Analysis of Circadian Rhythms in Embryonic Stem Cells

  • Jiffin K. Paulose
  • Edmund B. RuckerIII
  • Vincent M. CassoneEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1235)


Recent attention on the early development of circadian rhythms has yielded several avenues of potential study regarding molecular and physiological rhythms in embryonic stem cells (ESCs) and their derivatives. While general guidelines of experimental design are—as always—applicable, there are certain idiosyncrasies with respect to experiments involving circadian rhythms that will be addressed. ESCs provide a number of challenges to the circadian biologist: growth rates are normally much higher than in established cell culture systems, the cells’ innate drive towards differentiation and the lack of known synchronizing input pathways are a few examples. Some of these challenges can be addressed post hoc, such as normalization to total RNA or protein for transcript abundance studies. Most others, as outlined here, require special handling of the samples before and during experimentation in order to preserve any potential circadian oscillation that is present. Failure to do so may result in a disruption of endogenous oscillation(s) or, potentially worse, generation of an artificial oscillation that has no biological basis. This chapter begins with cultured ESCs, derived from primary blastocysts or in the form of cell lines, and outlines two methods of measuring circadian rhythms: the 2DG method of measuring glucose uptake (Sokoloff et al. J Neurochem 28:897–916, 1977) and real-time measurement of molecular rhythms using transgenic bioluminescence (Yoo et al. Proc Natl Acad Sci U S A 101:5339–5346, 2004).

Key words

Embryonic stem cells Circadian Clock genes ESCs 2-Deoxyglucose Real-time bioluminescence 


  1. 1.
    Pittendrigh CS (1993) Temporal organization: reflections of a Darwinian clock-watcher. Annu Rev Physiol 55:16–54PubMedCrossRefGoogle Scholar
  2. 2.
    Bell-Pedersen D, Cassone VM, Earnest DJ et al (2005) Circadian rhythms from multiple oscillators: lessons from diverse organisms. Nat Rev Genet 6:544–556PubMedCentralPubMedCrossRefGoogle Scholar
  3. 3.
    Pittendrigh CS (1960) Circadian rhythms and the circadian organization of living systems. Cold Spring Harb Symp Quant Biol 25:159–184PubMedCrossRefGoogle Scholar
  4. 4.
    Pittendrigh CS (1981) Circadian systems: entrainment. In: Aschoff J (ed) Biol. Rhythm. Springer, Boston, pp 95–124CrossRefGoogle Scholar
  5. 5.
    Pittendrigh CS (1954) On temperature independence in the clock system controlling emergence time in Drosophila. Proc Natl Acad Sci U S A 40:1018–1029PubMedCentralPubMedCrossRefGoogle Scholar
  6. 6.
    Buhr ED, Takahashi JS (2013) Molecular components of the Mammalian circadian clock. Handb Exp Pharmacol (217):3–27. doi: 10.1007/978-3-642-25950-0_1
  7. 7.
    Paulose JK, Rucker EB, Cassone VM (2012) Toward the beginning of time: circadian rhythms in metabolism precede rhythms in clock gene expression in mouse embryonic stem cells. PLoS One 7:e49555. doi: 10.1371/journal.pone.0049555 PubMedCentralPubMedCrossRefGoogle Scholar
  8. 8.
    Guido ME, Garbarino-Pico E, Contin MA et al (2004) Circadian regulation of phospholipid biosynthesis in chick retinal ganglion cells. ARVO Meet Abstr 45:3666Google Scholar
  9. 9.
    Sládek M, Sumová A (2013) Entrainment of spontaneously hypertensive rat fibroblasts by temperature cycles. PLoS One 8:e77010. doi: 10.1371/journal.pone.0077010 PubMedCentralPubMedCrossRefGoogle Scholar
  10. 10.
    Schwartz WJ, Gainer H (1977) Suprachiasmatic nucleus: use of 14C-labeled deoxyglucose uptake as a functional marker. Science 197:1089–1091PubMedCrossRefGoogle Scholar
  11. 11.
    Sokoloff L, Reivich M, Kennedy C et al (1977) The [14C]deoxyglucose method for the measurement of local cerebral glucose utilization: theory, procedure, and normal values in the conscious and anesthetized albino rat. J Neurochem 28:897–916PubMedCrossRefGoogle Scholar
  12. 12.
    Earnest DJ, Liang FQ, Ratcliff M et al (1999) Immortal time: circadian clock properties of rat suprachiasmatic cell lines. Science 283:693–695PubMedCrossRefGoogle Scholar
  13. 13.
    Paulose JK, Peters JL, Karaganis SP et al (2009) Pineal melatonin acts as a circadian zeitgeber and growth factor in chick astrocytes. J Pineal Res 46:286–294PubMedCentralPubMedCrossRefGoogle Scholar
  14. 14.
    Hastings JW, Sweeney BM (1958) A persistent diurnal rhythm of luminescence in Gonyaulax polyedra. Biol Bull 115:440–458CrossRefGoogle Scholar
  15. 15.
    Yamazaki S, Numano R, Abe M et al (2000) Resetting central and peripheral circadian oscillators in transgenic rats. Science 288:682–685PubMedCrossRefGoogle Scholar
  16. 16.
    Yoo S-HH, Yamazaki S, Lowrey PL et al (2004) PERIOD2::LUCIFERASE real-time reporting of circadian dynamics reveals persistent circadian oscillations in mouse peripheral tissues. Proc Natl Acad Sci U S A 101:5339–5346PubMedCentralPubMedCrossRefGoogle Scholar
  17. 17.
    Yagita K, Horie K, Koinuma S et al (2010) Development of the circadian oscillator during differentiation of mouse embryonic stem cells in vitro. Proc Natl Acad Sci U S A 107:3846–3851PubMedCentralPubMedCrossRefGoogle Scholar
  18. 18.
    Kowalska E, Moriggi E, Bauer C et al (2010) The circadian clock starts ticking at a developmentally early stage. J Biol Rhythms 25:442–449PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Jiffin K. Paulose
    • 1
  • Edmund B. RuckerIII
    • 1
  • Vincent M. Cassone
    • 1
    Email author
  1. 1.Department of BiologyUniversity of KentuckyLexingtonUSA

Personalised recommendations