Culture of hpDPSCs
All animal procedures were approved by the Animal Care and Use Committee of the National Center for Geriatrics and Gerontology, Research Institute and the Aichi Medical University (permission #30-19, #31-17). All procedures and methods were performed in accordance with relevant guidelines and regulations. Upper third incisors, a total of 12 teeth from 6 young female beagle dogs (Kitayama Lab, Ina, Japan) at 1-year-old were used for isolation of MDPSCs and hpDPSCs. MDPSCs based on their migratory response to G-CSF (Neutrogin, Chugai Pharmaceutical, Tokyo, Japan) were isolated at the 2nd passage from colony-derived primary DPSCs and cultured according to our previous study . For hpDPSCs, isolated DPSCs were cultured in a stable hypoxic condition in a closed container with a regular octahedron having 21cm2 of each surface (Animal Stem Cell, Tokyo, Japan), in which humidified gas mixtures of the composition of 5% O2-5% CO2-90% N2 were flushed. The pH of the hypoxic cultures was adjusted by adding HEPES buffer (Gibco, Dublin, Ireland) at a final concentration of 25 mM to Dulbecco’s Modified Eagle’s Medium (DMEM) (Sigma Aldrich, MO, USA) supplemented with 10 % fetal bovine serum (FBS, GE Healthcare, Little Chalfont, England). Each sensor chip (SP-LG1-SA-S, and SP-PSt3-SA, PreSens, Regensburg, Germany) was patched on the bottom of the container inside, respectively, and the pH and O2 concentration in the DMEM and the air were measured by non-contact pH meter (pH-1SMA LG1; PreSens) and non-contact oxygen analyzer (OXY-1SMA trace, PreSens).
The primary colony-derived DPSCs were expanded in the same one surface of the octahedron container at the 2nd passage of culture, and further cultured in the all surface of the container by rotating 45 degree every 1 min by a rotary equipment (Biomedica Solution, Ibaraki, Japan) at the 3rd passage. These hpDPSCs were detached and cryopreserved at 1 × 106 cells /mL in the stem cell banker (ZENOAQ Co., LTD., Fukushima, Japan) for further experiments.
The population doubling time was calculated by counting the cell number from the 2nd expansion to the 3rd expansion. The cells were stained with trypan blue and the viable cells were counted with a hemocytometer.
Real-time reverse transcription-polymerase chain reaction analysis
For real-time reverse transcription-polymerase chain reaction (RT-PCR) analysis, total RNA was extracted using TRIzol (Thermo Fisher Scientific, Waltham, MA, USA) from hpDPSCs and MDPSCs at the 5th to 6th passage of culture. First-strand cDNA syntheses were performed from 1 µg of total RNA by reverse transcription using ReverTra Ace-α (Toyobo, Tokyo, Japan). Real-time-PCR amplifications were performed using canine HIF-1α (forward) 5′-ACTGATGACCAACAACTTGAGG-3′ and (reverse) 5′-TTTGGAGTTTCAGAAGCAGGTA-3′. Canine stem cell markers, angiogenic/neurotrophic factors, and immunomodulatory factors were used as our previous studies [8, 25]. All primers were labeled with Power SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA) in 7500 RT-PCR system (Applied Biosystems). The relative mRNA expression was examined in hpDPSCs to MDPSCs after normalizing with β-actin.
Migration activity of hpDPSCs and MDPSCs
To determine the migratory activity in response to G-CSF, 1×105 of hpDPSCs or MDPSCs were seeded in 100 µl of DMEM on top of an insert membrane with 8 µm pore size in 24-well plates (Corning- Transwell- polycarbonate membrane cell culture inserts, Sigma-Aldrich, Missouri, USA). The lower compartment medium contained 2% FBS and was supplemented with or without G-CSF (100 ng/ml). After 24 h, cells were removed from the top of the membrane with cotton swabs. The migrating cells on the lower surface of the membrane were fixed with 95% methanol and stained with 0.5% Giemsa stain for 15 min. After washing, the stained cells were counted in 4 fields per well under an inverted bright-field microscope (Leica, 6000B-4, Leica Microsystems GmbH, Wetzlar, Germany) at × 100 magnification.
The effect of the CM on angiogenesis and neurite extension
For collecting the conditioned media (CM), the hpDPSCs and MDPSCs were cultured in the complete cultured medium. The medium was changed into DMEM without serum at 70% confluence, and the CM collected 48 h later and concentrated by Amicon Ultra-15 Centrifugal Filter (Millipore, Billerica, MA, USA). To compare the stimulative effect of the CM on endothelial cell differentiation, human umbilical vein endothelial cells (HUVEC, clone 7F3415, Lonza) were seeded on Matrigel (BD Biosciences, San Jose, CA, USA) in DMEM containing 2% FBS, 5 µg/ml heparin (Lonza, Basel, Switzerland), 5 µg/ml ascorbic acid (Lonza), 5 µg/ml hydrocortisone (Lonza) supplemented with the CM (5 µg/ml proteins). The mean length of networks of cords and tube-like structures was measured 5 h after cultivation under the inverted microscope (Leica) using ImageJ software (version 1.52, imagej.nih.gov). The same experiment was performed with 100 ng/ml G-CSF (Peprotech, London, UK) as a positive control.
For examining the effect on neurite outgrowth, human neuroblastoma cell line (TGW, clone JCRB 0618, Health Science Research Resources Bank, Japan) was cultured without serum overnight and then stimulated with the CM (5 µg/ml proteins) for 24 h. The mean neurite length was measured under the inverted microscope using ImageJ software (version 1.52, imagej.nih.gov). The same experiment was performed with 50 ng/ml Neurotrophin-3 (Peprotech, London, UK) as a positive control.
The combinatorial effect of the CM with G-CSF on migration
The migratory effects of the CM of hpDPSCs or MDPSCs together with G-CSF were compared with those of the CM only. Periodontal ligament cells (PDLCs) from young dog (10 months old) were isolated according to the previous study . PDLCs were cultured in DMEM supplemented with 10% FBS and cryopreserved at the 4th to 7th passage of culture. For the migratory activity, 1× 105 of PDLCs were seeded in 100 µl of DMEM on top of the insert membrane. The lower compartment medium containing 2% FBS were supplemented with 5 µg/ml CM with or without 100 ng/ml of G-CSF. G-CSF only and 2% FBS only were used as a positive control and as a negative control respectively. After 24 h, the migrating cells were stained as previously described.
Anti-apoptotic activity of the CM
To examine the anti-apoptotic effect of G-CSF, hpDPSCs or MDPSCs were incubated with 500 nM staurosporine (Sigma) in DMEM supplemented with 100 μg/ml of G-CSF. After 3 h, cells were harvested and the activity of caspase-3 was measured using APOPCYTOTM Caspase-3 Colorimetric Assay Kit (Medical and Biological Laboratories, Nagoya, Japan) according to the manufacturer’s protocol.
For examining the combinatorial effect of the CM with G-CSF on anti-apoptotic activity, canine PDLCs were cultured in DMEM with staurosporine and 50 μg/ml of CM with or without 100 ng/ml of G-CSF and the activity was measured as previously described.
Immunomodulation activity upon stimulating with interferon gamma (IFN-γ)
hpDPSCs and MDPSCs were stimulated with IFN-γ (PROSPEC, East Brunswick NJ, USA) at a concentration of 20 ng/ml in DMEM without serum for 24 h according to the previous studies [27, 28] with slight modification. Non-stimulated cells were used as a control. RNA was extracted using Trizol and the CM was collected and concentrated. The mRNA expression of immunosuppressive markers, IDO, TGF-β1, PTGE and IL-6 as our previous study  were examined by RT-PCR. The concentration of nitric oxide (NO) was examined by measuring its stable end product, nitrite, in the CM using a Griess reagent (Promega Corporation, Madison, WI, USA) according to manufacturer’s protocol. Absorbance at 540 nm was measured by microplate reader (SpectraMax Gemini XPS/EM, Molecular Devices, San Jose, CA, USA) and nitrite concentrations were calculated using a standard nitrite curve.
Comparison of trophic factor mRNA expressionbetween the rotating and stationary conditions
After validation the pH and O2 concentration, we examined the effect of the rotating culture. Freshly isolated pulp cells from an upper fourth incisor from one-year old dog were plated into three containers and the cultured hpDPSCs were further divided into the two containers at the 3rd passage of culture respectively; 1/9 of the total cells was plated in the stationary condition at one surface of the octahedron closed container with 5% O2 and the remaining 8/9 was plated in the rotating condition at eight surfaces of the container with 5% O2. The trophic factor mRNA expression was compared by RT-PCR between the stationary and rotating conditions.
Transplantation of hpDPSCs and MDPSCs into pulpectomized teeth in dogs
Upper first and second incisors, a total of 12 teeth from 3 young female beagle dogs (Kitayama Lab, Ina, Japan) at 1-year-old were used. Transplantation of hpDPSCs or MDPSCs at 5 × 105 cells together with G-CSF (Neutrogin) in 20 μl of atelocollagen scaffold (1% atelocollagen implant; Koken, Tokyo, Japan) was performed for pulp regeneration in pulpectomized teeth as described previously with slight modification . The teeth were extracted at 4 weeks after cell transplantation. Histological examination of the regenerated tissue was performed in the paraffin sections (5 μm in thickness) of the teeth. The regenerated tissue was outlined in on-screen image of the histological preparations of each 4 sections (n=6) by a binocular microscope (Leica, M 205 FA Leica Microsystems, Wetzlar, Germany) and its relative amount to the root canals was determined by using Leica Application Suite software (Leica, version 3.4.1). For neovascularization and innervations analyses, Fluorescein Griffonia (Bandeiraea) Simplicifolia Lectin 1/fluorescein-galanthus nivalis (snowdrop) or anti-PGP9.5 (Ultra Clone) (1: 10,000) were used respectively. The ratios of newly formed capillary area and neurite extension area to the regenerated pulp area were measured respectively by Dynamic cell count BZ-HIC (KEYENCE, Osaka, Japan).
For evaluation of safety of the hpDPSC transplantation, each upper left second incisor was extracted from 3 dogs at 9 to 12 months-old in Shin Nippon Biomedical Laboratories Ltd and transported to the National Center for Geriatrics and Gerontology to isolate and culture hpDPSCs. The cryopreserved hpDPSCs were transported by air to the animal facility in Shin Nippon Biomedical Laboratories Ltd, and autologously transplanted at 5 × 105 cells into pulpectomized upper right second incisors (n=3) as described previously. The dogs were observed in clinical signs, daily food consumption, and weekly weight change for toxicology assessment. Urinalysis were performed by Clinitek AtlasXL (Sparton Medical Systems, Strongsville, OH, USA) at 2 and 4 weeks. Blood tests and blood chemistry examinations were performed by ADIVIA 120 (Siemens Healthcare Diagnostics Manufacturing Ltd, Erlangen, Germany) and by JCA-BM6070 (Japan Electron Optical Laboratory, Tokyo, Japan), respectively at 1, 2 and 4 weeks. One dog without cell transplantation was used as a control. The transplanted teeth were extracted and all organs were weighed and macroscopically examined at 4 weeks followed by euthanization. They were further examined histopathologically in the paraffin sections stained with hematoxylin and eosin (HE).
All of the results were expressed as the means ± standard deviation (SD). The data were analyzed statistically using a t-test or one-way ANOVA with Tukey’s comparison test as a posttest using SPSS 25.0 (IBM, Armonk, NY).