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Cell Source-Dependent in Vitro Chondrogenic Differentiation Potential of Mesenchymal Stem Cell Established from Bone Marrow and Synovial Fluid of Camelus Dromedarius

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Cell Source-Dependent in Vitro Chondrogenic Differentiation Potential of Mesenchymal Stem Cell Established from Bone Marrow and Synovial Fluid of Camelus Dromedarius animals Article Cell Source-Dependent In Vitro Chondrogenic Differentiation Potential of Mesenchymal Stem Cell Established from Bone Marrow and Synovial Fluid of Camelus dromedarius Young-Bum Son 1, Yeon Ik Jeong 1, Yeon Woo Jeong 1, Mohammad Shamim Hossein 1, Per Olof Olsson 1 , Alex Tinson 2, Kuhad Kuldip Singh 2, Sang-Yun Lee 3 and Woo Suk Hwang 1,* 1 UAE Biotech Research Center, Abu Dhabi 30310, United Arab Emirates; [email protected] (Y.-B.S.); [email protected] (Y.I.J.); [email protected] (Y.W.J.); [email protected] (M.S.H.); [email protected] (P.O.O.) 2 Hilli E.T. Cloning and Surgical Centre Presidential Camels and Camel Racing Affairs, Al-Ain 17292, United Arab Emirates; [email protected] (A.T.); [email protected] (K.K.S.) 3 Department of Theriogenology and Biotechnology, College of Veterinary Medicine and Research Institute of Life Science, Gyeongsang National University, Jinju 52828, Korea; [email protected] * Correspondence: [email protected] Simple Summary: This is the first study to demonstrate the establishment and subsequent analysis of attributes, including the chondrogenic capacity of mesenchymal stem cells (MSCs) from bone marrow (BM) and synovial fluid (SF) from the same donor Camelus dromedarius. MSCs of SF origin were notably more efficient in their chondrogenic capacity and represent a potential source for camel Citation: Son, Y.-B.; Jeong, Y.I.; regenerative medicine addressing chondrocyte-related problems. Jeong, Y.W.; Hossein, M.S.; Olsson, P.O.; Tinson, A.; Singh, K.K.; Abstract: Mesenchymal stem cells (MSCs) are promising multipotent cells with applications for Lee, S.-Y.; Hwang, W.S. Cell cartilage tissue regeneration in stem cell-based therapies. In cartilage regeneration, both bone Source-Dependent In Vitro marrow (BM-MSCs) and synovial fluid (SF-MSCs) are valuable sources. However, the cellular Chondrogenic Differentiation characteristics and chondrocyte differentiation potential were not reported in either of the camel Potential of Mesenchymal Stem Cell stem cells. The in vitro chondrocyte differentiation competence of MSCs, from (BM and SF) sources Established from Bone Marrow and Synovial Fluid of Camelus dromedarius. of the same Camelus dromedaries (camel) donor, was determined. Both MSCs were evaluated on Animals 2021, 11, 1918. https:// pluripotent markers and proliferation capacity. After passage three, both MSCs showed fibroblast- doi.org/10.3390/ani11071918 like morphology. The proliferation capacity was significantly increased in SF-MSCs compared to BM-MSCs. Furthermore, SF-MSCs showed an enhanced expression of transcription factors Academic Editor: Ralf Einspanier than BM-MSCs. SF-MSCs exhibited lower differentiation potential toward adipocytes than BM- MSCs. However, the osteoblast differentiation potential was similar in MSCs from both sources. Received: 12 June 2021 Chondrogenic pellets obtained from SF-MSCs revealed higher levels of chondrocyte-specific markers Accepted: 21 June 2021 than those from BM-MSCs. Additionally, glycosaminoglycan (GAG) content was elevated in SF- Published: 28 June 2021 MSCs related to BM-MSCs. This is, to our knowledge, the first study to establish BM-MSCs and SF-MSCs from the same donor and to demonstrate in vitro differentiation potential into chondrocytes Publisher’s Note: MDPI stays neutral in camels. with regard to jurisdictional claims in published maps and institutional affil- Keywords: mesenchymal stem cells; Camelus dromedarius; bone marrow; synovial fluid; chondro- iations. cyte differentiation Copyright: © 2021 by the authors. 1. Introduction Licensee MDPI, Basel, Switzerland. This article is an open access article Cartilage damage to joint surfaces can result in osteoarthritis (OA), a condition where distributed under the terms and the bone under the articular cartilage is exposed and the synovial membrane around the conditions of the Creative Commons joint is inflamed [1]. When cartilage is damaged due to inflammation or trauma, it cannot Attribution (CC BY) license (https:// buffer between the bone, causing severe pain and deformation of the articular cartilage. creativecommons.org/licenses/by/ OA is a chronic musculoskeletal disorder frequently occurring in racing horses and camels 4.0/). that adversely affects their future racing careers [2,3]. Animals 2021, 11, 1918. https://doi.org/10.3390/ani11071918 https://www.mdpi.com/journal/animals Animals 2021, 11, 1918 2 of 13 Since cartilage has no distribution of blood vessels and nerves, it is difficult to regener- ate once damaged [4]. To treat damaged cartilage tissue, drugs (steroids and painkiller), chondroprotective agents (hyaluronic acid and glucosamine), and surgical approaches are used [5]. However, limited effects of non-specific alleviation of pain and inflammatory reactions are expected in drug treatment and chondroprotective agents only play a role in supplying nutrients to chondrocytes [1,6]. Microfracture and autologous chondrocyte im- plantation (ACI) have been performed as clinical surgical methods based on cell therapy [7]. Microfracture is a method of regenerating cartilage with blood clots containing mesenchy- mal stem cells (MSCs) by puncture or abrasion of damaged subchondral bone and has the disadvantages of regenerating into fibrocartilage rather than hyaline cartilage [8]. ACI, which is commonly used, induces the regeneration of cartilage tissue by transplanting autologous cartilage cell culture in vitro [8]. However, this method has specific problems, including the formation of cartilage defects in macroscopically normal-looking tissue donor sites. Furthermore, the phenotype can change due to de-differentiation during in vitro culture since the transplanted cells are already differentiated cells [9,10]. Therefore, therapeutic approaches have emerged for cartilage regeneration using stem cells. MSCs are a potent resource for therapeutic application with a high stemness ability, anti-inflammatory, and immunosuppressive effects [11,12]. MSCs also possess multi- lineage differentiation capacity including chondrocytes [13]. Bone marrow stem cells (BM-MSCs), which were the first to be discovered, can mainly be collected from the iliac crest of the pelvis, and have been reported for a long time. However, several disadvantages have been reported, such as the limited amount of harvest and the complicated collection process [14]. Furthermore, compared to other sources, low proliferative capacity and donor-age-dependent characteristics have been reported in BM-MSCs [15]. Considering the shortcomings of BM-MSCs, studies have been performed on alternative MSCs sources. Synovial fluid-derived MSCs (SF-MSCs) can be collected by the process of arthro- centesis and possess superior chondrogenic capacity [16]. SF-MSCs are the most similar source to articular cartilage, have a high expression of CD44 (hyaluronan receptors) and uridine disphosphoglucose dehydrogenase (UDPGD), and are required for hyaluronan synthesis [17]. Accordingly, several studies have reported on cartilage regeneration using SF-MSCs in various mammalians [18–20]. Human SF-MSCs derived from OA patients were transplanted with a collagen sponge into the joint of the anterior cruciate ligament transection of rats and cartilage defects were recovered [18]. SF-MSCs derived from equine sources have also been shown to have an analogous beneficial effect compared to BM- MSCs after implantation into articular cartilage defects in rat femurs [19]. An interesting study reported that after the transplantation of porcine SF-MSCs into collagen-induced arthritis mouse (CIA mouse) by intraperitoneal injection, cartilage damage in the joint was decreased, and inflammation was also reduced [20]. Nevertheless, a comparison of these two cell types is lacking in Camelus dromedarius (camel). In this study, we established a distinctive population of BM-MSCs and SF-MSCs derived from the same donor. Additionally, to evaluate chondrogenic potential, both camel MSCs were differentiated into chondrocytes under specific induction conditions. 2. Materials and Methods 2.1. Chemicals and Media Unless otherwise stated, all reagents were obtained from Sigma (St. Louis, MO, USA). 2.2. Collection and Culture of Bone Marrow and Synovial Fluid Derived Mesenchymal Stem Cells Institutional Animal Care and Use Committee (IACUC) guidelines were followed and all experiments were approved by the Management of Scientific Centers and Presidential Camels (Accession No: PC4.1.5). Four female camels aged between 5 and 7 years were used to establish and compare the various MSCs. After placing the camels in a seated position, they were sedated with ketamine hydrochloride (Ilium, Hlendenning, Australia; 0.25 mg/kg body weight) and xylazine (Ceva, Libourne, France; 0.25 mg/kg body weight) Animals 2021, 11, 1918 3 of 13 through intravenous injection using an 18-gauge needle [13]. The 2% lidocaine (Jeil, Daegu, Korea) was infiltrated subcutaneously on the iliac crest (2 cm × 2 cm). Approximately 5 mL of BM was extracted using the 11-gauge, 15-cm-long biopsy needle (Argon Medical Devices, Frisco, TX, USA). A heparin-coated 10 mL syringe, was used for BM extraction and subsequently added to the same quantity of Dulbecco’s phosphate-buffered saline (DPBS, Welgene, Gyeongsan, Korea). Centrifugation was performed at 400× g for 40 min, layered onto Ficoll-Paque (Amersham Biosciences, Uppsala, Sweden) without mixing. Mononuclear cells (MNCs) were harvested from the interface buffy layers and washed with DPBS. We collected the SF using syringe aspiration as previously reported with
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