logy
d
, O
de
ine
ptem
en
e
m
o i
ma
hi
2 (H
isolated from sorted SP and whole BM cells and exposed to DNase treatment. The relative
k
-
],
-
f
n
n
s
n
d
s
e
lmRNA levels of major clock genes mPer1, mPer2, mBmal1, mCry1, mClock, and mRev-erb a
were measured with real-time quantitative reverse transcription polymerase chain reaction
(Q-RT-PCR) and normalized to m36B4, used as a reference gene. The clonogenity of sorted SP
cells and whole BM; cells taken before and after sorting, were tested in colony-formation assay.
Results. Clock gene activity in sorted SP cells showed pronounced relative differences
compared with whole BM for mPer1 and mCry1. The high-speed sorting procedure did not
influence clock gene expression or cell clonogenity, even when this was performed with a delay
period up to 24 hours.
Conclusions. We demonstrated expression of six clock genes in mouse hematopoietic stem
cells. A combination of high-speed flow cytometric sorting and Q-RT-PCR was shown to be
useful and reliable for analysis of clock gene activity in small stem cell fractions.


2005
International Society for Experimental Hematology. Published by Elsevier Inc.
Adaptation of organisms to their periodically varying envi-
ronment is mediated through the entrainment to circadian
rhythms. A molecular and cellular basis of these rhythms
in mammals has recently been provided through identifica-
tion of clock genes and their products [1]. The clock mech-
anism involves transcription-translation feedback loops
composed of positive (mClock and mBmal1) and negative
(mPer1, mPer2, mCry1, mCry2, and mRev-erb a) elements
[2]. Most detailed description of clock gene expression
has been done for the suprachiasmatic nucleus cells—the
main circadian pacemaker in mammals, located in ventral
hypothalamus of the brain [2]. Recent studies have also
shown that peripheral tissues such as liver, heart, kidney,
lung, pancreas, skeletal muscles, oral mucosa, skin and bone
marrow (BM), and mononuclear leukocytes express cloc
genes giving rise to circadian rhythms with a different phas
ing from that observed in suprachiasmatic nucleus ([3–8
reviewed in [9]). Even cell tissue culture, such as rat fibro
blasts [7] or mouse smooth muscles [10], are capable o
generating circadian rhythmicity of clock gene expressio
when entrained by humoral signals.
Although it is well established that hematopoiesis i
general undergoes strong circadian variations in mammal
[11–14], clock gene regulation of stem cells has not yet bee
analyzed. So far only mPer1 and mPer2 were analyze
in mouse BM [15], while clock genes in primitive stem cell
have not been investigated. Hematopoietic stem cells ar
objects of considerable interest for research and clinicaExperimental Hemato
Clock gene expression in purifie
Oleg Tsinkalovsky
a
, Benedikte Rosenlund
b
a
Stem Cell Research Group, Department of Pathology, the Ga
b
Center for Medical Genetics and Molecular Medic
(Received 31 August 2004; revised 20 Se
Objective. Circadian genes have recently be
hematopoietic stem cells. These cells are rar
difficult to collect enough cells for detailed
without reduced RNA quality. The aim was t
gene expression analysis in purified mouse he
Methods. Stem cells were highly enriched by
side population (SP) cells from Hoechst 3334Offprint requests to: OlegTsinkalovsky,M.D., StemCell ResearchGroup,
the Gade Institute, Department of Pathology, Haukeland University Hospi-
tal, N-5021 Bergen, Norway; E-mail: oleg.tsinkalovsky@gades.uib.no
0301-472X/05 $–see front matter. Copyright


2005 International Society for E
doi: 10.1016/j .exphem.2004.09.00733 (2005) 100–107
mouse hematopoietic stem cells
le Didrik Laerum
a
, and Hans Geir Eiken
b
Institute, Haukeland University Hospital, Bergen, Norway;
, Haukeland University Hospital, Bergen, Norway
ber 2004; accepted 27 September 2004)
characterized in many tissues, but not in
in the bone marrow (BM), which makes it
olecular analysis in a short period of time
mprove methodology and reliability of clock
topoietic stem cells.
gh-speed flow cytometric cell sorting of the
oechst)-stained mouse BM. Total RNA wasxperimental Hematology. Published by Elsevier Inc.
purposes [16,17], especially in the last years, when their
high “plasticity” has been elucidated [17,18]. New data about
clock gene expression in primitive stem cells could bring
further insights into molecular mechanism controlling the

O. Tsinkalovsky et al. /Experimental Hematology 33 (2005) 100–107 101circadian rhythms in hematopoiesis and stem cell biology
in general.
The present lack of data on circadian gene expression
in stem cells could partly be explained by methodological
difficulties. Hematopoietic stem cells comprise a very
small fraction (0.1–0.001%) of whole BM, and the long time
necessary for enrichment of such rare cells in sufficient
quantities for further analysis could increase the risk of
RNA degradation. RNA quantity is critical for simultaneous
analysis of all major clock genes in the evaluation of clock
genes interactions. Moreover, when many samples have to
be collected within 24 to 27 hours, a delay in cell flow
cytometry analysis and sorting could also increase the risk
of RNA degradation. Therefore, the aim of our study was
to evaluate methodology for analysis of clock gene ex-
pression in primitive hematopoietic stem cells.
We employed a single-step Hoechst-33342 (Hoechst)-
staining method for stem cell isolation described recently
[19]. In Hoechst-stained BM a small fraction of the cells
defined as side population (SP), efflux Hoechst dye, and
express low Hoechst fluorescence in blue and red regions
of the spectrum [19]. The process is dependent on the ex-
pression of the Bcrp1 gene [20] and can be blocked by
verapamil. SP fraction was shown to be highly enriched for
long-term hematopoietic stem cells [19,21]. We performed
high-speed flow cytometric sorting of SP cells followed by
total RNA extraction and DNase treatment. This allowed
us to get RNA of high quality and specifically analyze for
the first time expression of the clock genes mPer1, mPer2,
mBmal1, mCry1, mClock, and mRev-erb a, in mouse hema-
topoietic stem cells by quantitative reverse transcription
polymerase chain reaction (Q-RT-PCR).
Materials and methods
Animals, cell preparation, and staining
Twenty-three C57 black male mice were synchronized to standard
lighting conditions (12-hour light/12-hour dark cycle) for 3 weeks
with a standard diet and tap water provided ad libitum. Mice
were sacrificed in the morning between 9:00 and 9:30 by neck
dislocation, and BM cells were obtained by crushing femurs and
tibiae in a mortar using a pestle in cold Hank’s balanced salt
solution (HBSS) containing 2% fetal calf serum (FCS) and 1 mM
HEPES (all purchased from Gibco, Invitrogen Corporation)—
HBSS


. Cells from all mice were pooled, filtered through 70-µm
nylon cell strainers BD Falcon (Becton Dickinson Labware-Europe,
Le Pont-de-Claix, France) and washed once. Cell pellets were
dissolved in 10 mL Lysing solution (a gift by DakoCytomation,
Denmark A/S) for 10 minutes in the dark. After centrifugation, cells
were resuspended in 31 mL cold HBSS


and counted. On average,
7.5 × 10
7
BM cells were recovered per mouse. Part of the cell
suspension (10
8
cells) was used for the experiments on the whole
BM, and remaining cells were stained with Hoechst (Molecular
Probes, Eugene, OR, USA).
Experiments on the whole BM samples. Approximately 10
8
cells
were used for analysis of gene expression and colony-forming assaybefore and after sorting of whole BM samples. Cells (2.5 × 10
6
)
were distributed into five 2-mL cryotubes (TPP AG, Trasadingen,
Switzerland) (5 × 10
5
per tube), and spun down (300g, 5 minutes
at
4C). Supernatants were discarded, pellets snap-frozen (30
seconds) in liquid nitrogen and placed at 80C until use. An
aliquot of the cells (2 × 10
5
) was used for colony-forming assay.
The rest of the whole BM suspension was kept on ice until the
sorting procedure.
Experiments on SP cell samples. Thirty milliliters of cell suspen-
sion were divided into three equal parts. One part was immediately
stained with fluorescent dye Hoechst. Two other parts were kept
at
4C for 12 hours and 24 hours, respectively, until staining.
Before staining, cells were spun down and resuspended in pre-
warmed Dulbecco’s modified Eagle’s medium with 2% FCS and
1 mM HEPES (all purchased from Gibco) at 10
6
cells/mL. BM
cells were stained with 5 µg/mL Hoechst for 90 minutes at 37C
as described elsewhere [19], spun down and resuspended in cold
HBSS


at 5–7 × 10
7
/mL. A parallel aliquot was stained, as
described, in the presence of 50 µM verapamil (Sigma-Aldrich,
St. Louis, MO, USA). For surface stem cell markers Sca-1 and c-Kit
analysis, an aliquot of 5 × 10
5
Hoechst-stained cells was washed,
resuspended in 80 µLcoldHBSS


and incubated for 30 minutes
on ice with 10 µL fluorescein isothiocyanate (FITC)-conjugated
Sca-1 and 10 µL R-phycoerythrin (RPE)-conjugated c-Kit anti-
bodies (both from PharMingen, San Diego, CA, USA) followed by
washing in HBSS


. Cells were kept on ice until flow cytometric
analysis and sorting. Propidium iodide (PI, 2 µg/mL; Sigma-Ald-
rich) was added to Hoechst-stained samples before flow cytometric
analysis to facilitate dead cell discrimination. Cell suspension was
filtered through a 50-µm Filcons filter device (DakoCytomation,
Denmark A/S) prior to flow cytometry analysis.
Flow cytometry and high-speed cell sorting
Cells were analyzed and sorted on theMoFlo cell sorter (DakoCyto-
mation, Denmark A/S; former Cytomation, USA), equipped with
Coherent Enterprise 621 dual-laser tuned to 488 nm (1 W) and
365 nm, UV (60 mW). Cells were sorted in “purify 1” mode with
high speed up to 30,000 events (cells) per second, and collected
in 5-mL polypropylene round-bottom Falcon tubes (Becton Dickin-
son Labware Europe, Le Pont-de-Claix, France), containing cold
HBSS with 10% FCS, that were placed on ice.
Experiments on the whole BM samples. Doublets were discrimi-
nated using a forward light scatter vs pulse width after 488-nm
excitation. Created region was gated into forward light scatter vs
side light scatter bivariate, and then all displayed nucleated BM
cells were sorted. Sorted cells were counted and 25 × 10
5
cells
were distributed into five 2-mL cryotubes (TPP AG, Trasadingen,
Switzerland), 5 × 10
5
per tube, and spun down (300g, 5 minutes
at
4C). Supernatants were then discarded; pellets snap-frozen
(30 seconds) in liquid nitrogen and placed at 80C until use; 2
× 10
5
cells were used for colony-forming assay.
Experiments on the SP cell samples. The Hoechst dye and PI were
excited at 365 nm. Hoechst fluorescence was measured by two
detectors through 450/20 BP (Hoechst “blue”) and 675 EFLP
(Hoechst “red” and PI) optical filters (Omega Optical, Brattleboro,
VT, USA). A 610 DMSP was used to separate the emission wave-
lengths. FITC and RPE emissions were observed through 530/40
and 580/30 filters, respectively, after 488-nm excitation.