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Protocols for Generation of Immortalized Human Brown and White Preadipocyte Cell Lines

  • Farnaz Shamsi
  • Yu-Hua Tseng
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1566)

Abstract

Human brown and white preadipocytes offer unique cell models to study human adipogenesis and thermogenesis. Here, we describe the detailed procedures for isolation of human brown and white predipocytes from deep and superficial neck fat. To grow these cells in vitro for a prolonged period of time, they should be immortalized following the procedure discussed here. We also provide the protocol for expansion, cryopreservation, and adipogenic differentiation of cells.

Key words

Brown and white adipose tissue Stromal vascular fraction (SVF) Preadipocytes Neck fat Immortalization Differentiation 

Notes

Acknowledgments

This work was supported in part by NIH grants R01 DK077097 and R01 DK102898 (to Y.-H.T.), and P30 DK036836 (to Joslin Diabetes Research Center).

References

  1. 1.
    Orava J, Nuutila P, Lidell ME, Oikonen V, Noponen T, Viljanen T, Scheinin M, Taittonen M, Niemi T, Enerback S, Virtanen KA (2011) Different metabolic responses of human brown adipose tissue to activation by cold and insulin. Cell Metab 14(2):272–279. doi: 10.1016/j.cmet.2011.06.012 CrossRefPubMedGoogle Scholar
  2. 2.
    Nedergaard J, Bengtsson T, Cannon B (2007) Unexpected evidence for active brown adipose tissue in adult humans. Am J Physiol Endocrinol Metab 293(2):E444–E452. doi: 10.1152/ajpendo.00691.2006 CrossRefPubMedGoogle Scholar
  3. 3.
    Cypess AM, Lehman S, Williams G, Tal I, Rodman D, Goldfine AB, Kuo FC, Palmer EL, Tseng YH, Doria A, Kolodny GM, Kahn CR (2009) Identification and importance of brown adipose tissue in adult humans. N Engl J Med 360(15):1509–1517. doi: 10.1056/NEJMoa0810780 CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    van Marken Lichtenbelt WD, Vanhommerig JW, Smulders NM, Drossaerts JM, Kemerink GJ, Bouvy ND, Schrauwen P, Teule GJ (2009) Cold-activated brown adipose tissue in healthy men. N Engl J Med 360(15):1500–1508. doi: 10.1056/NEJMoa0808718 CrossRefPubMedGoogle Scholar
  5. 5.
    Virtanen KA, Lidell ME, Orava J, Heglind M, Westergren R, Niemi T, Taittonen M, Laine J, Savisto NJ, Enerback S, Nuutila P (2009) Functional brown adipose tissue in healthy adults. N Engl J Med 360(15):1518–1525. doi: 10.1056/NEJMoa0808949 CrossRefPubMedGoogle Scholar
  6. 6.
    Cypess AM, White AP, Vernochet C, Schulz TJ, Xue R, Sass CA, Huang TL, Roberts-Toler C, Weiner LS, Sze C, Chacko AT, Deschamps LN, Herder LM, Truchan N, Glasgow AL, Holman AR, Gavrila A, Hasselgren PO, Mori MA, Molla M, Tseng YH (2013) Anatomical localization, gene expression profiling and functional characterization of adult human neck brown fat. Nat Med 19(5):635–639. doi: 10.1038/nm.3112 CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Pear WS, Nolan GP, Scott ML, Baltimore D (1993) Production of high-titer helper-free retroviruses by transient transfection. Proc Natl Acad Sci U S A 90(18):8392–8396CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Swift S, Lorens J, Achacoso P, Nolan GP (2001) Rapid production of retroviruses for efficient gene delivery to mammalian cells using 293T cell-based systems. Curr Protoc Immunol Chapter 10:Unit 10 17C. doi: 10.1002/0471142735.im1017cs31 PubMedGoogle Scholar
  9. 9.
    Schulz TJ, Huang TL, Tran TT, Zhang H, Townsend KL, Shadrach JL, Cerletti M, McDougall LE, Giorgadze N, Tchkonia T, Schrier D, Falb D, Kirkland JL, Wagers AJ, Tseng YH (2011) Identification of inducible brown adipocyte progenitors residing in skeletal muscle and white fat. Proc Natl Acad Sci U S A 108(1):143–148. doi: 10.1073/pnas.1010929108 CrossRefPubMedGoogle Scholar
  10. 10.
    Tseng YH, Kokkotou E, Schulz TJ, Huang TL, Winnay JN, Taniguchi CM, Tran TT, Suzuki R, Espinoza DO, Yamamoto Y, Ahrens MJ, Dudley AT, Norris AW, Kulkarni RN, Kahn CR (2008) New role of bone morphogenetic protein 7 in brown adipogenesis and energy expenditure. Nature 454(7207):1000–1004. doi: 10.1038/nature07221 CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Xue R, Lynes MD, Dreyfuss JM, Shamsi F, Schulz TJ, Zhang H, Huang TL, Townsend KL, Li Y, Takahashi H, Weiner LS, White AP, Lynes MS, Rubin LL, Goodyear LJ, Cypess AM, Tseng YH (2015) Clonal analyses and gene profiling identify genetic biomarkers of the thermogenic potential of human brown and white preadipocytes. Nat Med 21(7):760–768. doi: 10.1038/nm.3881 CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  1. 1.Section on Integrative Physiology and Metabolism, Joslin Diabetes CenterHarvard Medical SchoolBostonUSA
  2. 2.Harvard Stem Cell InstituteHarvard UniversityCambridgeUSA

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