Comparison of bone marrow and adipose tissue-derived canine mesenchymal stem cells

Background Bone marrow-derived mesenchymal stem cells (BM-MSCs) and adipose tissue-derived mesenchymal stem cells (AT-MSCs) are potential cellular sources of therapeutic stem cells. MSCs are a multipotent population of cells capable of differentiating into a number of mesodermal lineages. Treatment using MSCs appears to be a helpful approach for structural restoration in regenerative medicine. Correct identification of these cells is necessary, but there is inadequate information on the MSC profile of cell surface markers and mRNA expression in dogs. In this study, we performed molecular characterization of canine BM-MSCs and AT-MSCs using immunological and mRNA expression analysis. Results Samples were confirmed to be multipotent based on their osteogenic and adipogenic differentiation. And these cells were checked as stem cell, hematopoietic and embryonic stem cell (ESC) markers by flow cytometry. BM- and AT-MSCs showed high expression of CD29 and CD44, moderate expression of CD90, and were negative for CD34, CD45, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81. SSEA-1 was expressed at very low levels in AT-MSCs. Quantitative real-time PCR (qRT-PCR) revealed expression of Oct3/4, Sox2, and Nanog in BM- and AT-MSCs. There was no significant difference in expression of Oct3/4 and Sox2 between BM-MSCs and AT-MSCs. However, Nanog expression was 2.5-fold higher in AT-MSCs than in BM-MSCs. Using immunocytochemical analysis, Oct3/4 and Sox2 proteins were observed in BM- and AT-MSCs. Conclusion Our results provide fundamental information to enable for more reproducible and reliable quality control in the identification of canine BM-MSCs and AT-MSCs by protein and mRNA expression analysis.

MSCs are defined by their plastic adherent growth and subsequent expansion under specific culture conditions and by their in vitro and in vivo differentiation potential [1][2][3][4][5][6][7][8]. Induction of differentiation into osteoblasts and adipocytes under appropriate culture conditions has been extensively demonstrated [11]. However, MSC cultures are composed of heterogeneous cell populations. The proportion of pluripotent stem cells in bone marrow-derived whole cell cultures ranged from 1/10,000 to 1/100,000 [12,13]. The lack of common standards and precise definition of initial cell preparations remains a major obstacle in research on MSCs and their application. Current research aims to characterize MSCs and to find ways of expanding MSC cultures and maintaining the cells in the undifferentiated state [14][15][16][17][18][19][20].
In veterinary medicine, the use of MSCs for tissue repair is helpful and is likely to increase in future. However, there have been few studies on cell surface markers and mRNA expression profiles of canine MSCs. Here we evaluated the canine BM-and AT-MSC cell surface markers CD29, CD44, CD90, CD34, CD45, SSEA-1, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81 by flow cytometry. We also analyzed the mRNA expression profile of Oct3/ 4, Sox2, and Nanog in canine BM-and AT-MSCs by quantitative real-time PCR (qRT-PCR). Finally, we used immunocytochemistry to examine the expression and localization of Oct3/4 and Sox2. The aim of this study was the biological characterization of canine MSCs isolated from bone marrow and adipose tissue.

Cell isolation and culture
Adherent cells were observed at the bottom of the culture flasks within 2 days; these cells were isolated from both bone marrow and adipose tissue after plating. Bone marrow-derived cells formed several colonies and proliferated, taking various shapes, including discoidal flat, triangular, and elongated ( Figure 1A). Bone marrowderived cells were uniformly distributed immediately after passage, but thereafter, gradually formed colonies and proliferated. Unlike bone marrow-derived cells, adipose-derived cells were uniformly distributed and no colony formation was observed ( Figure 1B).

In vitro differentiation
Osteogenic differentiation was demonstrated histochemically using von Kossa stain ( Figure 1C

mRNA expression analysis using quantitative real-time PCR
Expression levels of canine Oct3/4, Sox2, and Nanog mRNA in BM-and AT-MSCs were examined by qRT-PCR ( Figure 3). Expression of Oct3/4 did not differ significantly difference between BM-MSCs and AT-MSCs. However, Sox2 expression tended to be higher in BM-MSCs than in AT-MSCs while Nanog expression in AT-MSCs was 2.5-fold higher than in BM-MSCs. (p < 0.01).

Immunocytochemistry
Immunocytochemistry was used to analyze Oct3/4 and Sox2 proteins ( Figure 4). In both BM-MSCs and AT-MSCs, Oct3/4 was detected mainly the nuclei, whereas Sox2 was detected in the cytoplasm.

Discussion
In this study, we confirmed the potential of MSCs to differentiate into osteoblasts or adipocytes, and evaluated the protein and mRNA expression profiles of these cells. In addition, we compared the expression patterns of ESC markers and germ layer markers in MSCs derived from bone marrow and adipose tissue. BM-MSC and AT-MSC populations expressed CD29, CD44, and CD90 with similar intensity. In particular, in both BMand AT-MSCs, more than 95% of the cell population expressed CD29 and CD44. Our results indicate that expression of CD90 was lower than that of other markers. However, many human studies have reported strong expression of CD90. This may be related to differences within species. Many human studies have reported positive expression of CD90 whereas many studies in mice have reported negative expression of CD90 [11,26]. In addition, the decline in CD90 expression may be influenced by the passage number of the cells. In early passage cells, CD90 showed variable expression [25,[27][28][29][30][31][32], and Riekstina et al. reported that CD90 expression decreased with increasing passage number [33]. CD34 and CD45 markers are usually associated with hematopoietic stem cells.
The qRT-PCR analysis revealed expression of the stem cell markers Oct3/4, Sox2, and Nanog in canine BMand AT-MSCs. In a human study, mRNAs of these markers were expressed in human MSCs [34]. These transcription factors mediate self-renewal and cell-fate specification and are downregulated when the cells are completely differentiated [35]. Our results reflect their   undifferentiated phenotype and high propensity for pluripotent differentiation and support the hypothesis that MSCs may be pluripotent stem cells deposited in tissues during development. Oct3/4 mRNA showed similar levels of expression in BM-and AT-MSCs. However, expression of Sox2 in BM-MSCs tended to be higher than in AT-MSCs. Sox2 belongs to the Sox subfamily, whose members are defined by the relationship of their HMG box [36]. In addition, Sox2 regulates Oct3/4 expression and maintains ESC pluripotency through upstream transcription factors [37] and through cooperative binding of OCT3/4 to DNA [38]. However, our results indicate Oct3/4 expression was similar in BM-and AT-MSCs, but Sox2 expression slightly differed between the two cell types. This is probably due to the additional expression of other Sox family members that act in a compensatory in MSCs [37]. In addition, expression of Nanog in AT-MSCs was 2.5-fold higher than in BM-MSCs. Nanog is required to maintain the undifferentiated state and for the self-renewal of stem cells. In ESCs, knockout or knockdown of Nanog abolishes both self-renewal and pluripotency, and results in differentiation of extraembryonic endoderm [39,40]. In a human study, comparison of proliferation in BM-and AT-MSCs revealed high proliferative ability in AT-MSCs [41]. These results suggest that AT-MSCs may maintain the undifferentiated state and that their self-renewal ability is greater than that of BM-MSCs. We performed immunostaining with Oct3/4 and Sox2 in MSCs. In both BM-and AT-MSCs, Oct3/4 was detected in the nuclear fraction, whereas Sox2 was detected in the cytoplasmic fraction. Several transcription factors are known to be localized in the cytoplasmic fraction, such as The Tead [42] and FOXO families [43]. These studies showed inhibitory activity of the transcription factors. Hence it is possible that they inactivate Sox2. In addition, Sox2 was localized in the nucleus in canine ESCs [23]. There are many functional differences between ESCs and MSCs. For example, ESCs form teratomas in the testis, but MSCs do not. We therefore suggest that restricted localization of Sox2 protein may lead to lack of proliferation of MSCs in vivo as well as maintenance of pluripotency of MSCs in vitro.

Conclusion
Our study reveals the protein and mRNA expression profiles of canine BM-and AT-MSCs. The two types of cells showed similar cell surface marker profiles. Quantitative real-time PCR revealed expression of mRNAs for Oct3/4, Sox2, and Nanog in BM-and AT-MSCs. The localization of Oct3/4 and Sox2 was demonstrated immunocytochemically. mRNA expression of Nanog was

MSC isolation and culture
Four young healthy female beagle dogs (1 year old, 9.5-11.3 kg body weight) were used. All animals were anesthetized with propofor (Hospira, Osaka, Japan) (7 mg/kg by intravenous injection) before tissue sample were taken. After incubation, anesthesia was maintained with isoflurane (1.5-2.0%) in oxygen. Animal experiments were carried out in accordance with the National Institutes of Health guidelines for the care and use of laboratory animals. The protocol of this study was approved by the University Committee for Animal Experimentation.

BM-MSCs
Under general anesthesia, bone marrow was aspirated from the proximal humerus using a general bone marrow biopsy technique. Briefly, a sterilized 13-gauge Jamshidi needle (Cardinal Health, McGaw Park, USA) was used to aspirate 5 ml of bone marrow into a syringe containing 5 ml of heparinized (1,000 units/ml) saline solution. Perioperative analgesic management was carried out by the pre-and post-operative administration of buprenorphine, which was administered twice daily for 3 consecutive days after surgery. In addition, ampicillin (25 mg/kg) was orally administered twice daily for up to 7 days after surgery.
The bone marrow collected was dissociated and then resuspended with a pipette. The suspension was centrifuged for 5 min at 300 × g and collected as a pellet. Marrow cells were then resuspended in 10 ml of 10% FBS-PBS (FBS, Invitrogen, Carlsbad, USA; PBS, Invitrogen). To obtain MSC-enriched nucleated cells, density separation (1.077 g/ml) was performed using Lymphoprep (Axis-Shield, Oslo, Norway). A suspension of marrow cells in FBS-PBS solution (10 ml) was carefully layered onto 5 ml of Lymphoprep. Separation was achieved by centrifugation at 800 × g for 30 min at room temperature. The nucleated cells collected from the PBS solution -Lymphoprep interface were then washed in PBS and transferred into T-75 cell culture flasks with 10 ml of control medium consisting of Dulbecco's modified Eagle's medium (Invitrogen), 10% FBS, and 1% antibiotic-antimycotic solution. Cells were plated at a density of 1-5 × 10 7 cells/plate and incubated at 37°C in a humidified 5% CO 2 incubator and the medium was changed twice weekly. When primary cultures reached 70%-80% confluency, attached cells were passaged by exposure to 0.25% trypsin, 1 mM EDTA (Invitrogen) for 3 min, and replated at a density of 8.0 × 10 3 cells/cm 2 for subsequent passage.

AT-MSCs
Adipose tissues were also harvested from each dog under general anesthesia. Subcutaneous fat pads (approximately 1.0 g) were harvested from the inguinal area. These pads were finely minced with scissors and then digested in 40 ml of PBS containing 0.15% collagenase (Invitrogen), with vigorous shaking for 60 min at 37°C. Samples were then filtered using 100 μm cell strainers (BD Biosciences, Franklin Lakes, USA) and washed with PBS. The cells obtained were seeded into T-75 cell culture flasks with 10 ml of control medium and incubated in the same manner as bone marrow cells.

In vitro differentiation
For osteogenic differentiation, passage 2 BM-MSCs and AT-MSCs were plated on 6-well culture plates at a density of 5.0 × 10 3 cells/cm 2 , and after incubation in control medium for 24 h, the medium was changed to osteogenic medium. The osteogenic medium was Canine Osteoblast Differentiation Medium purchased from Cell Applications (San Diego, USA). The medium was changed twice weekly. For osteogenic analysis, mineral deposits were quantitatively analyzed by von Kossa (Sigma, St. Louis, USA) staining at 14 days.
For adipogenic differentiation, passage 2 BM-and AT-MSCs were plated on 6-well culture plates at a density

Reverse transcription and quantitative real-time PCR (qRT-PCR)
Total RNA was obtained from cultured BM-MSCs and AT-MSCs in passage 2. Total RNA was extracted using TRIzol reagent (Invitrogen) according to the manufacturer's protocol. Total RNA was measured by spectrophotometry. Total RNA (1 μg) was reverse-transcribed at 42°C for 15 min in 20 μl with QuantiTect (Qiagen, Düsseldorf, Germany) after inactivation of reverse transcription by heating at 95°C for 3 min. The cDNA product was subjected to real-time PCR according to the user instructions for the Real-Time PCR System 7300 (Applied Biosystems, Foster City, CA). qRT-PCR was performed at 95°C for 5 s and 60°C for 34 s in 20 μl buffer containing SYBR premix ExTaq II and ROX Reference Dye (Takara Bio, Shiga, Japan) and 0.2 μM each of the primers (Table 2). Quantitative measurement was performed by establishing a linear amplification curve from serial dilutions of plasmid DNA containing each cDNA.

Immunocytochemistry
Immunofluorescent staining was used to assess expression of the transcription factors Oct4, Sox2, and Nanog in BM-and AT-MSCs. Passage 2 cells were cultured in 4-well chamber slides (Nunc, Roskilde, Denmark) until 50% confluency. The cells were washed twice with PBS and fixed with 4% paraformaldehyde at room temperature for 30 min. After washing thrice with PBS, the cells were incubated with blocking solution containing 0.4% Triton X-100 and 4% Block Ace (DS Pharma Biomedical, Osaka, Japan) in PBS at room temperature for 1 h. The cells were incubated with rabbit polyclonal primary antibodies against Oct3/4 (Santa Cruz Biotechnology, Santa Cruz, USA), Sox2 (Stem Cell Technologies, Vancouver, Canada), and Nanog (Peprotech, Rocky Hill, USA) [21] diluted in blocking solution at 4°C for 16 h. The negative control cells were incubated without primary antibody and isotype control cells were incubated with normal rabbit IgG antibody (R&D Systems). The cells were washed thrice with PBS and incubated with secondary anti-rabbit antibody labeled with Alexa Fluor 488 (Invitrogen) diluted in blocking solution at room temperature for 1 h in darkness. The cells were then washed thrice with PBS and slides were mounted in Vectashield Hard Mounting Medium with DAPI (Vector Laboratories, Burlingame, USA). The cells were analyzed under a Zeiss Axiovert 200 M fluorescence microscope (Carl Zeiss MicroImaging, Jena, Germany), and image overlay was performed using Axio Vision Rel.4.6 software (Carl Zeiss MicroImaging) (Additional file 1: Table S1).

Statistical analysis
Data were analyzed with an independent samples t-test, and a P-value of less than 0.01 was considered significant. Statistical analysis was performed using Prism software (GraphPad Software, San Diego, USA).

Competing interests
None of the authors has any financial or personal relationships that could inappropriately influence or bias the content of the paper.

Authors' contributions
HT and DZ designed the study, drafted the manuscript, and analyzed data; IY helped with editing and revision of the manuscript; YH helped with collection of samples and revision of the manuscript; MM helped with FACS analysis and revision of the manuscript; and TA contributed to the study design and helped with editing and revision of the manuscript. All authors read and approved the final manuscript.