Department of Oral Disease Research, National Institute for Longevity Sciences, National Center for Geriatrics and Gerontology, Obu, Aichi, Japan
Department of Oral Disease Research, National Institute for Longevity Sciences, National Center for Geriatrics and Gerontology, Obu, Aichi 474-8522, Japan. Telephone: 0562-44-5651 ext. 5063; Fax: 0562-46-8684
Cell therapy with stem cells and endothelial progenitor cells (EPCs) to stimulate vasculogenesis as a potential treatment for ischemic disease is an exciting area of research in regenerative medicine. EPCs are present in bone marrow, peripheral blood, and adipose tissue. Autologous EPCs, however, are obtained by invasive biopsy, a potentially painful procedure. An alternative approach is proposed in this investigation. Permanent and deciduous pulp tissue is easily available from teeth after extraction without ethical issues and has potential for clinical use. We isolated a highly vasculogenic subfraction of side population (SP) cells based on CD31 and CD146, from dental pulp. The CD31−;CD146− SP cells, demonstrating CD34+ and vascular endothelial growth factor-2 (VEGFR2)/Flk1+, were similar to EPCs. These cells were distinct from the hematopoietic lineage as CD11b, CD14, and CD45 mRNA were not expressed. They showed high proliferation and migration activities and multilineage differentiation potential including vasculogenic potential. In models of mouse hind limb ischemia, local transplantation of this subfraction of SP cells resulted in successful engraftment and an increase in the blood flow including high density of capillary formation. The transplanted cells were in proximity of the newly formed vasculature and expressed several proangiogenic factors, such as VEGF-A, G-CSF, GM-CSF, and MMP3. Conditioned medium from this subfraction showed the mitogenic and antiapoptotic activity on human umbilical vein endothelial cells. In conclusion, subfraction of SP cells from dental pulp is a new stem cell source for cell-based therapy to stimulate angiogenesis/vasculogenesis during tissue regeneration.
Disclosure of potential conflicts of interest is found at the end of this article.
Author contributions: K.I.: conception and design, provision of study material or patients, collection and/or assembly of data, data analysis and interpretation, manuscript writing, final approval of manuscript; L.Z.: provision of study material or patients, collection and/or assembly of data, data analysis and interpretation; H.W. and M.I.: provision of study material or patients, collection and/or assembly of data, data analysis and interpretation; J.N. and H.W.: provision of study material or patients; H.N.: financial support, provision of study material or patients; T.I.: provision of study material or patients; K.M.: financial support, final approval of manuscript; M.N.: conception and design, financial support, provision of study material or patients, collection and/or assembly of data, data analysis and interpretation, manuscript writing, final approval of manuscript.
The potential application of stem/progenitor cells to treat ischemia has generated genuine excitement in regenerative medicine [1, 2]. Endothelial progenitor cells (EPCs) are of utility to achieve corrective vasculogenesis to treat cardiac, cerebral, and limb ischemia. The characteristic features of EPCs are CD34-, CD133-, and vascular endothelial growth factor-2 (VEGFR2)-positive cells [3, –5]. In both embryonic and adult human aorta CD34+;CD31− cells differentiate into endothelial cells [6, 7]. Human adipose tissue-derived stromal-vascular fraction contains CD34+;CD31− cells with potential to differentiate into endothelial cells .
The human dental pulp is a highly vascular tissue that is enriched in stem/progenitor cells [9, –11]. The ready availability of dental pulp from teeth obtained during orthodontic treatment and extracted third molars circumvents any ethical concerns and is a definite advantage. In addition, their immunosuppressive properties  may be useful for allogeneic transplantation. Recent work in our laboratory identified stem/progenitor cells in porcine dental pulp by the use of fluorescent Hoechst dye 33342 to isolate side population (SP) cells . The subfractionation of SP cells that were CD34+;VEGFR2/Flk1+ into CD31−;CD146− and CD31+;CD146− cells revealed distinct properties, the former differentiated into endothelial cells in vitro. In addition, the CD31−;CD146− SP cell subfraction caused functional revascularization of hind limb ischemia in vivo and is the topic of this investigation.
Materials and Methods
Isolation by Flow Cytometry
The primary pulp cells from porcine tooth germ were separated and labeled with Hoechst 33342 (Sigma, St. Louis, http://www.sigmaaldrich.com) as previously described . Then the cells were preincubated with mouse BD Fc Block (BD Biosciences, San Jose, CA, http://www.bdbiosciences.com) for 30 minutes at 4°C to reduce nonspecific binding. The cells were further incubated with the mouse IgG1 negative control (MCA928) (AbD Serotec Ltd., Oxford, U.K., http://www.serotec.com), mouse IgG1 negative control (phycoerythrin [PE]) (MCA928PE) (AbD Serotec Ltd.), mouse IgG1 negative control (fluorescein isothiocyanate [FITC]) (MCA928FITC) (AbD Serotec Ltd.), mouse anti-porcine CD31 (PE) (LCI-4) (AbD Serotec Ltd.), and mouse anti-human CD146 (FITC) (OJ79c) (AbD Serotec Ltd.) in phosphate-buffered saline (PBS) with 20% fetal bovine serum (Invitrogen Corp., Carlsbad, CA, http://www.invitrogen.com) for 60 minutes at 4°C. Those were resuspended in HEPES buffer containing 2 μg/ml propidium iodide (Sigma). Analysis/sorting of cells was performed using a flow cytometer JSAN (Bay Bioscience, Kobe, Japan, http://www.baybio.co.jp).
We investigated the most suitable supplement of culture medium, EBM2 (Cambrex Bio Science Walkersville, Inc., Walkersville, MD, http://www.cambrex.com), including growth factors such as basic fibroblast growth factor (bFGF), insulin-like growth factor 1 (IGF1), epidermal growth factor (EGF), and vascular endothelial growth factor-A (VEGF-A) (Cambrex Bio Science, Inc.). The optimal concentration of porcine serum (JRH Biosciences, Inc., Lenexa, KS, http://www.jrhbio.com) was also determined to maintain all the sorted cells, CD31−;CD146− SP cells, CD31+;CD146− SP cells, and CD31+;CD146+ SP cells. Each cell fraction was plated into 35-mm collagen type I-coated dishes (Asahi Technoglass Corp., Funabashi, Japan, http://www.atgc.co.jp) in EBM2 supplemented with suitable growth factors. Medium was changed every 4–5 days. Once cells reached 50%–60% confluence, they were detached by incubation with 0.02% EDTA at 37°C for 10 minutes and subcultured at a 1:4 dilution under the same conditions for more than 20 passages.
Expression of Cell Surface Markers
To characterize the phenotype of the CD31− SP cells and CD31+ SP cells, the freshly isolated cells were double-stained with each antibody against CD146 (FITC) (OJ79c) (AbD Serotec Ltd.), CD11b (biotin) (M1/70) (BD Biosciences), CD14 (Alexa Flour 647) (TuK4) (AbD Serotec Ltd.), CD90 (Alexa Flour 647) (F15–42-1) (AbD Serotec Ltd.), CD117/c-kit (allophycocyanin [APC]) (A3C6E2) (Miltenyi Biotec, Bergisch Gladbach, Germany, http://www.miltenyibiotec.com), CD150 (FITC) (A12) (AbD Serotec Ltd.), CD271 (APC) (ME20.4–1H4) (Miltenyi Biotec) together with CD31 antibody after Hoechst 33342 labeling, and analyzed by flow cytometry. Streptavidin (PE-Cy7) (eBioscience, San Diego, http://www.ebioscience.com) was used for a secondary antibody of CD11b. In case of CD34 and VEGFR2/Flk1, the isolated CD31− SP cells and CD31+ SP cells were cultured for 5 days to remove the CD31 antibody bound on the cell surface, and the expanded secondary cells were immunolabeled with antibodies against CD34 (QBEnd-10) (Immunotech, Cedex, France, http://www.beckmancoulter.com/products/pr_immunology.asp) and VEGFR2/Flk1 (30457) (Upstate, Spartanburg, SC, http://www.upstate.com), respectively, and goat anti-rabbit IgG (Alexa 488) (Molecular Probes, Eugene, OR, http://probes.invitrogen.com) as the secondary antibody.
Real-Time Reverse Transcription-Polymerase Chain Reaction Analysis for Cell Surface Markers and Stem Cell Markers
To characterize the phenotype of the cell population, total RNA was extracted from the freshly sorted CD31−;CD146− SP cells and CD31+;CD146− SP cells using Trizol (Invitrogen Corp.). The number of these cells was normalized to 5 × 104 cells in each experiment. First-strand cDNA syntheses were performed from total RNA by reverse transcription using the SuperScript II preamplification system (Invitrogen Corp.). Real-time reverse transcription-polymerase chain reaction (RT-PCR) amplifications were performed at 95°C for 10 seconds, 62°C for 15 seconds, and 72°C for 8 seconds using macrophage/mononuclear cell markers, CD11b and CD14, hematopoietic cell marker, CD45, angioblast marker, CD133, neuronal progenitor marker, Sox2, and stem cell markers, CXCR4, Bcrp1, Stat3, Bmi1, and Tert (supplemental online Table 1 and Iohara et al. ) labeled with Light Cycler-Fast Start DNA master SYBR Green I (Roche Diagnostics, Pleasanton, CA, http://www.roche-applied-science.com) in Light Cycler (Roche Diagnostics). The design of the oligonucleotide primers was based on published porcine cDNA sequences. When porcine sequences were not available, human sequences were used. The RT-PCR products were subcloned into pGEM-T Easy vector (Promega, Madison, WI, http://www.promega.com) and confirmed by sequencing based on published cDNA sequences. The expression in CD31−;CD146− SP cells and CD31+;CD146− SP cells was compared with porcine pulp tissue after normalizing with β-actin.
Proliferation and Migration Assay
To measure proliferation of CD31−;CD146− SP cells compared with CD31+;CD146− SP cells and CD31+;CD146+ SP cells, these cells at third passage at the 103 cells per 96 well were cultured in EBM2 supplemented with 0.2% bovine serum albumin (Sigma) and bFGF (50 ng/ml; Invitrogen Corp.), VEGF-A (50 ng/ml; Peprotech Ltd., London, http://www.peprotech.com), EGF (50 ng/ml, Invitrogen Corp.), stromal cell-derived factor 1 (SDF1; 50 ng/ml) (Acris, Hiddenhausen, Germany, http://www.acris-antibodies.com), and IGF1 (50 ng/ml; Peprotech Ltd.). Tetra-color one (10 μl) (Seikagaku Kogyo, Co., Tokyo, http://www.seikagaku.co.jp) was added to the 96-well plate, and cell numbers were measured using spectrophotometer at 450 nm absorbance at 0, 12, 24, 36, 48, and 72 hours of culture. Wells without cells served as negative controls.
To examine the migration activity of CD31−;CD146− SP cells compared with CD31+;CD146− SP cells and CD31+;CD146+ SP cells, 5 × 104 cells were seeded on PET-membrane (BD Biosciences) inserted into 24-well assembly containing EBM2 supplemented with VEGF-A (Peprotech Ltd.) at the final concentration of 0, 5, 10, and 100 ng/ml. Twenty-four hours later, cells that passed through the membrane were counted after detaching the cells from the membrane with 0.2% trypsin-0.02% EDTA. The migration activity was also examined in the culture with SDF1 (Acris) or granulocyte colony-stimulating factor (G-CSF) (Peprotech Ltd.) and compared with VEGF-A at the final concentration of 50 ng/ml.
Induced Chondrogenic, Adipogenic, Neurogenic, and Odontogenic Differentiation
The differentiation of pulp CD31−;CD146− SP cells into adipogenic, chondrogenic, neurogenic, and odontoblastic cells was determined and compared with CD31+;CD146− SP cells by the previously described methods . Odontogenic potential in vivo was confirmed 28 days after autologous transplantation in a canine amputated model of pulp injury [13; K.I. manuscript submitted for publication]. The cells were transplanted in the form of a pellet (cellular aggregates) with collagen type I and type III after 1,1′-dioetadeeyl-3,3,3′,3′-tetramethylindocarboeyanine perchlorate (DiI) labeling on the amputated pulp.
Endothelial Differentiation In Vitro
The CD31−;CD146− SP cells, CD31+;CD146− SP cells, and CD31+;CD146+ SP cells at the third to fifth passage were seeded on the matrigel (BD Biosciences) in EGM2. Network formation was observed after 24-hour cultivation. The paraffin-embedded sections on day 10 were observed by in situ hybridization analysis  using porcine CEACAM1, CD146, and occludin antisense probes. The DIG-labeled probes were constructed out of the plasmids after subcloning the RT-PCR products into pGEM-T Easy vector using each primer pair (supplemental online Table 2). CD31−;CD146− SP cells were cultured for 14 days and then subcultured. The immunocytochemical analyses were performed with primary antibodies, anti-von Willebrand factor (vWF) (1:20) (H-300) (Santa-Cruz, Biotech, Santa Cruz, CA, http://www.scbt.com), anti-CD31 (1:20) (LCI-4) (AbD Serotec Ltd.), and anti-vascular endothelial (VE)-cadherin/CD144 (1:50) (123) (Acris). They were further stained with goat anti-mouse IgG-horseradish peroxidase (HRP) (Invitrogen Corp.) enhanced with TSA system rhodamine-conjugated tyramide (Invitrogen Corp.), goat anti-rabbit IgG-Alexa 568 (Invitrogen Corp.), and goat anti-rat IgG-HRP (GE Healthcare U.K. Ltd., Buckinghamshire, U.K., http://www.gehealthcare.com) enhanced with TSA system Alexa 488-conjugated tyramide (Invitrogen Corp.). The differential changes of expression were analyzed by a fluorescence microscope IX 71 (Olympus, Tokyo http://www.olympus-global.com) after counterstaining with Hoechst 33342.
To detect the endothelial function of histamine-mediated release of vWF, CD31−;CD146− SP cells at the third passage were cultured in EGM2 for 14 days. They were further incubated with 10 μM histamine (Sigma) for 60 minutes and stained with an antibody against vWF. The uptake of acetylated-low-density lipoprotein (LDL) (Biomedical Technologies, Inc., Stoughton, MA, http://www.btiinc.com) as an index of endothelial function was examined. The cells derived from CD31−;CD146− SP cells (104 cells/ml) at the third passage were cultured in EGM2 for 14 days. On day 17 and day 21, DiI-acetylated-LDL (Biomedical Technologies, Inc.) was added at the final concentration of 10 μg/ml for 2 hours.
Transplantation into Mouse Ischemic Hind Limbs
The potential of neovascularization of porcine pulp CD31−;CD146− SP cells and CD31+;CD146− SP cells was examined in a murine model of hind limb ischemia in 5-week-old severe combined immunodeficient mice (CB17; CLEA, Tokyo, http://www.clea-japan.com). PBS injection was also used as control. After inhalation anesthesia with isoflurane, the left proximal portion of femoral artery including the superficial and the deep branches and the distal portion of the saphenous artery were ligated as previously described . After 24 hours, 100 μl of PBS with or without 1 × 106 freshly detached CD31−;CD146− SP cells or CD31+;CD146− SP cells at the third to fifth passage with DiI (Sigma) labeling was injected intramuscularly. Laser Doppler imaging (Perimed AB, Stockholm, Sweden, http://www.perimed.se) was performed 14 days after cell transplantation. The blood vessels were decorated with perfused FITC-conjugated dextran (Sigma). Neovascularization and engraftment of the transplanted cells into the hind limb were examined by confocal microscope using FLUO VIEW FV1000 (Olympus) instrument. Three-dimensional structures were reconstructed by METAMORPH (Molecular Devices, Sunnyvale, CA, http://www.moleculardevices.com) and IMARIS (Bitplane AG, Zurich, Switzerland, http://www.bitplane.com). Isolated muscle tissues of ischemic hind limb were fixed and serial cryotome sections (12 μm) were stained with Fluorescein Griffonia (Bandeiraea) Simplicifolia Lectin 1/fluorescein-galanthus nivalis (snowdrop) lectin (20 μg/ml; Vector Laboratories, Inc., Youngstown, OH, http://www.vectorchemicals.com) to monitor the presence and localization of the transplanted cells in relation to newly formed blood vessels using a fluorescence microscope BIOREVO, BZ-9000 (KEYENCE, Osaka, Japan, http://www.keyence.co.jp). Microscopic digital images of six sections of every 120 μm were scanned in a frame composed of 500 μm × 380 μm rectangle and statistical analyses was performed using software, Dynamic cell count, BZ-HIC (KEYENCE). The experiment was repeated three times. The ultrathin sections of the hamstring muscles embedded in Epon were examined with an electron microscope (model 1010; JEOL, Tokyo, http://www.jeol.com) as previously described . The cryotome sections obtained on day 7 were observed by in situ hybridization analysis  using porcine G-CSF, granulocyte-macrophage colony-stimulating factor (GM-CSF), matrix metalloprotease (MMP)1, MMP3, VEGF-A, and CXCR4 antisense probes. The probes were constructed out of the plasmids after subcloning the PCR products using the same primers designed for real-time RT-PCR (supplemental online Table 1).
Analysis of Gene Expression of Cytokines and Enzymes by Real-Time RT-PCR
The mRNA expression of angiogenic (VEGF-A, hepatocyte growth factor [HGF]), chemotactic (G-CSF, GM-CSF, MCP1, CXCL2, MDCFI, MDCFII, TF), and proinflammatory (interleukin [IL]-1α, IL-6, IL-12A, leukemia inhibitory factor [LIF]) cytokines and matrix-degrading enzymes (MMP1, MMP2, MMP3, MMP9) and others (Arginase I, Lipoprotein lipase, Dipeptidyl peptidase IV, Hyaluronan synthase 2 [SHAS2], parathyroid hormone-like hormone [PTHLH], Integrin β-like protein 1, GP38K, and Calcitonin receptor-stimulating peptide [CRSP]) (supplemental online Table 1) was compared in pulp CD31−;CD146− SP cells with those in pulp CD31+;CD146− SP cells at third passage of culture by real-time RT-PCR. The RT-PCR products were confirmed by sequencing based on published cDNA sequences. The expression was compared with porcine pulp tissue after normalizing with β-actin.
Proliferation and Antiapoptotic Effect of Pulp CD31−;CD146− SP Cell-Conditioned Medium
At 50% confluence, culture medium was switched to EBM2 and the conditioned media from CD31−;CD146− SP cells and CD31+;CD146− SP cells were collected 48 hours later. Human umbilical vein endothelial cells (HUVECs) (KURABO Industries, Osaka, Japan, http://www.kurabo.co.jp) were cultured in EGM2 containing 2% fetal bovine serum (FBS) for 24 hours and further in EBM2 containing 0.2% bovine serum albumin (BSA) for 24 hours. Then, the medium was changed into EBM2 containing 2% FBS supplemented with 20% of conditioned medium from pulp CD31−;CD146− SP cells and CD31+;CD146− SP cells. Cell numbers were measured by Tetra-color one. The proliferation effects of these conditioned media were compared with those of MMP3 (Millipore, Billerica, MA, http://www.millipore.com), VEGF-A (Peprotech Ltd.), G-CSF (Peprotech Ltd.), and GM-CSF (Peprotech Ltd.) at final concentration of 50 ng/ml. Data were expressed as means ± SD at four determinations. To assess the effect of the conditioned medium of CD31−;CD146− SP cells on apoptosis, HUVECs at passage six or less were grown in EGM2 in 35-mm dish for 3 days and then incubated with 100 nM staurosporine (Sigma) in EBM2 supplemented with 20% of conditioned medium from CD31−;CD146− SP cells and CD31+;CD146− SP cells. As controls, MMP3, VEGF-A, G-CSF, and GM-CSF were added to the EBM2. After 8 hours, HUVECs were harvested, and the cell suspensions were treated with Annexin V-FITC (Roche Diagnostics) and propidium iodide for 15 minutes, and analyzed by flow cytometry. Data were expressed as means ± SD at three determinations.
Data are reported as means ± SD. P values were calculated using the unpaired Student's t test. The number of replicates in each experiment is indicated in the figure legends.
Isolation of CD31−; CD146− SP Cells from Dental Pulp
Flow cytometric analyses of the SP cells from porcine adult pulp tissues were performed using antibodies against CD31 and CD146 to isolate further distinct subpopulations. CD31 is known to be highly expressed in endothelial progenitor cells and endothelial cells and CD146, in smooth muscle cells and endothelial cells. The CD31− population was devoid of CD146+ and represented 50% of total SP cells. The CD31+ population contained both CD146− and CD146+ cells, and CD31+;CD146+ and CD31+;CD146− represented 2% and 48% of total SP cells, respectively (Fig. 1A, 1B). The CD31−;CD146− SP cells contained two types of cells: a stellate cell with long processes and a spindle-shaped cell. The stellate cells contained a large nucleus with nucleoli. The spindle-shaped cell was neuron-like cell with a long slender process and sparse cytoplasm (Fig. 1C). CD31+;CD146− SP cells were endothelial-like cells, which grew clonally and were contact inhibited (Fig. 1D). CD31+;CD146+ SP cells were irregularly shaped with short processes (Fig. 1E). To maintain the phenotype of CD31−;CD146− SP cells EBM2 supplemented with IGF1, EGF, and 10% porcine serum was used, and EBM2 with bFGF, VEGF, and 2% porcine serum was used for CD31+;CD146− SP cells.
The single CD31−;CD146− SP cell plated in 35-mm collagen type I-coated dish formed a colony in 8 days (data not shown), showing colony formation activity of these cells. The efficiency of attachment and growth of CD31−;CD146− SP cells was estimated to be 8.9%, whereas for CD31+;CD146− SP cells it was 7.7%. Limiting dilution analysis at third passage culture showed that the frequency of colony-forming unit in CD31−;CD146− SP cells was estimated to be 80%, whereas that in CD31+;CD146− SP cells was 30%.
Cell Surface Antigen Markers for Stem Cells
To characterize the “stemness,” hematopoietic lineage, and endothelial lineage of the porcine pulp CD31− SP cells and CD31+ SP cells, cell surface antigen markers were examined by flow cytometry and compared with CD31− SP cells and CD31+ SP cells derived from porcine bone marrow. Markers of monocyte/macrophage origin, CD11b and CD14 were negative in pulp CD31− SP cells and CD31+ SP cells. Few pulp CD31− SP cells and CD31+ SP cells expressed CD90, and none expressed CD117/c-kit or CD150 (supplemental online Table 3), whereas CD31− SP cells derived from porcine bone marrow expressed those at the ratio of 0%, 100%, and 1%, respectively (data not shown). Pulp CD31− SP cells expressed CD34 and VEGFR2/ Flk1 mRNA and proteins (supplemental online Tables 3, 4) and no CD133 mRNA (supplemental online Table 4), suggesting that pulp CD31−;CD146− SP cells were similar but not identical to bone marrow-derived endothelial progenitor cells. It is noteworthy that 94% of pulp CD31−;CD146− SP cells expressed CD271/LNGFR, a marker of neuronal progenitor cells (supplemental online Table 3). Sox2 mRNA was highly expressed in CD31−;CD146− SP cells compared with CD31+;CD146− SP cells (supplemental online Table 4), suggesting a neurogenic population in the former.
Expression of stem cell markers CXCR4, Stat3, Bmi1, and Tert mRNA was 8, 1.3, 1.5, and 37.5 times higher, respectively, in CD31−;CD146− SP cells than those in CD31+;CD146− SP cells detected by real-time RT-PCR (supplemental online Table 4). Lack of CD11b, CD14, and CD45 mRNA expression in CD31−;CD146− SP cells was also confirmed (supplemental online Table 4), suggesting that they are neither of monocyte/macrophage origin nor of hematopoietic lineage.
Proliferation Activity and Chemotaxis of CD31−;CD146− SP Cells
We first examined the proliferation activity of pulp CD31−;CD146− SP cells and compared them with CD31+;CD146− SP cells and CD31+;CD146+ SP cells. In the presence of 0.2% BSA without serum, all three cell populations proliferated similarly, doubled in 3 days. There is a progressive increase with time in the response to the various factors. Treatment with VEGF-A, bFGF, EGF, and SDF1 singly enhanced proliferation of CD31−;CD146− SP cells and CD31+;CD146− SP cells almost three times and two times more, respectively, compared with control 0.2% BSA on day 3 (Fig. 1F). IGF1 was less effective in proliferation of CD31−;CD146− SP cells and CD31+;CD146− SP cells but more effective in proliferation of CD31+;CD146+ SP cells compared with other growth factors (Fig. 1F).
VEGF-A (100 ng/ml) induced a chemotactic response in a dose-dependent manner in CD31−;CD146− SP cells, and induced 1.6 and 1.4 times more strongly than that in CD31+;CD146− SP cells and CD31+;CD146+ SP cells, respectively (Fig. 1G). SDF1 at the final concentration of 50 ng/ml also induced a two times stronger response than VEGF-A (Fig. 1H).
Multilineage Differentiation Potential Capability of SP Cells
The chondrogenic potential of CD31−;CD146− SP cells and CD31+;CD146− SP cells was examined in both chondrogenic and control media. The porcine pulp CD31−;CD146− SP cells and CD31+;CD146− SP cells from the fourth passage culture were maintained in pellet cultures for 30 days. The amount of cartilage proteoglycan stained with Alcian Blue was stronger in the pellets induced from CD31−;CD146− SP cells compared with those from CD31+;CD146− SP cells (Fig. 2A, 2B). The expression of chondrogenic markers aggrecan and type II collagen mRNA was much stronger in CD31−;CD146− SP cells than in CD31+;CD146− SP cells and SP cells 14 days after induction (data not shown). However, the expression of type II collagen (Fig. 2C) was similar in the two subfractions 21 days after induction. In control media there were no chondrocytes (Fig. 2C).
The adipogenic potential was examined in the third passage cultures that were cultured in adipogenic media for 28 days. Both CD31−;CD146− SP cells and CD31+;CD146− SP cells showed staining with oil red O (Fig. 2D, 2E), but in control media no staining was observed. Adipogenic markers aP2 and PPARγ mRNA were expressed in the CD31−;CD146− SP cells and CD31+;CD146− SP cells on day 28 in adipogenic media (Fig. 2F).
Next, the neurogenic potential was determined. Clusters of proliferating neurospheres were more prevalent in the CD31−;CD146− SP cells compared with CD31+;CD146− SP cells (Fig. 2G, 2H). Sox2 mRNA expression was similar in both groups (Fig. 2I). The neurospheres from both fractions were immunoreactive for neuromodulin 14 days after induction (Fig. 2J, 2K). The expression of neural markers neuromodulin, neurofilament, and sodium channel, voltage-gated, type Iα (Scn1A) mRNA was similar in the CD31−;CD146− SP cells as that in the CD31+;CD146− SP cells (Fig. 2L).
Finally, differentiation of CD31−;CD146− SP cells into odontoblast lineage was examined. The mineralized matrix was stained by alizarin red in both CD31−;CD146− SP cells and CD31+;CD146− SP cells 28 days after induction in vitro (Fig. 2M, 2N). The DiI-labeled CD31−;CD146− SP cells that attached to the dentinal wall in the cavity on the amputated pulp differentiated into odontoblasts and formed tubular dentin 28 days after autologous transplantation in the cavity on the canine amputated pulp in vivo (Fig. 2O–2Q). The mRNA expression of odontoblast markers, Dspp and enamelysin, was similar in CD31−;CD146− SP cells and CD31+;CD146− SP cells 14 days after induction in pellet culture (Fig. 2R).
Differentiation of CD31−;CD146− SP Cells into Endothelial Cells
The endothelial differentiation potential was assessed. CD31−;CD146− SP cells readily formed extensive networks of cords and tube-like structures as early as 12 hours (Fig. 3A), a phenotype typically associated with endothelial cells, suggesting an angioblast phenotype. On the other hand, CD31+;CD146− SP cells and CD31+;CD146+ SP cells formed only short strands (Fig. 3B, 3C). Capillary-like structures were also observed in cells cultured in matrigel (Fig. 3D). In situ hybridization analysis showed mRNA expression of CEACAM1 (Fig. 3E), CD146 (Fig. 3F), and Occludin (Fig. 3G), markers for endothelial cells.
We also examined whether CD31−;CD146− SP cells can differentiate into endothelial cells in monolayer culture. In the EBM2 supplemented with 2% porcine serum and 10 ng/ml VEGF-A and 10 ng/ml bFGF, endothelial marker vWF (Fig. 3H, 3K) was detected by immunocytochemistry in 3 days, whereas expression of CD31 (Fig. 3I, 3L) and VE-cadherin (Fig. 3J, 3M), a marker of more mature endothelial cells, was observed after 10 days and 21 days of culture, respectively.
Next, functional characteristics of endothelial cells induced from CD31−;CD146− SP cells with VEGF-A were investigated. In vitro release of vWF is stimulated by histamine treatment. vWF was distributed throughout the cytoplasm prior to histamine treatment and much decreased after treatment (Fig. 3N, 3O). The uptake of acetylated-LDL in CD31−;CD146− SP cells was high on day 21 (Fig. 3P, 3Q).
CD31−;CD146− SP Cells Induce Functional Neovascularization in Ischemic Hind Limb
Fourteen days after transplantation of CD31−;CD146− SP cells (Fig. 4A, 4D), the quantitative analysis of laser Doppler imaging revealed that the blood flow was significantly increased 1.3 and 1.6 times more in the ischemic hind limb compared with CD31+;CD146− SP cells (Fig. 4B, 4D) and PBS control without cells (Fig. 4C, 4D), respectively. Capillary density was increased in the ischemic hind limb after transplantation of CD31−;CD146− SP cells (Fig. 4E), to a greater extent compared with CD31+;CD146− SP cells (Fig. 4F) and PBS control (Fig. 4G). Quantitative analysis using serial sections revealed that capillary density in the ischemic region transplanted with CD31−;CD146− SP cells increased 13-fold higher than that with CD31+;CD146− SP cells (Fig. 4H–4K). Semithin sections of the ischemic lesion of transplantation of CD31−;CD146− SP cells (Fig. 4L) demonstrated numerous migrating cells among newly formed capillaries. Electron micrographs showed that intact capillaries with basement membrane and pericytes were surrounded by the migrating cells (Fig. 4M). Capillaries were functional with complete lumens. The migrating cells surrounding these intact capillaries were rich in cytoplasmic organelles with irregularly shaped nuclei. They were unlike the inflammatory polymorphonuclear cells or scavenging mononuclear cells (Fig. 4N). Confocal laser micrographs showed that CD31−;CD146− SP cells were present in close proximity to the vessel (Fig. 4O, 4P), suggesting they migrated to the ischemic region and stimulated neovascularization rather than functionally incorporating into vessels (Fig. 4O). However, CD31+;CD146− SP cells were not in close proximity of the vessels (Fig. 4Q).
Analysis of Gene Expression
The expression of angiogenic (VEGF-A, HGF), chemotactic (G-CSF, GM-CSF, MCP1, CXCL2, MDCFI, MDCFII, TF), and proinflammatory (IL-1α, IL-6, LIF) cytokines, matrix-degrading enzymes (MMP1, MMP3, MMP9), and others (Arginase I, Lipoprotein lipase, Dipeptidyl peptidase IV, Hyaluronan synthase 2, GP38K and CRSP) was stronger in CD31−;CD146− SP cells compared with CD31+;CD146− SP cells (Table 1). G-CSF, GM-CSF, MMP1, MMP3, VEGF-A, and CXCR4 were expressed in the DiI-labeled CD31−;CD146− SP cells in the ischemic region 7 days after transplantation (Fig. 5). The conditioned medium of CD31−;CD146− SP cells showed mitogenic (Fig. 6A) and antiapoptotic activities on HUVECs as MMP3, VEGF-A, and G-CSF (Fig. 6B). Thus, these results imply paracrine actions of proangiogenic and chemotactic cytokines in promoting neovascularization.
Table Table 1.. Relative mRNA expression of cytokines and enzymes by real-time reverse transcription-polymerase chain reaction in CD31−;CD146− side population (SP) cells, pulp CD31+ SP cells
The present investigation focused on subfractionation of SP cells from porcine dental pulp into CD31−; CD146− and CD31+; CD146− cells and assessment of their multilineage differentiation with special reference to vasculogenesis. There is increasing evidence of multilineage differentiation of tissue stem cells including SP cells. Previous work has demonstrated that SP cells from porcine dental pulp differentiated into adipocytes, chondrocytes, neuronal cells, and odontoblasts . Subfractionation of SP cells into CD31−;CD146− and CD31+;CD146− cells demonstrated that the former subfraction formed more neurospheres and expressed neurogenic marker CD271, suggesting a stronger neurogenic potential in CD31−;CD146− SP cells. On the other hand, the adipogenic, chondrogenic, and odontogenic differentiation potential was similar in the two subfractions of SP cells.
It is well known that bone marrow and peripheral blood contain EPCs with properties of embryonic angioblasts with potential to differentiate into mature endothelial cells [4, 16, –18]. The early angioblasts and EPCs express CD34, CD133, and VEGFR2. The expression of CD133 declines and that of CD146 increases in the differentiated endothelial cells [19, 20]. During maturation of bone marrow angioblasts to early EPCs, CD31 is expressed . In human embryonic aorta  and human adult vascular wall , the endothelial progenitors are CD34+ and CD31−. The adipose tissue-derived stromal-vascular fraction also contains CD34+;CD31− cells . It is noteworthy that the porcine pulp SP subfraction, CD31−;CD146− SP cells expressed CD34 and VEGFR2 as in EPCs. However, they lacked CD11b, CD14, and CD45, demonstrating that these cells are distinct from the hematopoietic lineage. In addition, it is noteworthy that pulp CD31−;CD146− SP cells did not express CD133 mRNA unlike the adipose tissue- and bone marrow-derived EPCs. Thus, the porcine pulp CD31−;CD146− SP cells are similar but not identical to EPCs and expressed stem cell markers, CXCR4, Stat3, Bmi1, and Tert.
Vasculogenic potential of CD34+;VEGFR2+;CD133+;CD90+ stem cells derived from human dental pulp has been reported in the induced bone tissue after subcutaneous transplantation  and in myocardial infarction . In the present study, the functional ability of neovascularization of CD31−;CD146− SP cells was determined and demonstrated to form extensive networks of cords and tube-like structures on matrigel. On the other hand, CD31+;CD146− SP cells were feeble in cord formation. Treatment of CD31−;CD146− SP cells with VEGF-A and bFGF resulted in VE-cadherin expression, histamine-induced vWF release, and uptake of acetylated-LDL, all hallmarks of endothelial differentiation. In addition, CD31−;CD146− SP cells exhibited neovascularization in the mouse hind limb ischemia model.
After injury, endothelial cells increase expression of VEGF, which induces SDF1 in the perivascular fibroblasts. SDF1 mobilizes CXCR4-positive cells to the perivascular site where they act in a paracrine fashion to enhance proliferation of resident endothelial cells . The regeneration potential for dentin-pulp complex in response to pulp injury may be attributed to pulp stem/progenitor cells migrating from perivascular region in the pulp tissue deeper from the injured site . The proangiogenic signals such as VEGF released from injured dental pulp cells  and endothelial cells  or from carious dentin  provide chemotactic signals to recruit pulp stem/progenitor cells in pulp tissue. CD31−;CD146− SP cells showed higher expression of CXCR4 compared with CD31+;CD146− SP cells. CD31−;CD146− SP cells were in proximity of vessel and close to neighboring cells expressing SDF1 (data not shown). CD31+;CD146− SP cells were at a distance from it in the ischemia model. CD31−;CD146− SP cells exhibits high migration activity by VEGF and SDF1 compared with CD31+;CD146− SP cells in the chemotaxis experiment. These results imply SDF1/CXCR4 system for migration of pulp CD31−;CD146− SP cells in the ischemic region.
It is important to note these CD31−;CD146− SP cells compared with CD31+;CD146− SP cells expressed more VEGF-A, G-CSF, and GM-CSF. G-CSF promotes endothelial migration and tubule formation in vitro, and local injection of G-CSF effectively augments ischemia-induced angiogenesis in vivo . GM-CSF induces vascular proliferation and improves blood flow in coronary artery disease and cerebral artery occlusion [30, –32]. MMPs are involved in degrading extracellular and basement membrane structures, allowing endothelial migration to occur. MMPs also promote the release of extracellular matrix-bound cytokines, such as VEGF, which can promote proliferation of EPCs and endothelial cells and regulate angiogenesis [33, , –36]. The higher gene and protein expression of MMP3 by pulp-derived CD31−;CD146− SP cells compared with CD31+;CD146− SP cells is noteworthy and may explain the anticipated invasive behavior during endothelial migration [33, 37, 38]. The conditioned medium of pulp-derived CD31−;CD146− SP cells enhanced proliferation and survival rate of HUVECs, suggesting the paracrine role of CD31−;CD146− SP cells on local vascular cells to create a permissive environment that enables rapid revascularization, proliferation, and survival of damaged cells [39, 40]. The isolation of EPCs from bone marrow, umbilical cord blood, peripheral blood, and adipose tissue is documented. To this list of sources of EPCs now dental pulp tissue-derived CD31−;CD146− SP cells can be added. It provides advantages for clinical use, since autologous pulp tissue is easily available from useless teeth after extraction with no ethical issues.
Dental pulp-derived CD31−;CD146− subfraction of SP cells is vasculogenic, and may induce vasculogenesis in vivo in the amputated pulp model. We are aware of the potential clinical utility to ameliorate ischemic disease and pulp regeneration in the cell therapy for endodontics and operative dentistry.
Disclosure of Potential Conflicts of Interest
The authors indicate no potential conflicts of interest.
The authors are grateful to Drs. N. Shibata and K. Adachi for their help. This work was supported by grants from the Collaborative Development of Innovative Seeds, Potentiality verification stage from Japan Science and Technology Agency; grants-in-aid for Scientific Research from the Ministry of Education, Science, Sports and Culture, Japan: number 17390509 (M.N.), number 19659499 (M.N.), and number 19791418 (K.I.); the Mitsubishi Pharma Research Foundation (2007, M.N.); the Japan Health Foundation (2007, M.N.); and Aichigakuin University High-Tech Research Center “Project for Private Universities: matching fund subsidy” from MEXT (Ministry of Education, Culture, Sports, Science and Technology), 2003–2007.