Although some growth factors, such as epi-dermal growth factor, glial cell line-derived neuro-trophic factor, leukemia inhibitory factor, and vascular Keywords embryonic neural stem or p
Trang 1stem ⁄ progenitor cells – role of hypoxia-inducible
transcription factor-1a
Tong Zhao1,*, Cui-ping Zhang1,*, Zhao-hui Liu1, Li-ying Wu1, Xin Huang1, Hai-tong Wu1,
Lei Xiong1, Xuan Wang2, Xiao-min Wang2, Ling-ling Zhu1and Ming Fan1,2
1 Department of Brain Protection and Plasticity, Institute of Basic Medical Sciences, Beijing, China
2 Key Laboratory for Neurodegenerative Disorders of the Ministry of Education and Department of Physiology, Capital Medical University, Beijing, China
Neural stem⁄ progenitor cells (NPCs), which exist in
the developing and adult mammalian brain, are
self-renewing and can differentiate into neurons, astrocytes
or oligodendrocytes in vitro [1–3] Stem cells derived
from the embryonic midbrain have been successfully
engrafted into the central nervous system (CNS) to
cure diseases such as stroke, ischemia and Parkinson’s disease [4–8] A recent encouraging report has raised hopes of using human NPCs for patients with brain trauma [9] Although some growth factors, such as epi-dermal growth factor, glial cell line-derived neuro-trophic factor, leukemia inhibitory factor, and vascular
Keywords
embryonic neural stem or progenitor cells;
HIF-1a; hypoxia; proliferation
Correspondence
L.-l Zhu, Department of Brain Protection
and Plasticity, Institute of Basic Medical
Sciences, Beijing, China
No 27 Taiping Rd, Beijing 100850, China
Fax: +86 10 6821 3039
Tel: +86 6821 0077 ext 931315
E-mail: linglingzhu@hotmail.com
M Fan, Department of Brain Protection and
Plasticity, Institute of Basic Medical
Sciences, Beijing, China
No.27 Taiping Rd, Beijing 100850, China
Fax: +86 10 6821 3039
Tel: +86 10 6821 4026
E-mail: fanming@nic.bmi.ac.cn
*These authors contributed equally to this
work
(Received 20 November 2007, revised 3
February 2008, accepted 15 February 2008)
doi:10.1111/j.1742-4658.2008.06340.x
We recently reported that intermittent hypoxia facilitated the proliferation
of neural stem⁄ progenitor cells (NPCs) in the subventricule zone and hip-pocampus in vivo Here, we demonstrate that hypoxia promoted the prolif-eration of NPCs in vitro and that hypoxia-inducible factor (HIF)-1a, which
is one of the key molecules in the response to hypoxia, was critical in this process NPCs were isolated from the rat embryonic mesencephalon (E13.5), and exposed to different oxygen concentrations (20% O2, 10% O2, and 3% O2) for 3 days The results showed that hypoxia, especially 10% O2, promoted the proliferation of NPCs as assayed by bromodeoxy-uridine incorporation, neurosphere formation, and proliferation index The level of HIF-1a mRNA and protein expression detected by RT-PCR and western blot significantly increased in NPCs subjected to 10% O2 To fur-ther elucidate the potential role of HIF-1a in the proliferation of NPCs induced by hypoxia, an adenovirus construct was used to overexpress HIF-1a, and the pSilencer 1.0-U6 plasmid as RNA interference vector targeting HIF-1a mRNA was used to knock down HIF-1a We found that over-expression of HIF-1a caused the same proliferative effect on NPCs under 20% O2 as under 10% O2 In contrast, knockdown of HIF-1a inhibited NPC proliferation induced by 10% O2 These results demonstrated that moderate hypoxia was more beneficial to NPC proliferation and that HIF-1a was critical in this process
Abbreviations
BrdU, bromodeoxyuridine; CKO, conditioned knockout; CNS, central nervous system; eGFP, enhanced green fluorescent protein;
HIF, hypoxia-inducible factor; mNPC, mouse neural precursor cell; m.o.i., multiplicity of infection; MSC, mesenchymal stem cell; NPC, neural stem ⁄ neural progenitor cell; PI, proliferation index; RNAi, RNA interference; shRNA, short hairpin RNA; siRNA, small interfering RNA; VEGF, vascular endothelial growth factor.
Trang 2endothelial growth factor (VEGF), can regulate the
proliferation of NPCs in vitro [10–12], the expansion
of NPCs in vitro is too slow to meet the huge demand
for NPCs, which can be used in clinical
transplanta-tion without side-effects The development of more
effective methods for expansion of NPCs has become
urgent and important both in vitro and in vivo
Recently, reports have shown that hypoxia can
reg-ulate the proliferation and differentiation of stem
cells, and that, especially, mild hypoxia has salutary
effects on stem⁄ progenitor cells [12–14]
Cytotropho-blasts proliferate at low O2 tensions and differentiate
into a highly invasive phenotype at high O2 tensions
[15,16] Mesenchymal stem cells (MSCs) from rat
bone marrow display enhanced colony-forming
capa-bility and increased proliferation at 5% O2 as
com-pared to those at 20% O2 [17] Studer and Morrison
reported that O2 lowered to more physiological levels
(3%) produced marked trophic and proliferative
effects on neural precursors and significantly changed
developmental kinetics and outcome as compared
with traditional culture conditions (20%) [13,14] We
also found that hypoxia (3% O2) increased the
prolif-eration of human MSCs, myoblasts, and neural stem
cells [12] These observations indicate that mild
hypoxia may be a useful tool for expansion of some
stem cells for clinical use in vitro at low cost
How-ever, the molecular mechanisms involved in
prolifera-tion of stem cells under hypoxic condiprolifera-tions are not
well understood
Hypoxia-inducible factor (HIF)-1 is one of the key
transcription factors in the response to hypoxia; it
mediates a variety of adaptive cellular and systemic
responses to hypoxia by upregulating the expression
of > 50 different genes to assist animals in their
adaptation and survival [18] HIF is a heterodimeric
DNA-binding complex consisting of a- and
b-sub-units, which are members of the bHLH-PAS
(PER-ARNT-SIM) superfamily of proteins [19,20]
The increase in HIF-1 activity is primarily due to the
hypoxia-induced stabilization and activation of
HIF-1a, which is degraded by the ubiquitin–proteasome
system under normoxic conditions [21] It has been
reported that a hypoxic environment is essential for
early development, and that HIF-1a induced by a
low O2 tension plays an important role in
maintain-ing the proliferative and undifferentiated phenotype
in human trophoblasts [15,16] In addition, HIF-1a
conditioned knockout (CKO) caused midbrain-specific
impairment Survival of mouse neural precursor cells
(mNPCs) and expression of VEGF mRNA was
reduced in HIF-1a CKO However, treatment of
HIF-1a CKO mNPCs with 50 ngÆmL)1 VEGF only
partially restored proliferation [22] On other hand, it was reported that low O2 could increase the expres-sion of fibroblast growth factor 8 and erythropoietin during proliferation of NPCs Furthermore, research findings showed that NPCs exposed to 250 ngÆmL)1 fibroblast growth factor 8 could partly recapitulate the proliferation–trophic effects of lowered O2 on CNS stem cells [13] Recently, we demonstrated that hypoxia promoted human bone barrow-derived MSC proliferation in vitro The gene profile assayed by using cDNA microarrays showed that only four genes among 282 differentially expressed genes were known
to be HIF-1-targeted genes [23] From the above, we wondered whether HIF-1a induced by lowered O2 is
a contributory factor in hypoxia-driven proliferation
of NPCs in vitro
In the present study, different O2 concentrations (20% O2, 10% O2, and 3% O2) were adopted for cul-turing NPCs to further assess the effect of hypoxia on NPC proliferation In an attempt to elucidate the role
of HIF-1a in the hypoxia-induced proliferative effect,
we investigated the expression of HIF-1a mRNA and protein during the proliferation of NPCs under hypoxia, and the effect of overexpression or knock-down of HIF-1a on NPC proliferation
Results
Hypoxia promotes the proliferation of NPCs Neurosphere formation
It has been reported that lowered O2 (3 ± 2%) pro-motes the survival and growth of neural stem cells derived from the neural crest and midbrain [13,14] In order to further elucidate the effect of hypoxia on NPC proliferation, different O2 concentrations were employed, and the neural stem cells derived from the embryonic mesencephalon (E13.5) were used in the present study Generally, neural stem cells were grown
to form as neurospheres in vitro The ability of stem cells to form neurospheres is one of the indicators for proliferation of neural stem cells in vitro The passaged neurospheres were dissociated into single cells and planted in four-well plates at a density of 5· 104cells per well, and then cultured under different O2 concen-trations (20% O2, 10% O2, and 3% O2) for 3 days The number of neurospheres formed in each well was counted blindly after 3 days of culture We found that the numbers of neurospheres in 10% O2 and 3% O2 were increased 2.5-fold and 1.5-fold, respectively, as compared with that in 20% O2 (Fig 1A–C) These data showed that hypoxia, especially 10% O2, signifi-cantly increased the number of neurospheres
Trang 3Proliferation index (PI)
We then determined the PI of NPCs by performing
a flow cytometric measurement of DNA distributions
of cells Phase fractions calculated from such
distri-butions are used to study the growth characteristics
of NPCs The NPCs were stained with propidium iodide after being subjected to different O2 concen-tration for 3 days Three cell subpopulations (G1, S and G2+ M) were estimated Under lower O2
0
200 400 600 800 1000
**
**
**
0
100 200 300 400 500 600 700 800 900 1000
0 5
10 15 20 25 30 35 40
20 % 10 % 3 %
20 % 10 % 3 %
**
*
D
DNA content
DNA content
I C
Fig 1 Hypoxia promoted the proliferation of embryonic NPCs (A) Phase contrast images of neurospheres formed under normoxic (20% O 2 ) conditions (B) Phase contrast images of neurospheres formed under hypoxic (10% O 2 ) conditions (C) The number of neuro-spheres produced under hypoxic conditions, especially 10% O2, increased significantly as compared with control Hypoxia promoted the for-mation of neurospheres (D, E) Flow cytometric analysis showed that hypoxia, especially 10% O2, led to more NPCs in the S phase and
G 2 ⁄ M phase of the cell cycle (F) Cartogram of flow cytometric analysis Hypoxia increased the PI BrdU was added to the culture medium (10 l M ), and NPCs were cultured in different O2concentrations (20% O2, 10% O2, and 3% O2) for 3 days (G) Representative photograph of BrdU-labeled cells in the control (20% O2) (H) Representative photograph of BrdU-labeled cells under hypoxia (10% O2) (I) Hypoxia signifi-cantly enhanced the number of BrdU-labeled cells The data are the means ± SD (n = 4) *P < 0.05, **P < 0.01, as compared with control (20% O2) Scale bar = 100 lm.
Trang 4conditions, especially under 10%, more NPCs were
in S phase and G2⁄ M phase of the cell cycle as
com-pared with NPCs grown in 20% O2 (PI values were
23.87 ± 0.5 in the 20% group, 31.08 ± 1.7 in
the 10% group, and 25.23 ± 0.3 in the 3% group;
Fig 1D–F) The data here suggested that more
neural stem cells entered the proliferative phase
under hypoxia, which was consistent with the above
data
Bromodeoxyuridine incorporation assay
Incorporation of Bromodeoxyuridine (BrdU) into
growing (DNA-synthesizing) S phase cells is more
accurate in determining phase fractions We further
used BrdU incorporation to determine whether
hypoxia affects the DNA synthesis phase of NPCs
BrdU (10 lm) was added to the culture medium, and
the NPCs were exposed to different O2 concentrations
(20% O2, 10% O2, and 3% O2) The number of
BrdU-positive cells among the newly divided cells was
measured after 3 days by BrdU
immunohistochemis-try The numbers of BrdU-positive cells under
10% O2 and 3% O2 showed an 88% and a 63%
increase, respectively, as compared with the control
(20% O2; Fig 1G–I) These results showed that
hypoxia could increase the number of BrdU-positive
NPCs
Expression of HIF-1a in NPCs under hypoxia (10%)
The above data demonstrate that hypoxia, especially
10% O2, promoted the proliferation of neural stem
cells in vitro We further investigated the expression
of HIF-1a during proliferation of NPCs, which is the
key molecular event in the response to hypoxia The
NPCs were exposed to 10% O2 for different periods
of time (1, 3, 6, 12, 24, 48, and 72 h) Then, the cells
were collected at different time points; the expression
of HIF-1a mRNA was detected by RT-PCR, and
HIF-1a protein was detected by western blot The
data showed that HIF-1a mRNA in NPCs was
expressed constantly under both normoxia and
hypoxia, and increased from 6 to 72 h (Fig 2A,B)
However, expression of HIF-1a protein was
undetect-able under normoxic conditions, whereas a strong
signal was observed in the hypoxic group The level
of HIF-1a protein expression increased from 6 h
onwards, and lasted for 3 days in the hypoxic group,
as compared with the control at the same time point
(Fig 2C,D) These results demonstrate that hypoxia
induced the expression of HIF-1a during the
prolifer-ation of NPCs
Overexpression of HIF-1a promotes NPC proliferation
To investigate the role of HIF-1a in hypoxia-driven proliferation of NPCs, an adenovirus construct con-taining CMV HIF-1a was used to overexpress HIF-1a
in NPCs [CMV-enhanced green fluorescent protein (eGFP) vector was used as control] As detailed in Experimental procedures, the NPCs were dissociated into single cells, and infected with adenovirus at a titer
of 50 multiplicity of infection (m.o.i.) for 2 h Then these cells were exposed to either hypoxia (10% O2) or normoxia (20% O2) for 3 days The adenovirus at a
C H C H C H C H C H C H C H
18SRNA HIF-1α
*
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8
Control Hypoxia
C H C H C H C H C H C H C H
β-Actin HIF-1α
0 0.2 0.4 0.6 0.8
1 1.2 1.4 1.6 1.8
1 h 3 h 6 h 12 h 24 h 48 h 72 h
*
* * *
*
A
B
C
D
Fig 2 Hypoxia increased the expression of HIF-1a in NPCs Cells were exposed to 20% O2or 10% O2for different periods of time, and then collected for RT-PCR and western blot assay (A) Repre-sentative photograph for HIF-1a mRNA tested by RT-PCR (C, con-trol; H, hypoxia) (B) The level of HIF-1a mRNA expression measured by densitometry analysis The HIF-1a mRNA expression value was normalized to that of 18S (C) Representative photograph for HIF-1a protein tested by western blot (C, control; H, hypoxia) A strong signal was observed in the groups exposed to hypoxia from
6 h to 3 days, whereas no expression of HIF-1a protein could be detected in the control.
Trang 5titer of 50 m.o.i resulted in an infection rate of almost
95%, with no significant increase in viral toxicity The
expression of HIF-1a protein showed an increase after
infection with Ad–HIF-1a (Fig 3A) The total number
of cells in NPCs infected with Ad–HIF-1a increased in
comparison to those in the Ad–eGFP group under
normal condition (Fig 3B) Flow cytometric analyses
showed that overexpression of HIF-1a enhanced the
PI of NPCs under normal conditions, and that the
Ad–HIF-1a groups had a significantly increased PI
(Fig 3C,D) These results suggest that overexpression
of HIF-1a could partially mimic the effect of hypoxia
on proliferation of NPCs in vitro
Knockdown of HIF-1a expression inhibits NPC proliferation
To knock down HIF-1a expression, the pSilencer 1.0-U6 plasmid was constructed as an HIF-1a-targeted RNA interference (RNAi) vector Three selected small interfering (si)RNAs targeting HIF-1a sequences were designed The efficiency of the RNAi was estimated by
con
β-Actin
0 5 10 15 20 25 30 35
con con/HIF 10% 10%/HIF
**
*
**#
0 50 100 150 200 250
4·mL –1 )
con con/HIF 10% 10%/HIF
**
**
#
**
D
con HIF
DNA content
DNA content
DNA content
DNA content
10%/HIF 10%
con/HIF
Fig 3 Overexpression of HIF-1a promoted proliferation of NPCs under normoxic conditions Cells infected with adenovirus at a titer of
50 m.o.i were exposed to either 10% or 20% O2for 3 days (A) Expression of HIF-1a protein was analyzed by western blot; expression of HIF-1a in NPCs infected with Ad–HIF under normoxia increased as compared with the control (B) Data for number of total cells counted by hematocytometer The total number of cells in the con ⁄ HIF group infected with Ad–HIF under normoxia increased significantly as compared with that in the control group (C) Representative flow cytometric analyses (D) Cartogram of flow cytometric analyses The data showed that overexpression of HIF-1a enhanced the PI of NPCs The data are the means ± SD **P < 0.01 as compared with the control group;
#
P < 0.01 as compared with the 10% O 2 group.
Trang 6testing the expression of HIF-1a after transfection We
found that RNAi could significantly reduce the
expres-sion of HIF-1a, and RNAi was consequently used for
the following experiment The NPCs were transfected
with the pSilencer 1.0-U6 plasmid by
Lipofecta-mine 2000, and then cells were exposed to either
norm-oxia (20% O2) or hypoxia (10% O2) for 3 days The
percentage of GFP-positive cells was about 70% of
that of total neural stem cells after 3 days of
transfec-tion (Fig 4A) The expression of HIF-1a was detected
by western blot, which showed that the level of
HIF-1a protein decreased in the RNAi group as compared
with the negative control in the hypoxic condition
(Fig 4B) Flow cytometric analyses showed that the PI
of GFP-positive cells decreased in the RNAi group as
compared with the negative control in the hypoxic
condition (Fig 4C,D) These results suggest that knockdown of HIF-1a could partially decrease the proliferation of NPCs induced by hypoxia (10% O2)
Discussion
In the present study, we demonstrated that hypoxia promoted the proliferation of NPCs in vitro and that HIF-1a played a key role in this process: (a) hypoxia, especially 10% O2, had a more potent proliferative effect on NPCs; (b) the level of HIF-1a mRNA and protein expression in NPCs increased significantly dur-ing proliferation of NPCs under hypoxia; and (c) over-expression of HIF-1a could mimic the hypoxia-driven proliferative effect in NPCs under 20% O2 Con-versely, lowering HIF-1a levels by RNAi reduced the
0 5 10 15 20 25 30 35
con conRNAi 10% 10%RNAi
**
**
HIF-1α β-actin
con conRNAi 10% 10%RNAi
D
10% RNAi 10%
DNA content
DNA content
DNA content
DNA content
Fig 4 Knockdown of HIF-1a repressed hypoxia-driven proliferation of NPCs (A) Photographs of NPCs transfected with empty vector (green: GFP; scale bar = 200 lm) (B) Evaluation of HIF-1a protein expression (lane 1, normoxia + empty vector; lane 2, normoxia + RNAi vector; lane 3, hypoxia + empty vector; lane 4, hypoxia + RNAi vector) Downregulation of HIF-1a decreased the enhanced PI of NPCs induced by hypoxia (A) Cytometry analysis indicates that the PI of NPCs was decreased by downregulation of HIF-1a (B) Graph of the PI of NPCs Each bar represents the mean ± SD **P < 0.01 as compared with control; ## P < 0.01, as compared with 10% O2.
Trang 7ability of hypoxia to induce proliferation These
sug-gest that mild hypoxia is a useful measure for
expan-sion of embryonic NPCs in vitro and that HIF-1a is
the causative molecule in this process
Lowered O2culture favors the proliferation of NPCs
Standard conditions for culture of mammalian cells
employ about 20% O2 in vitro Exposure of cells to
O2 deprivation in vitro has been shown to reduce
pro-liferation and⁄ or lead to programmed cell death
[24,25] However, there is considerable controversy in
the literature regarding cellular responses under
hypoxia [26–28], and most of the discrepancies can be
explained by differences in O2 concentration, exposure
time, and type of cells In general, O2 concentrations
over 1%, rather than arresting the growth of most
kinds of cells, promote the proliferation of some
types of cells There is increasing evidence that mild
hypoxia acts as a potent regulator of various types of
stem cells [12] Therefore, the effects of hypoxia
on the stem cells are extensive, cell-type specific, and
O2-regulated
The modulation of cell proliferation in NPCs is
believed to play a role in neuronal regeneration In
2000, Morrison and Studer reported for the first time
that culturing NPCs from E12 rat mesencephalon and
peripheral nerve crest in a decreased O2 (3 ± 2%)
environment promoted their survival, proliferation,
and differentiation [13,14] They also found that the
cells yielded greater numbers of precursors and showed
less apoptosis after being grown in low O2 (3 ± 2%)
for 6 days Storch and colleagues cultured human
me-sencephalic neural precursor cells in low O2 (3%), and
found long-term proliferation of these cells, which
could grow and survive for up to 11 months [11,30]
To mimic physiological or pathological hypoxia, in the
present study different O2concentrations were adopted
to investigate the effects of hypoxia on NPC
prolifera-tion in vitro
The results of BrdU administration, neurosphere
counting and PI determination demonstrated that
hypoxia (3% O2) promoted NPC proliferation in vitro,
which is consistent with previous reports [13,14] In
addition, we also found that 10% O2is more beneficial
to NPC proliferation in vitro than 3% O2 The above
data indicate that mild hypoxia promoted the
prolifer-ation of NPCs in both the peripheral nervous system
and CNS of rat and human in vitro These results
sug-gest that lowered O2 conditions favor neural NPCs,
and that a suitable level of hypoxia could be a useful
tool for expansion of NPCs for ex vivo cell therapy
and for a mechanism study of neural development
Possible role of HIF-1a in hypoxia-driven proliferation
of NPCs HIF-1 has been identified as an important transcription factor that mediates the cellular response to hypoxia, promoting either cellular survival or apoptosis under different conditions [24,25] Activation of HIF-1a under < 1% O2 in the pathogenesis of cancer cells has been widely studied, and the involvement of HIF-1a in antiproliferation, migration and invasion of cancer cells has been shown [31–35] However, the role of HIF-1a
in hypoxia-driven proliferation is less well understood HIF-1 is composed of two subunits: HIF-1a and HIF-1b HIF-1b, also called ARNT, is expressed con-stitutively in all cells and does not respond to changes
in O2tension, whereas HIF-1a is specific in its response
to hypoxia [36] It has been reported that hypoxia induces the transcription of HIF-1a mRNA, which increases the level of HIF-1a protein in the presence
of continued hypoxia [37,38] Expression of HIF-1 (protein level) was markedly upregulated by hypoxia [36,39] Consistent with the above, our data showed that the expression of HIF-1a mRNA in NPCs increased from 6 h to 72 h during exposure to hypoxia (Fig 2A) Under normoxic conditions, HIF-1a is con-stitutively synthesized and sent to be destroyed by the ubiquitin–proteasome pathway (half-life < 5 min), so HIF-1a protein is absent or nearly absent in most normoxic cells [40,41] Consistent with this, we did not detect the expression of HIF-1a protein in NPCs under normoxic conditions With the onset of hypoxia (10% O2), we found that HIF-1a protein in NPCs was highly expressed and that this lasted for at least 3 days Therefore, HIF-1a protein expression is stable under hypoxic conditions from 6 to 72 h as compared with the control at each time point (Fig 2C) From our quantification of the data in Fig 2D, the expression of HIF-1a protein indicates a very dynamic regulation pattern during hypoxia Under hypoxia, HIF-1a sub-units are stabilized, translocated to the nucleus, dimer-ized with the stable b-subunit ARNT, and promote
O2-regulated gene expression These results indicate that HIF-1a might play an important role in this process
To determine whether HIF-1a plays a key role in hypoxia-driven NPC proliferation, overexpression of HIF-1a and RNAi were applied to study the role of the HIF-1a gene in NPCs First, overexpression of HIF-1a
by transient adenovirus transfection caused the same proliferative effect under normal conditions as that in hypoxia HIF-1a overexpression could mimic the prolif-eration of NPCs under normal conditions This suggests that activation of HIF-1a is the primary hypoxia-driven
Trang 8signaling pathway in NPCs, as well as in human
pulmo-nary artery fibroblasts [26,39] and human vascular
smooth muscle cells [42] Second, specific inhibition of
HIF-1a by RNAi technology was achieved With this
approach, it was found that short hairpin RNA
(shRNA) targeting human HIF-1a was transferred into
human endothelial progenitor cells by an adenoviral
vector HIF-1a mRNA and protein expression were
dramatically and specifically downregulated after
siRNA–HIF-1a infection in cells under hypoxia
HIF-1a knockdown via adenoviral siRNA transfer
inhibited endothelial progenitor cell colony formation,
differentiation, and proliferation [43] Consistent with
this, this effect in our study persisted for at least 72 h
and was accompanied by suppression of HIF-1a protein
expression (Fig 4B) Knockdown of HIF-1a expression
inhibits NPC proliferation induced by 10% O2 These
observations suggest that HIF-1a plays an important
role in hypoxia-induced NPC proliferation The above
data support the conclusion that HIF-1a is critical in
hypoxia-induced NPC proliferation
Conclusion
Efficient generation of NPCs in vitro may serve as a
source of cells for brain repair and treatment of
neuro-degenerative diseases Moreover, to eliminate the risk
of transformation in culture, an ideal expansion
proto-col would produce rapid proliferation without the need
for prolonged passage in cell culture In this study, we
found that hypoxia provides an easily expandable
source of NPCs in vitro for transplantation, and we
confirmed for the first time that the HIF-1 signaling
pathway was activated and critical in hypoxia-driven
proliferation of NPCs On the basis of this result, we
believe that elucidation of the molecular mechanisms
mediating this phenomenon may stimulate new
strate-gies for expansion of NPCs
Experimental procedures
Animals
Pregnant 13.5-day-old Wistar rats were used The
Institu-tional Animal Care and Use Committee (IACUC) of the
Academy of Military Medical Science gave consent for the
use of rats in all of the experiments
Isolation and culture of NPCs
Cells derived from Wistar rat mesencephalon (E13.5) were
mechanically dissociated and grown in DMEM⁄ F-12 (1 : 1)
medium containing 2 mm l-glutamine, 5 IU of penicillin,
5 lgÆmL)1 streptomycin, 1% N2, 1% B27 (Invitrogen, Grand Island, NY, USA), 20 ngÆmL)1 EGF (Sigma, St Louis, MO, USA) and 20 ngÆmL)1 basic fibroblast growth factor (Invitrogen) The primary neurospheres were defined
as passage zero (P0) NPCs The NPCs were subcultured into two to five generations, and used in the following experiments
Hypoxic conditions
For decreased O2 conditions, an incubator chamber (Therm 3111; Billups-Rothenberg, Del Mar, CA, USA), which is adjustable for the desired O2 concentration, was used The incubator chamber was flushed with 5% CO2 (bal-ance N2) The actual concentrations of 20% O2, 10% O2
and 3% O2 inside the chamber were based on direct mea-surement with a microelectrode (Animus Corp., Malvern,
PA, USA) The time of hypoxia was calculated from the measurement indicating the desired O2concentration
Neurosphere formation
Cells were seeded in four-well plates at 5· 104cells per well (Costar, Cambridge, MA, USA; culture area per well 1.9 cm2) The total number of neurospheres (size>5 cells) in each well was counted, after exposure to hypoxia for 3 days The observers were blinded to the experimental conditions Each experiment was repeated three times independently
Cell counting
Cells infected with adenovirus were seeded at a density of
4· 105
cellsÆmL)1 in 35 mm plates (Costar) and then placed in either the hypoxic or normoxic condition for
3 days; the neurospheres were trypsinized, and cells were counted with a hemocytometer
Cell cycle analysis
The neural spheres under hypoxic or normal conditions were dissociated by using 0.25% trypsin⁄ EDTA Single cell suspensions were obtained and washed with NaCl⁄ Pithree times After fixation with 75% ethanol, cells were digested with DNase-free RNase in NaCl⁄ Pi containing 5 lgÆmL)1 propidium iodide for DNA staining (45 min at 37C) [44] The propidium iodide fluorescence and forward light scat-tering were detected with a flow cytometer (FACS scan; Beckton Dickinson) equipped with cellquest (Largo, FL, USA) software
BrdU administration and immunohistochemistry
Cells were plated on 35 mm dishes (Costar) precoated with polylysine BrdU (Sigma) (10 lm) was added to the
Trang 9medium, and cells were exposed to hypoxia for 3 days The
cells were then fixed with 4% paraformaldehyde at 4C for
2 h For BrdU immunohistochemistry, the cells were
pre-treated with 2 m HCl to denature the DNA and incubated
with a mouse mAb against BrdU (Molecular Probes, NY,
USA; diluted 1 : 1000) for 48 h at 4C After being washed
in 0.1 m phosphate buffer, the cells were incubated with
biotinylated anti-(mouse IgG; Vector Laboratories,
Burlin-game, CA, USA; diluted 1 : 1000) at 4C overnight
BrdU-positive cells were visualized as a black nuclear precipitate,
using a nickel-intensified 3,3V-diaminobenzidine procedure
[45]
RNA extraction and RT-PCR
Cultures were washed once with NaCl⁄ Pibefore
solubiliza-tion in Trizol (Invitrogen) and then stored at)80 C Total
RNA extraction was performed according to the
recom-mendations of the manufacturer The program for PCR
was as follows: primers for HIF-1a (5¢-TGCTTGGT
GCTGATTTGTGA-3¢ and 5¢-GGTCAGATGATCAGA
GTCCA-3¢) were used to yield a 209 bp product for
30 cycles at 58C; m18S rRNA primers (forward, 5¢-TT
ATGGTTCCTTTGGTCGCT-3¢; reverse, 5¢-ATGTGGTA
GCCGTTTCTCAG-3¢) were used to yield a 355 bp
prod-uct for 30 cycles at 56C The level of HIF-1a mRNA
expression was semiquantified relative to the endogenous
expression level of 18S rRNA
Protein extraction and western blot
Cells were harvested quickly after either hypoxic or
norm-oxic culture for the desired times, and the total protein was
extracted with lysis buffer, which contained 100 mm
Tris⁄ HCl (pH 7.5), 300 mm NaCl, 2% (v ⁄ v) Tween-20,
0.4% NP-40, and 20% glycerol, supplemented with
pro-tease inhibitors (1 lgÆmL)1 leupeptin and pepstatin,
2 lgÆmL)1 aprotinin, and 1 mm phenylmethanesulfonyl
fluoride) and phosphatase inhibitors (10 mm NaF and
1 mm Na3VO4) Then, western blot analyses were carried
out Extracts were quantified with a protein assay kit
(Bio-Rad, Hercules, CA, USA), fractionated by 6%
SDS⁄ PAGE, and transferred to a poly(vinylpyrrolidone
difluoride) membrane (Immobilon-P; Millipore, Bedford,
MA, USA) The membrane was blocked with NaCl⁄ Tris
containing 5% dry milk at room temperature for 2 h
Membranes were incubated with mouse mAb to HIF-1a
(Chemicon, Temecula, CA, USA; dilution 1 : 500) in
NaCl⁄ Tris containing 5% nonfat dry milk Membranes
were treated with secondary antibody, goat anti-(mouse
IgG), conjugated with horseradish peroxidase (Santa Cruz,
CA, USA; dilution 1 : 1000) in NaCl⁄ Tris containing 5%
nonfat dry milk Immune complexes on the membrane were
visualized by using an enhanced chemiluminescence
detec-tion system (Amersham Biosciences, Piscataway, USA)
Construction of the recombinant adenoviral vector Ad–HIF-1a
The recombinant adenovirus overexpressing the human HIF-1a gene was a kind gift from T Hong (Institute of Microbiology, Chinese Academy of Science) The AdEasy system was used to generate recombinant adenoviruses The complete cDNA of human HIF-1a with a length
of 3720 bp contained an ORF of 2478 bp and a 1242 bp 5¢-UTR and 3¢-UTR An ORF of 2478 bp, which encoded
a sequence of 826 amino acids, was constructed into the recombinant adenoviral vector The recombinant adenoviral vector, digested with PacI to linearize the plasmid and etha-nol-precipitated, was used for transfection into HEK293 cells The recombinant virus produced in HEK293 cells could then be further purified and then viral titers were assayed
Adenovirus infection assay
Neurospheres were dissociated into single cells before infec-tion with adenovirus After 2 h of incubainfec-tion, the virus-containing medium was replaced by fresh complete growth medium Then, the NPCs were cultured for 72 h, and the expression of HIF-1a was measured The modified con-structs contained HIF-1a coupled to GFP in separate expression cassettes The rate of infection with the adeno-virus was determined by the percentage of GFP-positive cells detected by flow cytometry
HIF-1a-targeted RNAi plasmid construction and transfection in NPCs
The sequence of HIF-1a mRNA was found in GenBank (GenBank accession no for rat HIF-1a: NM_024359) and segments of siRNA targeting HIF-1a mRNA were designed
by using siRNA-designing software The sense strand con-taining 19 nucleotides was followed by a short space (TTCAAGAGA), and the reverse complement of the sense strand was followed by six thymidines as an RNA polymer-ase III transcriptional stop signal The sequences were: for-ward, 5¢-GCCTTAACCTATCTGTCACTTCAAGAGAGT GACAGATAGGTTAAGGC TTTTTT-3¢; and reverse, 5¢-AATTAAAAAAGCCTTAACCTATCTGTCACTCTCT TGAAGTGACAGATAGGTTAAGGC GGCC-3¢ (reverse complement sequences to form stem-loop structure in RNAi are underlined) The oligonucleotides were annealed in the buffer [100 mmolÆL)1 potassium acetate, 30 mmolÆL)1 Hepes⁄ KOH (pH 7.4), magnesium acetate 2 mmolÆL)1], and the mixture was incubated at 90C for 3 min, and then at 37C for 1 h The double-stranded oligonucleotides were cloned into an ApaI–EcoRI site in the
pSilenc-er 1.0-U6 vector (Ambion, Austin, TX, USA), in which shRNAs were expressed under the control of the U6 promoter A negative control scrambled siRNA, which
Trang 10had no significant homology to rat gene sequences, was
designed to detect any nonspecific effects The plasmids
were transfected into NPCs by using Lipofectamin 2000
(Invitrogen, CA, USA), and the transfection rate was
determined by the percentage of GFP-positive cells
Statistical analysis
All the experimental data shown were from experiments
that were repeated at least three times, unless otherwise
indicated Data are presented as mean ± SD Statistical
analysis was performed by t-test A statistical probability of
P< 0.05 was considered to be significant
Acknowledgements
This work was supported by grants from the National
Basic Research Program of China (nos 2006CB504100
and 2006CB943703), the Nature and Sciences
Founda-tion of China (no 30393130), the Hi-tech Research
and Development Program of China (no
2006AA02A101) and Grant of Beijing for Tibet (no
Z0006342040191)
References
1 Reynolds BA, Tetzlaff W & Weiss S (1992) A
multi-potent EGF-responsive striatal embryonic progenitor
cell produces neurons and astrocytes J Neurosci 12,
4565–4574
2 McKay R (1997) Stem cells in the central nervous
sys-tem Science 276, 66–71
3 Gage FH (2000) Mammalian neural stem cells Science
287, 1433–1438
4 Svendsen CN, Clarke DJ, Rosser AE & Dunnett SB
(1996) Survival and differentiation of rat and human
epidermal growth factor-responsive precursor cells
fol-lowing grafting into the lesioned adult central nervous
system Exp Neurol 137, 376–388
5 Svendsen CN, Caldwell MA, Shen J, ter Borg MG,
Rosser AE, Tyers P, Karmiol S & Dunnett SB (1997)
Long-term survival of human central nervous system
progenitor cells transplanted into a rat model of
Parkinson’s disease Exp Neurol 148, 135–146
6 Storch A & Schwarz J (2002) Neural stem cells and
neurodegeneration Curr Opin Investig Drugs 3,
774–781
7 Storch A, Sabolek M, Milosevic J, Schwarz SC &
Schwarz J (2004) Midbrain-derived neural stem cells:
from basic science to therapeutic approaches Cell
Tissue Res 318, 15–22
8 Ishibashi S, Sakaguchi M, Kuroiwa T, Yamasaki M,
Kanemura Y, Shizuko I, Shimazaki T, Onodera M,
Okano H & Mizusawa H (2004) Human neural
stem⁄ progenitor cells, expanded in long-term neurosphere culture, promote functional recovery after focal ischemia
in Mongolian gerbils J Neurosci Res 78, 215–223
9 Zhu J, Zhou L & XingWu F (2006) Tracking neural stem cells in patients with brain trauma N Engl J Med
355, 2376–2378
10 Kuhn HG, Winkler J, Kempermann G, Thal LJ & Gage FH (1997) Epidermal growth factor and fibroblast growth factor-2 have different effects on neural
progenitors in the adult rat brain J Neurosci 17, 5820–5829
11 Storch A, Paul G, Csete M, Boehm BO, Carvey PM, Kupsch A & Schwarz J (2001) Long-term proliferation and dopaminergic differentiation of human mesence-phalic neural precursor cells Exp Neurol 170, 317–325
12 Zhu LL, Wu LY, Yew DT & Fan M (2005) Effects of hypoxia on the proliferation and differentiation of NSCs Mol Neurobiol 31, 231–242
13 Studer L, Csete M, Lee SH, Kabbani N, Walikonis J, Wold B & McKay R (2000) Enhanced proliferation, survival, and dopaminergic differentiation of CNS pre-cursors in lowered oxygen J Neurosci 20, 7377–7383
14 Morrison SJ, Csete M, Groves AK, Melega W, Wold B
& Anderson DJ (2000) Culture in reduced levels of oxygen promotes clonogenic sympathoadrenal differen-tiation by isolated neural crest stem cells J Neurosci 20, 7370–7376
15 Genbacev O, Zhou Y, Ludlow JW & Fisher SJ (1997) Regulation of human placental development by oxygen tension Science 277, 1669–1672
16 Caniggia I, Mostachfi H, Winter J, Gassmann M, Lye
SJ, Kuliszewski M & Post M (2000) Hypoxia-inducible factor-1 mediates the biological effects of oxygen on human trophoblast differentiation through TGFbeta(3)
J Clin Invest 105, 577–587
17 Lennon DP, Edmison JM & Caplan AI (2001) Cultiva-tion of rat marrow-derived mesenchymal stem cells in reduced oxygen tension:effects on in vitro and in vivo osteochondrogenesis J Cell Physiol 187, 345–355
18 Semenza G (1999) Regulation of mammalian O2
homeostasis by hypoxia-inducible factor1 Annu Rev Cell Dev Biol 15, 551–578
19 Wang GL & Semenza GL (1993) General involvement
of hypoxia-inducible factor1 in transcriptional response
to hypoxia Proc Natl Acad Sci USA 90, 4304–4308
20 Wang GL, Jiang BH, Rue EA & Semenza GL (1995) Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O2tension Proc Natl Acad Sci USA 92, 5510–5514
21 Wang GL & Semenza GL (1995) Purification and characterization of hypoxia-inducible factor1 J Biol Chem 270, 1230–1237
22 Milosevic J, Maisel M, Wegner F, Leuchtenberger J, Wenger RH, Gerlach M, Storch A & Schwarz J (2007)