Patient selection

DPPSC were isolated from healthy human third molars extracted for orthodontic and prophylactic reasons from 15 patients with ages between 14 and 21 years old. All patients (or their legal guardians) provided informed consent before obtaining the samples. This study was approved by the Committee on Ethics in Research (CER) of the Universitat Internacional de Catalunya (Spain) under the protocol code BIO-ELB-2013-04.

Isolation and culture of DPPSC

DPPSC were extracted and isolated as previously described [2]. Briefly, teeth were washed after extraction using gauze soaked in 70% ethanol and dental pulp was extracted from the teeth using a sterile nerve-puller file 15 and forceps (if the apexes were still open) or fracturing the teeth and taking the dental pulp using forceps. The dental pulp was placed in sterile 1X phosphate-buffered saline (PBS; Life Technologies, Carlsbad, CA, USA) with 5% of 0.25% trypsin-EDTA (Life Technologies) and 1% penicillin-streptomycin (Life Technologies) and transferred to the laboratory. The tissues were disaggregated by digestion with collagenase type I (3 mg/mL; Sigma-Aldrich, St. Louis, MO, USA) for 60 minutes at 37 °C. Obtained cells were cultivated in DPPSC medium, which consisted of 60% Dulbecco’s modified Eagle’s medium (DMEM)-low glucose (Life Technologies) and 40% MCDB-201 (Sigma-Aldrich) supplemented with 1 × insulin-transferrin-selenium (ITS; Sigma-Aldrich), 1 × linoleic acid-bovine serum albumin (LA-BSA; Sigma-Aldrich), 10-9 M dexamethasone (Sigma-Aldrich), 10-4 M ascorbic acid 2-phosphate (Sigma-Aldrich), 100 units of penicillin/1000 units of streptomycin (Life Technologies), 2% foetal bovine serum (FBS; Sigma-Aldrich), 10 ng/mL human PDGF-BB (Abcam, Cambridge, UK), 10 ng/mL epidermal growth factor (EGF; R&D Systems, Minneapolis, MN, USA), 1000 units/mL human leukemia inhibitory factor (LIF; EMD Millipore, Billerica, MA, USA), Chemically Defined Lipid Concentrate (Life Technologies), 0.8 mg/mL BSA (Sigma-Aldrich) and 55 mM β-mercaptoethanol (Sigma-Aldrich) in 650 mL flasks precoated overnight with 100 ng/mL fibronectin at 37 °C in a 5% CO2 incubator. During the 2 weeks of primary culture, the medium was changed every 4 days. To propagate DPPSC, the cells were detached at 30% confluence by adding PBS containing 0.25% trypsin-EDTA (Life Technologies) and replated at a density of 100–150 cells/cm2. Cell populations from different donors at passage 5 and passage 10 were negative for mycoplasma contamination (MycoAlert™ Mycoplasma Detection Kit, Lonza, Basel, Switzerland).

Culture of HUVECs

Human umbilical vein endothelial cells (HUVECs; Lonza; tested for mycoplasma, bacteria, yeast, fungi, HIV-1, hepatitis B and hepatitis C) were maintained in Endothelial Growth Medium 2 (EGM-2; Lonza). The medium was changed every 2 days and the cells were passaged when they reached 70–85% confluence using PBS containing 0.25% trypsin-EDTA (Life Technologies) and replated at a density of 2.5 × 103 cells/cm2.

Culture of C2C12 cells

The mouse immortalised myoblast cell line C2C12 was maintained using DMEM 4.5 g/L glucose supplemented with 10% FBS (Hyclone, South Logan, UT, USA), 1% glutamine (Sigma-Aldrich), 1% sodium pyruvate (Life Technologies) and 1% penicillin/streptomycin (Life Technologies). Cells were negative for mycoplasma contamination (MycoAlert™ Mycoplasma Detection Kit, Lonza).

Lentiviral transduction

DPPSC were seeded at a density of 500 cells/cm2 and transduced at 60% confluence with lentiviruses encoding for turbo green fluorescent protein (tGFP) from Pontellina Plumata for 24 hours. Briefly, incompetent viral particles were produced in packaging cells (HEK293T) by co-transfecting the tGFP lentiviral vector plasmid (Sigma-Aldrich shc003) with compatible Lentiviral Packaging Mix (Sigma-Aldrich). This method allowed generation of 100% GFP+ DPPSC that were used for the wound healing assay.

In vitro EC differentiation

DPPSC were seeded in 24-well plates at 2 × 104 cells/cm2 and differentiated using EGM-2 (Lonza) conditioned for 24 h with HUVECs (and then filtrated with 0.22-μm diameter pores to eliminate any HUVECs). The medium was changed every 2–3 days for 28 days. RNA extraction and Matrigel™ assay were performed at day 7, 14, 21 and 28 of differentiation. Immunofluorescence analysis was performed at day 28. For the Matrigel™ assay, DPPSC were detached using PBS containing 0.25% trypsin-EDTA (Life Technologies) and 50,000 cells were replated in EGM-2 medium in a 24-well coated with Matrigel™ (Basement Membrane Matrix Growth Factor Reduced; BD Biosciences, San Jose, CA, USA). Matrigel™ coating was performed following manufacturer’s instructions for the Thin Gel Method assay, adding 250 μl of Matrigel™ in a 24-well and keeping it 30 minutes at 37 °C. After 24 hours, tube-like structures were analysed and pictures were taken with an OX.3040 Euromex binocular microscope for phase contrast using the camera DC.10000c CMEX-10 digital 10 Mpix USB-2 CMOS (Euromex, Arnhem, The Netherlands).

In vitro SMC differentiation

DPPSC from two different donors and two passages (passage 5 and passage 10) were differentiated to SMCs using differentiation media consisting of High-Glucose DMEM (Life Technologies) supplemented with 2% horse serum (Life Technologies), 1% sodium pyruvate (Life Technologies), 1% glutamine (Life Technologies), 1% penicillin-streptomycin (Life Technologies) and 50 ng/mL TGF-β1 (Peprotech, Rocky Hill, NJ, USA). Cells were plated in 24-well plates at 500 cells/cm2 and differentiation was started when they reached 60% confluence. Medium was changed every 2–3 days for 10 days. tGFP+ DPPSC from one of the donors were also differentiated using the same conditions.

In vitro skeletal muscle differentiation

DPPSC from three different donors and two passages (passage 5 and passage 10) were differentiated to skeletal muscle using differentiation media consisting of High-Glucose DMEM (Life Technologies) supplemented with 2% horse serum (Life Technologies), 1% sodium pyruvate (Life Technologies), 1% glutamine (Life Technologies) and 1% penicillin-streptomycin (Life Technologies). Cells were differentiated alone or in direct co-culture with C2C12 cells at 1:1 ratio. The cells were plated in 24-well plates at a density of 1 × 104 cells/cm2 and induction was started when cells reached 70% confluence. Medium was changed every 2–3 days for 5–7 days. tGFP+ DPPSC from one of the donors were also differentiated in the same conditions. Fusion index was calculated as the ratio of the number of nuclei inside the MyHC+ myotubes to the number of total nuclei (percentage).

Wound healing assay

Eight-week-old male athymic nude mice (Foxn1, Charles River Laboratories, Wilmington, MA, USA) were treated with DPPSC (P10) or PBS (n = 5 for each condition). The mice were injected with anti-NK to eliminate natural killer cell activity 24 hours before starting the surgery. Full-thickness wounds (0.5-cm diameter) were made on the back of the mice, splinted with silicone rings and treated with tGFP+ DPPSC (1 × 106 cells per mouse) or PBS. A Tegaderm™ dressing (3M, Maplewood, MN, USA) was applied to prevent the wound area from drying out. All wounded mice were housed individually to avoid fighting and to prevent removal of the occlusive wound dressing. Every other day, digital pictures of the wounds were taken (using a NikonD1 camera and Camera-Control-Pro software; Nikon, Tokyo, Japan) under isoflurane anaesthesia and the dressings were renewed. Presence of DPPSC was monitored at day 5 and 10 using a Zeiss SteREO Discovery.V12 microscope and Axio Imaging software (Carl Zeiss, Oberkocken, Germany). Wound contraction was evaluated by comparing relative wound area (RWA) over time. RWA was calculated using ImageJ software (NIH, Baltimore, MD, USA) by dividing the healing wound area by the fixed reference area inside the silicone ring and expressing it as a percentage. To account for small inter-animal variations, for each time point, relative wound area of each individual animal was expressed as percentage compared to the relative wound area at day 0. Wound contraction (%) was calculated as the complement of relative wound area (100-RWA). At day 11 after wounding, mice were sacrificed and skin fragments including the wound area and a rim of normal skin were dissected out, fixed, separated in two pieces at the midline and processed for paraffin embedding. For all stainings and analyses, 7 μm microtome sections were used. Mouse procedures were approved by the ethics committee for use of animals in research from KU Leuven (Belgium) under the protocol code ECD N°P018/2015.

DPPSC injection in dystrophic mice

Ten 3-month-old Scid/mdx mice, five males and five females, and four 3-month-old Sgcb-null Rag2-null γc-null mice, three males and one female, were injected in the left tibialis anterior with 2.5 × 105 cells per mouse. DPPSC from P10 were used. Untreated right limbs were used as controls. After 20 days, mice were sacrificed and muscles were frozen and kept at -80 °C. The samples were then cut in 7 μm sections using a cryostat (Leica, Wetzlar, Germany). Mouse procedures were approved by the ethics committee for use of animals in research from KU Leuven (Belgium) under the protocol code ECD N°P095/2012.

Short-comparative genomic hybridisation

The short-comparative genomic hybridisation (sCGH) technique was performed as described in Rius et al. [30] catching single cells from a homogeneous DPPSC culture. All samples were analysed in triplicate. The DNA control used for the hybridisation was XXY.

Quantitative reverse transcription (qRT)-PCR analysis

Samples of total RNA were extracted using Trizol (Life Technologies) and isolated following manufacturer’s instructions. Two μg of total RNA with a ratio 260/280 between 1.8 and 2 were treated with DNase I (Life Technologies) and reverse-transcribed using Transcriptor First Strand cDNA Synthesis Kit (Roche, Basel, Switzerland). Polymerase chain reaction (PCR) was performed using the primers in Additional file 1: Table S1 for the amplification of the desired cDNA using FastStart Universal SYBR Green Master (Roche) for Real-Time PCR using a CFX96 Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). The expression levels of the genes of interest were normalised against the housekeeping gene GAPDH and the relative expression levels were then normalised to undifferentiated DPPSC at P5 or at day 0 of differentiation, which were assigned as 1. All analyses were performed using the 2(-ΔΔCT) method and three technical replicates.

Cytokine antibody arrays

Undifferentiated DPPSC (1.5 × 106 cells) from three different donors were cultured for 48 hours in DPPSC medium without FBS, LIF or BSA. tGFP+ DPPSC were also used. The presence of several growth factors and cytokines secreted in the medium was analysed using human cytokine antibody arrays containing 80 target proteins (Abcam ab133998). Briefly, the supplied membranes (spotted with 80 different antibodies) were blocked with the provided buffer and subsequently incubated at 4 °C overnight with 1 ml of the DPPSC medium in contact with DPPSC for 48 hours. One of the membranes was incubated with an uncultured medium and used as a control. Membranes were washed with the provided buffers and incubated again at 4 °C overnight with the supplied biotin-conjugated anti-cytokines. The arrays were then washed, incubated with horseradish-peroxidase-conjugated streptavidin for 2 hours at room temperature and washed. Chemiluminescence reaction was detected using the supplied detection buffers. Semi-quantification by relative densitometry was obtained using Quantity One software (Bio-Rad) and normalised to the positive control signals in each membrane for comparison of multiple arrays and to EGF.

The presence of cytokines in lysates from DPPSC-injected and control muscles of dystrophic mice (three Sgcb-null Rag2-null γc-null mice and one Scid/mdx mouse) was also analysed using mouse cytokine antibody arrays containing 22 target proteins (Abcam ab133993). Briefly, muscle lysates were obtained by mechanical homogenisation in RIPA buffer (Sigma-Aldrich) supplemented with and 1 mM phenylmethanesulfonyl fluoride, 0.5 mM sodium orthovanadate, 1:100 protease inhibitor cocktail and 10 mM sodium fluoride (Sigma-Aldrich) using a T10 basic Ultra-Turrax homogeniser (IKA, Staufen, Germany). Subsequently, samples were sonicated (Branson digital sonifier 250; Branson Ultrasonics, Danbury, CT, USA) two times for 10 seconds each, kept on ice for 30 minutes, centrifuged at 13000 × g for 10 minutes at 4 °C and supernatants were collected. Protein concentrations were determined using the Bradford assay (Bio-Rad) and equal amounts of protein (300 μg) were incubated at 4 °C overnight with the supplied membranes (previously blocked). Subsequent steps were performed following the same protocol used with the human cytokine arrays.

Immunofluorescence analyses

Cryosections and cells were fixed with 4% paraformaldehyde for 15 minutes or ethanol-acetone 1:1 for 4 minutes at room temperature and then, after three PBS washes, incubated with 1% BSA + 0.2% or 0.5% triton for 30–45 minutes at room temperature to increase permeability. Cells were then incubated for 30–60 minutes with donkey serum at room temperature and, after that, 2 hours at room temperature or 4 °C overnight with the primary antibody (Additional file 2: Table S2). The following day, after three PBS washes, they were incubated for 1–2 hours at room temperature with the secondary antibody and washed again three times. DAPI (1:3000) was used for nuclear counterstaining, washed three times and wells containing cells or slides were mounted using FluorSave™ mounting medium (EMD Millipore). Paraffin-embedded sections were deparaffinised and rehydrated, followed by an antigen recovery step (putting the slides in citrate buffer pH = 6 for 20 minutes in the microwave, in trypsin 1:80 in 0.01% CaCl2 at 37 °C for 7 minutes or in target retrieval solution (Dako, Glostrup, Denmark) for 20 minutes at 95 °C). Samples were washed with Tris-buffered saline (TBS), incubated for 20 minutes with MeOH-H2O2 and washed again. They were then permeabilised using 0.5% triton, washed with TBS, blocked in 20% donkey serum or Tris-NaCl blocking buffer and incubated with the primary antibody (Additional file 2: Table S2) 2 hours or overnight at room temperature. Samples were washed with Tris-NaCl-Tween buffer, incubated with the secondary antibody and washed again. CD31, laminin and collagen type I and III signals were amplified using TSA Fluorescein System (Perkin Elmer, Waltham, MA, USA). Samples were mounted using Prolong Gold with DAPI (Life Technologies). Pictures were taken with an Eclipse Ti Microscope (Nikon) and NIS-Elements AR 4.11 software. Images were merged and/or quantified using ImageJ software (NIH).

Alkaline phosphatase staining

SIGMAFAST™ BCIP®/NBT (Sigma-Aldrich) was used following manufacturer’s instructions. Briefly, one tablet was dissolved in 10 mL of water, and 1 mL was added in one 24-well plate containing undifferentiated DPPSC. The solution was kept for 2 hours at 37 °C and the wells were washed afterwards with PBS. Fibroblasts were used as a negative control.

Haematoxylin and eosin staining

Paraffin sections were heated at 57 °C for 60 minutes, deparaffinised and rehydrated. Cryosections were fixed with 4% paraformaldehyde for 15 minutes. The samples were then soaked in distilled water for 5 minutes, Harris haematoxylin for 4 minutes and washed afterwards in running tap water for 2 minutes. The sections were subsequently soaked for 1 minute each in acid alcohol, running water, bluing reagent, running water, eosin, 95% ethanol, 100% ethanol and histoclear. The slides were then mounted with DPX and left on a slide heater overnight. Pictures from paraffin sections were taken with a Leica DMRBE microscope connected with an AxioCam MRc5 camera (Zeiss). Pictures from cryosections were taken with a Nikon Eclipse Ti microscope (Nikon). Epithelial coverage and thickness and cross-sectional area were determined using ImageJ software (NIH).

Sirius red staining

Sirius Red solution was prepared by mixing 0.2 g of Direct Red 80 (Sigma-Aldrich) with saturated aqueous solution of picric acid (prepared mixing 8 g of picric acid in 200 mL of distilled water). Paraffin sections were deparaffinised and rehydrated and crysections were fixed with 4% paraformaldehyde for 15 minutes. Sections were put in tap water for 10 minutes, distilled water for 5 minutes and Sirius Red solution for 90 minutes. After that, the slides were washed with HCL 0.01 N for 2 minutes and dehydrated with ethanol 70% for 45 seconds and ethanol 100% for 5 minutes (twice). Lastly, samples were cleared in xylol for 5 minutes (twice), mounted with DPX and left on a slide heater overnight. Pictures were taken with a Leica DMRBE microscope connected with an AxioCam MRc5 camera (Zeiss). Collagen quantification was determined using ImageJ software (NIH).

Masson’s trichrome staining

Cryosections were fixed with 4% paraformaldehyde for 15 minutes and subsequently incubated for 15 min at 57 °C in Bouin solution. Afterwards, cryosections were stained in Working Weigert’s Iron Hematoxylin Solution for 5 minutes, washed in running tap water for 5 minutes and stained in Biebrich Scarlet-Acid Fucsin for 5 minutes. Slides were subsequently soaked in de-ionised water and in Working PhosphotungsticPhosphomolybdic acid solution for 5 minutes and stained in Aniline Blue solution for 5 minutes and acid acetic 1% for 2 minutes. Slides were mounted and left in a slide heater overnight. Pictures were taken on a Nikon Eclipse Ti microscope (Nikon). Fibrotic area was quantified using ImageJ software (NIH).

Esterase staining

Cryosections were rehydrated and left in PBS for 5 minutes. A staining solution of 5 mL containing 2 mg NADH (Sigma-Aldrich) with 4 mg NBT (Roche) in 4.5 mL H2O and 0.5 mL 1 M Tris-HCl pH = 7.5 was prepared. Drops of prepared solution were added to the sections, which were then incubated for 30–60 minutes at 37 °C incubator. Samples were washed with H2O twice, dehydrated in ascending scale of alcohol (50%, 80%, 90% and 100%) for 1 minute each and then alcohol was removed with histoclear. Slides were mounted with DPX and left to dry in a heater. Pictures were taken on a Nikon Eclipse Ti microscope (Nikon). Cross-sectional area was calculated using ImageJ software (NIH).

Statistical analyses

Data from the different experiments were analysed using the statistical program Statgraphics Centurion XVI (Statgraphics, Warrenton, VA, USA). Two-tailed t test or one-way ANOVA were used to compare interrelated samples, while two-way ANOVA was used to analyse multiple factors. Confidence intervals were fixed at 95% (p < 0.05), 99% (p < 0.01) and 99.9% (p < 0.001). GraphPad Prism (GraphPad Software, San Diego, CA, USA) was used to graph the results as the mean ± standard error of the mean (s.e.m.). Refer to figure legends for specific information regarding the statistical test used and the number of independent experiments or biological replicates.