differentially expressed genes in the deciduous and permanent teeth. Genes involved in cell division, mitosis, stemness and aging were differently expressed in favor of pulp cells from deciduous teeth. The results indicate a regeneration-favorable phenotype of pulp cells from deciduous teeth.
Somatic stem cells age with time, with a concomitant reduction in amounts, mitotic activity and self-renewable capacity [1,25,26]. This should be ascribed to age-dependent alterations in gene function. When mesenchymal cells from bone marrow were compared between adults and children, it was proposed that the bone marrow cells from the latter are more suitable for tissue engineering purposes because of age-related factors . HMGA2 is an example of a gene that confers self-renewal and proliferation capacity to stem cells. It is strongly expressed by neural stem cells/progenitors in the subventricular zone, but declines over time with age [27-29]. A recent study has shown that the senescence-related tumor suppressor gene p16 Ink4a , which diminishes the self renewal capacity of cells and also reduces the expression of HMGA2, is increased in older permanent teeth compared to young deciduous teeth . This agrees with our data, where HMGA2 was differentially expressed with a substantial level in cells from deciduous teeth but not detected in cells from permanent pulps. An overall age-dependent decline in cellular expression of HMGA2 outside brain and teeth is suggested by our immunohistochemical observations in human gingiva, where embryonic tissue expressed widespread HMGA2 immunoreactivity while none was observed in adult samples.
It should be noted, that our results show that not all stromal cells in deciduous pulps are HMGA2 positive. Instead, HMGA2+ cells constitute a subpopulation of approximately 16% of the total number. The full phenotype/s of this subpopulation remains elusive, although the HMGA2 expression suggests that it may contain progenitor cells. Other investigators have demonstrated that stem/progenitor cells in teeth are associated with blood vessels [7,12]. We did not find that HMGA2-positive cells were related to blood vessels, which suggests that we might have found another proliferative subpopulation in deciduous teeth.
We attempted to clarify the role of HMGA2 in deciduous teeth pulp cells by reducing the level through siRNA transfections. Due to stable expression of HMGA2 mRNA in the cells, we managed to suppress HMGA2 levels to approximately 50% of those siControl untreated samples (Figure 4A). This resulted in a significant reduction of mRNA levels of NANOG, which is thought to be a key factor in maintaining pluripotency. This finding gives further indications that HMGA2 has a key role in the regulation and maintenance of a subset of pulpal cells. SOX2 levels were not detectable, which is expected since dental stem cells that express SOX2 are epithelial and not mesenchymal. OCT4 levels were not affected by the depletion of HMGA2.
Our results indicate that deciduous teeth harbor a population of cells, with a higher self-renewal and proliferation capacity than those of permanent teeth. Thus, markers involved in cell division and mitosis were up-regulated in deciduous cells compared to cells from permanent teeth (Table 2). This could make primary pulp cells more suitable for tissue regeneration. In fact, from certain aspects, they might even be more useful than BMMSCs, since the levels of stem cell markers NANOG and OCT4 were similar between these cell types, but HMGA2 was slightly higher in deciduous pulp cells (Figure 5A-C).
Using specifically designed microarray panels, RT q-PCR, Western blot and immunohistochemistry we show that HMGA2, a stem cell-associated gene, is robustly expressed in deciduous pulp cells but not in permanent tooth pulp cells. Additionally, several genes associated with mitosis were highly expressed in deciduous pulp cells, while matrix genes and several signaling molecules were strongly expressed in the adult. These findings strongly suggest that there are molecular differences in cellular populations of different age and should serve as important basic information for understanding the therapeutic potential of pulpal cells. Specifically, primary pulp cells may be better suited for regenerative medicine and tissue engineering purposes than cells from permanent tooth pulps and based on this, are recommended for early banking.
bone marrow mesenchymal stem cells
human embryonic stem cells
high-mobility group AT-hook 2
Ingenuity Pathway Analysis
optimum cutting temperature
- RT q-PCR:
real time quantitative real-time polymerase chain reaction
small interfering RNA
This study has been supported by the Swedish Dental Society and we would also like to thank Knut and Alice Wallenberg Foundation (CLICK facility). Work in this manuscript is based on a PhD dissertation by Nina Kaukua at the Karolinska Institutet, with parts of experiments performed at Columbia University. We thank Ms. R. Burdie, Q. Guo, H. Keyes and Ms. J. Melendez, R. Bergström-Tengzelius, F. Holm, M. Barr Agholme, A.-M. Thelander, M-L Olsson and S. Kaukua for their technical and administrative assistance.
- Enwere E, Shingo T, Gregg C, Fujikawa H, Ohta S, Weiss S. Aging results in reduced epidermal growth factor receptor signaling, diminished olfactory neurogenesis, and deficits in fine olfactory discrimination. J Neurosci. 2004;24:8354–65.View ArticlePubMedGoogle Scholar
- Sharpless NE, DePinho RA. How stem cells age and why this makes us grow old. Nat Rev Mol Cell Biol. 2007;8:703–13.View ArticlePubMedGoogle Scholar
- Choumerianou DM, Martimianaki G, Stiakaki E, Kalmanti L, Kalmanti M, Dimitriou H. Comparative study of stemness characteristics of mesenchymal cells from bone marrow of children and adults. Cytotherapy. 2010;12:881–7.View ArticlePubMedGoogle Scholar
- Jung Y, Brack AS. Cellular mechanisms of somatic stem cell aging. Curr Top Dev Biol. 2014;107:405–38.View ArticlePubMed CentralPubMedGoogle Scholar
- Gronthos S, Mankani M, Brahim J, Robey PG, Shi S. Postnatal human dental pulp stem cells (DPSCs) in vitro and in vivo. Proc Natl Acad Sci U S A. 2000;97:13625–30.View ArticlePubMed CentralPubMedGoogle Scholar
- Shi S, Robey PG, Gronthos S. Comparison of human dental pulp and bone marrow stromal stem cells by cDNA microarray analysis. Bone. 2001;29:532–9.View ArticlePubMedGoogle Scholar
- Miura M, Gronthos S, Zhao M, Lu B, Fisher LW, Robey PG, et al. SHED: stem cells from human exfoliated deciduous teeth. Proc Natl Acad Sci U S A. 2003;100:5807–12.View ArticlePubMed CentralPubMedGoogle Scholar
- Sakai K, Yamamoto A, Matsubara K, Nakamura S, Naruse M, Yamagata M, et al. Human dental pulp derived stem cells promote locomotor recovery after complete transection of the rat spinalcord by multiple neuro regenerative mechanisms. J Clin Invest. 2012;3:80–90.Google Scholar
- Yang R, Chen M, Lee CH, Yoon R, Lal S, Mao JJ. Clones of ectopic stem cells in the regeneration of muscle defects in vivo. PLoS One. 2010;5, e13547.View ArticlePubMed CentralPubMedGoogle Scholar
- Ma L, Makino Y, Yamaza H, Akiyama K, Hoshino Y, Nonaka K, et al. Cryopreserved dental pulp tissues of exfoliated deciduous teeth is a feasible stem cell resource for regenerative medicine. PLoS One. 2012;7:12.Google Scholar
- Smith AJ, Smith JG, Shelton RM, Cooper PR. Harnessing the natural regenerative potential of the dental pulp. Dent Clin North Am. 2012;56:589–601.View ArticlePubMedGoogle Scholar
- Huang GT, Gronthos S, Shi S. Mesenchymal stem cells derived from dental tissues vs. those from other sources: their biology and role in regenerative medicine. J Dent Res. 2009;88:792–806.View ArticlePubMed CentralPubMedGoogle Scholar
- Menicanin D, Bartold PM, Zannettino AC, Gronthos S. Identification of a common gene expression signature associated with immature clonal mesenchymal cell populations derived from bone marrow and dental tissues. Stem Cells Dev. 2010;19:1501–10.View ArticlePubMedGoogle Scholar
- Gronthos S, Brahim J, Li W, Fisher LW, Cherman N, Boyde A, et al. Stem cell properties of human dental pulp stem cells. J Dent Res. 2002;81:531–5.View ArticlePubMedGoogle Scholar
- Tete S, Mastrangelo F, Scioletti AP, Tranasi M, Raicu F, Paolantonio M, et al. Microarray expression profiling of human dental pulp from single subject. Clin Invest Med. 2008;31:e55–61.PubMedGoogle Scholar
- Tranasi M, Sberna MT, Zizzari V, D'Apolito G, Mastrangelo F, Salini L, et al. Microarray evaluation of age-related changes in human dental pulp. J Endod. 2009;35:1211–7.View ArticlePubMedGoogle Scholar
- Govindasamy V, Abdullah AN, Ronald VS, Musa S, Ab Aziz ZA, Nain RB, et al. Inherent differential propensity of dental pulp stem cells derived from human deciduous and permanent teeth. J Endod. 2010;36:1504–15.View ArticlePubMedGoogle Scholar
- Suzuki T, Lee CH, Chen M, Zhao W, Fu SY, Qi JJ, et al. Induced migration of dental pulp stem cells for in vivo pulp regeneration. J Dent Res. 2011;90:1013–8.View ArticlePubMedGoogle Scholar
- Smyth G. Limma: linear models for microarray data. In: Gentleman R, Carey V, Huber W, Irizarry R, Dudoit S, editors. Bioinformatics and computational biology solutions using R and Bioconductor. Berlin: Springer-Verlag; 2005. p. 397–420.View ArticleGoogle Scholar
- Huang DW, Sherman BT, Lempicki RA. Systematic and integrative analysis of large gene lists using DAVID Bioinformatics Resources. Nature Protoc. 2009;4:44–57.View ArticleGoogle Scholar
- Huang DW, Sherman BT, Lempicki RA. Bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists. Nucleic Acids Res. 2009;37:1–13.View ArticlePubMed CentralGoogle Scholar
- Merico D, Isserlin R, Bader GD. Visualizing gene-set enrichment results using the Cytoscape plug-in enrichment map. Methods Mol Biol. 2011;781:257–77.View ArticlePubMedGoogle Scholar
- Shannon P, Markiel A, Ozier O, Baliga NS, Wang JT, Ramage D, et al. Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res. 2003;13:2498–504.View ArticlePubMed CentralPubMedGoogle Scholar
- Illingworth CM. Trapped fingers and amputated finger tips in children. J Pediatr Surg. 1974;9:853–8.View ArticlePubMedGoogle Scholar
- Maslov AY, Barone TA, Plunkett RJ, Pruitt SC. Neural stem cell detection, characterization, and age-related changes in the subventricular zone of mice. J Neurosci. 2004;24:1726–33.View ArticlePubMedGoogle Scholar
- Molofsky AV, Pardal R, Iwashita T, Park IK, Clarke MF, Morrison SJ. Bmi-1 dependence distinguishes neural stem cell self-renewal from progenitor proliferation. Nature. 2003;425:962–7.View ArticlePubMed CentralPubMedGoogle Scholar
- Nishino J, Kim I, Chada K, Morrison SJ. Hmga2 promotes neural stem cell self-renewal in young but not old mice by reducing p16Ink4a and p19Arf expression. Cell. 2008;135:227–39.View ArticlePubMed CentralPubMedGoogle Scholar
- Hammond SM, Sharpless NE. HMGA2, microRNAs, and stem cell aging. Cell. 2008;135:1013–6.View ArticlePubMed CentralPubMedGoogle Scholar
- Ashar HR, Chouinard Jr RA, Dokur M, Chada K. In vivo modulation of HMGA2 expression. Biochim Biophys Acta. 2010;1799:55–61.View ArticlePubMedGoogle Scholar
- Feng X, Xing J, Feng G, Huang D, Lu X, Liu S, et al. p16INK4A mediates age-related changes in mesenchymal stem cells derived from human dental pulp through the DNA damage and stress response. Mech Ageing Dev. 2014;141–142:46–55. doi:10.1016/j.mad.2014.09.004.View ArticlePubMedGoogle Scholar
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.