Acta Biochim Biophys Sin 2011, 43: 133 – 142 | ª The Author 2011. Published by ABBS Editorial Office in association with Oxford University Press on behalf of the
Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. DOI: 10.1093/abbs/gmq121.
Original Article
Human mesenchymal stem cells are sensitive to abnormal gravity and exhibit classic
apoptotic features
Rui Meng 1, Hui-yun Xu 1, Sheng-meng Di 1, Dong-yan Shi 2, Ai-rong Qian 1, Jin-fu Wang 2*, and Peng Shang 1 *
1
Key Laboratory for Space Bioscience and Biotechnology, Institute of Special Environmental Biophysics, Faculty of Life Sciences, Northwestern
Polytechnical University, Xi’an 710072, China
2
Institute of Cell Biology and Genetics, College of Life Science, Zhejiang University, Hangzhou 310058, China
*Correspondence address. Tel: þ86-29-88460391; Fax: þ86-29-88491671; E-mail: [email protected] (P.S.). Tel: þ86-571-88206592;
Fax: þ86-571-85128776; E-mail: [email protected] (J.W.)
The aim of the present study was to investigate the effects
of abnormal gravity on human mesenchymal stem cells
(hMSCs). Strong magnetic field and magnetic field gradient generate a magnetic force that can add to or subtract
from the gravitational force. In this study, this is defined
as a high-magneto-gravitational environment (HMGE).
The HMGE provides three apparent gravity levels, i.e.
hypogravity (mg), hypergravity (2g) and normal gravity
with strong magnetic field (1g) conditions. After hMSCs
were subject to HMGE for 12 h, the proliferation, morphology, structure and apoptosis were investigated.
Results showed that the proliferation of hMSCs was inhibited under mg condition. The abnormal gravity induced
morphologic characteristics of apoptosis cells, such as cell
shrinkage, membrane blebbing, nuclear chromatin condensation and margination, decreased cell viability, and
increased caspase-3/7 activity. The rate of apoptosis under
mg condition is up to 56.95%. The F-actin stress fibers
and microtubules were disrupted under abnormal gravity
condition. Under mg-condition, the expression of p53 at
mRNA and protein levels was up-regulated more than 9and 6 folds, respectively. The Pifithrin-a, an specific
inhibitor of p53, inhibited the apoptosis and prevented the
disruption of cytoskeleton induced by abnormal gravity.
These results implied that hMSCs were sensitive to abnormal gravity and exhibited classic apoptotic features,
which might be associated with p53 signaling.
Keywords
human mesenchymal stem cells; abnormal
gravity; sensitivity; apoptosis; p53
Received: September 6, 2010
Accepted: November 2, 2010
Introduction
Bone loss is a serious medical problem for astronauts
during long-term space flight [1]. It has been well
documented that bone tissue is sensitive to its mechanical
environment. Although it has been demonstrated that the
bone loss results from both the decrease in osteoblastic formation and the increase in osteoclastic bone resorption [2–
5], the mechanism of bone loss remains unclear. Bone
remodeling is a dynamic process that requires coordinated
cellular activities among osteocytes, osteoblasts, and osteoclasts. Bone marrow (BM) mesenchymal stem cells
(MSCs), as a population of stem cells in adult BM and the
precursor of osteoblasts, can replicate as undifferentiated
cells and have the potential to differentiate into different
lineages of mesenchymal tissues, including bone, cartilage,
fat, muscle, and marrow stroma [6]. They play an important
role in the development and regeneration of tissue. The morphology and function of hMSCs is regulated by biochemical
substances, mechanical stimuli, and cellular interactions.
Previous studies demonstrated that the simulated weightless environment inhibited the osteogenesis and increased the
adipogenesis of MSCs [7–11]. Zayzafoon et al. [7] reported
that mouse calvarial bone tissue cultured in Rotary Cell
Culture System (RCCS) for 7 days failed to mineralize and
the alkaline phosphatase (ALP) activity was decreased.
However, the inhibition of mineralization was neither due to
an increase in bone resorption nor due to an increase in the
osteoblasts death and apoptosis [7]. Subsequently, a marked
suppression of hMSC differentiation into osteoblast was
observed because the cells failed to express ALP, collagen 1,
and osteonectin. The expression of runt-related transcription
factor 2, which is essential for the development of osteoblasts
from mesenchymal precursors, osteoblast maturation, and
bone formation, was also inhibited [7]. Further studies
showed that the decreased osteoblastogenesis and increased
adipogenesis were associated with actin cytoskeleton [8,12].
Buravkova et al. [13] showed that the simulated weightlessness by long-term (4–7 days) slow clinorotation decreased
hMSCs proliferation, changed cell morphology and modified
F-actin cytoskeleton. Dai et al. [9] using clinostat also
Acta Biochim Biophys Sin (2011) | Volume 43 | Issue 2 | Page 133
hMSCs are sensitive to abnormal gravity
showed that weightlessness simulated for 1–4 days inhibited
the proliferation of rat BM MSCs (rMSCs) and altered cytoskeleton distribution. However, Zayzafoon et al. [7] indicated
that the proliferation of hMSCs was not affected by simulated
weightlessness. What’s more, F-actin stress fibers were disrupted in hMSCs by modeled weightlessness within 3 h and
were completely absent by 7 days, whereas monomeric
G-actin was increased. These results indicated that MSCs are
direct targets for weightlessness and may play an important
role in bone loss during long-term space flight.
Limited to the space flight frequency and high cost, many
ground-based models simulating various aspects of decreased
gravity have been designed and developed, such as random
positional machine (RPM), clinostat/rotation, RCCS, headdown bed rest, hindlimb unloading (HLU). The magnetic
levitation is a new technology to simulate weightless environment. It was firstly used to produce weightlessness in 1991
by Beaugnon and Tournier [14,15]. Since then, this new
technology has been attracting more and more interest and
providing a good opportunity for investigating mechanisms
of organism response to gravity. The effects of highmagneto-gravitational environment (HMGE) on biological
materials have been reported [16–20]. Qian et al. [20–22]
demonstrated that HMGE affected osteoblast morphology,
cytoskeleton architecture, and function. In this study, a
ground-based experimental platform that could produce an
abnormal gravitational environment by a large gradient high
magnetic field [23] was adopted and defined as HMGE.
Materials and Methods
Isolation and culture of hMSCs
hMSCs were isolated from BM of healthy adult human
donors at a median age of 25 years (21–29 years). BM
was kindly provided by the First People’s Hospital of
Zhejiang Province, and we were given consent to use the
BM for research purpose in accordance with the procedures
approved by the Human Experimentation Committee at
Public Health Bureau of Zhejiang Province, China. hMSCs
were isolated and cultured according to previous report
[11]. Isolated hMSCs have potential of multi-directional
differentiation and are positive for CD44 (Pgp-1/ly-24),
CD29 (integrin b1), CD90 (Thy-1), CD166 (activated leukocyte cell adhesion molecule, ALCAM), SH2, and SH3
(Src-homology domains). All the cells in this study were
used from passages 7 to 9.
Apparent gravity produced by HMGE
A superconducting magnet (JMTA-16 T50 MF, Japan
Superconductor Technology, Inc., Tokyo, Japan) with a
large gradient high magnetic field, which could produce
HMGE as described by Qian et al. [16], was used in this
study (Fig. 1). It could generate three different magnetic
Acta Biochim Biophys Sin (2011) | Volume 43 | Issue 2 | Page 134
Figure 1 HMGE produced by a large gradient high magnetic
field (A) Superconducting magnet. (B) Three different apparent gravity
level positions (z axis), mg (weightlessness), 1g and 2g (hypergravity) and
their corresponding intensity of magnetic induction 12, 16 and 12 T,
respectively.
force fields of 21360, 0, and 1312 T2/m in a 50 mm diameter room temperature bore, and the corresponding apparent
gravity levels as mg (weightlessness, 12 T), 1g (normal
gravity, 16 T), and 2g (hypergravity, 12 T). The normal
gravity in geomagnetic field was used as the control.
In order to develop a long time and stable ground-based
simulated platform for space life science research, some
equipment matched with HMGE was designed, including
the temperature control system, object stage, gas control
system, and supervisory system. hMSCs were placed in the
HMGE to detect the effects of abnormal gravity.
Cell proliferation assay
The cells were seeded in StripwellTM plates (Corning, Inc.,
Acton, USA) at 1 104 cells/well and pre-cultured for 24 h
at 378C in 5% CO2. Cell cycle synchronization was achieved
by serum starvation for 48 h. Then, the serum-free medium
was replaced by fresh a-modified minimal essential media
supplemented with 10% FBS. Cells were placed into three
gravity levels (mg, 1g, and 2g) in HMGE, respectively, and
continually cultured for 12 h at 378C. After the cells were
removed from HMGE, 10 ml 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) (5 mg/ml) was
added to each well, followed by incubation at 378C for 4 h.
Medium was aspirated, and the cells were lysed with
dimethyl sulfoxide. Absorbance of each well was measured
at 490 nm by a microplate reader. Data were presented as
means + SD. Three wells were performed for each treatment.
Hematoxylin– eosin staining
To observe the effect of HMGE on morphology of
hMSCs, H&E staining were carried out. hMSCs were
seeded on coverslips in 35 mm Petri dishes and precultured for 24 h at 378C in 5% CO2. Then, cells were
placed into HMGE at different gravitational levels of mg,
1g, and 2g. Controls were maintained in the incubator at
hMSCs are sensitive to abnormal gravity
the same temperature. After 12 h, cells were washed twice
with phosphate-buffered saline (PBS), and then removed
from superconducting magnet ( pH 7.4), and fixed in 95%
ethanol. Afterward, cells were stained with 20% hematoxylin and 0.5% eosin (H&E) for 5 min, respectively. After
dehydration by graded ethanol, cells were mounted and
imaged by microscope (Nikon 80i; Nikon, Tokyo, Japan).
Hoechst staining assay for apoptosis
hMSCs were seeded on the coverslips and cultured in
medium with or without 10 mM Pifithrin-a (PFT-a;
Merck, Darmstadt, Germany) at different gravities for 12 h.
Then, cells were washed twice with PBS and stained with
Hoechst 33258 (5 mg/ml) for 10 min and observed by fluorescence microscope (Nikon 80i). Quantification was performed by randomly selecting 10 fields of view and
counting the number of cells with apoptosis. Only those
Hoechst-labeled cells showing morphological features of
nuclear condensation were identified as apoptosis.
Cytoskeleton assay
Immunofluorescence staining was used to determine F-actin
and a-tubulin cytoskeletal structures of hMSCs. Briefly,
hMSCs subjected to abnormal gravity for 12 h were fixed
with 0.5% glutaraldehyde solution in PBS for 10 min at
room temperature. The cells were permeabilized with PBS
containing 0.1% Triton X-100 solution and blocked with
blocking buffer (PBS þ 0.1% Tween þ 1% serum) for
10 min. Then cells were incubated with primary anti-atubulin antibody (1:20; Sigma, St. Louis, USA) for 2 h,
followed by the incubation with FITC-labeled anti-IgG secondary antibody (1:20; Merck CalBiochem, Darmstadt,
Germany) and rhodamine phalloidin-labeled F-actin
(Invitrogen, Carlsbad, USA) for 1 h at room temperature.
Cells were washed three times with TBS-0.1% Triton-X100
for 5 min each and incubated with 0.5 mg/ml 4’,6-diamidino2-phenylindole (DAPI) (Invitrogen) for 5 min. The cells were
imaged by Nikon 80i fluorescence microscope.
Caspase-3/7 activity assay
The cells were seeded in poly-L-lysine-coated Stripwell
white plate at 1 104 cells/well and pre-cultured for 24 h
at 378C in 5% CO2. After cells were treated with the
HMGE for 12 h, caspase-3/7 activity was detected using
ApoLive-Glo multiplex assay kit (Promega, Madison,
USA) according to the manufacturer’s protocol. Briefly,
20 ml of GF-AFC substrate (Promega) were added to each
well, and briefly mixed by orbital shaking. After incubation
for 1 h at 378C, absorbance was measured at wavelengths
400Ex/505Em. Then 100 ml of Caspase-Glow 3/7 reagent
(Promega) were added to each well, and briefly mixed by
orbital shaking. After incubated for 1 h at room temperature, caspase-3/7 activity was measured by luminescence.
Quantitative RT– PCR
After hMSCs were exposed to HMGE for 12 h, total RNA
was extracted using TRIzol (Invitrogen) according to the
protocol. RNA was reverse-transcribed into cDNA, and
then RT–PCR was performed using TaKaRa SYBR
Premix Ex TaqTM (TaKaRa Biotechnology Co., Ltd.,
Dalian, China) on PTC-200 Peltier Thermol Cycler
(Bio-Rad, Hercules, USA). GAPDH was used as a reference gene to normalize target gene [24,25]. The thermal
cycler experimental run protocol was as follows: denaturing
at 948C for 5 min, 40 cycles of amplification and quantification at 948C for 30 s, 558C for 30 s, 728C for 30 s, 808C
for 2 s with a single fluorescence detection, melting at 70–
958C with a heating rate of 0.38C per second and a continuous fluorescence detection. The primer of p53 gene
was: 30 -GTCTACCTCCCGCCATAA-50 (sense); 30 -CATCT
CCCAAACATCCCT-50 (antisense). The method of 22DDCt
was adopted to analyze the relative changes of gene
expression [26].
Western blotting
Whole cell lysates (20 mg per lane) were separated by
SDS–PAGE and transferred to apolyvinylidene difluoride
Immobilon-P membrane (Millipore, Massachusetts, USA)
using a Bio-Rad wet transfer system. Transfer efficiency and
size determination were detected by comparison with prestained protein markers (Bio-Rad). For p53 detection, membranes was blocked with Bloto B (Santa Cruz
Biotechnology, Santa Cruz, USA) for 3 h at room temperature, followed by incubation overnight at 48C with primary
anti-human p53 antibody (R&D Systems, Inc., Minneapolis,
USA). Then the membrane was washed with PBS for five
times. The membrane was incubated with diluted IRDyeTM
800-labeled goat anti-mouse IgG (1:4000, LI-COR
Biosciences, Nebraska, USA) for 2 h. Signals were visualized using Odyssey Infrared Imaging System (LI-COR).
Statistical analysis
Statistical analysis was performed using GraphPad Prism 5
statistics software (GraphPad Software, Inc., San Diego,
USA). All experiments were repeated at least three times.
The data are expressed as the mean + SD. One-way
repeated measure ANOVA together with Tukey’s multiple
comparison test was used for paired observations. The statistical significance was defined as P , 0.05.
Results
Cell proliferation was inhibited by abnormal gravity
The effect of HMGE on the proliferation of hMSCs was
determined by MTT assay. As shown in Fig. 2, after
hMSCs were exposed to HMGE for 12 h, the difference in
cell proliferation was not significant between the control
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hMSCs are sensitive to abnormal gravity
Figure 2 Abnormal gravity decreased the proliferation of hMSCs
under HMGE After culturing hMSCs under HMGE for 12 h, 20 ml
MTT were added to microlon ELISA strips for 4 h, then the medium was
removed and 150-ml DMSO was added. The absorbance was detected by
microplate reader at 490 nm. **P , 0.01 and ***P , 0.001, n ¼ 3.
and 1g groups. However, cell proliferation was significantly suppressed under mg condition compared with the
control and 1g groups (**P , 0.01 and ***P , 0.001,
respectively). Cell proliferation was also decreased under
2g condition.
Apoptosis was induced by abnormal gravity
We first examined the effects of HMGE on morphology of
hMSCs by H&E staining. After hMSCs were exposed to
HMGE for 12 h, cell phenotype was altered under mg condition [Fig. 3(A)]. Classic apoptotic phenotype such as
round cell phenotype and plasma membrane blebbing was
observed under mg condition. Some round cell phenotype
were also observed under 2g condition.
To further confirm apoptosis induced by abnormal
gravity, nuclei were stained with DNA-binding fluorochrome Hoechst 33258 and examined by fluorescence
microscopy after hMSCs were exposed to HMGE for 12 h.
In the control and 1g groups, the nuclei blue fluorescence
uniformly distributed towards the nuclear membrane periphery. And chromatin condensation or margination was
not observed [Fig. 3(B), a, c]. However, irregular nuclear
morphology, chromatin condensation and margination
toward the nuclear membrane periphery were observed
under mg condition [Fig. 3(B), b]. Part of cells presented
apoptotic phenotype under 2g condition [Fig. 3(B), d].
Statistical analysis was performed by counting the number
of apoptotic cells under fluorescence microscope
[Fig. 3(C)]. The data showed that apoptosis rate was 56.95
and 3.53% in the mg and 2g groups, respectively. mg condition significantly induced apoptosis compared with
control, 1g-, and 2g conditions (***P , 0.001). Compared
with the control and 1g conditions, the apoptosis of cells
under 2g condition was also significantly increased.
However, there is no difference between the control and 1g
groups.
Acta Biochim Biophys Sin (2011) | Volume 43 | Issue 2 | Page 136
Cytoskeleton distribution was altered by abnormal
gravity
After hMSCs were exposed to HMGE for 12 h, the cytoskeleton distribution was investigated by fluorescence
microscopy. In the control and 1g groups, abundant F-actin
stress fibers extended across the cytoplasm and were well
distributed in whole cell [Fig. 4(A,K)]. However, F-actin
stress fibers disappeared almost completely from the cytoplasm and formed a peripheral ring in the perinuclear region
of the cell under mg condition [Fig. 4(F)]. Accompanied by
some cell shrinking, F-actin stress fibers were jumbled
inside the cell under 2g condition [Fig. 4(P)].
Microtubules cytoskeleton also underwent a marked collapse under abnormal gravity. a-tubulin cytoskeleton was
well organized in the cell, and microtubule was clearly
observed in the control and 1g groups [Fig. 4(B,L)].
However, microtubules were reorganized under mg condition [Fig. 4(G)]. a-Tubulin cytoskeleton was distributed
above the nucleus, membrane peripheral and membrane
blebbing. a-Tubulin cytoskeleton was also reorganized
under 2g condition [Fig. 4(Q)].
To observe the changes of chromatin after hMSCs were
exposed to HMGE for 12 h, the nuclei labeled with DAPI
were detected. It was shown that the nuclei were well distributed in the center of cells in the control and 1g groups
[Fig. 4(D,E,N,O)]. However, the fluorescence intensity of
nuclei was strengthened and the distribution of them
tended to one side of cell under mg condition [Fig. 4(I,J)].
Changes of some nuclei under 2g condition were similar to
that under mg condition [Fig. 4(S,T)].
PFT-a inhibited the apoptosis of hMSCs and
disruption of cytoskeleton induced by abnormal gravity
To examine whether the role of p53 is related to the cell
apoptosis induced by abnormal gravity, hMSCs were
treated with a specific p53 inhibitor, PFT-a. When cells
were cultured in medium supplemented with PFT-a, chromatin condensation or margination toward the nuclear
membrane periphery induced by abnormal gravity (mg- and
2g conditions) were not observed [Fig. 3(B), f, h].
Quantitative analysis showed that the apoptosis rate was
decreased from 56.95 to 12.73% after PFT-a addition
[Fig. 3(C)].
To determine the relationship between apoptosis and disruption of cytoskeleton induced by abnormal gravity, the
effects of PFT-a on cytoskeleton were examined during
hMSCs were exposed to HMGE. Whether PFT-a was
added into the culture medium or not, there were no
changes for F-actin stress fibers [Fig. 4 (A,A’,K,K’)] and
a-tubulin microtubulins [Fig. 4(B,B’,L,L’)] in the control
and 1g groups. However, when cells were cultured in
medium supplemented with PFT-a, the disruption of
F-acitn stress fibers [Fig. 4(F’,P’)] and a-tubulin
hMSCs are sensitive to abnormal gravity
Figure 3 Abnormal gravity altered hMSCs morphology and chromatin distribution under HMGE The cells were seeded on coverslips and
precultured for 24 h. (A), After culturing hMSCs under HMGE for 12 h, the coverslips were removed from HMGE, fixed in 95% ethanol and placed in
20:0.5% H&E for 5 min, respectively. Then cells were dehydrated by an ethanol gradient and mounted by Permount. a, control; b, mg; c, 1g; d, 2g. (B)
After culturing the cell in medium with or without 10 mM PFT-a under HMGE, the coverslips coated with cells were washed twice with pre-warmed
PBS, stained with Hoechst 33258 (1 mg/ml) for 5 min. a2d, medium without PFT-a; e2h, medium with PFT-a. Arrows indicate apoptotic cells. The
slides were observed and photographed by microscope. Bar ¼ 50 mm. (C) The rate of apoptosis cultured in medium without PFT-a. Apoptosis rate is
expressed as percentage of apoptotic cells of the total number of cells in the view field as justified by Hoechst staining. Ten view fields were randomly
selected to calculate the number of apoptotic cells. Treatment with 10 mmol/l PFT-a prevented the apoptosis induced by HMGE. ***P , 0.001, n ¼ 3.
Bar ¼ 50 mm.
microtubules [Fig. 4(G’,Q’)] induced by abnormal gravity
were not observed. These results showed that the disruption
of cytoskeleton induced by abnormal gravity was not only
associated with apoptosis, but also prevented by PFT-a.
Caspase-3/7 activity was increased by abnormal gravity
Cell viability and caspase activation events were assessed
after hMSCs were exposed to HMGE for 12 h. Cell viability was measured using a fluorgenic and cell-permeable
peptide substrate (glycylphenylalanyl-amino fluorocoumarin, GF-AFC), and the caspase-3/7 activity was
measured using caspase-3/7 cleavage of the luminogenic
substrate containing DEVD sequence. The results showed
that cell viability was significantly decreased under mg
condition (***P , 0.001) [Fig. 5(A)], however, the
caspase-3/7 activity was significantly increased compared
with the control, 1g, and 2g groups (***P , 0.001)
[Fig. 5(B)].
The expression of p53 in mRNA and protein levels were
up-regulated by abnormal gravity After hMSCs were
exposed to HMGE for 12 h, the results of quantitative RT–
PCR showed that p53 expression at mRNA levels was
increased to 8.9-, 5.8-, and 6.5 folds, respectively, under mg-,
1g-, and 2g conditions when compared with the control condition [Fig. 6(A)]. Western-blot analysis indicated that p53
expression at protein levels was up-regulated 5.5 fold under
mg condition compared with control condition [Fig. 6(B)].
Discussion
Unloading of skeleton due to the abnormal gravitational
environment during space flight results in bone loss. The
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hMSCs are sensitive to abnormal gravity
Figure 4 Abnormal gravity induced the disruption of cytoskeleton under HMGE The cells were seeded on coverslips and precultured for 24 h.
After culture under HMGE and normal gravity (control) with or without 10 mM PFT-a for 12 h, cells were labeled with rhodamine phalloidin to
visualize filamentous actin (red) and with primary anti-a-tubulin antibodies, FITC-labeled anti-IgG secondary antibodies to visualize the a-tubulin
(green). DAPI was used to label nucleus (blue). Images were acquired using a fluorescence microscope (Nikon 80i, Japan) by a 20 objective. Each
image is representative of three separate experiments. Bar ¼ 100 mm.
function of bone cells is affected during spaceflight and in
simulated weightless environment. hMSCs, as a population
of stem cells in adult BM, are important progenitor cells of
osteoblasts. Thus, biological response of hMSCs to abnormal gravity must be understood before the mechanism of
physiological changes that occur during spaceflight can be
identified.
Many ground-based models simulated various aspects of
decreased gravity (i.e. RPM, clinostat/rotation, head down
Acta Biochim Biophys Sin (2011) | Volume 43 | Issue 2 | Page 138
Bed rest, HLU) have been designed and developed. Some
cell culture systems, such as clinostat, random positioning
machine and rotary cell culture, also simulate weightlessness by gravity-vector averaging. These systems altered the
effects of gravity, but gravity cannot be reduced on Earth.
Drop tower and parabolic flight can realize the weightlessness realistically, but it is of short duration and samples
experience variable g-forces at the start point and end point
of the test. In this study, we adopted a ground-based
hMSCs are sensitive to abnormal gravity
Figure 5 Abnormal gravity decreased cell viability and increased caspases-3/7 activity After culture under HMGE for 12 h, 20 ml of viability
reagent was added to all wells and incubated at 378C for 1 h. The fluorescence at 400Ex/505Em was measured for cell viability using automatic microplate
reader (Bio-Rad). Caspase-Glo 3/7 reagent was added to all wells and incubated at 378C for 1 h. Luminescence was measured for caspase-3/7 activity
using automatic microplate reader (Bio-Rad). No-cell control: well with no cell culture; positive control: cells were induced apoptosis by tumor necrosis
factor alpha and cycloheximide; control: cells were cultured in normal condition. (A) Cell viability; B, caspase-3/7 activity. ***P , 0.001, n ¼ 3.
Figure 6 Abnormal gravity up-regulated p53 expression at mRNA and protein levels under HMGE (A) Quantitative RT –PCR analysis. Relative
gene expression from three experiments was normalized to 18S rRNA expression. ***P , 0.001. (B) Western blot analysis. After hMSCs were cultured
in the HMGE and in normal gravity (control), whole cell protein was extracted. Total protein was separated by 10% SDS– PAGE. Immunoblot was
probed using antibody directed against p53. The graph is representative of three separate experiments. The band intensities of p53 relative to GAPDH are
graphed as a percentage relative to controls. ***P , 0.001.
experimental platform that could provide HMGE [16]. That
is, the HMGE is a compound environment of magnetic
field and gravity and it provides three apparent gravity
levels as mg (weightlessness, 12 T), 1g (16 T), and 2g
(hypergravity, 12 T) stably and continually for diamagnetic
materials. Qian et al. [21–23] have performed some studies
about bone cells using this system and prove that it can be
used to simulate weightlessness. Although this system is a
complex environment, the effects induced by this environment can be analyzed in detail. The effects of weightlessness can be obtained by comparing mg condition with 1g
condition in the HMGE and the control group in a geomagnetic field. The effects of hypergravity could be
obtained by comparing 2g condition with 1g condition in
the HMGE and control. The effects of magnetic field can
be removed by comparing 1g condition with control in the
HMGE. The apoptosis rate was the same in control and 1g
conditions. These results showed that the effect of magnetic field on cell was not obvious. However, the proliferation was inhibited and apoptosis was induced under mg
condition. Our study demonstrated that hMSCs were sensitive to abnormal gravity produced by this HMGE. This
model can simulate weightlessness environment formed in
the spaceflight to a certain degree and provide an important
platform for the study of space biology effects.
To understand the effects of weightlessness on undifferentiated hMSCs, we investigated cell proliferation under
HMGE. Our results showed that the abnormal gravity
(hypogravity) could inhibit the proliferation of hMSCs statistically, although the differences were smaller than 10%
between the mg and control groups, as well as the mg and
1g groups (Fig. 2). Paulette et al. [27] reported a
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hMSCs are sensitive to abnormal gravity
population-doubling time of 33 h in vitro. So, the smaller
difference that we found in this study may be due to the
short time of exposing to HMGE (12 h). Similar results
have been reported previously. For example, Buravkova
et al. [13] and Merzlikina et al. [28] reported that the proliferative activity of hMSCs was significantly inhibited
under weightlessness simulated by long-term (4–7 days)
slow clinorotation and the cells became larger and flat.
Chen et al. [29] also reported that the proliferation of
hMSCs was inhibited by RCCS. Dai et al. [9] showed that
simulated weightlessness using clinostat for 1–4 days
inhibited the population growth of rat MSCs (rMSCs) and
cells were arrested in the G0/G1 phase of cell cycle.
Kostenuik et al. [30] demonstrated that rMSCs from
5 day-hindlimb-suspended rats had significantly decreased
proliferation capability when cultured in vitro. However,
McDonald et al. [7] showed that the proliferation of
hMSCs was not affected by simulated weightlessness using
RCCS. The discrepancy in proliferation of MSCs induced
by weightlessness may be due to different weightless
models, different cell sources, different functional status of
MSC (donors, passages, culturing time and conditions) or
others.
The responses of cells to weightlessness are dependent
upon cell type and time of exposure. To investigate the
effects of abnormal gravity on morphology and apoptosis
of hMSCs, H&E staining and Hoechst staining were used
to observe the phenotype and chromatin of hMSCs,
respectively. We demonstrated that hMSCs were sensitive
to abnormal gravity and exhibited classic apoptotic features: rounded cellular morphology, membrane blebbing,
nuclear condensation, decreased viability, increased
caspase-3/7 activity, and disruption of cytoskeleton related
to apoptosis. Although the increase of cell apoptosis is one
of the significant consequences in cell structure and function that occurs in microgravity [31,32], such a high apoptotic rate resulted from simulated weightlessness at such a
short time (for 12 h) has not been reported previously.
Qian et al. [17–21] demonstrated that the proliferation of
MC3T3-E1 (inhibited) and MG-63 (accelerated) osteoblasts
lines were affected by HMGE and the cell shape became
more flat under mg condition. However, the apoptosis was
not significant. These results suggest that hMSCs are
affected directly by abnormal gravity.
Many studies demonstrated that the cytoskeleton is
highly sensitive to real microgravity and simulated microgravity [32,33]. Most cells appeared to exhibit cytoskeleton
changes and became disorganized when firstly exposed to
real and simulated weightlessness environment [32,34,35].
The disorganization of actin cytoskeleton was observed in
cells cultured during spaceflight and in ground-based
models of weightless environment [36,37]. Our results
demonstrated that F-actin stress fibers were disrupted and
Acta Biochim Biophys Sin (2011) | Volume 43 | Issue 2 | Page 140
reorganized into a periphery ring in the mg condition. This
result is consistent with Meyers et al.’s report. Using
RCCS to simulated weightlessness, Meyers et al. [8]
showed that stress fibers of hMSCs were disrupted and cortical actin rings were formed in 3 h and stress fibers were
completely absent after 7 days. Buravkova et al. [13] also
showed that actin filaments thinned down and some of
stress fibers disappeared along with alterations of cellular
shape after hMSCs were cultured in clinorotation for 2–
4 h. The changes of actins occurred during weightlessness
in this study was in accordance with the disruption of actin
stress fibers occurred during apoptosis [38]. Therefore, the
changes of F-actin induced by abnormal gravity may relate
to apoptosis. But it is not known whether the changes of
F-actin resulted in apoptosis or apoptosis induced the
changes of F-actin under abnormal gravity.
Experiments of in vitro microtubule formation performed
by Papaseit et al. [39] have shown that self-assembly of
microtubules from tubulin is to a certain degree regulated
by gravity. It has been reported that in weightlessness
microtubules in vitro grow and organize in a homogenous
or random pattern and in the 1g condition microtubules
grow and spontaneously organize in a striped pattern [39].
In this study, we found that microtubules were reorganized
under mg condition. The altered distribution of microtubules in abnormal gravity is consistent with the function of
microtubule during apoptosis. Microtubules are depolymerized at the onset of the execution phase of apoptosis concomitant with disruption of pericentriolar components
( pericentrin, ninein, and g-tubulin) of the centrosomal
region [40–42]. Moss et al. [43] reported that the function
of microtubules maintained chromatin at the periphery by
resisting some form of retractile pressure and preserved
plasma membrane integrity during the execution phase of
apoptosis. So, the altered distribution of microtubules
induced by abnormal gravity also related to apoptosis.
These results also demonstrated that the altered microtubules in abnormal gravity were related to apoptosis.
Therefore, the disrupted microfilament and microtubule
cytoskeleton and their relationship with apoptosis under
HMGE implied that hMSCs were sensitive to abnormal
gravity.
p53, a tumor suppressor gene, regulates various cellular
processes, including apoptosis, differentiation, and genomic
integrity [44]. Therefore, it may be a strong candidate in the
apoptosis of hMSCs induced by abnormal gravity. In many
cell types, p53 plays a crucial role in controlling apoptosis
and cell cycle arrest when these cells are exposed to
stress-induced conditions [45]. p53 protein is normally
maintained at a low level within the cell. And when the cell
is exposed to different stresses including DNA damage and
hypoxia, p53 protein is elevated through post-translational
modifications that increase p53 stability [46]. In response to
hMSCs are sensitive to abnormal gravity
stress, p53 accumulates and transactivates downstream
target genes such as mdm2, p21, bax and puma [47]. To
determine the role of p53 signaling in apoptosis of hMSCs
induced by abnormal gravity, we examined the expression
of p53 at mRNA and protein levels. The up-regulated
expression of p53 at mRNA and protein levels showed that
p53 signaling did associate with the apoptosis induced by
abnormal gravity. PFT-a is a small molecule inhibitor of
p53 signaling. It can reversibly inhibit p53-transcriptional
activity and therefore inhibit p53-induced apoptosis, cell
cycle, and DNA-synthesis block [48]. The inhibition of
apoptosis and altered distribution of cytoskeleton by PFT-a
proved that p53 signaling could play an important role in
apoptosis induced by abnormal gravity.
In conclusion, classic apoptotic features and disruption
of cytoskeleton indicate that hMSCs may be sensitive to
abnormal gravity. It provides a new insight into the mechanisms of bone loss in humans under weightlessness
during spaceflight.
9
10
11
12
13
14
15
16
Acknowledgements
17
The authors thank Dr. Dan Shen in the First people’s
Hospital of Zhejiang Province for providing human BM.
18
Funding
19
This study was supported by the grant from the National
Nature Science Foundation of China (No. 30970689).
20
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