Allogeneic transplantation of mobilized dental pulp stem cells with the mismatched dog leukocyte antigen type is safe and efficacious for total pulp regeneration
Iohara et al. Stem Cell Research & Therapy
Allogeneic transplantation of mobilized dental pulp stem cells with the mismatched dog leukocyte antigen type is safe and efficacious for total pulp regeneration
Koichiro Iohara 0
Shinji Utsunomiya 2
Sakae Kohara 1
Misako Nakashima 0
0 Department of Stem Cell Biology and Regenerative Medicine, National Center for Geriatrics and Gerontology, Research Institute , 7-430 Morioka, Obu, Aichi 474-8511 , Japan
1 Preclinical Research Support Division, Shin Nippon Biomedical Laboratories Ltd , Kainan , Japan
2 Drug Safety Research Laboratories, Shin Nippon Biomedical Laboratories Ltd , Kagoshima , Japan
Background: We recently demonstrated that autologous transplantation of mobilized dental pulp stem cells (MDPSCs) was a safe and efficacious potential therapy for total pulp regeneration in a clinical study. The autologous MDPSCs, however, have some limitations to overcome, such as limited availability of discarded teeth from older patients. In the present study, we investigated whether MDPSCs can be used for allogeneic applications to expand their therapeutic use. Methods: Analysis of dog leukocyte antigen (DLA) was performed using polymerase chain reaction from blood. Canine allogeneic MDPSCs with the matched and mismatched DLA were transplanted with granulocyte-colony stimulating factor in collagen into pulpectomized teeth respectively (n = 7, each). Results were evaluated by hematoxylin and eosin staining, Masson trichrome staining, PGP9.5 immunostaining, and BS-1 lectin immunostaining performed 12 weeks after transplantation. The MDPSCs of the same DLA used in the first transplantation were further transplanted into another pulpectomized tooth and evaluated 12 weeks after transplantation. Results: There was no evidence of toxicity or adverse events of the allogeneic transplantation of the MDPSCs with the mismatched DLA. No adverse event of dual transplantation of the MDPSCs with the matched and mismatched DLA was observed. Regenerated pulp tissues including neovascularization and neuronal extension were quantitatively and qualitatively similar at 12 weeks in both matched and mismatched DLA transplants. Regenerated pulp tissue was similarly observed in the dual transplantation as in the single transplantation of MDPSCs both with the matched and mismatched DLA. Conclusions: Dual allogeneic transplantation of MDPSCs with the mismatched DLA is a safe and efficacious method for total pulp regeneration.
Allogeneic cell transplantation; Pulp regeneration; Mobilized dental pulp stem cells; Granulocyte-colony stimulating factor; Pulpectomy; Dog leukocyte antigen; Dual transplantation
Pulp/dentin complex in teeth has a critical function in
the maintenance of tooth homeostasis, and viable pulp is
essential for the longevity of the tooth [
]. The ultimate
goal for regenerative endodontics is to replace or restore
the impaired or damaged tissues with viable pulp tissue
in the case of pulpitis or apical periodontitis, leading to
the reestablishment of the physiologic, structural, and
mechanical integrity of the native dentin/pulp complex,
including function of pulp immunity, dentin formation,
pulp innervation, and vascular perfusion [
]. Stem cell
therapy has been suggested as an effective regenerative
technique for pulpitis and apical periodontitis.
Autologous transplantation of dental pulp stem cell (DPSC)
subsets, dental pulp-derived CD31− side population (SP)
cells, or CD105+ cells with SDF1 in orthotopic sites
demonstrated complete pulp regeneration [
Subsequently, colony-derived DPSCs with platelet-rich
plasma/fibrin (PRP/PRF) showed similar successful
]. The safety and efficacy of autologous DPSC
therapy were demonstrated in the preclinical study
harnessing DPSCs mobilized (MDPSCs) with
granulocytecolony stimulating factor (G-CSF) harvested in good
manufacturing practice conditions [
]. Furthermore, a
recent clinical study suggested autologous MDPSC
transplantation may be safe and efficacious for pulp
regeneration in humans [
]. The autologous DPSCs,
however, have certain limitations to overcome, such as
limited availability of human discarded teeth and the
high cost of the safety and quality control tests of
individual cell products before transplantation. The further
potential disadvantages of the autologous
mesenchymal stem cells (MSCs) are their decreased biological
activity from older patients and altered intrinsic stem
cell properties from patients with some systemic
diseases including diabetes and rheumatoid arthritis [
Transplantation of autologous mobilized
adiposederived or bone marrow-derived MSCs resulted in
lower volume of regenerated pulp tissue, less
angiogenesis, and reinnervation compared with MDPSCs
]. Furthermore, regenerated pulp tissues in adipose
and bone marrow MSC transplants were more
mineralized compared with MDPSC transplant, suggesting
pulp MSCs were an optimal cell source for pulp
regeneration. Thus, banked allogeneic DPSCs would be
highly advantageous to save time and costs and to
confirm high quality [
The use of allogeneic MSCs permits low
immunogenicity with immunomodulatory and immunosuppressive
properties. It is well known that MSCs have low
immunogenicity due to no expression of class II major
histocompatibility complex (MHC-II) proteins, and low
or modest expression of MHC-I proteins and
costimulatory molecules such as CD40, CD80, and CD86 on their
cell surface [
]. Therefore, MSCs are unable to
provoke a cytotoxic effect by allogeneic immune cells ,
and MSCs from MHC-mismatched donors may also be
used for cell therapy [
]. Many studies recently focused
on mechanisms of immunomodulation and
immunosuppression of MSCs, especially in reducing inflammation,
escaping from immune cell response, and modulating
Tcell proliferation. MSCs can interfere with different
pathways of the immune responses by means of direct
cell-to-cell interactions and secretion of soluble factors
such as transforming growth factor (TGF)-β, hepatocyte
growth factor (HGF), prostaglandin E2 (PGE2), nitric
oxide (NO), indoleamine-2,3 dioxygenase (IDO), tumor
necrosis factor (TNF)-α stimulated gene-protein 6,
interleukin (IL)-6, IL-10, semaphorin-3A, galectin (Gal)-1,
and Gal-9 [
]. MSCs also possess the ability to
generate regulatory T cells (Tregs) which suppress other
immune cells [
]. The whole range of mechanisms
of immunomodulation and immunosuppression
mediated by MSCs remains incompletely understood. The
immunosuppressive and immunomodulatory responses
are, however, properties shared by MSCs from a variety
of adult and fetal tissues including dental pulp [
A number of animal experiments have demonstrated
that allogeneic MSCs improve acute myocardial
infarction, chronic spinal cord injury, ischemic stroke, fracture
healing, and osteoarthritis by local injection or
intravenous infusion [
]. No adverse effects have been
noticed in 291 equine recipients over a period of up to
1 year after intravenous injection of allogeneic peripheral
blood-derived MSCs [
]. On the other hand, MHC
class I mismatched MSCs induced CD8+VE, CD16+VE,
and CD8+VE/CD16+VE lymphocyte subpopulations,
dependent on the dose of administered MSCs in
intracranial injection and the degree of antigenic
mismatch between donor and recipient [
bone marrow MSC transplantation into infarcted rat
myocardium improved ventricular function for 3 months
and a delayed immune rejection/response has been
reported within 5 months due to the shift from a
hypoimmunogenic to an immunogenic state of the
transplanted MSCs upon differentiation [
consistent results have not yet been obtained on the
therapeutic effects of allogeneic MSCs, depending on
routes, timing duration, and dosage of MSC administration
in vivo [
It is desirable for MSC transplantation into the root
canal of the tooth to be repeated for pulp regeneration
in patients with multiple caries. Repeated injection of
allogeneic adipose tissue-derived MSCs (AT-MSCs) or
bone marrow-derived MSCs (BM-MSCs) demonstrated
a lack of adverse effects. However, repeated BM-MSC
injections resulted in an increase in blood CD8+ T-cell
numbers and splenic regulatory T-cell numbers
compared with AT-MSCs in healthy horses, indicating a
mild alloantigen-directed cytotoxic response [
Repeated intravenous injection of allogeneic porcine bone
marrow MSCs or human umbilical cord blood-derived
MSCs also induced no immunological alterations
including T-cell proliferation, high levels of IFN-γ, TNF-α, and
human IgG and no adverse events due to low
]. Dual allogeneic MSC treatment by
transepicardial injection in the acute and the subacute
period after myocardial infarction improved ventricular
function with increased myocardial mass and anteriolar
density more than single MSC treatment in rats .
There are no reports on MHC-mismatched allogeneic
transplantation and on dual allogeneic MSC
transplantation in pulp regenerative therapy. Thus, there is a need
to address challenges to allogeneic MDPSC cell therapy
for total pulp regeneration. The aim of this investigation
is to assess the safety and efficacy of allogeneic
transplantation of canine MDPSCs into the pulpectomized
tooth. The dual consecutive transplantation was further
evaluated for safety and efficacy compared with the
Dog leukocyte antigen genotyping and matching analysis
We used beagle dogs (Kitayama Labes, Iwakuni and Ina,
Japan) owned by Shin Nippon Kagaku Biomedical
Laboratories Ltd (n = 26). Total genomic DNA was
extracted from whole blood of dogs by NuclesaseMag®96
Blood (Marcherey-Nagel, Düren, Germany) according to
the protocol. All of the dogs in the investigation were
not in a sibling relationship. Genotyping was performed
by direct sequencing and sequencing of the polymerase
chain reaction (PCR) product. PCR was performed using
primers DLA-88 exon 1–3 (1100 bp), DLA-DQA exon 2
(300 bp), DLA-DQB exon 2 (350 bp), and DLA-DRB
exon 2 (350 bp) [
] (Table 1) with KOD Fx
(TOYOBO Co., Ltd, Osaka, Japan) in a GeneAmp PCR
system 9700 (Thermo Fisher Scientific K.K., Yokohama,
5′–3′ DNA sequence
GG AC AG ATT C AGT G AAG AG A
Japan). PCR products were subcloned into a
ZeroBlunt®TOPO PCR Cloning Kit (Thermo Fisher Scientific K.K.).
Sequencing was carried out using a ABI PRISM BigDye
Terminator v3.0 Ready Reaction Cycle Sequencing Kit
(Thermo Fisher Scientific K.K.) with an ABI PRISM
3730 DNA Analyzer (Thermo Fisher Scientific K.K.),
and the raw data were analyzed by Sequencer Ver 4.8
(Gene Codes Corp., Ann Arbor, MI, USA). The allele
names were determined according to the universal
nomenclature found in the Immuno Polymorphism
Database (EMBL-EBI, Cambridge, UK).
Cell isolation and culture
Upper left lateral incisors were freshly extracted from each
beagle dog at 8 months of age. After making a longitudinal
cut, they were transported by air within 30 h to the
National Center for Geriatrics and Gerontology (NCGG)
from Shin Nippon Biomedical Laboratories Ltd, Drug
Safety Research Laboratories (Kagoshima, Japan), and
RaQualia Pharma Inc. (Rental Laboratories of NCGG) in
Hank’s balanced salt solution (Invitrogen, Carlsbad, CA,
USA) with 2.5 mg/ml amphotericin B (Bristol-Myers
Squibb, Tokyo, Japan) and 0.3% gentamicin (Nitten,
Nagoya, Japan). Mobilized dental pulp stem cells
(MDPSCs) were isolated using the similar procedure used
for a previous autologous preclinical study [
]. In brief,
dental pulp tissues isolated from the teeth were
enzymatically digested in 0.04 mg/ml Liberase (Roche, Mannheim,
Germany) for 30 min at 37 °C. The isolated pulp cells
were plated at 2 × 104 cells on T-25 (Asahi Technoglass,
Funabashi, Japan) in Dulbecco’s Modified Eagle’s Medium
(DMEM) (Sigma, St. Louis, MO, USA) supplemented with
10% autologous canine serum, 2.5 mg/ml amphotericin B,
and 0.3% gentamicin. They were detached by incubation
with TrypLE™ Select (Invitrogen) prior to 70% confluence.
The colony-formed DPSCs were further isolated by
GCSF-induced stem cell mobilization method with G-CSF
(Neutrogin; Chugai Pharmaceutical Co., Ltd, Tokyo,
Japan) at 100 ng/ml, with 2 × 104 cells/100 μl on the
Transwell (Corning, Lowell, MA, USA), and inserted into
24-well tissue culture plates for 48-h incubation [
isolated MDPSCs were detached by incubation with
TrypLE™ Select at 60–70% confluence and subcultured at a 1:
3 dilution into cell culture flasks (25 cm2 and further
75 cm2) (Asahi Technoglass) in DMEM supplemented
with 10% autologous canine serum without antibiotics.
The cells were cryopreserved at a cell concentration of
1 × 106 cells/ml in an extracellular cryoprotectant (CP-1;
Kyokuto Pharmaceutical Industrial Co., Ltd, Tokyo, Japan)
at the 7th passage of culture.
Characterization of mobilized dental pulp stem cells
The quality of cryopreserved MDPSCs was confirmed by
cell viability and proliferation abilities at the 7th passage
of culture. In brief, the cells stained with trypan blue
(Sigma-Aldrich, St. Louis, MO, USA) were counted
following thawing for the cell viability test, further plated
at 2.0 × 105 cells in 10-cm dishes (Corning, NY, USA),
and proliferation rates were calculated at 48 h as the
To further characterize the immunomodulation ability
of MDPSCs, mobilized adipose-derived stem cells
(MADSCs) were isolated from the abdominal
subcutaneous adipose tissue [
] from the same individual dog
similarly to MDPSCs as already described for comparison with
MDPSCs. Total RNA was extracted with TRIzol (Life
Technologies). First-strand cDNA syntheses were
performed on the total RNA of these cells by reverse
transcription using ReverTra Ace-a (Toyobo, Tokyo, Japan)
after DNase I treatment (Roche Diagnostics) at 37 °C for
20 min. Real-time RT-PCR was performed using primers
for the immunomodulatory factors (Table 2)
prostaglandin E synthase (PTGES), cyclooxygenase-2 (COX-2), IL-6,
TGF-β and indoleamine 2, 3-dioxygenase-1 (IDO-1)
labeled with AmpliTaq Gold master mix (Thermo Fisher
Scientific) in an Applied Biosystems® 7500 Real-Time PCR
(Life Technologies). After normalizing with β-actin, the
mRNA level of each immunomodulatory factor in
MDPSCs was compared with that in MADSCs derived
from the same individual dog (n = 3).
Evaluation of safety of first and second allogeneic transplantation for pulp regeneration
The cryopreserved MDPSCs were transported by air to
the operating room of the animal facility in Shin Nippon
Biomedical Laboratories Ltd under strict temperature
control. One day before transplantation, the root canal
was open to the apex with a #25 K file after pulpectomy
and shaped to 0.55 mm in width, 0.5 mm from the apex
in the upper right lateral incisors in 14 dogs. For the first
allogeneic transplantation, the dog leukocyte antigen
(DLA) matched and mismatched MDPSCs (n = 5,
respectively) were transplanted into the root canal, 5 × 105 cells
together with 20 μl of collagen scaffold (atelocollagen
implant; Koken, Tokyo, Japan) and 150 ng of G-CSF
(Neutrogin), respectively (Fig. 1). For toxicology assessment,
clinical signs of dogs were observed and their food
consumption was measured daily, and their weights were
recorded weekly. Urine chemistry examinations by Clinitek
AtlasXL (Sparton Medical Systems, Strongsville, OH,
USA) were performed at 4 and 12 weeks, and blood tests
by ADIVIA 120 (Siemens Healthcare Diagnostics
Manufacturing Ltd, Erlangen, Germany) and blood chemistry
examinations by JCA-BM6070 (Japan Electron Optical
Laboratory, Tokyo, Japan) were performed at 4 and 12 weeks
after transplantation. Blood tests demonstrate the red
blood cell count (RBC) and hematocrit (Ht) for
homeostasis of blood cells, the platelet count (Plt) for inflammation,
and the white blood cell count (WBC) for inflammation
and infection. Blood chemistry examinations demonstrate
aspartate transaminase (AST) and alanine transaminase
(ALT) for abnormality of the liver, albumin and
globulin for protein metabolism, total cholesterol
(Tcho) for the lipid profile, and glucose for abnormality
of hormone. After extraction of the upper right lateral
incisors at 12 weeks, the second allogeneic
transplantation of the same matched and mismatched MDPSCs
as the first transplantation was performed in
pulpectomized lower right third incisors in the same dogs,
respectively (n = 5). The safety tests were further
performed at 4 and 12 weeks. The transplanted teeth
were extracted at 24 weeks followed by euthanization.
All organs were weighed and macroscopically
examined. Furthermore, histopathological examination of
all organs and tissues including the transplanted teeth
with surrounding periodontal tissue were also
PTGES prostaglandin E synthase, COX-2 cyclooxygenase-2, IL interleukin, TGF transforming growth factor, IDO-1 indoleamine 2,3-dioxygenase 1.
performed in the paraffin
hematoxylin and eosin (HE).
Efficacy of first and second allogeneic transplantation for pulp regeneration
Morphological examination of the regenerated tissue
was performed in the paraffin sections (5 μm in
thickness) of the teeth. The relative amounts of
regenerated tissue were measured in the sections of the first
transplants of matched (n = 5) and mismatched MDPSCs
(n = 5), and the second transplants of matched (n = 4)
and mismatched MDPSCs (n = 5). On-screen image
outlines of regenerated tissue in the root canals were traced
by capturing images of the histological preparations on a
binocular microscope (M 205 FA; Leica) and the surface
area of these outlines was determined using Leica
Application Suite software (version 3.4.1; Leica).
For neovascularization and innervations analyses,
5μm-thick paraffin sections were deparaffinized and
stained with Fluorescein Griffonia (Bandeiraea)
Simplicifolia Lectin 1/fluorescein-galanthus nivalis (snowdrop)
lectin (BS-1 lectin) (20 μg/ml; Vector laboratories, Inc.,
Youngstown, OH, USA) and anti-PGP9.5 (Ultra Clone)
(1:10,000), respectively, as described previously [
ratios of the BS-1 lectin-positive newly formed
capillaries were calculated respectively using a Dynamic cell
count BZ-HIC (KEYENCE, Osaka, Japan) in the first
transplants of matched (n = 4) and mismatched MDPSCs
(n = 4), and in the second transplants of matched (n = 5)
and mismatched (n = 5) MDPSCs.
For quantitative analysis of matrix formation, the
sections from each four teeth at 12 weeks after the first and
second transplantation of matched and mismatched
MDPSCs were stained with Masson trichrome staining
(Muto Pure Chemicals Co., Ltd, Tokyo, Japan). The
relative amounts of matrix formation area were measured in
the sections of the first transplants of matched (n = 5)
and mismatched MDPSCs (n = 5), and the second
transplants of matched (n = 4) and mismatched MDPSCs
(n = 3). On-screen image outlines were traced by
capturing images of the histological preparations on a
binocular microscope (M 205 FA; Leica). The positive
area was quantitatively analyzed using Leica
Application Suite software (version 3.4.1; Leica).
Data are reported as mean ± SD. P values were
calculated using Tukey’s multiple comparison test method in
SPSS 21.0 (IBM, Armonk, NY, USA).
DLA genotyping and matching analyses in 26 dogs
demonstrated a four homozygous allele profile (nine dogs), a
three homozygous and one heterozygous allele profile
(three dogs), a two homozygous and two heterozygous
allele profile (four dogs), a one homozygous and three
heterozygous allele profile (one dog), and a four
heterozygous allele profile (nine dogs). In the four homozygous
allele profile group, eight dogs had eight completely
matched alleles (Group A) out of nine dogs. In the two
homozygous and two heterozygous allele profile group,
four dogs had seven matched alleles. In the four
heterozygous haplotype group, four dogs had seven matched
alleles (Group B) out of nine dogs (Table 3). We
selected five identical and almost identical donors of
the allele profiles (four dogs from Group A, one dog
from Group B) and five nonidentical donors with at
least four mismatched alleles for allogeneic
transplantation (Table 4).
The isolated canine MDPSCs
The isolated and cryopreserved MDPSCs at the 7th
passage of culture were stellate with short processes or
spindle-shaped. The cell viability was more than 90%
following thawing of the frozen cells. The doubling time was
approximately 30 h as previously isolated from canine
teeth transported by land within 1 h [
], suggesting that
the transportation of the extracted teeth by air within 30 h
did not affect the cell proliferation ability. The mRNA
expression levels of PTGES, COX-2, IL-6, TGF-β, and IDO-1
were similar in MDPSCs and MADSCs derived from the
same individual dog (Table 5), suggesting similar
immunomodulatory/immunosuppressive function of MDPSCs to
Safety of allogeneic transplantation
Toxicology assessment showed no adverse effects on
appearance, clinical signs, food consumption, and body
weight for 12 weeks after allogeneic first transplantation
of the MDPSCs from four DLA-nonidentical donors as
well as those from three DLA-identical and one almost
DLA-identical donors. The blood test demonstrated no
increase of white blood cell and platelet numbers
(Table 6), demonstrating no alloreaction toward the
transplanted cells. Serum and urine chemistry
parameters showed values within normal ranges at 4 and
12 weeks after both first and second allogeneic
transplantation (Table 6). Furthermore, there was also no
evidence of toxicity or adverse events at 4 and 12 weeks
after second DLA-nonidentical and DLA-identical
transplantation of the same type of MDPSCs as in the first
transplantation. No abnormalities were caused by the
allogeneic transplantation in any organ or tissues
assessed by histopathological examinations at 12 weeks
after the second transplantation. These results
demonstrate that DLA mismatched transplantation might be
safe for pulp regeneration for 12 weeks in dogs not only
the first time but also the second time.
Pulp regeneration after allogeneic transplantation
We next compared the regenerated tissue after DLA
mismatched MDPSC transplantation with matched MDPSC
transplantation into the pulpectomized root canal (Fig. 2).
Pulp-like tissues with well-developed vasculature were
observed at 12 weeks in both the allogeneic first transplants
(Fig. 2a, b, d, e and Additional file 1: Figure S1). Similar
pulp-like tissues in cell morphology, cell density, and
architecture of the extracellular matrix with a few
inflammatory cells were also regenerated at 12 weeks in both the
DLA mismatched and matched second transplants
(Fig. 2g, h, j, k). Odontoblast-like cells with a long process
and osteodentinoblast-like cells were attached to the wall
of newly formed osteo/tubular dentin (Fig. 2c, f, i, m) and
neither inflammation nor internal/external resorption was
detected (Fig. 2j, n). The regenerated tissue was filled in
the root canal more than 80% to the dentin–enamel
junction in all four transplants. The statistical analysis
demonstrated no difference among the four transplants (Fig. 2o).
Vascularization demonstrated by BS-1 lectin staining was
also similar in density and orientation in the four groups
of transplants (Fig. 3a–d), and there was no significant
differences in the capillary density among them (Fig. 3e).
Nerve fibers stained by PGP9.5 antibody were similarly
observed, indicating similar reinnervation (Fig. 3f–i).
Dentin-like mineralized tissue formation was similarly
observed along the dentinal wall (Fig. 3j–m). There was no
significant difference in the matrix formation among the
four transplants by morphometric analysis of the Masson
trichrome positively stained area (Fig. 3n). These results
suggest no qualitative and quantitative differences in the
regenerated tissue between the DLA mismatched and
matched transplants and between first and second
The aim of the present investigation was to evaluate the
safety and efficacy of allogeneic transplantation of DLA
matched and mismatched MDPSCs in pulpectomized
teeth with complete apical closure for pulp/dentin
regeneration in dogs. A crucial challenge, however, is the
limitation of genotyping major histocompatibility with
relevance for humans using animal models in a
preclinical study. The dog is a suitable animal model for pulp
regenerative therapy, where the incisor tooth and its
dental pulp tissue are similar in anatomy and
developmental biology to humans [
]. Tissue regeneration in
the dog may also be influenced by similar factors as in
humans, including the immune system [
] and genetics
]. Thus, dogs have served as an effective, directly
translatable model for MSC transplantation [
Major histocompatibility complex (MHC) genes in
mammals include class I and class II genes that are
highly polymorphic and their donor–recipient matching
is important for cell transplantation . The genes for
the dog MHC or DLA have been defined as a
sequencebased nomenclature [
]. There are four complete
class I genes: DLA-88, DLA-12, DLA-64, and DLA-79,
in which DLA-88 is highly polymorphic (more than 72
alleles) and the others are less polymorphic. In the class
MPDSC mobilized dental pulp stem cell, MADSC mobilized adipose derived
stem cell, PTGES prostaglandin E synthase, COX-2 cyclooxygenase-2, IL
interleukin, TGF transforming growth factor, IDO-1 indoleamine 2,3-dioxygenase 1
2.6 ± 3.5
0.9 ± 0.7
1.3 ± 0.6
1.6 ± 0.7
1.5 ± 1.9
II region, there are DLA-DQA1 (nine alleles),
DLADQB1 (20 alleles), DLA-DRB1 (at least 24 alleles), and
DLA-DRA (monomorphic) [
]. In the present work,
DLA genotyping and matching analysis were performed
in 26 dogs by PCR for the four genes orthologous to the
human genes including DLA-88, DLA-DQA, DLA-DQB,
and DLA-DRB . The results demonstrated eight dogs
with four completely matched alleles (four homozygous
allele profile), four dogs with three completely matched
alleles (two homozygous and two heterozygous allele
profile), and four dogs with three completely matched
alleles (four heterozygous allele profile). The similarity of
the allele profiles was not due to related siblings, and all
of the MDPSCs were transplanted into unrelated
recipients. Based on an analysis of canine DLA diversity, the
three-locus DLA haplotype, DQA1 00101;DQB1 00201;
DRB1 00102 represented 40.3% and DQA1 00101;DQB1
00201;DRB1 00101 represented 11.9% in the beagle [
which is higher than the present rate: eight dogs out of
26 (30.7%) and only one dog out of 26 (3.8%),
respectively, in the present study, suggesting the possibility of a
distinct breed. The true extent of diversity of DLA genes
in canines, especially of the class I gene DLA-88,
however, is still unknown [
A variety of animal and human studies have
demonstrated that stem cell-based therapy with allogeneic
MSCs is a potential therapeutic option to regenerate
damaged tissue/organ [
]. The low immunogenicity
and immunomodulatory/immunosuppressive properties
of allogeneic MSCs may contribute to a reduced
immune response [
]. We have previously demonstrated
lack of expression of MHC class II and costimulatory
molecules, such as CD40, CD80 (B7-1), and CD86
(B72), although MHC-I is expressed in human MDPSCs
]. We have also demonstrated that human and canine
MDPSC conditioned medium inhibits allogeneic
peripheral blood mononuclear cell (PBMC) proliferation and
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demonstrates a dose-dependent inhibition of PBMC
immune responses in mixed lymphocyte reaction (MLR)
], confirming the work in DPSCs . IFN-γ
secreted by activated PBMCs induce the expression of
soluble factors by DPSCs, which may play an important
role in the immunosuppressive process [
of PGE2, TGF-β, indolamine-2, 3-dioxygenase-1 (IDO1),
IL-6, IL-10, and COX2 triggers the immunosuppressive
activity of DPSCs [
]. The present study
demonstrated that genes related to immunomodulation
(See figure on previous page.)
Fig. 3 Histochemical analyses of regenerated pulp tissue. Immunostaining with (a–d) BS-1 lectin, (f–i) PGP 9.5 (arrows indicate neurite extension),
and (j–m) Masson trichrome staining (hatched lines indicate newly formed osteo/tubular dentin (d)). a, b, f, g, j, k First transplantation. c, d, h, i,
l, m Second transplantation. a, c, f, h, j, l DLA matched transplant. b, d, g, i, k, m DLA mismatched transplant. e Ratio of positively stained area
by BS-1 lectin in a frame 310 μm × 240 μm in size. Data are mean ± standard deviation (n = 4). Tukey’s multiple comparison test method. n Ratio
of Masson trichrome positively stained area to pulp regenerated area. Data are mean ± standard deviation (n = 3). Tukey’s multiple comparison
including prostaglandin E synthase (PTGES), COX-2,
IL6, TGF-β, and IDO1 were similarly expressed in canine
MDPSCs compared to canine mobilized adipose-derived
MSCs. Fas ligand (FasL) associated with the
immunoregulatory properties of DPSCs in the context of inducing
T-cell apoptosis [
] was also expressed in MDPSCs
(data not shown). The present results are consistent with
the previous studies in human MSCs [
demonstrating low immunogenicity of MDPSCs and potential
to induce immune tolerance in the hosts.
Furthermore, MSCs can inhibit the immune response
not only by suppressing T cells and by inducing Tregs
but also by converting macrophages into a regulatory
]. Polarization of macrophages toward the
anti-inflammatory M2 phenotype has been reported
after DPSC transplantation in diabetic peripheral nerves
]. In addition, similar findings were seen by injection
of conditioned medium from stem cells of human
exfoliated deciduous teeth (SHED) into acute lung injury [
autoimmune encephalomyelitis [
], and rheumatoid
]. Further investigation is necessary to
examine the shift in the macrophage phenotype from M1 to
M2 in the regenerated pulp and periapical tissue after
allogeneic DPSC transplantation.
MSCs express detectable levels of MHC class I and
low levels of MHC class II to avoid recognition by a
host immune system in allogeneic therapies [
Several animal and clinical studies, however, have
demonstrated that MSCs are weakly immunogenic in
vivo in the case of transplantation across MHC class
I barriers and that MSCs are rejected to induce
chronic immune responses [
responses could restrict the effectiveness of repeated
transplantation of allogeneic MSCs . Syngeneic
and minor mismatched transplantation of synovial
MSCs demonstrated more optimal meniscus
regeneration compared with major mismatched
transplantation in a meniscectomized model [
]. On the other
hand, the correlation between the number of donor–
host MHC mismatches and the efficacy of treatment
was not detected in local injection in osteoarthritis
and degenerative disc disease [
osteogenically differentiated MSCs are
immunomodulatory and lack immunogenicity, demonstrating
potential use in bone repair. However, maintenance of
these properties in vivo is still open to question since
immunogenic markers are upregulated after
transplantation of the differentiated MSCs [
]. In the
present study there was no correlation between the
number of donor–host mismatches and efficacy,
demonstrating the lack of immune response. This may be
due to reduced host immune responses by the
transplanted MDPSCs and effective confinement of these
cells into the root canal of the tooth. Another
possibility is that the transplanted MDPSCs are not
differentiated into any host cells in the regenerated pulp
tissue. Our previous study demonstrated that injected
DiI-labeled autologous MDPSCs did not differentiate
into host cells and induced pulp regeneration by
secreting trophic factors to elicit migration and
proliferation and inhibit apoptosis of endogenous MSCs
]. We further demonstrated that transplanted
porcine pulp MSCs were not directly incorporated into
endothelial cells, neuronal cells, or host pulp cells in
mouse ectopic tooth transplantation models [
Although MSCs are known to be immunoprivileged,
repeated transplantation of mismatched MSCs has been
reported to lead to alloimmunization and subsequent
refractoriness in mice [
]. Multiple intravenous
injections of allogeneic MSCs are well tolerated in healthy
horses, indicating no clinical signs of organ toxicity and
systemic inflammatory response. A mild
alloantigeninduced cytotoxic response, however, is suggested by an
increased numbers of circulating CD8+ T cells . Dual
injection of allogeneic MSCs into joint and articular
cartilage induces an adverse clinical response, suggesting
immune recognition of allogeneic MSCs after the
second infection [
]. Allogeneic MSCs are weakly
immunogenic when transplanted across MHC
boundaries in rhesus monkeys, indicating negative
impact by dual transplantation , while repeated
intravenous injection of human umbilical cord
bloodderived MSCs has low immunogenicity and no adverse
events detected in mice [
] and humans [
Furthermore, there are no toxicological abnormalities and no
obvious pathological changes although CD3+ and IL-6
levels are significantly increased after repeated
intravenous injection of monkey umbilical cord MSCs [
the present study, there were no toxicological
abnormalities and no significant difference in tissue volume of
regenerated dental pulp and inflammatory cell
infiltration between the first and second transplants for both
DLA matched and mismatched MDPSCs. This result
demonstrates that MDPSCs are immunologically safe for
use in allogeneic applications.
In this preclinical study, the safety of allogeneic
mismatched MDPSC transplantation in pulpectomized teeth
was demonstrated. Regenerated pulp tissues including
neovascularization and neuronal extension were similar in
the DLA mismatched transplants compared to the DLA
matched transplants even after dual transplantation of
MDPSCs, suggesting efficacy for total pulp regeneration.
Additional file 1: Figure S1. Histochemical analyses of normal pulp tissue. Immunostaining with (A) BS-1 lectin and (B) PGP 9.5. Neurite extension (arrow). (PDF 155 kb)
G-CSF: Granulocyte colony-stimulating factor; MDPSC: Mobilized dental pulp stem cell
The authors thank Mr Takahiro Nakamura from Shin Nippon Biomedical
Laboratories Ltd for technical assistance of allogeneic matched and mismatched transplantation.
This work was supported by the Budget for Promoting Science and Technology
in Japan, directly following the policy of the Council for Science and Technology
Policy (CSTP) chaired by the Prime Minister (to MN).
Availability of data and materials
Please contact author for data requests.
KI contributed to provision of study materials, collection and/or assembly of
data, data analysis, and manuscript writing. SU contributed to collection of
data and data analysis and interpretation. SK contributed to collection of
data and data analysis and interpretation. MN contributed to conception and
design, financial support, collection and/or assembly of data, data analysis
and interpretation, manuscript writing, and final approval of the manuscript.
All authors read and approved the final manuscript.
This study was approved by the Animal Care and Use Committee of the
National Center for Geriatrics and Gerontology (NCGG) (animal 25-24: national guideline) and Shin Nippon Biomedical Laboratories, Ltd (IACUC 860-013, 015: international guideline). All experiments were conducted using the strict guidelines of DNA Safety Programs.
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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