Canine Bone Marrow-derived Mesenchymal Stem Cells: Genomics, Proteomics and Functional Analyses of Paracrine Factors.

Adult stem cells have become prominent candidates for treating various diseases in veterinary practice. The main goal of our study was therefore to provide a comprehensive study of canine bone marrow-derived mesenchymal stem cells (BMMSC) and conditioned media, isolated from healthy adult dogs of different breeds. Under well-defined standardized isolation protocols, the multipotent differentiation and specific surface markers of BMMSC were supplemented with their gene expression, proteomic profile, and their biological function. The presented data confirm that canine BMMSC express important genes for differentiation toward osteo-, chondro-, and tendo-genic directions, but also genes associated with angiogenic, neurotrophic, and immunomodulatory properties. Furthermore, using proteome profiling, we identify for the first time the dynamic release of various bioactive molecules, such as transcription and translation factors and osteogenic, growth, angiogenic, and neurotrophic factors from canine BMMSC conditioned medium. Importantly, the relevant genes were linked to their proteins as detected in the conditioned medium and further associated with angiogenic activity in chorioallantoic membrane (CAM) assay. In this way, we show that the canine BMMSC release a variety of bioactive molecules, revealing a strong paracrine component that may possess therapeutic potential in various pathologies. However, extensive experimental or preclinical trials testing canine sources need to be performed in order to better understand their paracrine action, which may lead to novel therapeutic strategies in veterinary medicine.


INTRODUCTION
Regenerative medicine has gradually gained a significant position in current clinical veterinary practice. Stem cells have become prominent candidates for treating various diseases in companion animals (dogs and cats) where traditional treatments have proven unsuccessful (1). As part of cell therapy, various tissue-specific stem cells (TSSCs) have a dominant strategic role, mainly because they can be obtained from different adult tissues (2). However, the diversity of TSSC sources suggests that these cells will also have different properties and possibly different healing effects. Currently, we know that bone marrow-derived mesenchymal stem cells (BMMSC) are suitable candidates for repairing locomotor disorders, particularly hard tissue such as bone, cartilage and tendons (1,3,4). The ability of BMMSC to replace damaged bone and cartilaginous tissue is closely related to their mesodermal origin and natural multi-line differentiation.
BMMSC, on the other hand, are also assigned immunomodulatory and paracrine functions because of their ability to affect the immune system and produce a wide variety of growth and proliferation factors (1,5). These immuno-trophic properties are used to suppress strong inflammatory processes, vascularize ischemic tissue (myocardium, nerve tissue), but also to rejuvenate plasticity and regenerate nerve tissue (6). However, not all BMMSC-based treatments are able to evoke these beneficial properties. Mesenchymal stem cells (MSCs) isolated from bone marrow and adipose tissue (ATMSCs) are most commonly used as autologous or allogenic transplants also in veterinary medicine (7). However, MSCs have also been isolated from other tissues as well as from donors of various ages. Because there are around 400 dog breeds worldwide, the high interbreed genetic diversity may also influence the available pool of mesenchymal stem cells.
The main objective of our study was to compare BMMSC isolated from healthy adult dogs of different breeds using optimized protocols ensuring good quality of the isolated cells.
Under these conditions we analysed mesenchymal CD markers, their multipotent potential, gene expression and proteomic profile, and their biological function. Our results confirm that canine BMMSC express not only genes for hard tissue differentiation, but also genes associated with neurotrophic, angiogenic and immunomodulatory properties. Furthermore, using proteomics we identified for the first time the dynamics of molecules released from canine BMMSC conditioned medium and studied their pro-angiogenic effect in a CAM model. Finally, we suggest that optimized and standardized guidelines should be respected by guest on July 17, 2020 https://www.mcponline.org

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Canine bone marrow mesenchymal stem cells characterization 5 when choosing suitable donors for specific stem cell therapies. Moreover, further studies testing different canine MSC sources need to be performed in order to better understand the individual differences between healthy donors which need to be considered prior to their inclusion in treatment studies.

Isolation of bone marrow and peripheral blood
Bone marrow and peripheral blood were obtained from healthy adult dogs (n = 6) after informed consent of the owners was obtained. In this study we included the following large Handelsges. mbH, Germany) as premedication and a 3mg/kg i.v. bolus of Propofol (Propofol 1% MCT/LCT Fresenius, Fresenius Kabi, Germany) as induction. Anaesthesia was maintained with Propofol as required. Radiological control was used to evaluate the right position of the standard Jamshidi™ Bone Marrow Biopsy Needles (16ga). Briefly, the epiphysis of the humerus was palpated and aseptically prepared. A single stab incision was made with a No. 11 scalpel blade through the skin and subcutaneous tissue over the intended bone marrow aspiration site. The bone marrow biopsy needle was inserted into the bone with firm pressure and twisting motion. After penetration of the bone cavity, we aspirated 20 ml of bone marrow. Post-procedural analgesia was provided with single dose of meloxicam 0.2mg/kg s.c. (Meloxidyl 5mg/ml, Ceva Sante Animale, France).
Peripheral blood samples of venous blood were collected from each dog (3 ml) from the jugular vein, and one part (1 ml) was used for standard haematology profiling (IDEXX ProCyte Dx Haematology Analyzer, Idexx Laboratories, U.S.A.) and 2 ml as control for PCR array.

Experimental Design and Statistical Rationale
A total of six dogs were included in the study for flow cytometry (n=6), multilineage differentiation (n=6), proteomics, genomic and functional studies (n=3), all of the latter being by guest on July 17, 2020 https://www.mcponline.org

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Canine bone marrow mesenchymal stem cells characterization 6 biological replicates. The statistical analysis carried out for BMMSC cultivated at different time points (14h, 24h, 48h) was based on multiple-sample ANOVA testing with p-value=0.01.
Normalization was achieved using the Z-score. These analyses were carried out on Perseus Software.

In vitro culture of bone marrow-derived mesenchymal stem cells
From each dog we obtained 2 -5 ml of bone marrow suspension, which was diluted in sterile phosphate-buffered saline solution supplemented with antibiotic (PBS; Gibco; Switzerland) and centrifuged at 500 × g for 10 min. The bone marrow cells (including erythrocytes) were counted using the trypan blue exclusion method and plated at a density of 5 × 10 7 cells/cm 2 in alpha MEM medium (Gibco; Switzerland) supplemented with 10% fetal bovine serum (FBS), 100 units/ml penicillin, 100 mg/ml stre Switzerland) in tissue culture flasks T75 and incubated at 37°C, and 5% CO2. After 2 -3 days, non-adherent cells were removed, and adherent mesenchymal stem cells (MSCs) were washed and then cultured under the above-mentioned conditions until they reached 80% confluence. MSCs were washed 2 times with Dulbecco's Phosphate-Buffered Saline (DPBS) and dissociated from the adherent surface with Trypsin-EDTA (0.05%, Invitrogen, 0.25 % Trypsin-EDTA, Thermo Fisher Scientific; USA). The trypsin was neutralized with cell culture media supplemented with 10% FBS, the cells were centrifuged, counted using the trypan blue exclusion method and plated at 3.6 × 10 3 cells/cm 2 in T75 flasks. This procedure was repeated until 2 -3 passages were completed. Non-adherent cells were removed after 4 -5 days by means of medium change and the remaining cells were fed twice per week. When the cultures reached 80% of confluence, the cells were passaged with 0.25% trypsin/0.53mM ethylene diamine tetra acetic (EDTA) (Gibco; Switzerland), centrifuged, and re-plated at a density of 5000 cells/cm 2 .
BMMSC conditioned media preparation. BMMSC at passage 3 cultured in Dulbecco's modification of Eagle's medium (DMEM) with low glucose and without FBS were incubated in a humidified atmosphere with 5% CO2 at 37°C for 14h, 24h and 48h and used for BMMSC conditioned media (BMMSC CM). BMMSC phenotype was confirmed by means of flow cytometry with specific CD markers and three lineage differentiation, and afterwards the samples were processed for PCR array profiling, proteomic analyses and biological function in CAM assay.

Analysis of gene expression in BMMSCs
Quantitative real-time RT-PCR was used to analyse gene expression in BMMSC, and PBMCs were used as comparator ("calibrator").  Array results were analyzed using the webmethod with amplification efficiency = 2) provided by the manufacturer (SABiosciences/Qiagen, (software version 3.5, available on:www.qiagen.com/sk/shop/genesand-pathways/data-analysis-center-overview-page/ ). HPRT1 transcript quantity was used for normalization of target gene quantity, and the fold change in gene expression (transcript upor down-regulation) in MSCs compared to PBMCs was calculated.

Protein extraction
From each BMMSC CM (n=3) and time course (14h, 24h, 48h) 1 mL was collected, vacuumdried and then taken up in 100 μL of extraction buffer (4% SDS, Tris 0.1M, pH 7.8). The samples were heated at 95°C for 15 min and then sonicated for 15 min. Centrifugation was performed at 16,000 x g, 20°C, for 10 min, and then the supernatant was collected. After the extraction was complete, a Bradford assay was performed to determine the protein concentration in each sample. The samples were kept at -80°C until further experimentation.

FASP method
The samples were processed using a shotgun bottom-up proteomic approach. All samples were normalized with a final protein concentration of 1 mg/ml in 50 μL. An equivalent volume of reduction solution (Dithiothreitol DTT 0.1 M) was added to each sample followed by an incubation step at 56°C for 40 min. Then the samples were processed following the filteraided sample preparation (FASP) protocol (8, 9) using a filter with a nominal molecular weight limit of 30,000 KDa (Amicon Ultra-0.5 30K, Millipore). Briefly, each sample was

LC-MS/MS
The analysis of digested proteins was performed using a nano Acquity UPLC system (Waters) coupled with a Q-Exactive Orbitrap mass spectrometer (Thermo Scientific) via a nanoelectrospray source. The samples were separated by means of online reversed-phase, using a pre-concentration column (nanoAcquity Symmetry C18, 5 μm, 180 μm x 20 mm) and an analytical column (nanoAcquity BEH C18, 1.7 μm, 75 μm x 250 mm). The peptides were separated by applying a linear gradient of acetonitrile in 0.1% formic acid (5%-35%) for 2 hours, at a flow rate of 300 nl/min. The Q-Exactive was operated in data-dependent mode were defined as variable modifications. The Carbamidomethylation of cysteine was chosen as fixed modifications. The Label-free quantification (LFQ) was done keeping the default parameters of the software. As for initial mass tolerance, 6 ppm was selected for MS mode, and 20 ppm was set for fragmentation data with regard to MS/MS tolerance. The identification parameters of the proteins and peptides were performed with a false discovery rate (FDR) at 1%, and a minimum of 2 peptides per protein in which 1 was unique. The statistical analysis was done by Perseus software (version 1.6.2.1). Briefly, the LFQ intensity of each sample were downloaded in Perseus and the data matrix was filtered by removing the potential contaminants, reverse and only identified by site. The data was then transformed using the log2(x). Before statistical analysis, 3 groups were defined with 3 replicates per group. A Multiple-sample tests was performed using ANOVA with a p-value of 0.01, and the results were normalized by Z-score and represented as a hierarchical clustering.

Angiogenic assays
Chorioallantoic membrane (CAM) assay CAM assay was used to evaluate the angiogenic response to CM derived from BMMSC.
Fertilized chicken hybrid (Gallus gallus) eggs, Ross 308 (n = 30) were purchased from a commercial farm (Parovske Haje, Nitra,SK) and delivered via courier in a temperaturecontrolled manner to ensure egg viability and quality. The eggs were incubated blunt end up in a forced-draft, constant-humidity incubator at 37.5°C and 60% relative humidity until embryonic day (ED) three of the incubation period. At ED3 the eggs were windowed on the blunt end, and the albumen (egg white, cc 2ml) and the inner shell membrane (membrana papyracea) were carefully removed. The windows were closed using insulation tape and returned to a still-draft incubator for re-incubation until the day of implantation (37.5°C; 70% relative humidity, without rocking). On ED6, a sterilized silicone ring (d=5mm) was gently situated on the chorioallantoic membrane using suture tying forceps. In the control group space bordered by silicone ring. For visual evaluation of vascular density we used a stereomicroscope (Leica MZ125) fitted with a DSLR camera (Nikon D7000, Nikon, Tokyo, Japan) for photo documentation. Photo documentation of vascular density was done at the moment of application 0h and again after 72 h.
The number of vessels growing within the area outlined by the ring where BMMSC CM was applied was counted blinded using program FIJI ImageJ. Firstly, we had to convert each image to 8bit quality, and we used the AutoLocalTrashold and Otsu function, which highlights the vessels. To count the numbers of vessels and their branches in the experimental area (bordered by the silicone ring) we used the Cell Counter function, which counts every marked object (vessel or branch) automatically. Statistical analysis was carried out using Graph Pad InStat software. Data are presented as mean ± SD from 10 CAM. Mean values in different groups were statistically compared using one-way ANOVA and Tukey's post hoc tests. Values of P < 0.05 were considered statistically significant (*P value of < 0.05, **P value of < 0.01, ***P < 0.001).

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Canine bone marrow mesenchymal stem cells characterization 13 by the representative flow cytometry results of canine BMMSC in Figure 1A. The cultured cell population at passage 3, day in vitro 1 (DIV 1) contained attached spindle-shaped and fibroblastic-like cells (Figure 1B) which reached confluence approximately after 4 -6 days of cultivation (Figure 1C).

Multilineage potential
Three-lineage potential was tested with commercial StemPro® Differentiation Kits. We confirmed that canine BMMSC after 14 days of incubation in specific differentiation medium underwent a high degree of biomineralizing osteogenesis with visual staining of calcium deposits expressing Alizarin Red positivity (Figure 2A). Furthermore, chondrocytes migrating from spherical chondrocyte-like aggregates revealed intense Alcian Blue staining, which is typical for chondrogenesis ( Figure 2B). In contrast to the high osteogenic and chondrogenic differentiation of canine MSCs, we found a low degree of adipogenesis, with limited vacuole formation and Oil Red O staining (Figure 2C). System biology analyses revealed that the proteins in Cluster 1 were involved in neurite outgrowth, cell growth, vascularization and cell migration (Supp. Figure 1). Cluster 2 proteins were involved in ischemia and exocytosis (Supp. Figure 1), Clusters 3 and 4 were involved in cell differentiation, migration and adhesion and developmental process (Supp. Figure 1). Reactome analysis (14) revealed that hedgehog ligand biosynthesis as well as the immune response pathways were the most predominant, with an e-value of 3.10 -11 . Among the proteins identified, some were of interest such as Mesencephalic astrocyte-derived neurotrophic factor (MANF), which is known to selectively promote the survival of dopaminergic neurons in the ventral mid-brain (15) Karyopherin-2, a member of the importin family, serves as an adaptor protein in the mediation of cargo-specific synapse-to-nucleus transport (16). Glia maturation factor (GMF) is a neurotrophic factor implicated in nervous system development, angiogenesis and immune function (17). Osteonectin (SPARC) is involved in embryogenesis and can modulate cytokines (18). Data 7), specific for CM collected at 48h contained proteins involved in the TGF beta pathway, e.g. S-phase kinase-associated protein 1 (SKIP1), Follistatin and Latent transforming growth factor beta binding protein 1 and 4 by guest on July 17, 2020 https://www.mcponline.org

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Canine bone marrow mesenchymal stem cells characterization 16 (LTBP1, LTBP4). Proteins involved in the regulation of the insulin growth factor were also detected e.g. Protease serine 23, Follistatin-like 1, follistatin and LTBP1. Interestingly, Dickkopf WNT signalling pathway inhibitor 3 (DKK3), known to be involved in embryonic development through its interactions with the Wnt signaling pathway, was found with NENF (Neudesin neurotrophic factor), TIMP-2, neurogenesis and neurotophic factors (19). The Taken together, in the four Clusters obtained nfrom proteomic analysis, several growth factors linked to mesenchymal stem cells were detected. However, compared to transcriptomic data only TGF Beta 1 was detected.

Effects of BMMSC CM on angiogenesis in chorioallantoic membrane (CAM) assay.
The changes in the vasculature of the CAM treated with BMMSC CM showed a positive response, with enhanced density of vessels (47.250 ± 9.7) after 72 h (Figures 5B, 5B´, Figure   6). The number of vessels and bifurcations was higher compared to control (medium only, 27.625 ± 7.981) after same time (Figures 5A, 5A´, Figure 6). In addition, the intervascular distance was lower and the large vessels seemed to further enhance their diameter ( Figure   5B´). Mean values in different groups were statistically compared using one-way ANOVA and Tukey's post hoc tests, with significant differences comparing values for CTR 0h vs EXP 72h (*P value of < 0.05); EXP 0h vs EXP 72h (**P value of < 0.01) and CTR72h vs EXP 72h (***P < 0.001) (Figure 6).  (Figure 7). Proteomic data revealed the presence of angiogenic factors only 14h after incubation. Along these factors MMP2 was detected, which like MMP9 is known to be involved in angiogenesis (23). The protein Myo1c, which is necessary to recruit to blood vessels together with vascular endothelial growth factor VEGFR2 (24), was also present, as well as the fibroblast-activating protein Alpha (FAPwhich is known to be associated with and to upregulate VEGF-A expression (25). VCAM1 and AngptL3, 2 factors involved directly in blood vessel formation (26,27) were also detected.

Angiogenesis-related genes expression in CAM
Altogether, BMMSC CM are clearly associated with the genes and proteins involved in angiogenesis.

DISCUSSION
Cell therapy in veterinary practice is becoming common practice, but it is often accompanied with ambiguities and contradictory results. This may be caused by the delivery of uncharacterized canine stem cell sources targeting a wider range of different diseases (1). by guest on July 17, 2020 https://www.mcponline.org

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Canine bone marrow mesenchymal stem cells characterization 18 Nowadays we know that procedures for isolation, cultivation, freezing and thawing of canine MSCs differ between laboratories (2), and therefore to achieve successful therapeutic efficacy of cell-based therapies, we need to know their bioactive potential under well-defined standardized protocols. Currently, MSCs isolated from fat tissue, synovia, umbilical cord and placenta are being applied for hard tissue repair as well as for cardiovascular or neurological disorders (1). However, the conditioned medium isolated from canine MSCs is more privileged, since it contains extracellular vesicles filled with a cargo of proteins, as well as important regulatory molecules (mRNA and miRNA). Although cell therapy is developing, there is still a lack of detailed studies unravelling the biological properties of MSCs derived from different sources, while cultivated under specific conditions. In our study, therefore, under well-defined standardized protocols the minimal characteristics of BMMSC (adherence, CD markers and three-line differentiation) were combined with their gene expression, their proteome profile and their biological function. We believe that it is necessary to standardize the isolation protocols ensuring the quality of both cells and their conditioned media. In our study we determined the basic criteria for donors, such as age, sex, breeds of dogs, heath parameters, bone marrow source (humerus) and maximum time for tissue processing. Some of the most popular medium and large breeds of dogs were included.
They were chosen because of their relatively long life-span and the possibility of obtaining from them enough bone marrow and peripheral blood for further studies. Firstly we selected ten dogs with matching age (between 2-3y), weight (over 10kg) and gender criterion (males), from which two dogs were excluded due to changes in their complete blood count (CBC) from normal patterns (eosinophilia, allergies), and another two dogs because of their poor in vitro proliferation rate. Although the final number of dogs enrolled in our study was decreased to six, these dogs served as good candidates for multi-characterisation of these popular breeds in order to confirm their similarities/ differences. We isolated BM from the humerus using standard biopsy sets. The operations were performed by the same surgeon, using identical guidelines for bone marrow withdrawal for each donor.
The time for completion of in vitro tissue processing was a maximum of five hours from collection.
We did not notice any side effects which might be related to the BM withdrawal procedure, such as pain, motor-sensory impairment or inflammation. Morphologically, there were no significant differences between the cells. Initially, they exhibited spindle shape, leading into by guest on July 17, 2020 https://www.mcponline.org

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Canine bone marrow mesenchymal stem cells characterization 19 homogenous fibroblastic populations. We observed some differences in flow cytometry parameters, with some decrease in the CD90 surface marker with increasing passage number (above P3), but CD29 and CD45 markers remained stable. We assume that the canine BMMSC surface marker CD90 is more sensitive to enzymatic digestion and may be degraded after passaging (28). However, this change did not mean that the canine MSCs were losing their multipotency, which was confirmed by three-lineage characteristics. Here we used a commercial differential kit which confirmed marked osteogenic and chondrogenic differentiation. On the other hand, we were unable to induce adipogenesis, similarly as in another study (4).  (38). In addition, infusion of GMFB into the cavities of injured nervous tissue stimulated dendritic outgrowth and hypertrophy of specific neurons (38,39).
NENF is known to be expressed abundantly in the developing brain and spinal cord in embryos, and its neurotrophic activity may provide new insights into the development and maintenance of neurons (40). TIMP-2 is a member of the matrix metalloproteinases (MMPs) inhibitor family (41). MMP-2 for instance is inhibited by TIMP-2. MSCs are known to inhibit high levels of exogenous MMP-2 and MMP-9 through TIMP-2 and TIMP-1 respectively.
Canine MSCs are revealed to be a good source of TIMP-mediated MMP inhibition, which can be highly useful for treating pathologies such as cancer. Antigen protein CD109 shows an interesting role. It is a GPI-anchored cell-surface glycoprotein and is a member of the alpha-2-macroglobulin/C3,C4,C5 family (42). CD109 is known to be expressed at least by CD34+ bone marrow mononuclear cells and mesenchymal stem cells in humans (42,43). Proteins related to osteonectin, osteomudulin and Spondin-2 are known to be implicated in osteoblastogenesis through the Wnt pathway (44). However, Spondin-2 is also involved in