To establish the influence of fetal bovine serum (FBS) and bovine serum lipids (BSL) on cell differentiation marker expression, bovine adipose-derived stem cells from subcutaneous tissue were incubated for 14 days in 4 types of differentiation media containing 10% FBS and 10 µL/mL BSL (TRT-1), no FBS and 10 µL/mL of BSL (TRT-2), 10% FBS and no BSL (TRT-3), or no supplements (TRT-4). Cells were subjected to Nile red staining, immunocytochemistry (CD73, CD90, CD105, DLK1, FabP4), and quantitative real-time PCR (CD73, CD90, CD105, FabP4). The number of cells presenting FabP4 and the percentage of mature adipocytes with large lipid droplets were increased in TRT-2, accompanied by a robust increase in FabP4 mRNA abundance and a decrease in DLK1-positive cells. In preadipocytes, CD73 was present around the nucleus and translocated towards cell membranes during differentiation. Although the percentage of CD73-positive cells was not different among treatments, its mRNA abundance, immunocytochemical staining intensity, and translocation towards cell membranes were decreased when the medium contained no FBS (TRT-2 and TRT-4). All cells showed a diffuse distribution of CD90 and CD105 and remained positive for these markers irrespective of the treatment. However, the CD90 and CD105 mRNA abundance was decreased in TRT-2 and TRT-4; i.e., in media containing no FBS. The presence of FBS increased the absolute number of cell nuclei as assessed by DAPI fluorescence. Our results suggest that bovine subcutaneous preadipocytes display typical stem cell markers. The differentiation into mature adipocytes is promoted by BSL, whereas FBS endorses cell proliferation.

Keywords:
Adipocytes, Adipose stem cells, Cattle, Immunocytochemistry
1.
Anderson, P., A.B. Carrillo-Gálvez, A. García-Pérez, M. Cobo, F. Martín (2013) CD105 (Endoglin) negative murine mesenchymal stromal cells define a new multipotent subpopulation with distinct differentiation and immunomodulatory capacities. PLoS One 8: e76979.
2.
Barbara, N.P., J.L. Wrana, M. Letarte (1999) Endoglin is an accessory protein that interacts with the signaling receptor complex of multiple members of the transforming growth factor-β super family. J Biol Chem 274: 584-594.
3.
Beloor, J., H.K. Kang, Y.S. Moon (2010) Serum lipids can convert bovine myogenic satellite cells to adipocytes. Asian Australas J Anim Sci 23: 1519-1526.
4.
Chang, C.B., S.A. Han, E.M. Kim, S. Lee, S.C. Seong, M.C. Lee (2013) Chondrogenic potentials of human synovium-derived cells sorted by specific surface markers. Osteoarthr Cartil 21: 190-199.
5.
Choy, L., J. Skillington, R. Derynck (2000) Roles of autocrine TGF-β receptor and Smad signaling in adipocyte differentiation. J Cell Biol 149: 667-682.
6.
De Francesco, F., G. Ricci, F. D'Andrea, G.F. Nicoletti, G.A. Ferraro (2015): Human adipose stem cells: from bench to bedside. Tissue Eng Part B Rev 21: 572-784.
7.
Djian, P., A.K. Roncari, C.H. Hollenberg (1983) Influence of anatomic site and age on the replication and differentiation of rat adipocyte precursors in culture. J Clin Invest 72: 1200-1208.
8.
Dominici, M., K. Le Blanc, I. Mueller, I. Slaper-Cortenbach, F. Marini, D. Krause, R. Deans, A. Keating, D. Prockop, E. Horwitz (2006) Minimal criteria for defining multipotent mesenchymal stromal cells: the International Society for Cellular Therapy position statement. Cytotherapy 8: 315-317.
9.
Elmasri, H., C. Karaaslan, Y. Teper, E. Ghelfi, M. Weng, T.A. Ince, H. Kozakewich, J. Bischoff, S. Cataltepe (2009) Fatty acid binding protein 4 is a target of VEGF and a regulator of cell proliferation in endothelial cells. FASEB J 23: 3865-3873.
10.
Gao, Z., K. Dong, H. Zhang (2014) The roles of CD73 in cancer. Bio Med Res Int 2014: 460654.
11.
Garces, C., M.J. Ruiz-Hidalgo, E. Bonvini, J. Goldstein, J. Laborda (1999) Adipocyte differentiation is modulated by secreted delta-like (dlk) variants and requires the expression of membrane-associated dlk. Differentiation 64: 103-114.
12.
Gesta, S., Y.H. Tseng, C.R. Kahn (2007) Developmental origin of fat: tracking obesity to its source. Cell 131: 242-256.
13.
Grant, A.C., G. Ortiz-Colòn, M.E. Doumit, D.D. Buskirk (2008) Optimization of in vitro conditions for bovine subcutaneous and intramuscular pre-adipocyte differentiation. J Anim Sci 86: 73-82.
14.
Greenspan, P., E.P. Mayer, S.D. Fowler (1985) Nile red: a selective fluorescent stain for intracellular lipid droplets. J Cell Biol 100: 965-973.
15.
Gstraunthaler, G (2003) Alternatives to the use of fetal bovine serum: serum-free cell culture. ALTEX 20: 275-281.
16.
Kingham, P.J., D.F. Kalbermatten, D. Mahay, S.J. Armstrong, M. Wiberg, G. Terenghi (2007) Adipose-derived stem cells differentiate into a Schwann cell phenotype and promote neurite outgrowth in vitro. Exp Neurol 207: 267-274.
17.
Kumar, A., A. Bhanja, J. Bhattacharyya, B.G. Jaganathan (2016) Multiple roles of CD90 in cancer. Tumour Biol 37: 11611-11622.
18.
Lehnert, S.A., A. Reverter, K.A. Byrne, Y. Wang, G.S. Nattrass, N.J. Hudson, P.L. Greenwood (2007) Gene expression studies of developing bovine longissimus muscle from two different beef cattle breeds. BMC Dev Biol 7: 95.
19.
Livak, K.J., T.D. Schmittgen (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods 25: 402-408.
20.
Mitterberger, M.C., S. Lechner, M. Mattesich, A. Kaiser, D. Probst, N. Wenger, G. Pierer, W. Zwerschke (2012) DLK1(PREF1) is a negative regulator of adipogenesis in CD105+/CD90+/CD34+/CD31−/FABP4− adipose-derived stromal cells from subcutaneous abdominal fat pats of adult women. Stem Cell Res 9: 35-48.
21.
Moreno-Navarrete, J.M., J.M. Fernández-Real (2012) Adipocyte differentiation; in Symonds, M.E. (ed.) Adipocyte Tissue Biology. Berlin, Springer Science, pp 17-38.
22.
Nicoletti, G.F., F. De Francesco, F. D'Andrea, G.A. Ferraro (2015) Methods and procedures in adipose stem cells: state of the art and perspective for translation medicine. J Cell Physiol 230: 489-495.
23.
Priya, N., S. Sarcar, A.S. Majumdar, S. SundarRaj (2014) Explant culture: a simple, reproducible, efficient and economic technique for isolation of mesenchymal stromal cells from human adipose tissue and lipoaspirate. J Tissue Eng Regen Med 8: 706-716.
24.
Oida, T., H.L. Weiner (2010) Depletion of TGF-β from fetal bovine serum. J Immunol Meth 362: 195-198.
25.
Ong, W.K., C.S. Tan, K.L. Chan, G.G. Goesantoso, X.H.D. Chan, E. Chan, J. Yin, C.R. Yeo, C.M. Khoo, J.B.Y. So, A. Shabbir, S.A. Toh, W. Han, S. Sugii (2014) Identification of specific cell-surface markers of adipose-derived stem cells from subcutaneous and visceral fat depots. Stem Cell Reports 2: 171-179.
26.
Ozpur, M.A., E. Guneren, H.I. Canter, M.V. Karaaltin, E. Ovali, F.N. Yogun, E.G. Baygol, S. Kaplan (2016) Generation of skin tissue using adipose tissue-derived stem cells. Plast Reconstr Surg 137: 134-143.
27.
Rosen, E.D., O.A. MacDougald (2006) Adipocyte differentiation from the inside out. Nat Rev Mol Cell Biol 7: 885-896.
28.
Salazar, K.D., S.M. Lankford, A.R. Brody (2009) Mesenchymal stem cells produce Wnt isoforms and TGF-β1 that mediate proliferation and procollagen expression by lung fibroblasts. Am J Physiol Lung Cell Mol Physiol 297: L1002-L1011.
29.
Sampaio, R.V., M.R. Chiaratti, D.C.N. Santos, F.F. Bressan, J.R. Sangalli, A.L.A. Sá, T.V.G. Silva, N.N. Costa, M.S. Cordeiro, S.S.D. Santos, C.E. Ambrosio, P.R. Adona, F.V. Meirelles, M.S. Miranda, O.M. Ohashi (2015) Generation of bovine (Bos indicus) and buffalo (Bubalus bubalis) adipose tissue derived stem cells: isolation, characterization and multipotentiality. Genet Mol Res 14: 53-62.
30.
Shan, T., W. Liu, S. Kuang (2013) Fatty acid binding protein 4 expression marks a population of adipocyte progenitors in white and brown adipose tissues. FASEB J 27: 277-287.
31.
Shebaby, W., E. K. Abdalla, F. Saad, W. H. Faour (2016) Data on isolating mesenchymal stromal cells from human adipose tissue using a collagenase-free method. Data Brief 6: 974-979.
32.
Smas, C.M., L. Chen, H.S. Sul (1997) Cleavage of membrane-associated pref-1 generates a soluble inhibitor of adipocyte differentiation. Mol Cell Biol 17: 977-988.
33.
Spiegelman, B.M., M. Frank, H. Green (1983) Molecular cloning of mRNA from 3T3 adipocytes: regulation of mRNA content for glycerophosphate dehydrogenase and other differentiation dependent proteins during adipocyte development. J Biol Chem 258: 10083-10089.
34.
Zuk, P.A., M. Zhu, P. Ashjian, D.A. De Ugarte, J.I. Huang, H. Mizuno, Z.C. Alfonso, J.K. Fraser, P. Benhaim, M.H. Hedrick (2002) Human adipose tissue is a source of multipotent stem cells. Mol Bio Cell 3: 4279-4295.
© 2017 S. Karger AG, Basel
2017
Copyright / Drug Dosage / Disclaimer
Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher.
Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug.
Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.
You do not currently have access to this content.