Abstract
Adipocytes are terminally differentiated cells derived from fibroblastic preadipocyte precursors. Here, we describe a method for the isolation and proliferation of preadipocytes from murine subcutaneous white adipose tissue, followed by differentiation in culture to mature adipocytes; we refer to these cells as primary preadipocytes differentiated in vitro (PPDIVs). Compared to adipogenic cell lines, PPDIV metabolism and adipokine secretion more closely resemble in vivo adipocyte biology. While primary mature adipocytes have the greatest in vivo relevance, their fragility and buoyancy make them unsuitable for many cell culture-based methods. PPDIVs can also take advantage of transgenic and knockout mouse models to produce genetically modified adipocytes. Thus, PPDIVs are a valuable resource for studying adipocyte biology in cell culture.
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References
Rodbell M (1964) Metabolism of isolated fat cells. I. Effects of hormones on glucose metabolism and lipolysis. J Biol Chem 239:375–380
Sugihara H, Yonemitsu N, Miyabara S, Yun K (1986) Primary cultures of unilocular fat cells: characteristics of growth in vitro and changes in differentiation properties. Differentiation 31(1):42–49. https://doi.org/10.1111/j.1432-0436.1986.tb00381.x
Zhang HH, Kumar S, Barnett AH, Eggo MC (2000) Ceiling culture of mature human adipocytes: use in studies of adipocyte functions. J Endocrinol 164(2):119–128. https://doi.org/10.1677/joe.0.1640119
Hollenberg CH, Vost A (1969) Regulation of DNA synthesis in fat cells and stromal elements from rat adipose tissue. J Clin Invest 47(11):2485–2498. https://doi.org/10.1172/JCI105930
Bjorntorp P, Karlsson M, Pertoft H, Pettersson P, Sjostrom L, Smith U (1978) Isolation and characterization of cells from rat adipose tissue developing into adipocytes. J Lipid Res 19(3):316–324
Ng CW, Poznanski WJ, Borowiecki M, Reimer G (1971) Differences in growth in vitro of adipose cells from normal and obese patients. Nature 231(5303):445. https://doi.org/10.1038/231445a0
Van RL, Bayliss CE, Roncari DA (1976) Cytological and enzymological characterization of adult human adipocyte precursors in culture. J Clin Invest 58(3):699–704. https://doi.org/10.1172/JCI108516
Green H, Kehinde O (1975) An established preadipose cell line and its differentiation in culture. II. Factors affecting the adipose conversion. Cell 5(1):19–27. https://doi.org/10.1016/0092-8674(75)90087-2
Green H, Meuth M (1974) An established pre-adipose cell line and its differentiation in culture. Cell 3(2):127–133. https://doi.org/10.1016/0092-8674(74)90116-0
Todaro GJ, Green H (1963) Quantitative studies of the growth of mouse embryo cells in culture and their development into established lines. J Cell Biol 17:299–313. https://doi.org/10.1083/jcb.17.2.299
Cawthorn WP, Scheller EL, MacDougald OA (2012) Adipose tissue stem cells meet preadipocyte commitment: going back to the future. J Lipid Res 53(2):227–246. https://doi.org/10.1194/jlr.R021089
Halvorsen YC, Wilkison WO, Gimble JM (2000) Adipose-derived stromal cells–their utility and potential in bone formation. Int J Obes Relat Metab Disord 24(Suppl 4):S41–S44. https://doi.org/10.1038/sj.ijo.0801503
Halvorsen YD, Franklin D, Bond AL, Hitt DC, Auchter C, Boskey AL, Paschalis EP, Wilkison WO, Gimble JM (2001) Extracellular matrix mineralization and osteoblast gene expression by human adipose tissue-derived stromal cells. Tissue Eng 7(6):729–741. https://doi.org/10.1089/107632701753337681
Zuk PA, Zhu M, Mizuno H, Huang J, Futrell JW, Katz AJ, Benhaim P, Lorenz HP, Hedrick MH (2001) Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Eng 7(2):211–228. https://doi.org/10.1089/107632701300062859
Rosenthal N, Brown S (2007) The mouse ascending: perspectives for human-disease models. Nat Cell Biol 9(9):993–999. https://doi.org/10.1038/ncb437
Kang S, Kong X, Rosen ED (2014) Adipocyte-specific transgenic and knockout models. Methods Enzymol 537:1–16. https://doi.org/10.1016/B978-0-12-411619-1.00001-X
Djian P, Roncari AK, Hollenberg CH (1983) Influence of anatomic site and age on the replication and differentiation of rat adipocyte precursors in culture. J Clin Invest 72(4):1200–1208. https://doi.org/10.1172/JCI111075
Kirkland JL, Hollenberg CH, Gillon WS (1990) Age, anatomic site, and the replication and differentiation of adipocyte precursors. Am J Phys 258(2 Pt 1):C206–C210. https://doi.org/10.1152/ajpcell.1990.258.2.C206
Kirkland JL, Hollenberg CH, Kindler S, Gillon WS (1994) Effects of age and anatomic site on preadipocyte number in rat fat depots. J Gerontol 49(1):B31–B35. https://doi.org/10.1093/geronj/49.1.b31
Forman BM, Tontonoz P, Chen J, Brun RP, Spiegelman BM, Evans RM (1995) 15-Deoxy-delta 12, 14-prostaglandin J2 is a ligand for the adipocyte determination factor PPAR gamma. Cell 83(5):803–812. https://doi.org/10.1016/0092-8674(95)90193-0
Ohno H, Shinoda K, Spiegelman BM, Kajimura S (2012) PPARgamma agonists induce a white-to-brown fat conversion through stabilization of PRDM16 protein. Cell Metab 15(3):395–404. https://doi.org/10.1016/j.cmet.2012.01.019
Bagchi DP, MacDougald OA (2019) Identification and dissection of diverse mouse adipose depots. J Vis Exp 149. https://doi.org/10.3791/59499
Grandl G, Muller S, Moest H, Moser C, Wollscheid B, Wolfrum C (2016) Depot specific differences in the adipogenic potential of precursors are mediated by collagenous extracellular matrix and Flotillin 2 dependent signaling. Mol Metab 5(10):937–947. https://doi.org/10.1016/j.molmet.2016.07.008
Gregoire F, Todoroff G, Hauser N, Remacle C (1990) The stroma-vascular fraction of rat inguinal and epididymal adipose tissue and the adipoconversion of fat cell precursors in primary culture. Biol Cell 69(3):215–222. https://doi.org/10.1016/0248-4900(90)90348-7
Hauner H, Wabitsch M, Pfeiffer EF (1988) Differentiation of adipocyte precursor cells from obese and nonobese adult women and from different adipose tissue sites. Horm Metab Res Suppl 19:35–39
Meissburger B, Perdikari A, Moest H, Muller S, Geiger M, Wolfrum C (2016) Regulation of adipogenesis by paracrine factors from adipose stromal-vascular fraction –a link to fat depot-specific differences. Biochim Biophys Acta 1861(9 Pt A):1121–1131. https://doi.org/10.1016/j.bbalip.2016.06.010
Mizuno H, Itoi Y, Kawahara S, Ogawa R, Akaishi S, Hyakusoku H (2008) In vivo adipose tissue regeneration by adipose-derived stromal cells isolated from GFP transgenic mice. Cells Tissues Organs 187(3):177–185. https://doi.org/10.1159/000110805
Van RL, Roncari DA (1982) Complete differentiation in vivo of implanted cultured adipocyte precursors from adult rats. Cell Tissue Res 225(3):557–566. https://doi.org/10.1007/BF00214804
Acknowledgments
We thank Omer Keinan, Joseph M. Valentine, and Preetveer Dhillon for their contributions in developing this protocol. This work was supported by the National Institutes of Health R01DK126944 to S.M.R.
The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
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DeLuca, J.H., Reilly, S.M. (2023). Culture and Differentiation of Primary Preadipocytes from Mouse Subcutaneous White Adipose Tissue. In: Lodhi, I.J. (eds) Thermogenic Fat. Methods in Molecular Biology, vol 2662. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3167-6_2
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DOI: https://doi.org/10.1007/978-1-0716-3167-6_2
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