Side Population Cells Isolated from Porcine Dental Pulp Tissue with Self-Renewal - 《stemcells》干细胞学杂志干细胞之家



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发表于 2009-3-5 00:00 |只看该作者 |倒序浏览 |打印

作者：Koichiro Ioharaa, Li Zhenga, Masataka Itob, Atsushi Tomokiyoc, Kenji Matsushitaa, Misako Nakashimaa作者单位：aLaboratory of Oral Disease Research, National Institute for Longevity Sciences, National Center for Geriatrics and Gerontology, Aichi, Japan;bDepartment of First Anatomy, National Defense Medical College, Tokorozawa, Japan;cDepartment of Oral Rehabilitation, Faculty of Dental Science, Kyushu Univer + w6 y( k/ q" Y+ l1 g6 b) Y7 Z) `, f
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      【摘要】: X! {2 Z/ B6 z, O
   Dental pulp has the potential to form dentin as a regenerative response to caries. This regeneration is mediated by stem/progenitor cells. Thus, stem cell therapy might be of potential utility in induction of reparative dentin. We isolated side population (SP) cells from dental pulp based on the exclusion of the DNA binding dye Hoechst 33342 by flow cytometry and compared its self-renewal capacities and multipotency with non-SP cells and primary pulp cells. The cumulative cell number of the SP cells was greater than the non-SP cells and primary pulp cells. Bmi1 was continuously expressed in SP cells, suggesting longer proliferative lifespan and self-renewal capacity of SP cells. Next, the maintenance of the multilineage differentiation potential of pulp SP cells was investigated. Expression of type II collagen and aggrecan confirmed chondrogenic conversion (30%) of SP cells. SP cells expressed peroxisome proliferator-activated receptor  and adaptor protein 2, showing adipogenic conversion. Expression of mRNA and proteins of neurofilament and neuromodulin confirmed neurogenic conversion (90%). These results demonstrate that pulp SP cells maintain multilineage differentiation potential. We further examined whether bone morphogenetic protein 2 (BMP2) could induce differentiation of pulp SP cells into odontoblasts. BMP2 stimulated the expression of dentin sialophosphoprotein (Dspp) and enamelysin in three-dimensional pellet cultures. Autogenous transplantation of the Bmp2-supplemented SP cells on the amputated pulp stimulated the reparative dentin formation. Thus, adult pulp contains SP cells, which are enriched for stem cell properties and useful for cell therapy with BMP2 for dentin regeneration.
      【关键词】 Dental pulp stem cells Side population cells Dentin regeneration Bone morphogenetic proteins Neurogenesis Chondrogenesis Adipogenesis
   INTRODUCTION6 C6 m8 O: X) M' w/ V

Caries is a common problem in dentistry, characterized by damaged dentin and the consequent exposure of the dental pulp. Regeneration and repair of the damaged dentin to protect the pulp is the goal of operative dentistry and endodontology. Regenerative dentistry is based on the triad of stem or progenitor cells, and morphogens and a scaffold of extracellular matrix .0 j3 j9 h* R4 ^: _) T

The results of the present investigation demonstrated that SP cells isolated from porcine dental pulp tissue have the plasticity to differentiate into adipogenic, chondrogenic, and neurogenic lineages. Their potential to differentiate into odontoblasts in response to BMP2 was also demonstrated. Furthermore, autogenous transplantation of the pulp SP cells treated with BMP2 resulted in regenerative dentin formation on the amputated dental pulp.

MATERIALS AND METHODS% M/ L( R5 x' _# M; K
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Isolation of Pulp SP Cells

The pulp cells from human, bovine, canine, and porcine pulp tissues were isolated enzymatically with slight modification of the described method in bovine pulp cells . Normal human adult third molars were extracted from patients 16¨C25 years old under approved guidelines set by Kyushu University, Faculty of Dental Science. Red blood cells were lysed with IOTest3 lysing solution (Beckman Coulter, Fullerton, CA, http://www.beckmancoulter.com). The cells passing through 40-µm nylon mesh (Cell Strainer; BD Biosciences, San Jose, CA, http://www.bdbioscience.com) were suspended at 1 x 106cells per milliliter in prewarmed Dulbecco's modified Eagle's medium (DMEM) (Invitrogen, Carlsbad, CA, http://www.invitrogen.com) to 37¡ãC with 2% fetal calf serum containing 5 µg/ml Hoechst 33342 (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com) and labeled for 70 minutes at 37¡ãC. Cells were resuspended in Hanks' balanced saline solution (Invitrogen) with 2% fetal calf serum and 10 mM HEPES buffer containing 2 µg/ml propidium iodide (PI) (Sigma-Aldrich) for the discrimination of dead cells. Analysis/sorting of cells was performed on JSAN (Bay Bioscience, Kobe, Japan, http://www.baybio.co.jp) equipped with laser lines of 375 nm (for UV), 488 nm, and 635 nm. Hoechst/PI red and Hoechst blue fluorescence signals were displayed on a linear, dual-fluorescence dot plot. A rectangular gate was drawn to exclude PI  dead cells and unstained debris. In parallel, 106 cells were stained in the presence of 50 µM verapamil (Sigma-Aldrich), an inhibitor of ABCG2/BCRP involved in dye efflux of Hoechst 33342 to set the gate for the isolation of SP cells. A non-SP gate was drawn that excluded SP cells. Part of the freshly sorted cells were analyzed for mRNA expression of Bcrp1, Stat3, Bmi1, and Telomerase reverse transcriptase (Tert) by real-time reverse transcription-polymerase chain reaction (RT-PCR).
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Characterization of Porcine Pulp SP Cells4 T: Q; g' O2 h9 M, T
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After flow cytometry of the primary pulp cells from porcine tooth germ, isolated SP cells, non-SP cells, and the original primary pulp cells without flow cytometry were plated into 35-mm collagen type I-coated dishes (Asahi Technoglass Corp., Funabashi City, Japan, http://www.atgc.co.jp) in proliferation medium: DMEM high-glucose (Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum (JRH Biosciences, Inc., Lenexa, KS, http://www.jrhbio.com), penicillin-streptomycin (Invitrogen), and basic fibroblast growth factor (bFGF) (5 ng/ml; Invitrogen). At subconfluence, the cells were passaged by enzymatic digestion with 0.2% trypsin containing 0.02% EDTA. Total RNA were extracted from these cells at each passage using Trizol (Invitrogen). First-strand cDNA syntheses were performed from 1 µg of total RNA by reverse transcription using the SuperScript II preamplification system (Invitrogen). Real-time RT-PCR amplifications were performed at 95¡ãC for 10 seconds, 62¡ãC for 15 seconds, and 72¡ãC for 8 seconds using porcine Bcrp1, Stat3, Bmi1, Tert, and ß-actin primers (Table 1) labeled with Light Cycler-Fast Start DNA master SYBR Green I (Roche Diagnostics, Pleasanton, CA, http://www.rochediagnostics.com) in Light Cycler (Roche Diagnostics). The RT-PCR products were subcloned into pCR2.1-TOPO vector (Invitrogen) and confirmed by sequencing on the basis of published cDNA sequences.
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Table 1. Porcine primers for real-time reverse transcription-polymerase chain reaction% x4 q1 l. l- n! M  b5 s
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The expression of cell-surface antigen markers for tissue stem cells, CD31, CD34, CD45, CD90, CD105, CD146, and CD150 mRNAs, in SP cells and non-SP cells was examined by real-time RT-PCR. The number of SP and non-SP cells was normalized to 5 x 104 cells in each experiment. The mRNA expression of the vascular pericyte markers smooth muscle (SM) -actin, desmin, and NG2 proteoglycan and the endothelial cell marker vascular endothelial growth factor receptor 2 (Vegfr2) was also analyzed (Table 2). The design of the oligonucleotide primers was based on published cDNA sequences of porcine. If porcine sequences were not available, human or mouse sequences were used (Table 1). The RT-PCR products were subcloned into pGEM-T Easy vector (Promega, Madison, WI, http://www.promega.com) and confirmed by sequencing. The absence of the differentiation markers for odontoblasts alkaline phosphatase (ALP), 1(I) collagen, 1(III) collagen, dentin sialophosphoprotein (Dspp), and enamelysin/MMP20 indicated the stemness of isolated SP cells. The expression in SP cells and non-SP cells were compared with porcine tooth germ control after normalizing with ß-actin.
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Table 2. mRNA expression of cell surface antigen markers for stem cells and odontoblast differentiation markers by real-time reverse transcription-polymerase chain reaction analysis in porcine pulp SP cells and non-SP cells compared with tooth germ
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In situ hybridization for RNA in sections was carried out as described previously ) cDNA linearized with NcoI was used as a probe.

Fifteen micrometers of frozen porcine tooth germ sections were dried and fixed in 100% acetone at ¨C20¡ãC for 5 minutes. After being treated with 2% peroxidase in methanol for 20 minutes and 1% blocking reagent (PerkinElmer) for 2 hours at room temperature, they were incubated with primary anti-mouse BCRP1 monoclonal antibody (BXP-53; 1:50; ABCAM Plc., Cambridge, U.K., http://www.abcam.com) for 2 hours at room temperature. Anti-rat IgG (1:100; HRP-conjugated; GE Healthcare Bio-Sciences Corp., Piscataway, NJ, http://www.amersham.com) enhanced with TSA system Alexa Fluor 488-conjugated tyramide (Molecular Probes Inc., Eugene, OR, http://probes.invitrogen.com) was used as secondary antibody. The stained sections were observed under a fluorescence microscope IX 71 (Olympus, Tokyo, Japan, http://www.olympus.co.jp) after being counterstained with Hoechst 33342.8 O6 l2 K8 w1 L( G9 m; o

Differentiation of Pulp SP Cells into Odontoblast Lineage in Three-Dimensional Pellet Culture; T) ]1 X# v- ^

Pulp SP cells were cultured in a three-dimensional pellet (cellular aggregates) supplemented with and without recombinant human BMP2 (kindly provided by Yamanouchi Pharmaceutical Co., Ltd., Tokyo, Japan, http://informagen.com) at a final concentration of 50 ng/ml, 10% heat-inactivated fetal bovine serum (JRH Biosciences, Lenexa, KS), and 50 µg/ml L-ascorbic acid 2-phosphate (Wako Pure Chemical Industries, Ltd., Osaka, Japan, http://www.wako-chem.co.jp/english), and penicillin-streptomycin. The medium was changed twice a week. To determine the effect on proliferation, the pellets were digested by trypsin and total cell numbers were calculated on days 0, 1, 3, 6, 10, and 14. On days 1, 3, 7, 10, and 14, the pellets was sonicated in 100 µl of 10 mM Tris-HCl buffer, pH 7.4, containing 0.1% Triton X-100 at 20 kHz for 5 minutes. Alkaline phosphatase activity was assayed by the method of Lowry et al. . Pellets were fixed on days 14 and 35, and the paraffin-embedded sections were stained in Masson trichrome for extracellular matrix formation and alizarin red for calcification, respectively. Total cellular RNA was isolated at each point on days 10, 14, and 28, and real-time RT-PCR was performed using porcine Dspp, enamelysin/MMP20, dentin matrix protein 1 (Dmp1), and 1(I) collagen as differentiation markers of odontoblasts.

Induced Chondrogenic, Adipogenic, and Neurogenic Differentiation of Pulp SP Cells

The differentiation of pulp SP cells into adipogenic, chondrogenic, or neurogenic cells was determined by the method of Pittenger et al. . For chondrogenic differentiation, 2.5 x 105 porcine pulp SP cells, non-SP cells, and primary pulp cells at the third passage were centrifuged in 15-ml conical polypropylene tube (Asahi Techno Glass) at 1,000 rpm for 5 minutes. The pellets were maintained in DMEM supplemented with 10% heat-inactivated bovine calf serum, 10 µg/ml insulin-transferin-selenite X (Invitrogen), 5.35 µg/ml linoleic acid (Sigma-Aldrich), 1.25 µg/ml bovine serum albumin (Sigma-Aldrich), 1.0 µg/ml dexamethason, 10 µg/ml L-ascorbic acid 2-phosphate (Wako Pure Chemical Industries), and 10 ng/ml transforming growth factor ß3 (Peprotech, London, http://www.peprotechec.com). Medium was changed every 3 days, and cultures were maintained for 28 days. Pellets were fixed in 4% paraformaldehyde overnight, and the paraffin-embedded sections (4¨C5 µm thick) were stained by Alcian Blue. The total cellular RNA of the pellets was isolated on day 28 for analysis of 1(II) collagen and aggrecan (Table 1) by RT-PCR., I+ K, m9 e4 ]
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For adipogenic differentiation, cells were seeded at 2 x 105cells per milliliter in 35-mm collagen type I-coated dishes in DMEM high-glucose supplemented with 10% heat-inactivated fetal calf serum. The third-passage culture of the SP cells was grown to confluence in DMEM low-glucose supplemented with 10% heat-inactivated fetal calf serum and 10 µg/ml L-glutamine (Invitrogen). Thereafter, adipogenic differentiation was induced by subjecting confluent monolayers to three rounds of adipogenic treatment. Each round had two steps: incubation with induction medium of Poietics hMSC Differentiation Media Bullet Kit-Adipogenic (Cambrex, Walkersville, MD, http://www.cambrex.com) for 3 days and incubation with maintenance medium for 3 days. Cells were cultured for another 7 days in maintenance medium. As a control, cells were cultured only in maintenance medium. The cell layers were stained with oil red O, and the mRNA levels of adaptor protein 2 (aP2) and peroxisome proliferator-activated receptor  (PPAR) (Table 1) were analyzed by RT-PCR.! I3 p5 I5 |* k) ?" |! g
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For neurogenic differentiation, pulp SP cells were cultured in noncoated 35-mm dishes in neurosphere medium (Neurobasal A; Invitrogen) containing B27 supplement (Invitrogen), penicillin-streptomycin, L-glutamine, endothelial growth factor (EGF) (Invitrogen), and bFGF (Invitrogen) for 15 days, designated for neurosphere formation. The neurospheres were triturated using polished glass pipettes, and the obtained single-cell suspension was cultured in gelatin-coated 35-mm dishes in neurodifferentiation medium one (Neurobasal A) containing 1 µg/ml laminin (Invitrogen), 5 µg/ml fibronectin (Nitta Gelatin, Osaka, Japan, http://nitta-gelatin.co.jp), 2 mM L-glutamine, 10 µg/ml N2 supplement (Invitrogen), 20 ng/ml bFGF, and 40 ng/ml EGF. The medium was changed to neurodifferentiation medium 2 (Neurobasal A) containing 1 µg/ml laminin, 5 µg/ml fibronectin, 2 mM L-glutamine, 10 µg/ml N2 supplement, 20 ng/ml neurotrophin-3 (Peprotech) after 24 hours of cultivation. The medium was changed every 3 days. Immunocytochemical analysis was performed 28 days after cultivation. The cells were fixed for 30 minutes in cold 4% paraformaldehyde in PBS, treated with 0.1% Triton-X for optimal penetration of cell membrane, and incubated at room temperature in a blocking solution (protein block; Dako, Glostrup, Denmark, http://www.dako.com) for 30 minutes. After an overnight incubation at 4¡ãC with a primary antibody anti-neuromodulin (GAP-43) (Clone91E12) (1:100; Roche Diagnostics), the cells were incubated for 30 minutes at room temperature with a secondary antibody (Envision ; Dako) and stained with diaminobenzidine substrate kit (Vector Laboratories, Burlingame, CA, http://www.vectorlabs.com). The mRNA expression of neuromodulin and neurofilament (Table 1) was analyzed by RT-PCR.6 O# q, m+ ~9 V- A" h

Canine Pulp SP Cells and In Vivo Transplantation on Amputated Pulp
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The ultimate utility of the SP cell-derived stem cells is the in vivo differentiation into dentin-forming odontoblasts. A switch was made to an experimental model of canine pulp partial removal and transplantation of SP cell-derived pulp stem cell population. Adult dogs were obtained from Narc (Chiba, Japan, http://www.narc.co.jp). The canine SP pulp cells were isolated from the pulp tissues surgically extracted from the incisors by flow cytometry as performed in porcine pulp tissue. Canine autogenous pulp pellets (cellular aggregates) were prepared from third-passage SP cells supplemented with and without recombinant human BMP2. Anesthesia for surgery was performed by intravenous administration of pentobarbital sodium. Surgical amputation was carried out in the canine teeth of the dog, and the pellets were implanted autogenously on the amputated pulp. The cavity was sealed with zinc phosphate cement and composite resin with the caution of using minimal pressure. The teeth were obtained by extraction 1 month after transplantation, fixed in 4% paraformaldehyde at 4¡ãC overnight, and demineralized in 10% formic acid. Reparative dentin formation was evaluated by histology in serial paraffin sections. For in situ hybridization in sections, after dewaxing and inhibition of endogenous peroxidase activity by treatment with 0.3% H2O2 in methanol solution for 30 minutes, the procedure described above was performed. The first-strand cDNA was synthesized from 2 µg of total RNA isolated from canine primary pulp pellet cultures on day 28, and RT-PCR amplifications were performed at 95¡ãC for 10 seconds, 58¡ãC for 15 seconds, and 72¡ãC for 8 seconds using canine Dspp primers (forward, 5'-GTCCTAGTGGGAATGGAGCA-3'; reverse, 5'-TCTTCAGGGCCATCA-TCTTC-3'), and enamelysin primers (forward, 5'-TATTCACCGTTGCTGCTCAC-3'; reverse, 5'-TACAATGCCTGGAT-CCCTTT-3'). RT-PCR products of Dspp (190 bp) and enamelysin (151 bp) were subcloned into pGEM-T Easy vector (Promega) and confirmed by sequencing based on published cDNA sequences. The canine Dspp and enamelysin cDNA, linearized with SpeI and NcoI, respectively, were used for making RNA probes. The animal experiments were conducted using the strict guidelines of the Kyushu University Animal Protocol Committee and DNA Safety Program.2 b- h3 y$ O' V

RESULTS; I- }! z, p; h  A* }& g! Y5 @3 P: a
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Isolation of SP Cells from Dental Pulp from Various Species

Pulp cells isolated from human, bovine, canine, and porcine adult pulp tissues and stained with Hoechst 33342 identified approximately 0.2% of the population with relatively lower Hoechst 33342 fluorescence (SP cells) (Fig. 1A¨C1D). Although some markers (CD90 and CD117) reacted with human pulp cells (data not shown), the markers required to subfractionate pulp SP cells are not well-defined due to small yields of isolated human SP cells. Therefore, sensitivity to verapamil, an inhibitor of multidrug resistance (MDR) and MDR-like transporter, was used to set the SP gate (Fig. 1D, 1E). This SP subpopulation has been shown to be a more homogeneous population than primary pulp cells and non-SP cells. Some of the single SP cells plated in a 35-mm, collagen type I-coated dish formed a colony in 7 days (Fig. 1F, 1G).
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Figure 1. Isolation of side population (SP) cells from dental pulp from various species. SP cells were isolated from human (A), bovine (B), canine (C), and porcine (D) dental pulp. It is noteworthy that in the presence of 50 µM verapamil, few SP cells were obtained from porcine (E) (compare ). The growth of a cell colony derived from single porcine SP cell is shown on day 1 (F) and on day 7 (G). SP cells could form a colony at a rate of 0.2%. Scale bars = 50 µm.

Self-Renewal Capability of Pulp SP Cells) ]3 d, f5 o, z$ Z4 o1 H6 l2 E
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The cumulative cell number of porcine pulp SP cells was much higher, and the proliferative life span of SP cells was longer than that of pulp non-SP cells and primary pulp cells (Fig. 2A). At first, the non-SP cells and primary cells were passaged every 3¨C4 days from the second to the 15th passage. The rate of proliferation progressively became slower. The cells became senescent by passage 26, on day 116 and by passage 27, on day 132, respectively. Most of SP cells proliferated and maintained a spindle-like appearance until the 42nd passage. The molecular basis for the SP phenotype has been attributed to multidrug-resistance transporter (ABCG2/Bcrp 1). To characterize pulp SP cells, the expression of the markers associated with other stem cells (Bcrp1, Stat3, Bmi1, and Tert mRNA) was analyzed by the real-time RT-PCR. Those levels of expression were much higher in SP cells compared with non-SP cells (Fig. 2B), suggesting that this population was highly enriched for stem cells. Stat3 and Bmi1 mRNA were still expressed in SP cells on day 116 and disappeared in non-SP cells and primary pulp cells (Fig. 2C), suggesting the self-renewal and replicative capacity of SP cells. The expression of Bcrp1 mRNA and BCRP1 protein was detected by in situ hybridization and immunohistochemistry in the perivascular region of porcine dental pulp tissue (Fig. 2D, 2E).
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Figure 2. Characterization of porcine pulp SP cells from dental pulp. (A): The cumulative cell number of pulp SP cells, non-SP cells, and primary pulp cells up to 140 days. (B): The relative expression (fold increases) of Bcrp1, Stat3, Bmi1, and Tert mRNA in porcine SP cells compared with that of non-SP cells. (C): The expression of Stat3 and Bmi1 mRNA on days 14 and 116 in SP cells, non-SP cells, and primary porcine dental pulp cells. Note that SP cells demonstrate Stat3 and Bmi1 mRNA up to 116 days. (D, E): The in situ expression of Bcrp1 mRNA (D) and BCRP1 protein (E) in vascular region in porcine dental pulp sections. Counterstain (blue) is Hoechst 33342. Scale bars = 50 µm. Abbreviations: Bcrp1, breast cancer resistant protein 1; prim, primary; SP, side population; Tert, telomerase reverse transcriptase." U) i7 U, T3 p2 N# i/ {
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Cell Surface Antigen Markers for Stem Cells

To examine the "stemness" of the porcine pulp SP cells, mRNA expression of cell surface antigen markers for stem cells were examined by real-time RT-PCR, since antibodies cross-reacting against CD are not available for porcine cells. CD105 and CD150 mRNA were highly expressed in SP cells compared with non-SP cells like other tissue stem cells (Table 2). Much lower expression of CD45, a gene typical of hematopoietic cells, was detected in SP cells than in non-SP cells. Expression of the differentiation markers Col1(I) and Col1(III) was much lower in SP cells than in non-SP cells. Markers of odontoblast differentiation Dspp and enamelysin were not detected in SP cells. These results suggest that SP cells contain an undifferentiated population, as do other tissue stem cells. CD146 is known to be highly expressed in smooth muscle cells and endothelial cells, as are CD31 and Vegfr2 in endothelial cells and NG2, desmin, and -smooth muscle cell actin in microvascular pericytes. The expression of -smooth muscle cell actin in SP cells was much lower than in non-SP cells. Endothelial markers CD31 and Vegfr2 were expressed in SP cells (Table 2).

Differentiation of SP Cells into Odontoblast Lineage
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Total cell numbers were rapidly decreased in BMP2-supplemented pellets and control non-supplemented pellets of pulp SP cells by day 1. They gradually decreased until day 10 and did not change thereafter. There was no difference in cell number between BMP2 pellets and control pellets (Fig. 3A). The alkaline phosphatase activity gradually increased after day 7 and was significantly higher on day 10 in BMP2 pellets than in control pellets (Fig. 3B). The collagenous extracellular matrix stained by Masson trichrome was stronger in the BMP2-supplemented pellets than those in control pellets starting on day 14 (Fig. 3C, 3D). Alizarin red staining showed mineralization in the BMP2 pellets on day 35 (Fig. 3E, 3F). We next investigated the functional effect of BMP2 application on the expression of 1(I) collagen and odontoblast markers Dspp, enamelysin, and Dmp1 on days 10, 14, and 28. Quantitative analysis by real-time RT-PCR showed that the expression of 1(I) collagen, Dspp, and enamelysin was significantly increased in the BMP2-supplemented pellet cultures compared with the control on days 14 and 28 (Fig. 3G). These results indicated that BMP2 supplement enhanced differentiation of pulp SP cells into odontoblasts.2 e! X5 ^$ i8 T! D

Figure 3. Differentiation of pulp SP cells into odontoblast lineage in the three-dimensional pellet culture. (A): The changes in cell numbers of porcine pulp SP cells in pellet culture treated with BMP2 compared with control. Each point is expressed as the mean ¡À SD of six determinations. (B): The changes in alkaline phosphatase activity in pellet culture treated with BMP2 and control. Each point is expressed as the mean ¡À SD of six determinations. In pellet cultures treated with BMP2, there was increased alkaline phosphatase activity compared with control. Morphological changes in the pellet were observed on day 14 (Masson trichrome stain). Shown are results of treatment with BMP2 (C) and untreated control (D). Scale bars = 50 µm. Pellets treated BMP2 had increased collagen matrix compared with control. Alizarin red staining shows mineralization of BMP2 supplemented pellet on day 35. Shown are BMP2-treated pellet (E) and control pellet (F). Scale bars = 50 µm. Note that mineralization was increased in the pellet supplemented with BMP2 compared with control. (G): Real-time reverse transcription-polymerase chain reaction, analysis of Dspp, enamelysin/matrix metalloproteinase 20, Dmp1, and 1(I)collagen expression in the pellet culture treated with BMP2 compared with control. The experiment was repeated four times, and one representative experiment is presented. Abbreviations: BMP, bone morphogenetic protein; Dmp1, dentin matrix protein 1; Dspp, dentin sialophosphoprotein; prim, primary; SP, side population.
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Multidifferentiation Capability of Pulp SP Cells
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The chondrogenic potential of SP cells was examined. The porcine pulp SP cells obtained from the fourth passage culture were maintained in pellet cultures for 45 days. The amount of cartilage proteoglycan stained with Alcian Blue was much greater in the pellets induced from SP cells compared with those from non-SP cells, primary pulp cells (Fig. 4A¨C4C), and noninduction pellet (data not shown). The expression of aggrecan and type II collagen mRNA was much stronger in the SP cells than that in the primary pulp cells. Almost 30% of SP cells were converted into chondrocytes. No expression was seen in the non-SP cells and uninduced SP cells (Fig. 4D).1 Z1 r3 m# y+ D5 U
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Figure 4. Induced differentiation of chondrogenic, adipogenic, and neurogenic lineages of dental pulp SP cells. Histological appearance of the pellet cultures of SP cells on day 28 (A), non-SP cells (B) and primary cells (C) stained by Alcian blue. Scale bars = 50 µm. (D): The expression of Col1 (II) and Aggrecan mRNA, markers of chondrogenic differentiation is prominent in SP cells compared with non-SP cells. (E): Oil red O staining of adipogenic differentiation on day 28. Note the presence of adipocytes in the SP cell fraction (E) but not in non-SP cells (F) and a few in primary cells (G). Scale bars = 100 µm. Markers of adipogenic differentiation aP2 and PPAR mRNA were observed only in SP cells (H). (I): Appearance of SP cells on day 3. Neurosphere formation on day 15 was seen only in SP cells (J) and not in non-SP cells (K) or primary cells (L). Scale bars = 500 µm. The expression of Sox2 mRNA confirms the neurosphere formation from SP cells only (M). A neuron-like cell induced after dissociation of the neurosphere from SP cells and plating on the gelatin-coated dish for 13 days (N). Immunostaining of neuromodulin (O) and the expression of neuromodulin and neurofilament mRNA were observed in SP cells (P). Scale bars = 5 µm. Abbreviations: aP2, adaptor protein 2; Col1, collagen 1; PPAR, peroxisome proliferator-activated receptor ; prim, primary; SP, side population.
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Next, the adipogenic potential of pulp SP cells was investigated in the third-passage cultures, which were cultured under the adipogenic condition for 28 days. The pulp SP cells showed intense staining with oil red O compared with non-SP and primary pulp cells (Fig. 4E¨C4G). aP2 and PPAR mRNA was expressed in SP cells on day 28, but not in uninduced SP cells, non-SP cells, or primary pulp cells (Fig. 4H).
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Finally, the neurogenic potential of SP cells was examined. The neurospheres, which were proliferating clusters of cells detached, were seen 15 days after neuronal induction of the SP cells from third-passage culture (Fig. 4I, 4J). Ninety percent of SP cells formed neurospheres. Sox2 mRNA was expressed in the neurospheres induced from SP cells but not in cells from non-SP cells or primary cells (Fig. 4M). Those neurosphere cells from SP cells were dissociated and seeded to the gelatin-coated dish to adhere and immunostained with antibody to neuromodulin after subconfluence (Fig. 4N). They were immunoreactive for neuromodulin in cytoplasm (Fig. 4O). The cells had rounded soma and a few of long, thin processes with neuronal morphology. These processes contacted each other at the end or soma and showed synapse-like morphology. The expression of neuromodulin and neurofilament mRNA was also detected in SP cells but not in uninduced SP cells (Fig. 4P).
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Dentin Regeneration by Transplantation of Bmp2-Transduced Cells In Vivo
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The autologous transplantation of the pellet (cell aggregate) of SP cells was performed on the amputated pulp of canine teeth 14 days after three-dimensional culture supplemented with BMP2. The pulp SP cells differentiated into osteodentinoblasts and secreted osteodentin matrix around them 1 month after surgery (Fig. 5A, 5B). A higher amount of osteodentin was observed in the BMP2 pellet than in the control pellet (Fig. 5A¨C5D). The in situ hybridization in serial sections showed mRNA expression of Dspp and enamelysin (Fig. 5E, 5F) in odontoblasts/osteodentinoblasts confined in osteodentin matrix stained with eosin (Fig. 5G) and Masson trichrome (Fig. 5H). There was no expression of Dspp or enamelysin in the control pellet (data not shown). These results suggest that the pulp SP cells responded to BMP2 to differentiate into odontoblasts/osteodentinoblasts.
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Figure 5. The autogenous transplantation in vivo of the pellet culture of canine side population (SP) cells on the canine amputated pulp (Masson trichrome stain). (A, B): Formation of OD in amputated pulp 1 month after autogenous transplantation of SP cell pellet treated with BMP2. (C, D): Small amount of osteodentin formation (OD) in the control. Scale bars = 500 µm (A, C), 50 µm (B, D). Shown is in situ hybridization analysis of canine tooth transplanted with SP cell pellet treated with BMP2 dentin sialophosphoprotein (E) and enamelysin (F). Adjacent sections were stained with H&E (G) and Masson trichrome (H). Note the odontoblasts/osteodentinoblasts (arrows) confined in osteodentin matrix. Scale bars = 50 µm. Abbreviations: BMP, bone morphogenetic protein; OD, osteodentin.( h+ z/ q) C8 s. s* C' \4 F) ?
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The present work describes the isolation and characterization of SP cells from dental pulp and their multilineage differentiation. SP cells were detected in adult pulp tissue from several species, including human, bovine, canine, and porcine species. They were purified by flow cytometry on the basis of efficient efflux of vital fluorescent dye Hoechst 33342 . These results suggest that the pulp SP cells have self-renewal capacity.
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The human pulp mesenchymal stem cells are currently demonstrating the similarity in positive expression of mesenchymal progenitor-related antigens, such as SH2, SH3, SH4, CD29, and CD166, to bone marrow mesenchymal stem cells .

Accumulating evidence indicates that tissue stem cells have the potential to differentiate into other unrelated organs. For example, the multipotency of bone marrow stem cells to engraft into cardiac muscle, vascular endothelium, liver, and skeletal muscle has been described  were not. It is possible that the SP cells were more in the hierarchy of stem cell lineage. The expression of odontoblast differentiation markers Dspp and enamelysin was increased in the BMP2-treated pellet cultures (cellular aggregates) compared with the untreated control, indicating that BMP2 enhanced differentiation of pulp SP cells into odontoblasts. Osteodentin matrix formation was also increased in the BMP2-treated pellet cultures compared with the control.% @" A% u- q- ~) t' m7 p5 t( g: m
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Finally, odontoblastic differentiation in vitro from pulp SP cells has to be extended to in vivo experiments to demonstrate osteodentin and tubular dentin formation. To experimentally investigate the dentinogenesis in vivo, a switch was made to the dog, as previously our laboratory has demonstrated the BMP-induced dentinogenesis in the canine teeth of dogs . Thus, the response of the SP cells to BMP2 was similar to that of the mixed population of cells in the canine dental pulp.
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The utility of pulp SP cells to induce in vivo dentin formation in dogs deserves additional comments with special reference to regeneration of tissues. SP cells derived from the liver and bone marrow were successfully used to regenerate liver . The results in the present investigation of dentin formation by SP cells in surgically amputated teeth, along with earlier results in liver, lung, and skeletal muscle, demonstrate the regenerative potential of SP cells. In conclusion, pulp-derived SP cells induced to differentiate with morphogens such as BMP2 may be of potential clinical utility in the cell therapy for endodontics and dentistry.$ {; o7 F! Q9 u  E) K

DISCLOSURES

The authors indicate no potential conflicts of interest.

ACKNOWLEDGMENTS
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We are grateful to Drs. A. Akamine and M. Ishikawa for help. This work was supported by Grants-in-Aid for Scientific Research 15390577 and 17390509 from the Ministry of Education, Science, Sports and Culture of Japan.' ~9 }/ R& C/ w, _: n
      【参考文献】' ]/ r  W: A  H, n# f& |

Reddi AH. Role of morphogenetic proteins in skeletal tissue engineering and regeneration. Nat Biotechnol 1998;16:247¨C252.- `' M: n% D" g- t

Nakashima M, Reddi AH. The application of bone morphogenetic proteins to dental tissue engineering. Nat Biotechnol 2003;21:1025¨C1032.

Nakashima M. Bone morphogenetic proteins in dentin regeneration for potential use in endodontic therapy. Cytokine Growth Factor Rev 2005;16:369¨C376." l. X4 f3 P' T! p! d
# w, v6 r  [( B" `2 O. r1 p( U. [# H
Yamamura T. Differentiation of pulpal cells and inductive influences of various matrices with reference to pulpal wound healing. J Dent Res 1985;64:530¨C540.7 N9 q$ w# K0 h/ R+ `- W7 d
2 I' _0 m4 r5 j# h3 q& L0 s
Nakashima M, Establishment of primary cultures of pulp cells from bovine permanent incisors. Arch Oral Biol 1991;36:655¨C663.! H* x+ O5 C% l
: L  w0 z( H: v; x; f2 A
Kuo MY, Lan WH, Lin SK et al. Collagen gene expression in human dental pulp cell cultures. Arch Oral Biol 1992;37:945¨C952.
' R$ e, s  l$ [* I7 B
Tsukamoto Y, Fukutani S, Shin-Ike T et al. Mineralized nodule formation by cultures of human dental pulp-derived fibroblasts. Arch Oral Biol 1992;37:1045¨C1055.
; s# \1 M3 S1 f; n7 @
Nakashima M, Nagasawa H, Yamada Y et al. Regulatory role of transforming growth factor-&szlig;, bone morphogenetic protein-2, and protein-4 on gene expression of extracellular matrix proteins and differentiation of dental pulp cells. Dev Biol 1994;162:18¨C28.

Kettunen P, Karavanova I, Thesleff I. Responsiveness of developing dental tissues to fibroblast growth factors: Expression of splicing alternatives of FGFR1, -2, -3, and of FGFR4; and stimulation of cell proliferation by FGF-2, -4, -8, and -9. Dev Genet 1998;22:374¨C385.
8 G( t, `9 A6 u8 a
Shiba H, Fujita T, Doi N et al. Differential effects of various growth factors and cytokines on the syntheses of DNA, type I collagen, laminin, fibronectin, osteonectin/secreted protein, acidic and rich in cysteine (SPARC), and alkaline phosphatase by human pulp cells in culture. J Cell Physiol 1998;174:194¨C205.
8 F. f! Z+ ?) E$ e' }% @) [; K
Yokose S, Kadokura H, Tajima Y et al. Establishment and characterization of a culture system for enzymatically released rat dental pulp cells. Calcif Tissue Int 2000;66:139¨C144.

Gronthos S, Mankani M, Brahim J et al. Postnatal human dental pulp stem cells (DPSCs) in vitro and in vivo. Proc Natl Acad Sci U S A 2000;97:13625¨C13630.

Gronthos S, Brahim J, Li W et al. Stem cell properties of human dental pulp stem cells. J Dent Res 2002;81:531¨C535.8 j( z1 e' a# G( o- L' z; T, D

Pierdomenico L, Bonsi L, Calvitti M et al. Multipotent mesenchymal stem cells with immunosuppressive activity can be easily isolated from dental pulp. Transplantation 2005;80:836¨C842.
% r$ ?. k% c; }
Miura M, Gronthos S, Zhao M et al. Stem cell from human exfoliated deciduous teeth. Proc Natl Acad Sci U S A 2003;100:5807¨C5812.3 f; ?6 X- p& R8 O' P# b: Y
5 }8 ^7 \7 u( z: l: i
Goodell MA, Brose K, Paradis G et al. Isolation and functional properties of murine hematopoietic stem cells that are replicating in vivo. J Exp Med 1996;183:1797¨C1806.

Jackson KA, Mi T, Goodell MA. Hematopoietic potential of stem cells isolated from murine skeletal muscle. Proc Natl Acad Sci U S A 1999;96:14482¨C14486." D1 f$ k- g% a

Asakura A, Rudnicki MA. Side population cells from diverse adult tissues are capable of in vitro hematopoietic differentiation. Exp Hematol 2002;30:1339¨C1345.. j# E; c5 o5 z  B. [
8 @8 }" m$ F8 t: d
Hulspas R, Quesenberry PJ. Characterization of neurosphere cell phenotypes by flow cytometry. Cytometry 2000;40:245¨C250.
: u$ ?" G7 K6 G' Y/ V2 [
Shimano K, Satake M, Okaya A et al. Hepatic oval cells have the side population phenotype defined by expression of ATP-binding cassette transporter ABCG2/BCRP1. Am J Pathol 2003;163:3¨C9.! ~2 W' c: v/ X2 Q' ^* S& w5 G5 q

Lechner A, Leech CA, Abraham EJ et al. Nestin-positive progenitor cells derived from adult human pancreatic islets of Langerhans contain side population (SP) cells defined by expression of the ABCG2 (BCRP1) ATP-binding cassette transporter. Biochem Biophys Res Commun 2002;293:670¨C674.( f, y7 m( F9 U4 |- m

Summer R, Kotton DN, Sun X et al. Side population cells and Bcrp1 expression in lung. Am J Physiol Lung Cell Mol Physiol 2003;285:L97¨CL104.

Martin CM, Meeson AP, Robertson SM et al. Persistent expression of the ATP-binding cassette transporter, Abcg2, identifies cardiac SP cells in the developing and adult heart. Dev Biol 2004;265:262¨C275.# ~' U& Z7 f; s) C7 t

Iwatani H, Ito T, Imai E et al. Hematopoietic and nonhematopoietic potentials of Hoechst(low)/side population cells isolated from adult rat kidney. Kidney Int 2004;65:1604¨C1614.! D  ?" B6 I  T. |' U: u
5 P  E# u7 p/ ?" K* \
Zhou S, Schuetz JD, Bunting KD et al. The ABC transporter Bcrp1/ABCG2 is expressed in a wide variety of stem cells and is a molecular determinant of the side-population phenotype. Nat Med 2001;7:1028¨C1034.

Matsuzaki Y, Kinjo K, Mulligan RC et al. Unexpectedly efficient homing capacity of purified murine hematopoietic stem cells. Immunity 2004;20:87¨C93.. k  `$ l8 X$ X; r! a, q1 G. A
7 i3 M" K% ?  Y0 Y" y
Nakashima M. Induction of dentin formation on canine amputated pulp by recombinant human bone morphogenetic protein (BMP)-2 and -4. J Dent Res 1994;73:1515¨C1522.$ `% L& X1 v, w
  _) v9 q; W% H8 V9 z2 C
Rutherford RB, Gu K. Treatment of inflamed ferret dental pulps with recombinant bone morphogenetic protein-7. Eur J Oral Sci 2000;108:202¨C206.

Smith AJ, Lesot H. Induction and regulation of crown dentinogenesis: Embryonic events as a template for dental tissue repair? Crit Rev Oral Biol Med 2001;12:425¨C437.5 q2 K) K  s/ O
6 w( i, R; p+ r+ K; S3 x
Varga AC, Wrana JL. The disparate role of BMP in stem cell biology. Oncogene 2005;24:5713¨C5721.
7 Q- o4 e: d" N8 j# A7 ^
Iohara K, Nakashima M, Ito M et al. Dentin regeneration by dental pulp stem cell therapy with recombinant human bone morphogenetic protein 2. J Dent Res 2004;83:590¨C595.

Platt KA, Michaud J, Joyner AL. Expression of the mouse Gli and Ptc genes is adjacent to embryonic sources of hedgehog signals suggesting a conservation of pathways between flies and mice. Mech Dev 1997;62:121¨C135.( o8 B3 N1 e% v$ ]5 L

Lowry OH, Roberts NR, Wu ML et al. The quantitative histochemistry of brain. II. Enzyme measurement. J Biol Chem 1954;207:19¨C37.
7 u' O" H7 c$ `) g9 V, Z5 r
Pittenger MF, Mackay AM, Beck SC et al. Multilineage potential of adult human mesenchymal stem cells. Science 1999;284:143¨C147.  C# F4 n/ D) x+ N: D" x$ s

Goodell MA, McKinney-Freeman S, Camargo FD. Isolation and characterization of side population cells. Methods Mol Biol 2005;290:343¨C352.
1 q1 l3 J& I4 T( ?/ N: k2 q& M
Park IK, Qian D, Kiel M et al. Bmi-1 is required for maintenance of adult self-renewing haematopoietic stem cells. Nature 2003;423:302¨C305.
, k& _4 x; T8 u
Rao M. Conserved and divergent paths that regulate self-renewal in mouse and human embryonic stem cells. Dev Biol 2004;275:269¨C286.

Lee HW, Blasco MA, Gottlieb GJ et al. Essential role of mouse telomerase in highly proliferative organs. Nature 1998;392:569¨C574.  t6 j& W- q. U0 l# l
7 n9 m% m: p, |# I, z2 J
Autexier C, Lue NF. The structure and function of telomerase reverse transcriptase. Annu Rev Biochem 2006;75:493¨C517.9 C: P( E* u1 M$ H; c) M

Goodell MA, Rosenzweig M, Kim H et al. Dye efflux studies suggest that hematopoietic stem cells expressing low or undetectable levels of CD34 antigen exist in multiple species. Nat Med 1997;3:1337¨C1345.- H. C& I3 K4 J+ P! W, @
3 ?  x- E/ n8 L) `$ [2 @! ^
Scharenberg CW, Harkey MA, Torok-Storb B. The ABCG2 transporter is an efficient Hoechst 33342 efflux pump and is preferentially expressed by immature human hematopoietic progenitors. Blood 2002;99:507¨C512.1 W! }$ D5 `+ F/ \/ V6 t2 l. ^, I
: k% s* u. f, L& N/ V
Dekaney CM, Rodriguez JM, Graul MC et al. Isolation and characterization of a putative intestinal stem cell fraction from mouse jejunum. Gastroenterology 2005;129:1567¨C1580., A) u) I' l+ H
# E, S" N3 ~! Z5 j6 m
Musina RA, Bekchanova ES, Sukhikh GT. Comparison of mesenchymal stem cells obtained from different human tissues. Cell Tech Biol Med 2005;1:504¨C509.) R5 i6 k2 x1 o; j% K
6 x1 |& x: F& k6 M/ [
Kiel MJ, Yilmaz OH, Iwashita T et al. SLAM family receptors distinguish hematopoietic stem and progenitor cells and reveal endothelial niches for stem cells. Cell 2005;121:1109¨C1121.
9 a$ a) P2 c3 O- |8 Q
Shi S, Gronthos S. Perivascular niche of postnatal mesenchymal stem cells in human bone marrow and dental pulp. J Bone Miner Res 2003;18:696¨C704.; I! C+ c& x% W! d& I6 }' W9 Q% m
4 @4 [' S+ ]+ a$ i# a
Jackson KA, Majka SM, Wang H et al. Regeneration of ischemic cardiac muscle and vascular endothelium by adult stem cells. J Clin Invest 2001;107:1395¨C1402.
$ ~  |& _* X- e7 p
Camargo FD, Green R, Capetanaki Y et al. Single hematopoietic stem cells generate skeletal muscle through myeloid intermediates. Nat Med 2003;9:1520¨C1527.

Kotton DN, Summer R, Fine A. Lung stem cells: New paradigms. Exp Hematol 2004;32:340¨C343.
6 g, `; y9 C; l; u
Wulf GG, Luo KL, Jackson KA et al. Cells of the hepatic side population contribute to liver regeneration and can be replenished with bone marrow stem cells. Haematologica 2003;88:368¨C378.
5 {8 e9 l8 g' |8 l- z2 q; x- H
Reyes M, Lund T, Lenvik T et al. Purification and ex vivo expansion of postnatal human marrow mesodermal progenitor cells. Blood 2001;98:2615¨C2625., p$ S& O8 U; @+ D

Caplan AI. Mesenchymal stem cells: Cell-based reconstructive therapy in orthopedics. Tissue Eng 2005;11:1198¨C1211.
- i2 t3 i$ U$ u$ ~( ]2 ]1 P
Nakashima M. Induction of dentine in amputated pulp of the dogs by recombinant human bone morphogenetic proteins-2 and -4 with collagen matrix. Arch Oral Biol 1994;39:1085¨C1089.5 U; P+ v7 O* g% y  X8 s: @

Rutherford RB, Wahle J, Tucker M et al. Induction of reparative dentin formation in monkeys by recombinant human osteogenic protein-1. Arch Oral Biol 1993;38:571¨C576.4 p  Y9 O6 W; z8 X+ }; |4 i
8 v! k4 i6 Q2 X/ g
Rutherford RB, Spangberg L, Tucker M et al. The time-course of the induction of reparative dentin formation in monkeys by recombinant human osteogenic protein-1. Arch Oral Biol 1994;39:833¨C838./ q- y# S& H  u9 c

Abe S, Lauby G, Boyer C et al. Transplanted BM and BM side population cells contribute progeny to the lung and liver in irradiated mice. Cytotherapy 2003;5:523¨C533." y4 `* c/ l7 R0 T/ _3 m
6 P4 i9 W% v, }
MacPherson H, Keir P, Webb S et al. Bone marrow-derived SP cells can contribute to the respiratory tract of mice in vivo. J Cell Sci 2005;118:2441¨C2450.

Montanaro F, Liadaki K, Volinski J et al. Skeletal muscle engraftment potential of adult mouse skin side population cells. Proc Natl Acad Sci U S A 2003;100:9336¨C9341.

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