animals

Article Comparative Study of Biological Characteristics, and Differentiation of Mesenchymal Stem Established from Camelus dromedarius , Dermal Skin, and Adipose Tissues

Young-Bum Son 1, Yeon Ik Jeong 1, Yeon Woo Jeong 1, Mohammad Shamim Hossein 1, Alex Tinson 2, Kuhad Kuldip Singh 2 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.) 2 Hilli E.T. Cloning and Surgical Centre Presidential Camels and Camel Racing Affairs, Al-Ain P.O. Box 17292, Unitied Arab Emirates; [email protected] (A.T.); [email protected] (K.K.S.) * Correspondence: [email protected]

Simple Summary: Mesenchymal stem cells (MSCs) can be isolated in various types of tissues and exhibit different characteristics. In this study, MSCs were established from skeletal muscle, dermal skin, and from a single Camelus dromedarius donor. We also identified an efficient   source for osteoblast differentiation and analyzed biological characteristics.

Citation: Son, Y.-B.; Jeong, Y.I.; Jeong, Abstract: Mesenchymal stem cells (MSCs) showed in vitro -lineage differentiation and Y.W.; Hossein, M.S.; Tinson, A.; Singh, self-renewal capacity. However, no comparative study was reported on the biological characteristics K.K.; Hwang, W.S. Comparative of stem cells derived from skeletal muscle (SM-MSCs), dermal skin (DS-MSCs), and adipose tissues Study of Biological Characteristics, (A-MSCs) from a single donor in camels. The present study aimed to evaluate the influence of and Osteoblast Differentiation of MSCs source on characteristics. We evaluated proliferation capacity and mesoderm- Established from Camelus dromedarius Skeletal lineage differentiation potential from SM-MSCs, DS-MSCs, and A-MSCs. They showed spindle-like Muscle, Dermal Skin, and Adipose morphology after homogenization. The proliferation ability was not significantly difference in any Tissues. Animals 2021, 11, 1017. of the groups. Furthermore, the portion of the cell cycle and expression of pluripotent markers https://doi.org/10.3390/ani11041017 (Oct4, Sox2, and Nanog) were similar in all cell lines at passage 3. The differentiation capacity of A-MSCs into was significantly higher than that of SM-MSCs and DS-MSCs. However, Academic Editor: the osteoblast differentiation capacity of A-MSCs was significantly lower than that of SM-MSCs and Ioanna-Katerina Aggeli DS-MSCs. Additionally, after osteoblast differentiation, the alkaline (ALP) activity and content significantly decreased in A-MSCs compared to SM-MSCs and DS-MSCs. To the Received: 24 February 2021 best of our knowledge, we primarily established MSCs from the single camel and demonstrated Accepted: 2 April 2021 their comparative characteristics, including expression of pluripotent factors and proliferation, and Published: 4 April 2021 in vitro differentiation capacity into adipocytes and .

Publisher’s Note: MDPI stays neutral Keywords: mesenchymal stem cells; Camelus dromedarius; skeletal muscle; dermal skin; adipose with regard to jurisdictional claims in tissue; differentiation published maps and institutional affil- iations.

1. Introduction The Camelus dromedarius is a domesticated camel and is a unique animal providing milk Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. and meat in desert areas because of its physiological adaptation to high temperature, intense This article is an open access article sunlight, and adaptability to water shortages [1,2]. Furthermore, camel racing is a very distributed under the terms and popular sport in the gulf areas and has great economic value [2]. As these racing camels are conditions of the Creative Commons trained from a young age, diseases, including bone fractures, are becoming a problem, Attribution (CC BY) license (https:// which is pointed out as a major cause of lameness in animals [3]. Generally, minor bone creativecommons.org/licenses/by/ defects due to fractures frequently appear, and most of them are regenerated into original 4.0/). tissues, but extensive bone defects due to severe trauma are difficult to treat [4]. Autogenous

Animals 2021, 11, 1017. https://doi.org/10.3390/ani11041017 https://www.mdpi.com/journal/animals Animals 2021, 11, 1017 2 of 12

bone graft has been attempted as a treatment for these extensive bone defects [5]. However, collecting autogenous accompanied by additional invasive surgery increases the risk of infection and pain [6]. Furthermore, the quantity of bones required for the grafts may be difficult to harvest and the quality of bone is also unpredictable [6,7]. Accordingly, attention has been focused on research for overcoming these bone diseases using stem cells. Mesenchymal stem cells (MSCs) are a promising source of multipotent cells hav- ing self-replication, and multi-lineage capacity, including adipocytes, , and osteoblasts [8–11]. They are suitable for stem cell therapeutic applications without any ethical issues and immune-reaction [10,11]. Accordingly, several studies were reported on cell-based bone using MSCs in various mammalians [12–14]. Studies have found stem cells were transplanted into bond defects resulted in fusion [12]. Canine allogenic -derived MSCs (BM-MSCs) transplanted into the site of critical-sized segmental bone defect, and the defect site was filled with new bone [13]. Furthermore, interesting studies confirmed that when human stem cells were transplanted with a scaffold into a miniature pig, the formation of new bones was confirmed at the transplant site, and it was reported that no immune rejection reaction was observed even when the xenogeneic stem cells were transplanted [9,14]. Applying an efficient cell source is essential for bone regeneration [15]. Therefore, there is increasing interest in improving the efficiency of differentiation into osteoblasts for bone regeneration. Several studies were reported on the isolation and culture of MSCs using various tissues including bone barrow [16–19]. However, BM-MSCs have lower proliferation and differentiation capacity than other sources of stem cells and show a small number of cells and donor-age-dependent characteristics [20]. Recently, many studies have been conducted on the source of MSCs that can replace bone marrow, and it has been reported that mesenchymal stem cells can be obtained from many tissues, including skeletal muscle, dermal skin, and adipose tissue [17–19]. Additionally, MSCs from skeletal muscle, dermal skin, and adipose tissue were reported for the presence of osteoblast differentiation capacity [21–23]. However, there was no comparative study of different sources of single donor-derived MSCs for the osteoblast differentiation in camel. In the present study, the stem cells derived from a single donor of skeletal muscle (SM-MSCs), dermal skin (DS-MSCs), and adipose tissues (A-MSCs) were successfully established. We also evaluated osteoblast differentiation capacity on the basis of evaluation under the same induction conditions.

2. Materials and Methods 2.1. Chemicals and Media We purchased all chemicals from Sigma (St. Louis, MO, USA) unless otherwise noted.

2.2. Establishment and Culture of Skeletal Muscle, Dermal Skin, and Adipose Tissue-Derived Mesenchymal Stem Cells The isolation and culture of mesenchymal stem cells from skeletal muscle (SM-MSCs), dermal skin (DS-MSCs), and adipose tissues (A-MSCs) were conducted following previ- ously reported techniques with minor modification [8,10]. All animal procedures were conducted following the animal study guidelines, which were approved by the Institu- tional Animal Care and Use Committee (IACUC) at the Management of Scientific Centers and Presidential Camels (Accession No: PC4.1.5). In the present study, a total of four female camels aged from 4 to 5 years and weighing approximately 400 kg were used. The animals 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) through intravenous injection using an 18-gauge needle. A meniscus pattern of 2 cm × 1 cm was infiltrated with local anesthetic on the left flank of the abdomen in front of the anterior crest of the ilium using 2% Lidocaine (Jeil, Daegu, Korea). The skin, fat, and muscle were exposed and obtained through a longitudinal incision in a meniscus shape (1.5 cm; depth and 2 cm × 1 cm diameter) using a combination of the scalpel blade, forceps, and Animals 2021, 11, 1017 3 of 12

surgical scissors. The incision site was covered with two layers using 2-0 vicryl and 2-0 nylon sutures. The obtained tissues washed two times in Dulbecco’s -buffered saline (DPBS; Welgene, Gyeongsan, Korea) supplemented with 1% antibiotic-antimycotic (Thermo fisher scientific, Waltham, MA, USA). We minced the samples into small pieces with a surgical blade. They were dissociated with in high-glucose Dulbecco’s modified Eagle’s medium (DMEM; Thermo fisher scientific, Waltham, MA, USA) containing 0.1% collagenase type I (Thermo fisher scientific, Waltham, MA, USA) at 38 ◦C in a humidified atmosphere of 5% CO2 and O2 for 4 h. The cells released with collagenase were washed with high-glucose DMEM containing 10% fetal bovine serum (FBS; Invitrogen, Carlsbad, California, USA) by centrifugation at 300× g for 3 min and filtered through 40 µm nylon strainer (Falcon, Franklin, NJ, USA). After that, we cultured these cells in 60 mm culture dishes with high-glucose DMEM supplemented with 10% FBS, 1% nonessential amino acid (Thermo fisher scientific, Waltham, MA, USA), 1% antibiotic-antimycotic, and 0.1% β-mercaptoethanol (Thermo fisher scientific, Waltham, MA, USA) at 38 ◦C in a humidified incubator with 5% CO2 and O2. The culture media was changed every 48 h until confluence was reached 80%, and they were sub-cultured with 0.25% trypsin EDTA solution (Gibco, Paisely, UK) and cryopreserved in high-glucose DMEM containing 20% FBS and 10% dimethyl sulfoxide (DMSO).

2.3. Cell Proliferation Analysis We conduct a population doubling time (PDT) assay to evaluate the cell proliferation capacity [8]. All cells were seeded into six-well plates (Nunc, NY, USA). Cells were counted using a hematocytometer for every passage at 72-h intervals. We calculated the PDT of the MSCs following the formula PDT = log2 × T/(logNC—logNI), where T is the time, logNC is the cultured cell number, and logNI is the initial cell number.

2.4. Cell Cycle Analysis The culture SM-MSCs, DS-MSCs, and A-MSCs were fixed with 70% ethanol for 1 h at passage 3. The fixed cells were washed twice with DPBS and mixed with propidium iodide solution (10 µg/mL) and RNase A (100 µg/mL) for 15 min. The stained cells were analyzed by flow cytometry (BD FACSVerseTM, BD Biosciences, Franklin Lakes, New Jersey, USA) and categorized into G0/G1, S, and G2/M phases.

2.5. In Vitro Differentiation into , and To confirm the mesoderm-lineage differentiation, the SM-MSCs, DS-MSCs, and A- MSCs were differentiated at passage three into adipocyte, and chondrocytes as previously described with minor modifications [24]. Briefly, the cells were cultured with adipogenic- specific induction media for 21 days. The adipogenic induction medium consisted of DMEM supplemented with 10% FBS, 100 µM indomethacin, 10 µM insulin, and 1 µM . Adipogenic differentiation was confirmed by staining lipid droplets using oil red O staining. The chondrocyte differentiation was performed using conventional pellet culture techniques. In brief, 1 × 106 cells at passage three were suspended with 1 mL of STEMPRO chondrogenesis differentiation media with 10% supplement in a 15 mL tube. The cell pellets were cultured for 3 weeks. To confirm the deposition of , the cell pellets were embedded paraffin section after dehydration. They were stained with 1% Alcian blue solubilized in 3% acetic acid for 10 min, with counterstaining of 0.1% nuclear fast red solution for 1 min. Animals 2021, 11, 1017 4 of 12

2.6. In Vitro Differentiation into Osteoblast The osteoblast differentiation was induced as previously reported with minor mod- ifications [24]. All types of MSCs were cultured in DMEM containing 10% FBS, 10 nM dexamethasone, 50 µg/mL ascorbic acid, and 10 mM sodium ß-glycerophosphate for three weeks. The osteoblast induction media was changed every 48 h. Differentiated osteoblasts were fixed with 4% paraformaldehyde and stained with silver nitrate solution (Von kossa staining) and alizarin red S to confirm the mineralization and calcium depositions.

2.7. Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR) Analysis The expression of pluripotent markers and lineage-related genes was analyzed using real-time quantitative polymerase chain reaction (RT-qPCR). Total RNA was extracted using an easy-spin Total RNA Extraction Kit (Intron, Seongnam, Korea) and quantified using a Nanodrop 1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). To synthesize complementary DNA (cDNA), reverse transcription was conducted from total purified RNA (2 µg) using HisenScript RT PreMix kit (Intron, Seongnam, Korea) with 10 µM OligodT primer at 42 ◦C for 50 min. The RT-qPCR was conducted using a Rotor-Gene Q cycler (Qiagen, Hilden, Germany) and RealMODTM Green AP 5 × qPCR mix (Intron, Seongnam, Korea) containing 200 nM of forward and reverse primers. The amplification setting included denaturation at 95 ◦C for 60 s followed by 50 cycles of 95 ◦C for 10 min, 60 ◦C for 6 s, and 72 ◦C for 4 s. Gene expression was normalized to the mRNA levels of a reference gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The information of primers used in this study is listed in Table1.

Table 1. Lists of camel primers used in real-time quantitative polymerase chain reaction (RT-qPCR) analysis.

Gene Name (Symbol) Primers Sequence Product Size (bp) Anneal. Temp (◦C) POU class 5 homeobox 1 F: CGAGAGGATTTTGAGGCTGC 122 60 (OCT4) R: GAGTACAGTGTGGTGAAGTGAG Sex determining region Y-box F: CTCGCAGACCTACATGAACG 144 60 2 (SOX2) R: TGGGAGGAAGAGGAAACCAC F: AGCACAGAGAAGCAGGAAGA Nanog homeobox (NANOG) 213 60 R: CCACCGCTTACATTTCATTC F: GAGAGTGTTACCTACACCAA Lipoprotein lipase (LPL) 248 60 R: GCCTTTACTCTGATCTTCTC fatty acid-binding 4 F: GTGACCATCAGTGTGAATG 152 60 (FABP4) R: GCACCTCCTTCTAAAGTTAC the type X gene F: TATCCAGCTATAGGCAGTC 194 60 (COL10A1) R: TCGTAGGTGTACATTACAGG F: TGTGGAGGGTGTTACTGAAC Agreecan (ACAN) 154 60 R: GACTGATGACCCTTCTACCC Runt-related transcription F: GACAGAAGCTTGATGACTCT 166 60 factor 2 (Runx2) R: GTAATCTGACTCTGTCCTTG F: AGTGAGATGGTGAAGAGACT 176 60 R: TAGGTTGTGCCGTAGAAG Glyceraldehyde 3-phosphate F: GCTGAGTACGTTGTGGAGTC 133 60 dehydrogenase (GAPDH) R: TCACGCCCATCACAAACATG Animals 2021, 11, 1017 5 of 12

Animals 2021, 11, x 5 of 12

2.8. Activity Alkaline phosphatase (ALP) activity was assessed using a Tartrate-resistant acid phosphatasewith DPBS and (TRACP) treated and with ALP 50 µL assay of extraction kit (Takara, solution Noji higashi, and substrate Japan) solution. according The to reac- the manufacturer’stion was stopped instructions. using 50 µL On of day stop seven solution of osteoblast (0.5 N NaOH) differentiation,, and absorbance cells were was washed evalu- withated DPBSusing and405 n treatedm by the with VERSAmax 50 µL of microplate extraction reader solution (Molecular and substrate Devices, solution. San Jose, The CA, reactionUSA). The was cellular stopped using 50wereµL ofdetermined stop solution using (0.5 the N BCA NaOH), protein and assay absorbance kit (Thermo was evaluatedFisher Scientific, using 405 Waltham, nm by the MA, VERSAmax USA). We microplate calculated reader ALP activity (Molecular values Devices, as IU/mg San to Jose, the CA,total USA). cellular The protein. cellular proteins were determined using the BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA). We calculated ALP activity values as IU/mg to the total2.9. cellularCalcium protein.Colorimetric Assay

2.9. CalciumWe measured Colorimetric calcium Assay deposition using a calcium colorimetric assay kit (Biovision, Milpitas, San Francisco, CA, USA) according to the manufacturer’s instructions. The in- We measured calcium deposition using a calcium colorimetric assay kit (Biovision, duced osteoblasts were treated with 0.6 N HCl for 24 h at 20 °C . After that, the supernatant Milpitas, San Francisco, CA, USA) according to the manufacturer’s instructions. The was moved to each well of 96-well plates and mixed with 60 µl of calcium assay buffer induced osteoblasts were treated with 0.6 N HCl for 24 h at 20 ◦C. After that, the supernatant and 90 µL of chromogenic reagent at 20 °C for 10 min. Absorbance at 575 nm was deter- was moved to each well of 96-well plates and mixed with 60 µL of calcium assay buffer and mined using the VERSAmax microplate reader (Molecular Devices, San Jose, CA, USA). 90 µL of chromogenic reagent at 20 ◦C for 10 min. Absorbance at 575 nm was determined We measured cellular proteins using the BCA protein assay kit (Thermo Fisher Scientific, using the VERSAmax microplate reader (Molecular Devices, San Jose, CA, USA). We Waltham, MA, USA). Calcium values were calculated as µg/mg to the total cellular pro- measured cellular proteins using the BCA protein assay kit (Thermo Fisher Scientific, tein. Waltham, MA, USA). Calcium values were calculated as µg/mg to the total cellular protein.

2.10.2.10. Statistical Statistical Analysis Analysis AllAll data data were were analyzed analyzed by by one-way one-way analysis analysis of varianceof variance (ANOVA) (ANOVA) using using SPSS SPSS version ver- 23sion (IBM), 23 (IBM), and Tukey’sand Tukey’s test wastest was conducted conducted for for between-group between-group comparisons. comparisons. Data Data were were representedrepresented as as mean mean± ± standardstandard deviationdeviation (SD),(SD),and andp p< < 0.050.05 waswas consideredconsidered significant.significant.

3.3. Results Results 3.1.3.1. Establishment Establishment of of MSCs MSCs Derived Derived from from Skeletal Skeletal Muscle, Muscle Dermal, Dermal Skin, Skin and, And Adipose Adipose Tissues Tissues MSCsMSCs derived derived from from skeletal skeletal muscle muscle (SM-MSCs), (SM-MSCs), dermal dermal skin skin (DS-MSCs), (DS-MSCs), and adiposeand adi- tissuepose tissue (A-MSCs) (A-MSCs) from afrom single a single donor donor were were isolated isolated and and cultured. cultured. All theAll the cells cells showed showed a spindle-likea spindle-like morphology morphology and and were were confirmed confirmed homogenous homogenous adherent adherent morphology morphology after after passagepassage three three (Figure (Figure1). 1). The The proliferation proliferation capacity capacity of of cells cells was was evaluated evaluated in in passages passage one,s one, two,two, three, three, and and four. four. There There was was no no difference difference in in population population doubling doubling time time (PDT) (PDT) among among thethe cells cells (Figure (Figure2a). 2a). To To assess assess the the cell cell viability, viability, we we also also analyzed analyzed cell cell cycle cycle characteristics characteristics inin all all cells cells at at passage passage three. three. OurOur datadata showedshowed thatthat thethe portionportion ofof thethe G0/G1G0/G1 phase, phase, and and S Sphase phase,, was was the the same same in in all all groups groups (Figure (Figure 2b).2b). All All SM SM-MSCs,-MSCs, DS DS-MSCs,-MSCs, and and A A-MSCs-MSCs ex- expressedpressed pluripotent pluripotent factors factors (OCT4, (OCT4, SOX2, and NANOG). ThereThere waswas nono differencedifference in in the the expression of mRNA levels among the cells (Figure3). expression of mRNA levels among the cells (Figure 3).

FigureFigure 1. 1.Cellular Cellular morphologymorphology of m mesenchymalesenchymal stem stem cells cells (MSCs (MSCs)) derived derived from from skeletal skeletal muscle muscle (SM-MSCs),(SM-MSCs), dermal dermal skin skin (DS-MSCs), (DS-MSCs), and and adipose adipose tissue tissue (A-MSCs) (A-MSCs) was was observed observed at passageat passage three three by by phase-contrast microscope. Scale bar = 100 µm. phase-contrast microscope. Scale bar = 100 µm. Animals 2021, 11, 1017 6 of 12 Animals 2021, 11, x 6 of 12

Animals 2021, 11, x 6 of 12

(a) (b) (a) (b) FigureFigure 2. 2. CellularCellular proliferation proliferation and cell cyclecycle analysisanalysis ofof skeletalskeletal muscle muscle (SM-MSCs), (SM-MSCs), dermal dermal skin skin (DS- Figure 2. Cellular proliferation and cell cycle analysis of skeletal muscle (SM-MSCs), dermal skin (DSMSCs),-MSCs), and and adipose adipose tissue tissue (A-MSCs). (A-MSCs). (a). ( Populationa). Population doubling doubling time time (PDT) (PDT) analysis analysis was was performed per- formed(DS-MSCs), in SM and-MSCs, adipose DS-MSCs, tissue and(A-MSCs). A-MSCs. (a) .There Population was no doubling significant time difference (PDT) analysis during wasthe pas-per- in SM-MSCs, DS-MSCs, and A-MSCs. There was no significant difference during the passage. (b). sage.formed (b) .in The SM portion-MSCs, of DS the-MSCs, cell cycle and wasA-MSCs. analyzed There in was SM -noMSCs, significant DS-MSCs, difference and A -duringMSCs. Thethe pas- cell The portion of the cell cycle was analyzed in SM-MSCs, DS-MSCs, and A-MSCs. The cell cycle arrest cyclesage. arrest (b). The and portion DNA replication of the cell cyclewere wassimilar analyzed in all three in SM types-MSCs, of MSCs.DS-MSCs, The andvalues A- MSCs.were ex- The cell pressedandcycle DNA arrest as replicationmean and ±DNA SD wereof replication four similar independent were in all similar three experiments. types in all of three MSCs. types The of values MSCs. were The expressedvalues were as ex- mean ± SDpressed of four as independentmean ± SD of experiments. four independent experiments.

Figure 3. The expression levels of pluripotent markers of MSCs are derived from skeletal muscle (SMFigureFigure-MSCs), 3.3. TheThe dermal expressionexpression skin (DS levels levels-MSCs), of of pluripotent pluripotent and adipose markers markers tissue of of (A MSCs MSCs-MSCs). are are derived There derived was from from no skeletal significant skeletal muscle muscle differ- (SM- enceMSCs),(SM among-MSCs), dermal all dermal three skin (DS-MSCs),typesskin (DS of MSCs.-MSCs), and Data adiposeand are adipose represented tissue tissue (A-MSCs). (A by- MSCs).the There mean There was± SD was no of significantfour no significant independent difference differ- experiments.amongence among all three all three types types of MSCs. of MSCs. Data Data are are represented represented by by the the mean mean ±± SDSD of of four four independent independent experiments.experiments. 3.2. In Vitro Adipogenic and Chondrogenic Lineage Differentiation Capacity of MSCs 3.2.3.2. InIn VitroVitro AdipogenicAdipogenic andand ChondrogenicChondrogenic LineageLineage DifferentiationDifferentiation CapacityCapacity ofof MSCsMSCs The mesoderm-lineage (adipocyte and chondrocyte) differentiation potential of SM- MSCs,TheThe DS mesoderm-lineage-mesodermMSCs, and- lineageA-MSCs (adipocyte(adipocyte was evaluated. and chondrocyte) Cytochemical differentiation staining confirmed potential that of cells SM-SM - inducedMSCs,MSCs, DS-MSCs,DS into-MSCs, adipocyte andand A-MSCs Aand-MSCs chondrocyte, waswas evaluated.evaluated. as verified CytochemicalCytochemical by the intracellular stainingstaining confirmedconfirmed lipid droplets thatthat cellsandcells accumulationinducedinduced into adipocyteof adipocyte and and chondrocyte, chondrocyte,by oil red O, as and as verified verified Alcian by blue by the the intracellularstaining, intracellular respectively lipid lipid droplets droplets(Figure and 4a).andaccumulation accumulation of proteoglycan of proteoglycan by oil by red oil O, red and O, Alcian and Alcianblue staining, blue staining, respectively respectively (Figure (Figure4a). The4 a).adipocyte- and chondrocyte-specific genes were analyzed before and after the differentiationTheThe adipocyte-adipocyte by RT- - andandqPCR chondrocyte-specificchondrocyte (Figure 4b)-.specific The adipocyte genesgenes werewere-specific analyzedanalyzed genes before,before i.e., lip andandoprotein afterafter thetheli- differentiation by RT-qPCR (Figure4b). The adipocyte-specific genes, i.e., lipoprotein pasedifferentiation (LPL) and byfatty RT - acidqPCR-binding (Figure protein 4b). The 4 adipocyte (FABP4), -showedspecific genes significantly, i.e., lip (oproteinp < 0.001 li-) lipase (LPL) and fatty acid-binding protein 4 (FABP4), showed significantly (p < 0.001) higherpase (LPL)expression and fatty in differentiated acid-binding adipocytes protein 4 (FABP4)compared, showed to the non significantly-differentiated (p < cells. 0.001 ) higher expression in differentiated adipocytes compared to the non-differentiated cells. Furthermore,higher expression our data in differentiated showed that theadipocytes expression compared of LPL toand the FABP4 non-differentiated was significantly cells. Furthermore, our data showed that the expression of LPL and FABP4 was significantly increasedFurthermore, in the our differentiated data showed cells that derived the expression from A-MSCs of LPL compared and FABP4 to those was derived significantly from increased in the differentiated cells derived from A-MSCs compared to those derived from SMincreased-MSCs, in and the DSdifferentiated-MSCs. The cells expression derived from of type A-MSCs X collagen compared gene to those (COL10A1) derived from and SM-MSCs, and DS-MSCs. The expression of type X collagen gene (COL10A1) and agreecan agreecanSM-MSCs, (ACAN), and DS chondrogenesis-MSCs. The expression markers, in of all typethe three X collagen types of geneMSCs (COL10A1)significantly and (p (ACAN), chondrogenesis markers, in all the three types of MSCs significantly (p < 0.001)

(a) (b)

FigureFigure 4. 4.In In vitro vitromesoderm-lineage mesoderm-lineage (adipocyte (adipocyte and and chondrocyte) chondrocyte) differentiation differentiation of MSCsof MSCs from from three typesthree of types tissues. of tissues. (a). Adipocytes (a). Adipocytes and chondrocytesand chondrocytes differentiation differentiation were were confirmed confirmed oil redoil red O (for O adipocytes)(for adipocytes) and Alcian and Alcian blue (for blue chondrocytes) (for chondrocytes) staining. staining. (b). The (b) gene. The expression gene expressi of adipocyteson of adipo- and chondrocytes-specificcytes and chondrocytes markers-specific in differentiatedmarkers in differentiated cells. Data are cells. represented Data are byrepresented the mean ±bySD the of mean four ± independentSD of four independent experiments. experiments Asterisks indicate. Asterisks significant indicate difference significant between difference the undifferentiated between the undif- cells andferentiated differentiated cells and cells differentiated (*** p < 0.001). cells White (*** bar: p < undifferentiated0.001). White bar: cells; undifferentiated black bar: differentiated cells; black cells; bar: differentiated cells; U: undifferentiated cells; A: adipogenic differentiated cells; C: chondrogenic U: undifferentiated cells; A: adipogenic differentiated cells; C: chondrogenic differentiated cells. differentiated cells. 3.3. In Vitro Osteogenic Differentiation Capacity of MSCs 3.3. In Vitro Osteogenic Differentiation Capacity of MSCs The SM-MSCs, DM-MSCs, and A-MSCs were successfully differentiated into os- teoblasts.The TheSM-MSCs, calcified DM extracellular-MSCs, and matrixA-MSCs formation were successfully was confirmed differentiated by Alizarin into red osteo- S, andblasts. Von The kossa calcified staining extracellular (Figure5a). matrix The expression formation of was osteoblast-specific confirmed by Alizarin markers red (Runx2, S, and andVon Osteocalcin) kossa staining was (Figure significantly 5a). The higher expression in the of osteogenic-induced osteoblast-specific MSCsmarkers than (Runx2, the non- and differentiatedOsteocalcin) was cells si (Figuregnificantly5b). higher Additionally, in the osteogenic upon the- inductioninduced MSCs of osteogenesis, than the non the-dif- expressionferentiated of cells Runx2 (Figure and 5b). Osteocalcin Additionally, was significantly upon the induction (p < 0.01) of higher osteogenesis, in SM-MSCs the expres- and DS-MSCs,sion of Runx2 compared and Osteocalcin to A-MSCs. was We significantly also assessed (p < ALP 0.01) activity higher andin SM calcium-MSCs content.and DS- TheMSCs ALP, compared activity significantly to A-MSCs. (Wep < also 0.01) assessed increased ALP differentiated activity andosteoblasts calcium content. derived The from ALP SM-MSCsactivity significantly and DS-MSCs, (p < 0.01) compared increased to those differentiated derived fromosteoblasts A-MSCs derived at seven from days SM- afterMSCs osteogenic-differentiationand DS-MSCs, compared (Figureto those6). derived Furthermore, from A we-MSCs also at confirmed seven days that after calcium osteogenic con- - tentdifferentiation was also significantly (Figure 6). (Furthermore,p < 0.001) increased we also in confirmed SM-MSCS that and calcium DS-MSCs content 21 days was after also differentiationsignificantly ( intop < 0.001) osteoblast increased (Figure in 6SM). -MSCS and DS-MSCs 21 days after differentiation into osteoblast (Figure 6).

(a) (b) Animals 2021, 11, x 7 of 12

(a) (b)

Figure 4. In vitro mesoderm-lineage (adipocyte and chondrocyte) differentiation of MSCs from three types of tissues. (a). Adipocytes and chondrocytes differentiation were confirmed oil red O (for adipocytes) and Alcian blue (for chondrocytes) staining. (b). The gene expression of adipo- cytes and chondrocytes-specific markers in differentiated cells. Data are represented by the mean ± SD of four independent experiments. Asterisks indicate significant difference between the undif- ferentiated cells and differentiated cells (*** p < 0.001). White bar: undifferentiated cells; black bar: differentiated cells; U: undifferentiated cells; A: adipogenic differentiated cells; C: chondrogenic differentiated cells.

3.3. In Vitro Osteogenic Differentiation Capacity of MSCs The SM-MSCs, DM-MSCs, and A-MSCs were successfully differentiated into osteo- blasts. The calcified formation was confirmed by Alizarin red S, and Von kossa staining (Figure 5a). The expression of osteoblast-specific markers (Runx2, and Osteocalcin) was significantly higher in the osteogenic-induced MSCs than the non-dif- ferentiated cells (Figure 5b). Additionally, upon the induction of osteogenesis, the expres- sion of Runx2 and Osteocalcin was significantly (p < 0.01) higher in SM-MSCs and DS- MSCs, compared to A-MSCs. We also assessed ALP activity and calcium content. The ALP activity significantly (p < 0.01) increased differentiated osteoblasts derived from SM-MSCs and DS-MSCs, compared to those derived from A-MSCs at seven days after osteogenic- differentiation (Figure 6). Furthermore, we also confirmed that calcium content was also Animals 2021, 11, 1017 8 of 12 significantly (p < 0.001) increased in SM-MSCS and DS-MSCs 21 days after differentiation into osteoblast (Figure 6).

Animals 2021, 11, x 8 of 12 (a) (b)

Figure 5. The results of in vitro osteoblast differentiation of MSCs derived from skeletal muscle Figure(SM-MSCs), 5. The dermalresults of skin in (DS-MSCs),vitro osteoblast and differentiation adipose tissue of (A-MSCs). MSCs derived (a). Cytochemicalfrom skeletal muscle staining of (SMdifferentiated-MSCs), dermal osteoblasts. skin (DS Scale-MSCs), bar = 200andµ adiposem. (b). Resultstissue (A of-MSCs). RT-qPCR (a) for. Cytochemical osteoblast-specific staining markers of differentiatedin differentiated osteoblasts. cells. All Scale osteoblast-specific bar = 200 µm. (b markers). Results were of RT significantly-qPCR for osteoblast higher in-specific differentiated mark- ers in differentiated cells. All osteoblast-specific markers were significantly higher in differentiated osteoblast from SM-MSCs and DS-MSCs than that from A-MSCs. Data are represented by the osteoblast from SM-MSCs and DS-MSCs than that from A-MSCs. Data are represented by the mean ± SD of four independent experiments. Asterisks indicate significant difference between the mean ± SD of four independent experiments. Asterisks indicate significant difference between the undifferentiatedundifferentiated cells cells and and differentiated differentiated cells cells (** (** pp << 0.01; 0.01; *** *** pp << 0.001). 0.001). White White bar: bar: undifferentiated undifferentiated cells;cells; black black bar: bar: differentiated differentiated cells; cells; U: U: und undifferentiatedifferentiated cells; cells; O: O: osteogenic osteogenic differentiated cells.

FigureFigure 6. 6. TheThe levels of alkaline phosphatasephosphatase (ALP)(ALP) activity activity and and calcium calcium content content of of MSCs MSCs derived derived from fromskeletal skeletal muscle muscle (SM-MSCs), (SM-MSCs), dermal dermal skin (DS-MSCs), skin (DS-MSCs), and adipose and adipose tissue (A-MSCs).tissue (A-MSCs). The differentiated The dif- ferentiated osteoblasts from SM-MSCs and DS-MSCs showed significantly (p < 0.05) increased the osteoblasts from SM-MSCs and DS-MSCs showed significantly (p < 0.05) increased the ALP activity ALP activity (day 7) and calcium content (day 21) compared to that from A-MSCs. Data are repre- (day 7) and calcium content (day 21) compared to that from A-MSCs. Data are represented by the sented by the mean ± SD of four independent experiments. Asterisks indicate significant difference ± betweenmean theSD ofundifferentiated four independent cells experiments. and differentiated Asterisks cells indicate (** p < 0 significant.01; *** p < 0.001). difference White between bar: the undifferentiatedundifferentiated cells; cells andblack differentiated bar: differentiated cells (** cells;p < U: 0.01; undifferentiated *** p < 0.001). Whitecells; O: bar: osteogenic undifferentiated differ- entiatedcells; black cells. bar: differentiated cells; U: undifferentiated cells; O: osteogenic differentiated cells. 4. Discussion 4. Discussion The bone is a hard calcified organ that consists of proteins such as collagen and The bone is a hard calcified organ that consists of proteins such as collagen and hy- [25]. Unlike other tissues, bones undergo mineralization process and droxyapatite [25]. Unlike other tissues, bones undergo mineralization process and remod- remodeling process [25,26]. Osteoblasts play important roles in producing proteins that eling process [25,26]. Osteoblasts play important roles in producing proteins that form form bone and accumulating minerals [27]. Bone-related problems, including bone and accumulating minerals [27]. Bone-related problems, including bone fracture and and bond defect, can become fatal unless properly treated. Traditionally, these disorders can bond defect, can become fatal unless properly treated. Traditionally, these disorders can be treated by using biological bone grafts [28]. Although bone grafts can yield satisfactory beresults, treated clinical by using use biological is limited duebone to grafts the lack [28]. of Although available bone,bone grafts additional can yield invasive satisfactory surgery, results, clinical use is limited due to the lack of available bone, additional invasive surgery, and risk of infections. Therefore, stem cell-based therapy has emerged as an alternative method for bone regeneration. As described above, bone marrow-derived MSCs (BM-MSCs) were the first known stem cells, but stemness and multi-potency differentiation capacity are limited depending on donor aging [20]. Furthermore, until now, studies on biological characteristics, includ- ing the establishment of stem cells, and osteoblast differentiation from various tissues of camels, has been limited. The present study was performed to evaluate the stemness and multi-potency of MSCs derived from different camel tissues. We successfully isolated and cultured SM-MSCs, DS-MSCs, and A-MSCs from a single donor to confirm the most effi- cient source for osteoblast differentiation. We compared the morphology, proliferation, expression of pluripotent markers, and in vitro differentiation into mesoderm-lineage in- cluding osteoblasts in SM-MSCs, DS-MSCs, and A-MSCs. All types of MSCs showed spin- dle-like morphology under identical conditions. The pluripotent factors play an important role in the proliferation and differentiation of MSCs [29,30]. In this study, MSCs from skel- etal muscle, dermal skin, and adipose tissues expressed critical pluripotent markers such as OCT4, SOX2, and NANOG at mRNA levels. Our data also showed that there was no difference in proliferation rate and expression of pluripotent markers among the cells. The Animals 2021, 11, 1017 9 of 12

and risk of infections. Therefore, stem cell-based therapy has emerged as an alternative method for bone regeneration. As described above, bone marrow-derived MSCs (BM-MSCs) were the first known stem cells, but stemness and multi-potency differentiation capacity are limited depending on donor aging [20]. Furthermore, until now, studies on biological characteristics, including the establishment of stem cells, and osteoblast differentiation from various tissues of camels, has been limited. The present study was performed to evaluate the stemness and multi-potency of MSCs derived from different camel tissues. We successfully isolated and cultured SM-MSCs, DS-MSCs, and A-MSCs from a single donor to confirm the most efficient source for osteoblast differentiation. We compared the morphology, proliferation, expression of pluripotent markers, and in vitro differentiation into mesoderm-lineage including osteoblasts in SM-MSCs, DS-MSCs, and A-MSCs. All types of MSCs showed spindle-like morphology under identical conditions. The pluripotent factors play an important role in the proliferation and differentiation of MSCs [29,30]. In this study, MSCs from skeletal muscle, dermal skin, and adipose tissues expressed critical pluripotent markers such as OCT4, SOX2, and NANOG at mRNA levels. Our data also showed that there was no difference in proliferation rate and expression of pluripotent markers among the cells. The expression of pluripotent markers revealed the stemness capacity of MSCs derived from skeletal muscles, dermal skin, and adipose tissues. We demonstrated that SM-MSCs, DS-MSCs, and A-MSCs were successfully differen- tiated into adipocytes under specific culture conditions (Figure4a). The gene expression of adipogenic markers, LPL, and FABP4 were significantly increased in differentiated adipocytes compared to the non-differentiated cells. Additionally, adipose tissue-derived MSCs revealed higher differentiation potential into adipocytes compared to skeletal muscle, and dermal skin-derived MSCs. This finding is similar to previous studies [31,32]. Stem cells derived from certain tissue show elevated differentiation efficiency depending on the environment where tissues are located [31]. Similarly, all MSCs differentiated into chondrocytes and expressed chondrocyte-related markers, i.e., the type X collagen gene (COL10A1) and agreecan (ACAN), which are following the previous study [31]. The present study also assessed the differentiation potential of SM-MSCs, DS-MSCs, and A-MSCs toward osteoblasts. Several studies demonstrated the capacity of various camel stem cells differentiated toward osteoblasts [2,33,34]. However, the comparative evaluation of osteoblast differentiation ability of different regions to derive MSCs in camels from an osteogenic lineage has not been reported. In the process of osteoblast differentia- tion, it is reported that the expression of collagen type I and Runx2 increases in the initial proliferative phase, and calcium accumulates with an increased in the expression of ALP and osteocalcin (OCN) during the formation of mineral matrix formation [28,35]. We also confirmed that after osteoblast differentiation, the osteoblast-specific genes (Runx2, and Osteocalcin) were significantly higher in differentiated cells compared to undifferentiated cells. Furthermore, the differentiated osteoblasts from SM-MSCs and DS-MSCs showed significantly higher expression of osteoblast-specific markers than that from A-MSCs. ALP activity, mineral matrix formation, and calcium deposition are crucial markers on osteoblast [35–37]. The transporting of calcium ions in osteoblast-induced mineralization and calcium deposition meant mineralization process in osteoblast differentiation [38,39]. Our data showed that the expression of ALP was significantly increased in differentiated osteoblast compared to non-differentiated cells. Interestingly, osteoblasts from SM-MSCs and DS-MSCs exhibited significantly higher expression of ALP activity than that from A- MSCs. Similarly, the levels of calcium content were also lowest in induced A-MSCs groups. Although additional studies will be necessary to evaluate the underlying mechanisms, the present study suggests that MSCs from different tissues have varied differentiation capacity into different lineage even if their identical donor origin. Animals 2021, 11, 1017 10 of 12

5. Conclusions MSCs established from a single donor of skeletal muscle, dermal skin, and adipose tissues showed similar characteristics related to cellular morphology, proliferation, ex- pression of pluripotent marker, and differentiation into chondrocytes. All types of MSCs successfully differentiated toward adipocyte and osteoblasts. However, the expression of adipocyte-specific markers revealed that adipose tissue-derived MSCs are notable sources for differentiation toward adipocytes. Furthermore, the expression of osteoblast-specific markers, ALP activity, and calcium content revealed that SM-MSCs and DS-MSCs are prominent sources for stem cell-based therapy. These in vitro results provide for the poten- tial treatment of Camelus dromedarius utilizing MSCs with excellent osteoblast differentiation capacity. MSCs pre-establishment can allow for direct treatment following bone-related problems, including bone defects and fractures due to severe trauma. This approach is warranted to promote animal welfare by providing immediate relief to injuries, amelio- rating suffering time, and reducing the risk for secondary injuries. To our knowledge, this is the first study in which the osteoblast differentiation capacity of MSCs from three different tissues from a single donor has been compared and a prominent MSCs source for the generation of osteoblasts has been demonstrated.

Author Contributions: Conceptualization, Y.-B.S., and W.S.H.; Methodology, Y.-B.S., Y.I.J., and Y.W.J.; Investigation, Y.-B.S.; Validation, Y.W.J.; Formal analysis, Y.-B.S.; Resources, Y.I.J., and A.T., and K.K.S.; Writing—original draft preparation, Y.-B.S.; Writing—review and editing, Y.-B.S., and M.S.H.; Funding acquisition, W.S.H. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Institutional Review Board Statement: All animal procedures were conducted following the animal study guidelines which were approved by the ethics committee at the Management of Scientific Centers and Presidential Camels (Accession No: PC4.1.5). Informed Consent Statement: Not applicable. Data Availability Statement: Data access can be requested on demand with the corresponding au- thor. Acknowledgments: This project was supported by the Patronage of H.H. Sheikh Mansour bin Zayed Al Nahyan, Deputy Prime Minister of the U.A.E. and the Minister of Presidential Affairs, we acknowledge his support and inspiration in the initiation and mentoring of this project, without whom this project would not have been possible. Conflicts of Interest: The authors declare that they have no competing interests.

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