Basic ResearchBiology

Isolation and Identification of CXCR4-positive Cells from

Human Dental Pulp Cells
Long Jiang, DDS, MDS, Wei-Wei Peng, DDS, MDS, Li-Fen Li, DDS, MDS,
Ya Yang, DDS, PhD, and Ya-Qin Zhu, DDS, PhD
Abstract
Introduction: In previous studies, we found expression of stromal cellderived factor-1a (SDF-1a)/CXC
chemokine receptor 4 (CXCR4) in human dental pulp
and the SDF-1aCXCR4 axis might play a role in
the recruitment of CXCR4-positive dental pulp cells
(CXCR4+ DPCs) toward the damaged sites. However,
the specific function of CXCR4+ DPCs in the injured
dental pulp was still unknown. The purpose of this
study was to isolate CXCR4+ DPCs from dental pulp
cells in vitro to pave the way for further study of
their characteristics. Methods: CXCR4+ DPCs were isolated with magnetic-activated cell sorting (MACS).
Freshly isolated CXCR4+ DPCs were identified by immunohistochemistry with light microscopy or confocal
microscopy. Then the phenotypes CXCR4, stromal cell
surface marker-1 (STRO-1), CD146, and CD34 in 3
groups (ie, CXCR4+ DPCs, CXCR4 DPCs, or nonsorted DPCs) were analyzed by flow cytometry after
they were cultured and expanded in vitro. Results:
The results indicated the isolated subpopulation of
DPCs was enriched with CXCR4+ DPCs, and the positive rates of STRO-1 and CD146 in CXCR4+ DPCs group
were higher than CXCR4 DPCs or non-sorted DPCs
groups (P < .05). There was no expression of CD34
in each group. Conclusions: We can isolate CXCR4+
DPCs from DPCs with MACS and identify them by immunohistochemistry and flow cytometry. (J Endod
2012;38:791795)

Key Words
CXCR4, dental pulp stem cells, magnetic-activated cell
sorting

t has been confirmed that there are stem cells in human dental pulp tissue (13). The
dental pulp stem cells (DPSCs), which usually remain quiescent, are thought to
respond during injury (4). After damage of the tooth, these DPSCs will be activated
to migrate to the injury sites and differentiate into odontoblast-like cells, which synthesize and excrete an extracellular matrix to form the reparative dentin (5, 6).
Furthermore, the DPSCs are believed to be promising clinical treatments for dental
disease in tissue engineering because of the potential capability to repair or even
regenerate a new tooth to replace the diseased tooth.
Many attempts have been made to isolate stem/progenitor cells from dental pulp
cells (DPCs). The stromal cell surface marker-1 positive (STRO-1+) cells, with high
colony-forming efficiency, have been isolated from dental pulp (7). Furthermore,
STRO-1+ cells from rat dental pulp cells by fluorescence-activated cell sorting were
able to differentiate into the odontogenic pathway, whereas the negative fraction in
the separation failed to differentiate (8). It has been reported that a side cell population
with characteristic features of stem cell, which cannot be stained by the DNA-binding dye
Hoechst 33342, were isolated from dental pulp (9). Recently, the cells expressing high
level of b1 integrin or expressing the embryonic neural crest cell mark, low-affinity
nerve growth factor receptor (P75), were selected by magnetic-activated cell sorting
(MACS) (10). Those studies showed that the stem/progenitor cells isolated from dental
pulp have high proliferation and multilineage differentiation potential. In addition, it has
been demonstrated that the dentin/pulp-like complex is formed by them in vivo.
However, the mechanisms underlying the recruitment of stem/progenitor cells toward
the injury site remain elusive.
In previous studies, we found the expression of stromal cellderived factor-1a
(SDF-1a) and CXC chemokine receptor 4 (CXCR4) in dental pulp. The SDF-1a
CXCR4 axis was believed to play a role in the recruitment of CXCR4-positive dental
pulp cells (CXCR4+ DPCs) toward the damaged sites (11). Generally, CXCR4 was
considered as a surface marker expressed in stem cells (12). Besides hematopoietic
stem cells, CXCR4 has also been expressed in other tissue-specific stem cells such as
skeletal muscle satellite cells, which are responsible for the migration of satellite cells
toward an SDF-1 gradient (13). Consequently, we hypothesized that CXCR4 might be
one of the surface markers of DPSCs, and CXCR4+ DPCs might be a homogeneous
stem/progenitor cell population. To verify this hypothesis, we must isolate CXCR4+
DPCs and analyze the characteristics of CXCR4+ DPCs, CXCR4 DPCs, and nonsorted DPCs in vitro. In this study, we successfully isolated the CXCR4+ DPCs by
MACS and identified them. The findings of this study are conducive with the systematic
characterization of CXCR4+ DPCs.

From the Department of General Dentistry, 9th Peoples Hospital, Shanghai JiaoTong University, School of Medicine, Shanghai Key Laboratory of Stomatology,
Shanghai, PR China.
Supported by grants from the Science and Technology Commission of Shanghai Municipality (grant no. 08DZ2271100, 08JC1414500), the Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry (grant no. 2008-0890-09), Innovation Program of Shanghai Municipal Education Commission (grant no. 09ZZ116), and the selection and cultivation for outstanding young teachers in scientific special fund of Shanghai university (grant no. JDY09051).
Address requests for reprints to Dr Ya-Qin Zhu, Department of General Dentistry, 9th Peoples Hospital, Shanghai JiaoTong University, School of Medicine, Shanghai
Key Laboratory of Stomatology, Shanghai 200011, PR China. E-mail address: zyq1590@163.com
0099-2399/$ - see front matter
Copyright 2012 American Association of Endodontists.
doi:10.1016/j.joen.2012.02.024

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Materials and Methods
Cell Cultures
Dental pulp tissue was removed from extracted third or premolars
for orthodontic treatments of patients between 18 and 29 years old. An
informed consent was obtained from each patient before the extractions. The 9th Peoples Hospital Ethics Committee approved the procedure and the research protocol for tooth extractions. Primary human
DPCs were cultured as described previously (11). Briefly, the pulp
tissue was cut into small pieces, and then these tissue fragments were
covered by plastic coverslips. The tissue fragments were maintained
in medium in 37 C incubator with a humidified atmosphere of 5%
CO2 in air. The culture medium was Dulbecco modified Eagle medium
(DMEM) (Gibco, Grand Island, NY) supplemented with 2 mmol/L
glutamine (Sigma, St Louis, MO), 100 IU/mL penicillin, 100 mg/mL
streptomycin (Gibco), 0.25 g/mL amphotericin B (Fungizone; Gibco),
and 20% fetal bovine serum (FBS) (Gibco). The culture medium was
changed at 5-day intervals. Confluent cultures were dissociated by trypsinization (0.2% trypsin and 0.02% ethylenediaminetetraacetic acid;
Gibco) and subcultured with DMEM plus 10% FBS.
MACS
The procedure was a modification of the manufacturers manual.
Briefly, the single-cell suspension of the third passage DPCs (1  107/
mL) was incubated with 5 mg of biotin-labeled anti-human CXCR4 antibody (eBioscience, San Diego, CA) for 45 minutes at room temperature.
The cells were washed by using 2 mL phosphate-buffered saline (PBS)
with 5% FBS and centrifuged at 1000 rpm for 5 minutes. Then the supernatant containing unbound primary antibody was completely removed.
The 90 mL of single-cell suspension was resuspended in 10 mL of streptavidin linked micro-Beads (Miltenyi Biotec, Inc, Auburn, CA) and incubated for 45 minutes on ice. Then the DPCs were sorted by using a mini
MACS magnetic column (Miltenyi Biotec, Inc) in the magnetic field according to the protocol. CXCR4+ cells were collected and seeded onto
6-well plates in DMEM supplemented with 20% FBS. Once cells were
cultured to 80% confluency, they were detached by trypsinization and
subcultured at 1:4 dilution for more than 10 passages. CXCR4 cells
and non-sorted DPCs were also collected and cultured under the
same conditions and used as controls for the following experiments.
Immunohistochemistry
The 3 types of cell populations were seeded immediately after
being sorted onto the coverslips in the 6-well plates respectively.
Then they were cultured in DMEM supplemented with 20% FBS. The
cells were fixed with 4% paraformaldahyde for 20 minutes when they
were 60% confluent. The primary antibody was mouse anti-CXCR4 (immunglobulin [Ig] G, 10 mg/mL; R&D, Minneapolis, MN) or mouse antiSTRO-1 (IgM, 1:25; Santa Cruz Biotechnology, Inc, Santa Cruz, CA). The
incubation was performed overnight at 4 C. The immunostaining was
revealed by using the EnVision Detection kit (Dako, Copenhagen,
Demark) according to the manufacturers instructions. PBS replaced
primary antibodies that were used to serve as the negative controls.
Five fields under 100-fold magnification were randomly selected, and
immunoreactive cells were counted. The positive rate was the number
of stained cells/the total number of cells  100%.
CXCR4+ cells grown on the coverslips were fixed and stained with
mouse anti-CXCR4 IgG (10 mg/mL) and mouse anti-STRO-1 IgM
(1:25), followed by goat anti-mouse IgG-PE (1:200; Santa Cruz Biotechnology, Inc) and goat anti-mouse IgM-FITC (1:200; Santa Cruz Biotechnology, Inc). After washing 3 times with PBS, the nuclei were stained
with 40 ng/mL Hoechst 33342 (Invitrogen, Carlsbad, CA) for 10
minutes. Slides were rinsed and mounted by glycerin. Images were ob792

Jiang et al.

tained by confocal microscope (TCS SP2; Leica, Mannheim, Germany).

In control groups, PBS replaced primary antibodies.

Flow Cytometry
When the sorted cells had been expanded, the single-cell suspensions of the 3 cells were adjusted to 1  105 cells in 100 mL and
incubated with mouse anti-CXCR4 IgG (10 mg/mL) or 4 mL of mouse
anti-STRO-1 IgM for 30 minutes at 4 C. The secondary antibody was
goat anti-mouse IgG-FITC (1:200; Santa Cruz Biotechnology, Inc) or
goat anti-mouse IgM-FITC (1:200). After incubating according to
protocol, those cells were resuspended in PBS. Analysis of cells was performed by using a flow cytometer (FACSCalibur; BD, Providence, RI).
Mouse anti-human CD146:FITC (1:10; ABD Serotec, Oxford, UK) and
mouse anti-human CD34:FITC (1:20; eBioscience) were also used to
test. All DPCs were from 25 patients teeth.
Statistical Analysis
Data analysis was performed by using SAS6.12 (SAS Institute Inc,
Raleigh, NC). Statistical significance between groups was determined by
using one-way analysis of variance. A P value <.05 was considered
significant.

Results
Immunohistochemistry
All isolated cells had a typical fibroblast-like morphology, without
apparent difference observed among CXCR4+ DPCS, CXCR4 DPCs, and
non-sorting DPCs by inverted phase-contrast microscopy. A significantly
greater percentage of immunoreactive CXCR4+ was detected in the
sorted subpopulation of cells (P < .05). Many more positively stained
cells were present in CXCR4+ DPCs group (Fig. 1A and D). For CXCR4
cells group, positively stained cells were almost absent (Fig. 1B and E).
The average positive rate (n = 5) of CXCR4 and STRO-1 in CXCR4+ DPCs
group was approximately 83% and 89%, respectively.
The results of immunofluorescence observed by confocal microscopy showed CXCR4 and STRO-1 were coexpressed by CXCR4+ DPCs
(Fig. 1GI).
Flow Cytometry
The flow cytometric analyses of the expanded CXCR4+ DPCs,
CXCR4 DPCs, and non-sorted DPCs showed differences among the
3 cell groups (Fig. 2). The average positive rates (n = 25) of CXCR4
were 7.32%, 2.52%, and 4.43%, respectively. The expression of
STRO-1 in CXCR4+ DPCs, CXCR4 DPCs, and non-sorted DPCs was
approximately 14%, 3%, and 5%, respectively. The expression of
CD146 in CXCR4+ DPCs, CXCR4 DPCs, and non-sorted DPCs groups
was 57%, 34%, and 47%, respectively. The positive rate of CXCR4,
STRO-1, or CX146 in CXCR4+ group was higher than in CXCR4 group
or non-sorted group (P < .05). However, the expressions of CD34 in
the 3 cell groups were negative, and there were no differences in the
3 groups (Fig. 2JL).

Discussion
CXCR4 is the receptor of SDF-1 (14). Compelling evidence is accumulating that stem cells of different organs and tissues resemble hematopoietic stem cells (HSCs) expressing functional CXCR4 on their
surface (15, 16). In damaged organs, SDF-1 is up-regulated to attract
CXCR4+ stem cells that are mobilized from their niches in response to
the stimulation related to tissue/organ damage (17). Our previous work
has implicated CXCR4 expression in some DPCs. Furthermore, the
immunohistochemical results showed that CXCR4+ DPCs were localized
to the perivascular area in physiological conditions, and they could be
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Figure 1. Immunostaining of cultured CXCR4+ DPCs, CXCR4 DPCs, and non-sorted DPCs. (A) CXCR4+ DPCs were stained with anti-CXCR4 monoclonal antibody
(MoAb). (B) CXCR4 DPCs were stained with anti-CXCR4 MoAb. No staining was observed. (C) Non-sorted DPCs were stained with anti-CXCR4 MoAb. A few DPCs
were positive. (D) CXCR4+ DPCs were stained with anti-STRO-1 MoAb. (E) CXCR4 DPCs were stained with anti-STRO-1 MoAb, No staining was observed. (F) Nonsorted DPCs were stained with anti-STRO-1 MoAb. A few DPCs were positive. (AF) Original magnification, 10. (G) Red fluorescence presented CXCR4 was expressed by CXCR4+ DPCs. (H) Green fluorescence presented STRO-1 was expressed by CXCR4+ DPCs. (I) Was compounded by (G) and (H). (JL) Negative control.

recruited toward the damaged site along an SDF-1 gradient (11).

Another study also demonstrated inflammation and hypoxia might
recruit DPSCs to participate in reparative dentinogenesis with regulating
the SDF-1aCXCR4 axis (18). However, the exact function of CXCR4+
DPCs in the area of injury remains unknown. We also do not know
whether CXCR4+ DPCs have properties of DPSCs.
In general, mesenchymal stem cells have the capacity of selfrenewal and multilineage differentiation (19). DPSCs have been further
characterized by a single colony isolation method, demonstrating selfrenewal capability, multipotent differentiation, and differentiation into
JOE Volume 38, Number 6, June 2012

odontoblasts forming tubular dentin in vivo after transplantation

with hydroxyapatite/tricalcium phosphate into immunocompromised
mice (20). To investigate the characteristics of CXCR4+ cells, the
successful isolation of them from DPCs is a critical step.
At present, stem cells can be identified and isolated from a variety
of cells by using 4 methods:
1. Fluorescence-labeled antibody to recognize and a flow cytometer to
sort the cells with specific surface antigen markers in a process
called fluorescent antibody cell sorting (FACS)

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Figure 2. Immunophenotype analysis of CXCR4+ DPCs, CXCR4 DPCs, and non-sorted DPCs by flow cytometry. CXCR4+ cells were labeled with antibody against
CXCR4 (A), STRO-1 (D), CD146 (G), or CD34 (J), respectively. CXCR4 cells were labeled with antibody against CXCR4 (B), STRO-1 (E), CD146 (H), or CD34 (K),
respectively. Non-sorted cells were labeled with antibody against CXCR4 (C), STRO-1 (F), CD146 (I), or CD34 (L), respectively. Blue area represents control
immunoglobulin.

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2. MACS
3. Immunohistochemical staining
4. Physiological and histologic criteria, including phenotype (appearance), chemotaxis, proliferation, differentiation, and mineralizing
activity (21)
In this study, we isolated CXCR4+ DPCs by MACS and examined the
efficacy of MACS. Three groups of cells obtained were also assessed for
CXCR4+ cell content by immunohistochemistry. The results demonstrated significant difference in the 3 types of cells. After culture expanding in vitro, the flow cytometric analyses of CXCR4+ DPCs showed the
positive rate of CXCR4 was 7%8%. Although it was 2%3% of CXCR4
DPCs and 4%5% of non-sorted DPCs, it was obviously discrepant with
the results of immunohistochemistry, which showed approximately
80%90% of cells were CXCR4+ in light microscope. The very low
percentage of CXCR4+ cells of the flow cytometric analyses could be
due to the long time of culture in normal DMEM after selection was performed. The cells applied to immunohistochemistry were cultured for
only 2 or 3 days after being sorted. However, the cells that were analyzed
by flow cytometry must be cultured longer than 2 weeks after being
sorted, so that the number of cells can suffice for the requirement of
the experiment. Further studies are needed for maintaining the stable
expression of CXCR4 in vitro.
The expression of STRO-1 as an important marker of DPSCs and
bone marrow stromal cells (2225) was similar to CXCR4 in the 3 types
of cells. The STRO-1+ fraction in CXCR4+ DPCs was approximately 14%
and more than 3% in CXCR4 DPCs or 5% in non-sorted DPCs. Earlier
experiments showed that DPSCs isolated from the pulp of human exfoliated deciduous teeth by a clonogenic method exhibited 9% positivity
for STRO-1 (2). This implied we could obtain much more positivity
for the STRO-1 cells from CXCR4+ DPCs population by MACS. CD146
is also considered as a marker for DPSCs, although the exact function
of CD146 is not known (7). CD34 is expressed in hematopoietic stem
cells (26). Most studies demonstrated that DPSCs did not express CD34
(1, 22, 27). In our study, the results indicated that CXCR4+ DPCs
comprised the most CD146+ DPCs, and CD34 was almost not
expressed in the 3 groups.
In conclusion, the results presented here demonstrated that
CXCR4+ DPCs can be isolated from DPCs by MACS, and STRO-1 is coexpressed by CXCR4+ DPCs, although the positive rates would be
decreased after some time of culture in vitro. Further studies will focus
on the detection of proliferation and multilineage differentiation
capacity of CXCR4+ DPCs.

Acknowledgments
The authors thank Dong-xia Ye, Xiu-li Zhang, and Han-bing Fu
for their excellent technical support.
The authors deny any conflicts of interest related to this study.

References
1. 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;97:1362530.

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2. Miura M, Gronthos S, Zhao M, et al. SHED: stem cells from human exfoliated deciduous teeth. Proc Natl Acad Sci U S A 2003;100:580712.
3. Pivoriu
unas A, Surovas A, Borutinskaite V, et al. Proteomic analysis of stromal cells
derived from the dental pulp of human exfoliated deciduous teeth. Stem Cells Dev
2010;19:108193.
4. Scadden DT. The stem-cell niche as an entity of action. Nature 2006;441:10759.
5. 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:
42537.
6. Goldberg M, Smith AJ. Cells and extracellular matrices of dentin and pulp: a biological basis for repair and tissue engineering. Crit Rev Oral Biol Med 2004;15:
1327.
7. Shi S, Gronthos S. Perivascular niche of postnatal mesenchymal stem cells in human
bone marrow and dental pulp. J Bone Miner Res 2003;18:696704.
8. Yang X, van den Dolder J, Walboomers XF, et al. The odontogenic potential of
STRO-1 sorted rat dental pulp stem cells in vitro. J Tissue Eng Regen Med 2007;
1:6673.
9. Iohara K, Zheng L, Ito M, et al. Side population cells isolated from porcine dental
pulp tissue with self-renewal and multipotency for dentinogenesis, chondrogenesis,
adipogenesis, and neurogenesis. Stem Cells 2006;24:2493503.
10. Waddington RJ, Youde SJ, Lee CP, et al. Isolation of distinct progenitor stem cell
populations from dental pulp. Cells Tissues Organs 2009;189:26874.
11. Jiang L, Zhu YQ, Du R, et al. The expression and role of stromal cell-derived factor1alpha-CXCR4 axis in human dental pulp. J Endod 2008;34:93944.
12. Majka M, Kucia M, Ratajczak MZ. Stem cell biology: a never ending quest for understanding. Acta Biochim Pol 2005;52:3538.
13. Ratajczak MZ, Majka M, Kucia M, et al. Expression of functional CXCR4 by muscle
satellite cells and secretion of SDF-1 by muscle-derived fibroblasts is associated with
the presence of both muscle progenitors in bone marrow and hematopoietic stem/
progenitor cells in muscles. Stem Cells 2003;21:36371.
14. Nagasawa T, Hirota S, Tachibana K, et al. Defects of B-cell lymphopoiesis and bonemarrow myelopoiesis in mice lacking the CXC chemokine PBSF/SDF-1. Nature 1996;
382:6358.
15. Ratajczak MZ, Kucia M, Reca R, et al. Stem cell plasticity revisited: CXCR4-positive
cells expressing mRNA for early muscle, liver and neural cells hide out in the bone
marrow. Leukemia 2004;18:2940.
16. Kucia M, Ratajczak J, Ratajczak MZ. Bone marrow as a source of circulating CXCR4+
tissue-committed stem cells. Biol Cell 2005;97:13346.
17. Imitola J, Raddassi K, Park KI, et al. Directed migration of neural stem cells to sites
of CNS injury by the stromal cell-derived factor 1alpha/CXC chemokine receptor 4
pathway. Proc Natl Acad Sci U S A 2004;101:1811722.
18. Gong QM, Quan JJ, Jiang HW, et al. Regulation of the stromal cell-derived factor1alpha-CXCR4 axis in human dental pulp cells. J Endod 2010;36:1499503.
19. Bakopoulou A, Leyhausen G, Volk J, et al. Comparative analysis of in vitro osteo/
odontogenic differentiation potential of human dental pulp stem cells (DPSCs)
and stem cells from the apical papilla (SCAP). Arch Oral Biol 2011;56:70921.
20. Nakashima M, Iohara K, Sugiyama M. Human dental pulp stem cells with highly
angiogenic and neurogenic potential for possible use in pulp regeneration. Cytokine
Growth Factor Rev 2009;20:43540.
21. Murray PE, Garcia-Godoy F, Hargreaves KM. Regenerative endodontics: a revised of
current statue and a call for action. J Endod 2007;33:37790.
22. Shi S, Bartold PM, Miura M, et al. The efficacy of mesenchymal stem cells to regenerate and repair dental structures. Orthod Craniofac Res 2005;8:1919.
23. Yang X, Zhang W, van den Dolder J, et al. Multilineage potential of STRO-1+ rat
dental pulp cells in vitro. J Tissue Eng Regen Med 2007;1:12835.
24. Yang X, Walboomers XF, van den Beucken JJ, et al. Hard tissue formation of STRO-1selected rat dental pulp stem cells in vivo. Tissue Eng Part A 2009;15:36775.
25. Gronthos S, Graves SE, Ohta S, et al. The STRO-1+ fraction of adult human bone
marrow contains the osteogenic precursors. Blood 1994;84:416473.
26. Mao M, Fu G, Wu JS, et al. Identification of genes expressed in human CD34(+)
hematopoietic stem/progenitor cells by expressed sequence tags and efficient
full-length cDNA cloning. Proc Natl Acad Sci U S A 1998;95:817580.
27. Karaoz E, Dogan BN, Aksoy A, et al. Isolation and in vitro characterisation of dental
pulp stem cells from natal teeth. Histochem Cell Biol 2010;133:95112.

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