J. Biomedical Science and Engineering, 2011, 4, 454-461 JBiSE
doi:10.4236/jbise.2011.46057 Published Online June 2011 (http://www.SciRP.org/journal/jbise/).
Published Online June 2011 in SciRes. http://www.scirp.org/journal/JBiSE
Hypoxia promotes growth of stem cells in dental follicle cell
populations
Yuntao Dai, Hongzhi He, Gary E. Wise, Shaomian Yao*
Department of Comparative Biomedical Sciences, School of Veterinary Medicine Louisiana State University, Baton Rouge, USA.
Received 8 April 2011; revised 15 May 2011; accepted 29 May 2011.
ABSTRACT
Adult stem cells (ASC) have been found in many tis-
sues and are of great therapeutic potential due to
their capability of differentiation. However, ASC
comprise only a small fraction of the tissues. In order
to use ASC for therapeutic purposes, it is important
to obtain relatively pure stem cells in large quantities.
Current methods for stem cell purification are mainly
based on marker-dependent cell sorting techniques,
which have various technical difficulties. In this study,
we have attempted to develop novel conditions to
favor the growth of the dental follicle stem cells
(DFSC) such that the resultant cell populations are
enriched in stem cells. Specifically, a heterogeneous
dental follicle cell (H-DFC) population containing
stem cells and homogenous non-stem cell dental fo lli-
cle cell population were cultured at 1% or 5% hy-
poxic conditions. Only the heterogeneous population
could increase proliferation in the hypoxic condition
whereas the homogenous DFC did not change their
proliferation rate. In addition, when the resultant
cells from the heterogonous population were sub-
jected to differentiation, they appeared to have a
higher capacity of adipogenesis and osteogenesis as
compared to the controls grown in the normal at-
mosphere (normoxic condition). These hypoxia-
treated cells also express higher levels of some stem
cell markers. Together, these data suggest that stem
cells are enriched by culturing the heterogeneous cell
populations in a reduced O2 condition.
Keywords: Hypoxia; Dental Follicle (DF); Stem Cells;
Proliferation; Gene Expression
1. INTRODUCTION
Stem cells in adult tissues, adult stem cells (ASC), function
as replenishment or repair systems for maintaining the
integrity of the given tissue. ASC are unspecialized cells
that are capable of differentiating into specialized cells.
Due to this characteristic, ASC have great potential in
medical applications such as in tissue regeneration and
tissue engineering.
The dental follicle is a loose connective tissue sac
surrounding the unerupted tooth and its presence is re-
quired for the tooth to erupt [1,2]. Our previous studies
revealed that the dental follicle plays a critical role in
regulating alveolar bone resorption and bone formation
for tooth eruption by regulating formation of osteoclasts
and osteoblasts [3,4]. Stem cells have been found in the
dental follicle, and dental follicle stem cells (DFSC) are
capable of multilineage differentiation including osteo-
genesis [5]. Thus, we proposed that stem cells in the
dental follicle may differentiate into osteoblasts to pro-
mote some of the bone formation needed for tooth erup-
tion. Therefore, studies of DFSC might help understand
the cell and molecular mechanisms of tooth eruption.
Moreover, the mutilineage differentiation potential of the
DFSC suggests that they would be valuable for tissue
engineering applications.
Isolation of ASC has often relied on their plastic ad-
herence preference. Because many non-stem cells can
also adhere to and grow on plastic surfaces, the cell pop-
ulation isolated based on plastic adherence contains
many non-stem cells. For example, adherent single cell-
derived colonies of progenitors isolated from bone mar-
row display a wide variation in cell morphology and
growth potential [6]. Alternatively, stem cells may be
isolated based on their colony-forming properties, a me-
thod which has been used to isolate pulp stem cells from
human third molars [7] and from human exfoliated de-
ciduous teeth [8]. However, such isolation techniques are
labor-intensive, time-consuming and skill-demanding.
Colonies derived from heterogeneous dental pulp cells
also show diversity in cell surface markers [7], suggest-
ing that the technique does not result in a pure popula-
tion of stem cells.
Cell marker-dependent sorting techniques, including

Y. Dai et al. / J. Biomedical Science and Engineering 4 (2011) 454-461 455
fluorescence-activated cell sorting (FACS) and mag-
netic-activated cell sorting (MACS) have been devel-
oped for isolation of stem cells from various tissues.
Such techniques require the presence of stem cell spe-
cific markers, especially surface markers. The major
drawback for application of the sorting techniques is the
lack of clear stem cell specific markers. If no unique
stem cell-specific surface marker is present, selection
must be repeated with multiple markers, which greatly
increases the cost while reducing the efficacy of the
methods. Therefore, such techniques do not allow puri-
fying stem cells on a large scale. In vitro expansion of
the primary isolated ASC still is required to obtain a
large quantity for clinical applications.
It is believed that stem cells in adult tissues function
as the repair/regeneration systems to regenerate damaged
or defective tissues/organs. Tissue damage can occur in
several situations, most often in a disease state. For ex-
ample, many diseases can cause a shortage of oxygen
supply in the tissues (i.e., hypoxia), and in turn may
cause tissue damage that requires the tissue stem cells to
function for regeneration. Theoretically, the stem cells
must remain intact in the hypoxic condition and respond
to hypoxia. It has been reported that stem cells grow
more rapidly and form more colonies in hypoxic condi-
tions than they do in normal oxygen atmosphere [9,10].
Thus, it is our hypothesis that DFSC are more responsive
to hypoxia than normal somatic cells, and hypoxia may
activate the stem cells from quiescence to begin rapid
growth. We further propose that this unique feature can
be used to develop conditions that favor stem cell
growth such that one could enrich stem cells from het-
erogeneous cell populations.
To that end, a homogeneous dental follicle cell (DFC)
population containing only fibroblast-like cells and a
heterogeneous dental follicle cell (H-DFC) population
containing fibroblast-like cells and stem cells were in-
cubated under hypoxic conditions. The effects of hy-
poxia on cell proliferation, differentiation and selected
stem cell marker gene expression were studied. The pos-
sibility of enrichment of stem cells by growing the cells
in hypoxic conditions is also discussed.
2. MATERIALS AND METHODS
2.1. Cell Culture
Dental follicles were isolated from the first mandibular
molars of rat pups at postnatal days 5-7, and then trypsi-
nized to obtain the primary dental follicle cell suspen-
sion. The primary cells were cultured in Eagle’s mini-
mum essential medium containing 10% newborn calf
serum and 1mM sodium pyruvate [11]. Cells were
passed at confluency until the desired passage to obtain a
pure fibroblast-like cell population containing no stem
cells [5]. This population is referred to as dental follicle
cells (DFC) in this manuscript. To obtain the heteroge-
neous cell population containing dental follicle stem
cells (DFSC) and non-stem cells, the primary cells were
cultured in alpha minimal essential medium (Invitrogen)
mixed with 20% fetal bovine serum (Atlanta Biologicals,
Lawrenceville, GA) and also passed at confluence. The
cell population obtained in this condition is heterogene-
ous containing stem cells and non-stem cells [9] and is
referred to as heterogeneous dental follicle cells (H-
DFC). All cultures were incubated at 37℃ with 5% CO2
atmosphere unless otherwise specified.
Hypoxic cultures were achieved using the MIC-101
hypoxia chamber (Billups-Rothenberg, Inc.). The cells
were seeded in flasks or plates and then the flasks or
plates were placed in the chambers. The chambers were
then filled with a gas mixture containing the designated
concentrations of O2 and 5% CO2, balanced with N2.
Next, the chambers were moved into the 37℃ incubator.
O2 concentrations were monitored daily with the O2 me-
ter inside the chambers. Cell culture media were
changed every other day.
2.2. Gene Expression Study
Gene expression was determined using real-time RT-
PCR, Western blotting or cytochemistry staining after 7
days of incubation with the designated treatments. For
real-time RT-PCR, total RNA was extracted with RNeasy
Mini Kit (Qiagen); RNA concentration and quality was
measured with a Nanodrop 8000 spectrophotometer
(Thermo scientific). RNA (2 µg per sample) was reverse
transcribed into 20 µl cDNA with random primers and
MLV reverse transcriptase. Next, SYBR green real-time
PCR was conducted with 0.5 µl cDNA of each sample
using gene specific primers to determine the expression
of Nt5e (forward: 5’ACTCCACCAAG TGCCTCAAC3’;
reverse 5’ GTCCTTCCACACCGTTA TCAA3’) and
Prom1 (forward: 5’GGGAGC GAGATG TTACTTTGA
G3’; reverse: 5’CAGC AGGACACT GAATAC GAGA3’).
For Western blotting, total protein was extracted from
the cells with the Cytobuster Protein Extraction Reagent
(Novagen), and quantified with the BCA Protein Assay
Kit (Pierce). An equal amount of total protein (20 µg)
from each sample was loaded onto a SDS-polyacryla-
mide gel for electrophoresis. The proteins were trans-
ferred to a membrane after the electrophoresis. The
membrane was hybridized with 12.5 µg/ml rabbit poly-
clonal anti-Prom1 or anti-Nt5e antibodies (Abcam) after
treatment of the membrane with 5% dry milk to block
nonspecific binding. The membrane was washed with
PBS-T for 6 times to remove unbound antibodies, and
then hybridized with anti-rabbit secondary antibody
conjugated to horseradish peroxidase. The protein signal

Y. Dai et al. / J. Biomedical Science and Engineering 4 (2011) 454-461 456
was detected by an enhanced chemiluminescence kit
(Santa Cruz) with a Kodak X-ray film.
For alkaline phosphatase (ALP) staining, H-DFC and
DFC were seeded in 6 well plates and cultured at the
designated hypoxic condition. After 7 days of incubation,
cells were stained for cell membrane ALP using Stem-
TAGTM Alkaline Phosphatase Staining Kit (Cell Biolabs,
Inc.) according to the manufacturer’s protocol. Cells
maintained in the normal culture condition (i.e., nor-
moxia) were used as the controls.
2.3. Determination of Cell Growth
Because the Alamar blue assay is a non-toxic method
that allows us to continuously monitor the cell growth
during a period of time in culture, this method was used
to monitor cell growth. To do that, DFC and H-DFC
were seeded in the wells of 6-well plates. Alamar blue
assays were conducted at days 1, 3, 5, and 7 of culture.
At the time of assay, the culture medium was removed
from the wells and assay medium containing 10% Ala-
mar blue was added to each well. After 2 hours of incu-
bation, 100 µl assay medium was pipetted into a well of
the 96-well plate in triplicate for each sample. Fresh cell
culture medium and fresh assay medium not subjected to
cell incubation were used as the controls. The 96-well
plate was placed into a Bio-Rad microplate reader (mod-
el 550) and OD values were obtained. Alamar blue re-
duction was calculated according to the manufacturer’s
formula.
2.4. Cell Cycle Analysis
Cell cycle analysis was conducted to determine if the
H-DFC cultured under hypoxic conditions divided
more than under normoxic conditions. Approximately 3
× 105 cells from each of the treatments were seeded per
75 cm2 T-flask in 20 ml medium with a change of me-
dium at 2- day intervals. The cells were collected on
day 4 of culture for cell cycle analysis using flow cy-
tometry. Briefly, the cells were pre-treated with cold
methanol followed by treatment with Rnase A and Pro-
pidium iodide. DNA content of the cells was deter-
mined using a Becton Dickinson FACScan flow cy-
tometer (San Jose). Percentages of cells in G0/G1, S,
and G2/M phases of the cell cycle were determined for
each sample.
2.5. Cell Differentiation
To determine the effect of hypoxia on differentiation of
resultant H-DFC, H-DFC were collected after 7 days of
hypoxic incubation, and seeded onto 6-well plates with
an concentration of 2 × 104 cells per well with 3 ml dif-
ferentiation medium for induction of either adipogenesis
or osteogenesis as described in our previous publication
[5]. The plates were incubated at 37℃ and 5% CO2 for
2 weeks. Next, the cells were stained with Alizarin Red
Solution (Sigma-Aldrich) for assessing osteogenesis, and
with Oil Red O (Sigma-Aldrich) for adipogenesis.
2.6. Statistical Analysis
All experiments were repeated three times. Treatment
effects were compared by analysis of variance with SAS
version 9.1, and means were separated using least sig-
nificant difference (LSD) at P ≤ 0.05.
3. RESULTS
3.1. Increase of cell Growth by Hypoxia
After 7 days of culture, H-DFC showed higher cell
density in both 1% and 5% hypoxia treatments as
compared to the normoxia control whereas similar cell
density was seen for DFC in all treatments (Figure
1(a)). We conducted an Alamar blue assay to monitor
cell proliferation during 7 days of culture. H-DFC had
an overall higher Alamar blue reduction than DFC in
all treatments after 5 and 7 days of incubation (Figure
1(b)), indicating that H-DFC proliferated more rapidly
than DFC. Alamar blue reduction (i.e., cell prolifera-
tion) was significantly higher when H-DFC were in-
cubated under 1% and 5% O2 hypoxic conditions than
under normoxic conditions after 5 and 7 days of incu-
bation. The maximum Alamar blue reduction was seen
at 5% O2, indicating that H-DFC grew most rapidly at
5% O2 (Figure 1(b)). In contrast to H-DFC, culture of
DFC in different conditions (hypoxic or normoxic)
resulted in a similar Alamar blue reduction within each
given day of incubation, suggesting that DFC did not
respond to hypoxia treatment.
To further study cell division, cell cycle analysis was
carried out by flow cytometry. Cell cycle analysis of
H-DFC showed that the average percentage of cells in
S-phase was 19.11% in 1% O2, 22.37% in 5% O2 and
16.07% in the control (normoxia) at day 4 of culture
(Figure 1(c)). The difference was statistically significant
(P ≤ 0.05), suggesting that a greater number of cells was
in the dividing stage under hypoxic conditions as com-
pared to under the normoxic condition.
3.2. Increase of Differentiation Capability after
Hypoxic Treatment
After 1 week of culture in hypoxia, the resultant H-DFC
populations were induced for adipogenesis and osteo-
genesis. Adipogenesis and osteogenesis were determined
by Oil Red O staining or Alizarin Red staining, respec-
tively. This study showed that the cell populations grown
in 1% and 5% hypoxia had greater osteogenesis and
adipogenesis than did the control cells grown in nor-

Y. Dai et al. / J. Biomedical Science and Engineering 4 (2011) 454-461 457
Figure 1. Effects of hypoxia on the culture of H-DFC and DFC. Note that only H-DFC responded to the hy-
poxia as shown by the cell density change after a 1 week of incubation (a); and by Alamar blue cell prolifera-
tion assay during 1 week period of incubation (b). Cell cycle analysis was conducted by flow cytometry show-
ing that hypoxia treatment resulted in a significantly higher percentage of cells in S-phase (c, d). Bars labeled
with different letters indicate a statistically significant difference at P ≤ 0.05.
moxia (Figure 2). Maximum staining was seen in the
cells that had been subjected to 5% hypoxic incubation
(Figure 2).
3.3. Enhancement of Stem Cell Marker Gene
Expression after Hypoxia Treatment
Real-time RT-PCR analysis showed that expression of
stem cell marker genes Nt5e and Prom1 were signifi-
cantly enhanced in H-DFC incubated in hypoxic treat-
ments (1% and 5% O2) as compared to the controls
maintained under normoxic condition (Figure 3). Cells
from 5% O2 treatment had the highest expression of
Nt5e, which was also significantly higher than 1% O2
treatment and controls (Figure 3(a)). For Prom1, al-
though expression was higher in both 1% and 5% O2
hypoxia treatments than in the controls, no difference
was detected between 5% and 1% O2 treatments (Figure
3(b)).
Western blotting determined that H-DFC from hy-
poxia treatment produced a greater amount of Prom1 and
Nt5e proteins than did the controls. Thus, this result
suggests that the mRNA level determined by the
real-time RT-PCR corresponds to the protein expression
(Figure 3(c)).

Y. Dai et al. / J. Biomedical Science and Engineering 4 (2011) 454-461 458
(a)
(b)
Figure 2. Enhancement of differentiation capability of H-DFC after hypoxia treat-
ments. Increased Oil Red O staining was seen in cells subjected to hypoxic incubation
as compared to the normoxic O2 (control), indicating increased adipogenesis in hy-
poxia-treated cells (a); Osteogenesis was determined by Alizarin Red staining, and in-
creased staining was seen from hypoxic treated H-DFC cells as compared to the nor-
moxic control (b). Note that the greatest adipogenesis and osteogenesis were seen in
cells resulting from 5% O2 treatment.
Figure 3. Enhanced expression of Nt5e and Prom1 genes after hypoxic treatment as determined by
real-time RT-PCR (a, b) and Western blotting (c). Note that increased expression of both genes was sta-
tistically significant in hypoxic treatments as compared to the normoxic control. Bars with different let-
ters indicate significant differences at P ≤ 0.05. Western blot results indicated that the transcription and
translation of the marker genes are consistent (a, b, c).
Normoxic O2 (Control)
Normoxic O2 Control
Normoxic O2
(
Cont r o l
)

Y. Dai et al. / J. Biomedical Science and Engineering 4 (2011) 454-461 459
Figure 4. Alkaline phosphatase (ALP) staining of H-DFC and DFC to detect
membrane ALP after 4 days of incubation in hypoxia. ALP staining was only
seen in the H-DFC population, and not in the DFC. Note that the numbers of
cells stained positive for ALP dramatically increased in the H-DFC popula-
tion after hypoxic incubation as compared to the H-DFC population not sub-
jected to hypoxia (Normoxic O2).
H-DFC from different treatments were also stained for
membrane ALP. The results showed that a greater num-
ber of cells stained for membrane ALP in hypoxia-
treated cells as compared to the cells grown under nor-
moxic condition (Figure 4). Comparing 1% and 5% O2
treatment, 5% O2 treatment resulted in more cells show-
ing strong ALP staining than 1% O2 treatment. In con-
trast, no ALP staining was observed in the DFC (non-
stem cells) incubated either under hypoxic or normoxic
conditions (Figure 4).
4. DISCUSSION
Sufficient quantities of stem cells must be obtained be-
fore they can be used for clinical treatments. It is diffi-
cult to collect large numbers of adult stem cells from
primary isolation because 1) stem cells are rare in adult
tissues; 2) there are limitations of current methods for
stem cell purification; and 3) large tissue samples are
often unavailable. The last is especially true for isolating
stem cells from tissues of small size, such as the dental
follicle. Thus, in vitro expansion of primary isolated
stem cells is required in most cases in order to have suf-
ficient numbers of cells for clinical treatments.
We have previously shown that stem cells can be iso-
lated from the dental follicle by growing cells in α-MEM
plus 20% FBS medium, and the cell population is het-
erogeneous (i.e., H-DFC) with some stem cells and a
majority of non-stem cells [5]. However, if the cells are
grown and passaged in MEM plus 10% NCS, a ho-
mogenous fibroblast-like cell population containing no
stem cells can be achieved after several passages [5,11].
In this study, we show that the H-DFC population
showed accelerated proliferation under hypoxic condi-
tions as compared to the control incubated under nor-
moxic conditions (Figure 1). In contrast, the homoge-
nous DFC population (containing only non-stem cells)
did not show notable change of its proliferation rate un-
der either hypoxic or normoxic conditions; i.e., the DFC
did not respond to hypoxic treatment. Because both
populations originate from the same tissue source (the
dental follicle), and because the non-stem cell homoge-
neous population does not increase growth rate in re-
sponse to hypoxia, the increase of proliferation rate in
response to hypoxia seen in the heterogeneous popula-
tion may be due to more rapid growth of the stem cells
than of the non-stem cells in the population.
If only the stem cells increase their proliferation such
that their growth rate exceeds the growth of non-stem
cells under hypoxic conditions, the ratio of stem cells to
non-stem cells in the heterogeneous population should
shift in favor of stem cells. To test this, the H-DFC
populations were collected after 1 week of incubation
under hypoxia and normoxia (control). The resultant
cells were evaluated for stem cell properties of differen-
tiation and marker gene expression. Enhancement of
adipogenesis and osteogenesis was observed in the cell
populations with 1% O2 and 5% O2 incubation (Figure
2), as compared to the control. The expression of several
stem cell markers including Nt5e, Prom1 and ALP also
were increased in hypoxia-treated cells (Figures 3 and
4).
ALP, Prom1 and Nt5e have been shown to be specific
markers for certain stem cells. Specifically, expression
Normoxic O2 (Control)
Normoxic O2 (Control)

Y. Dai et al. / J. Biomedical Science and Engineering 4 (2011) 454-461 460
of cell surface ALP is associated with undifferentiated
pluripotent stem cells such as undifferentiated human
pluripotent stem cells [12]. Induced pluripotent stem
cells also show strong ALP activity [13,14]. In this study,
H-DFC resulting from 1 week hypoxia incubation show
increased activity of membrane ALP.
Prom1 (also known as CD133) is well-known as a
stem cell marker used for identification and isolation of
adult stem cells. Prom1 was originally classified as a
marker of primitive hematopoietic and neural stem cells
in humans and mice [15,16]. Cells forming neurosphere
or neural stem cells capable of multilineage differentiation
were isolated based on Prom1 expression [17-19]. Prom1
expression in adult stem cells has been shown to help in
maintaining the stem cell properties by suppressing their
differentiation [20].
Nt5e (also known as CD73) has been reported to be
present on mesenchymal stem cells [21]. Expression of
this gene is enhanced by hypoxia in endothelial cells
[22,23]. In this study, there was an increased expression
of Nt5e after hypoxia treatment of the H-DFC population,
suggesting that hypoxia may also enhance the expression
of Nt5e in stem cells. It has been proposed that increased
expression of Nt5e may have protective roles during
hypoxia [22,24].
Based on the evidence of increased differentiation ca-
pabilities (Figure 2) and marker gene expression after
hypoxia treatment of H-DFC (Figures 3 and 4), it can be
suggested that the ratio of stem cells to non-stem cells in
the H-DFC populations subjected to hypoxia was in-
creased; i.e., greater numbers of stem cells were achieved
by incubating H-DFC in hypoxia than in normoxia. This
is likely due to the differential growth of the stem cells
and non-stem cells in response to hypoxia. In particular,
stem cells significantly accelerate their proliferation rate
in response to hypoxia whereas the non-stem cells (DFC)
do not notably increase their proliferation rate in hypoxia
(Figure 1).
Hypoxia can be a medical condition in which tissues
do not receive sufficient O2 supply. Cells in a hypoxic
condition undergo anaerobic metabolism, in which they
accumulate lactic acid [25]. Another effect of hypoxia is
stimulation of inflammation [26,27] with cell death and
tissue damage occurring under severe and prolonged
hypoxia. Under such conditions, the stem cells residing
in the tissue may function to regenerate the damaged
tissue. Because the stem cells exist in a quiescent state
under normal physiological conditions and become ac-
tive only when needed [28], tissue damage or the hostile
conditions causing the damage may serve as signals to
activate the stem cells to proliferate. This would explain
why the DFSC in a H-DFC population increase their
growth under hypoxic conditions (Figure 1). Increased
proliferation is also seen when mesenchymal stem cells
are subjected to hypoxic conditions [9].
In conclusion, incubation of heterogeneous dental fol-
licle cell populations containing stem cells and non-stem
cells in hypoxia can result in a greater increase of stem
cell numbers in the population than incubation of the
cells in normoxia. In particular, a 5% O2 concentration
may be optimal for growth of dental follicle stem cells
(DFSC). This differential growth of the stem cells and
non-stem cells in response to hypoxia may be exploited
for enrichment of dental tissue stem cells from hetero-
geneous cell populations.
5. ACKNOWLEDGEMENTS
We thank Ms. Marilyn Dietrich for help with the flow cytometry study.
This research was supported by NIH grant 1R03DE018998 to S. Yao.
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