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' W7 {1 f5 p6 _1 d _! w- \' u& D, F9 @iPS细胞培养与分化研究用石墨烯平台" G7 \8 L7 `$ d' e' u
A graphene-based platform for induced pluripotent stem cells culture and differentiation- G.-Y. Chena, 1,
- D.W.-P. Panga, 1,
- S.-M. Hwangb,
- H.-Y. Tuana,
 , - Y.-C. Hua, ,
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- a Department of Chemical Engineering, National Tsing Hua University, Hsinchu 300, Taiwan
- b Bioresource Collection and Research Center, Food Industry Research and Development Institute, Hsinchu 300, Taiwan
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- Received 9 September 2011. Accepted 27 September 2011. Available online 18 October 2011.
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AbstractInduced pluripotent stem cells (iPSCs) hold great promise as a cell source for regenerative medicine yet its culture, maintenance of pluripotency and induction of differentiation remain challenging. Conversely, graphene (G) and graphene oxide (GO) have captured tremendous interests in the fields of materials science, physics, chemistry and nanotechnology. Here we report on that G and GO can support the mouse iPSCs culture and allow for spontaneous differentiation. Intriguingly, G and GO surfaces led to distinct cell proliferation and differentiation characteristics. In comparison with the glass surface, iPSCs cultured on the G surface exhibited similar degrees of cell adhesion and proliferation while iPSCs on the GO surface adhered and proliferated at a faster rate. Moreover, G favorably maintained the iPSCs in the undifferentiated state while GO expedited the differentiation. The iPSCs cultured on both G and GO surfaces spontaneously differentiated into ectodermal and mesodermal lineages without significant disparity, but G suppressed the iPSCs differentiation towards the endodermal lineage whereas GO augmented the endodermal differentiation. These data collectively demonstrated that the different surface properties of G and GO governed the iPSCs behavior and implicate the potentials of graphene-based materials as a platform for iPSCs culture and diverse applications. 9 s$ s, ~( e) L; U. h) }! ?. S' W
Keywords- Graphene;
- Graphene oxide;
- Induced pluripotent stem cells;
- Differentiation;
- Proliferation;
- Stem cells1 u+ B( l: p4 H: a. @6 g- v
 Fig. 1. FTIR spectra (1000–3750 cm−1) and XPS spectra of GO and G. (a) FTIR spectra. (b) XPS spectra of GO, (c) XPS spectra of G. The FTIR absorption bands at 1042 cm−1 and 1730 cm−1 demonstrated C–O and C O stretching of COOH group, respectively; the 1620 cm−1 band indicated the absorptions of O–H bending vibration, epoxide groups and skeletal ring vibrations; C–O vibration band of epoxide groups was shown at 1170 cm−1. In the G spectrum, a new band of 1560 cm−1 was attributed to the skeletal vibration of G sheets, indicating the higher degree of graphitic domain. The GO spectrum showed a more prominent broad peak than G spectrum near 3380 cm−1 due to the O-H stretching vibration and the resultant adsorbed water molecules on the GO surface. The deconvolution spectrum of GO showed four different peaks centered at 285 eV, 286.4 eV, 287.1 eV and 289 eV, which corresponded to C–C/CC, C–OH, CO and O=C–OH, respectively. Except the aromatic C–C/CC at 285 eV, peaks were diminished in the case of G, confirming the reduction of GO to G. View Within Article Fig. 2. AFM analyses of GO. (a) Images of as-prepared GO on a silicon substrate. (b) Height profile of the square area shown in (a). The lateral size of GO was ≈2–6 μm. Height difference between the GO sheet and substrate (the cursor pair in (b)) was 1.319 nm, consistent with the thickness of the single layer GO sheet. The crumpled silk wave observed under the AFM was characteristic of very thin sheets of GO layer covered on the substrate. View Within Article/ A0 X: z/ h8 P4 Y9 E9 _
: X4 G' S5 `( E4 F a) V5 z* t Fig. 3. SEM images of GO and G. (a, d) Silicon wafer, (b, e) GO and (c, f) G on the silicon substrate. Dense coverage of GO and G on the substrate is revealed in (b, c). The thin sheet morphology of GO and G is clearly observed in (e, f). View Within Article
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0 L* n' | [" q' K# l4 ?9 P! u Fig. 4. AFM images of (a) GO and (b) G sheets immobilized on silicon substrates. Surface roughness parameters, including the average deviation from mean (Ra), the root–mean-square deviation (Rq) and the peak-to-peak distance (Rz), are shown in Table S2. View Within Article
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; P, X) z/ b$ M& Q" o- i! x3 U Fig. 5. G and GO enabled iPSCs attachment and proliferation. (a) Photograph of blank, G- and GO-coated glass coverslips, (b) cell growth, (c) cell morphology. The cells were seeded at the same density (1 × 104 cells/cm2) onto the unmodified, G-coated and GO-coated coverslips and cultured in the LIF-containing medium to facilitate the maintenance of undifferentiated state. Bar, 100 μm. View Within Article
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6 [/ |# i1 ^7 m& }+ d Fig. 6. G and GO led to differences in the iPSCs differentiation state. (a) Confocal microscopic observation of GFP expression. (b) mRNA levels of pluripotency markers Nanog and Oct4. (c) Immunohistochemical staining against Oct4. iPSCs were cultured on the 3 different substrates and analyzed at days 5 and 9. For confocal microscopy, the cells were counterstained by DAPI. Bar, 100 μm. View Within Article' t% o9 [* b9 d+ C
. X6 B' v4 C6 s) R Fig. 7. G and GO resulted in discrepancies in the iPSCs propensity of differentiation. iPSCs were cultured on the 3 different substrates and analyzed for the expression levels of lineage-specific marker genes: (a) endodermal markers (Gata4 and Ihh), (b) ectodermal markers (Fgf5 and Nestin) and (c) mesodermal markers (T and Bmp4). The expression levels were measured by qRT-PCR and normalized against those at day 1. View Within Article5 S- R) ]2 `' L: ?
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 Fig. 8. iPSCs cultured on the G- or GO-coated substrates remained amenable to gene transfer. (a) Microscopic observation. (b) Quantitative analyses of gene expression. The iPSCs cultured on the 3 substrates were transduced with a recombinant baculovirus expressing DsRed and continued to be cultured. Mock-transduced cells were cultured in parallel and served as the negative control. The cells were observed under the phase contrast microscope (upper panel in (a)) or the confocal microscope (lower panel in (a)), or measured by flow cytometry for the total fluorescence intensities (FI) at 1 day post-transduction. The total FI represent the averages of 3 independent culture experiments and are expressed in arbitrary units (a.u.). The total FI are similar for the 3 groups (glass, G and GO) without statistical difference.
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http://www.sciencedirect.com/science/article/pii/S0142961211011471
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发表于 2011-5-18 19:41
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