Circadian Rhythms of Whole Bone Marrow and Lineage Negative Stem Cells of the Mouse.

Circadian rhythms underlie most biological processes. In mammals circadian control of physiology and behavior is mediated via a central master oscillator, in the supra-schismatic nuclei of the hypothalamus. At the cellular level this oscillator is composed of an auto-regulatory transcription-translation loop of clock genes.

The Period2 (Per2) gene is one of the clock genes which plays a key role in controlling the circadian rhythm in mammals. Mice with mutations in Per2 become arrhythmic.

Expression of clock genes is also present in many peripheral tissues, including the bone marrow.

Stem cell engraftment has been shown to vary with cell cycle transit (Habibian et al, 1998). A diurnal circadian variation in the ability of bone marrow to engraft sub-lethally irradiated mice has been previously shown by our laboratory. An increase in numbers of progenitors in S-phase underlined the engraftment nadirs.

The host’s ability to accept incoming cells did not show circadian variation.

To further study the interplay of circadian rhythm with cell cycle in bone marrow and populations of engraftable stem cells, we utilized a transgenic mouse model for the Per2 gene. The mouse Period2 (mPer2) real time gene expression reporter of circadian dynamics, constructed by Takahashi et al., was employed for these studies. In this reporter a Luciferin (Luc) gene was fused in frame to the 3′ of the promoter of the endogenous mPer2 gene. This system allows for detection of Per2 gene expression in the presence of luciferase, by recording light given off, during the luciferase catalyzed conversion of Luciferin to Oxyluciferin.

We have detected circadian rhythm in whole bone marrow and Lineage negative cells i.e. whole bone marrow mononuclear cells depleted of B220, Ter119, GR1, CD4, CD8 and CD11b, with one peak every 24 hours for up to 14 days, from as few as 500,000 cells. Dissociated lung cells also show a circadian rhythm as do Lineage Negative Sca+ marrow stem cells. The later show an intermittent rhythm, for up to 10 days.

The best rhythms were obtained from cells grown on a 12mm dish bathed in 4 mls of media or a 1 ml drop of media, the later covered with mineral oil. The media was DMEM with L Glutamine, low glucose no phenol red, 1% Penicillin, 10,000 U/ml/streptomycin, 4.18mM NaHCO3, 10mM Hepes, 0.019mM D-glucose pH 7.2 supplemented with 1×B27, 5–15% HIFCS and either stem cell factor alone (50ng/ml) or IL3, IL6, IL11 and stem cell factor (steel) (50 ng or units/ml).

Feeding of the cells after 7 days increased the amplitude of the rhythm.

Absence of cytokines dampened the rhythm, especially for Lineage Negative Sca+ cells. Steel in the presence of HIFCS induces some rhythm, but is not as effective as a cocktail of IL3, IL6, IL11 and steel, together with HIFCS.

Given work from our laboratory on synchronized progenitor cells entering and progressing through cell cycle in cytokine cocktails, including IL3, IL6, IL11 and steel, and the correlation of engraftment potential with cell cycle phase and adhesion molecule phenotype, the appearance of the best circadian rhythm in proliferating cytokine cocktails, in this system, is intriguing. The cell cycle kinetics of the marrow cells exhibiting circadian rhythmicity, are being explored further, in this culture system. The links between expression of cell cycle control molecules and adhesion molecules in Lineage Negative Sca+ cells and circadian rhythms for engraftment are also under investigation.