Cerium oxide nanoparticles inhibit adipogenesis in rat mesenchymal stem
cells: Potential therapeutic implications
Antonella Rocca
a
a,b,*
a
a
, Virgilio Mattoli , Barbara Mazzolai , Gianni Ciofani
a,*
Istituto Italiano di Tecnologia, Center for Micro-BioRobotics @SSSA, Viale Rinaldo Piaggio
34, 56025 Pontedera (Pisa), Italy
b
Scuola Superiore Sant'Anna, The BioRobotics Institute, Viale Rinaldo Piaggio 34, 56025
Pontedera (Pisa), Italy
*
Corresponding Authors
Antonella Rocca, antonella.rocca@iit.it
Gianni Ciofani, gianni.ciofani@iit.it
Istituto Italiano di Tecnologia, Center for Micro-BioRobotics @SSSA
Viale Rinaldo Piaggio 34, 56025 Pontedera (Pisa), Italy
Tel. +39050883089
Fax +39050883497
1
Supplementary Material
Reactive oxygen species (ROS) are well known to play a key role during adipogenesis [S1S4]. In order to verify their production in our cell model and to confirm their inhibition
following cerium oxide nanoparticles (nanoceria, NC) treatment, we measured intracellular
ROS after 24 hours since differentiation induction in differentiating cultures, in differentiating
cultures treated with 50 µg/ml of NC and, as anti-oxidant positive control, in differentiating
cultures treated with 5 mM of N-acetyl-L-cysteine (NAC) [S5]. Proliferating cells were
considered as reference control.
Rat mesenchymal stem cells (MSCs) were plated at a density of 6,000 /cm2 in 6-well plates.
At 24 hours since seeding, differentiating samples were provided with high glucose DMEM
supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, 100 mg/ml
streptomycin, 200 mM L -glutamine, 5 µg/ml insulin, 1 µM dexamethasone, 20 µM
indomethacin, and 500 µM 3-isobuty-l-methyl-xanthine. Two experimental groups were
moreover provided with NC and NAC, respectively, as previously mentioned. Control
proliferating samples were maintained in standard medium (DMEM supplemented with 10%
FBS, 100 U/ml penicillin, 100 mg/ml streptomycin, and 200 mM glutamine).
ROS
generation
was
measured
with
the
fluorescent
dye
6-carboxy-2′,7′-
dichlorodihydrofluorescein diacetate bis(acetoxymethyl)-ester (C-DCF-DA; Molecular
Probes), following standard procedures [S6]. Briefly, MSC cultures were treated as described
for 24 h, then washed with phenol red-free Hanks'-buffered saline and incubated with C-DCFDA (25 μM) for 30 min at 37°C, in Hank's buffer. After this step, cells were washed and
scraped off into 1 ml of distilled water, sonicated and centrifuged. The fluorescence of the
supernatants was measured with a microplate reader (Victor3, Perkin Elmer) at 485 nm
excitation and 525 nm emission, and data were expressed as % variation with respect to the
control (proliferating cells).
Three independent experiments were carried out, and statistical analysis was performed with
KaleidaGraph (Sinergy Software), using one-way analysis of variance (ANOVA) followed by
post-hoc Bonferroni's test.
Results are reported in Figure S1. A significant increment (~13%, p < 0.05) of ROS
production is highlighted in differentiating samples with respect to the proliferating ones.
However, ROS levels are drastically reduced in differentiating samples treated both with
2
NAC (same level of the proliferating cultures, p > 0.05) and NC (non-significant increment of
about 5%, p > 0.05 with respect to the proliferating samples), thus demonstrating efficiency of
these treatments in the inhibition of ROS production in MSCs and, as a consequence, of their
maturation toward adipocytes.
ROS level (% of control)
20
*
15
10
5
0
Proliferating
Differentiating
Differentiating
50 µg/ml NC
Differentiating
5 mM NAC
Figure S1 - ROS levels, expressed as % variation with respect to the control proliferating cells, in MSC differentiating
cultures after 24 hours since adipogenesis induction. Nanoceria and NAC treatments inhibit ROS production
occurring in differentiating samples.
Supplementary References
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S3
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S6
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