Supporting Information Anti-oxidant activity reinforced reduced graphene oxide/alginate microgels: mesenchymal stem cell encapsulation and regeneration of infarcted hearts Goeun Choe 1† , Seon-wook Kim 2† , Junggeon Park 1 , Junha Park 1 , Semin Kim 1 , Yong Sook Kim 3 , Youngkeun Ahn 4 , Da-Woon Jung 2 *, Darren Reece Williams 2 *, and Jae Young Lee 1 * 1 School of Materials Science and Engineering, Gwangju Institute of Science and Technology (GIST), Gwangju, 61005, Republic of Korea 2 School of Life Science, Gwangju Institute of Science and Technology (GIST), Gwangju, 61005, Republic of Korea 3 Biomedical Research Institute, Chonnam National University Hospital, Gwangju, 61469, Republic of Korea 4 Division of Cardiology, Department of Internal Medicine, Chonnam National University Medical School, Gwangju, 61469, Republic of Korea † These authors contributed equally to this work.
Figure S1. Morphology and size examination of used graphene oxide. (A) TEM image and
(B) DLS analysis. Morphology of graphene oxide (GO) used in this study was studied using
transmission electron microscopy (TEM, Technai G2 S-Twin, 300 Kev). Its size and
distribution was measured by dynamic light scattering (DLS, ELSZ, Photal Otsuka
electronics).
Circularity AR Roundness0.0
0.5
1.0
1.5
Figure S2. Circularity, AR (aspect ratio), and roundness analysis of the fabricated
GO/alginate microgels. The circularity, AR and roundness were analyzed from the optical
images using image J software.
Figure S3. (A) SEM image of hydrogels. (B) Pore size of various hydrogels analyzed by
Image J based on SEM images. A scanning electron microscope (SEM, Hitachi S-4700) was
used to study the internal structures of the hydrogels. Hydrogels were washed with DIW and
lyophilized for 3 days. After platinum coating using a plasma sputter for 35 s, the samples
were analyzed. Scale bar = 50 μm.
Figure S4. (A) Tensile stress and (B) shear modulus of various hydrogels. Tensile stress of
hydrogel was analyzed by using tensile mode of Universal Testing Machine with a 100 N
load cell (UTM/TO-100-IC, testone, Siheung, Korea). Hydrogels were prepared in a
rectangular shape with a gauge length 50 mm, a width 15 mm and a thickness 2 mm. The
tensile mode was performed at a rate of 100 mm/min at 25 ° C and tensile stress was obtained
at the failure point. The shear modulus of bulk hydrogel was measured by a rheometer
(Kinexus lab+, Malvern, Worcestershire, UK) with a frequency sweep mode using a 10 mm
diameter plate geometry and 1 mm gap. The gap was set to 1 mm using a 10 mm diameter
plate geometry.Frequency was changed from 0.1 to 10 Hz at constant 0.5 % strain at 25 ºC.
Note that internal structures and mechanical properties of the gels were examined using bulk
hydrogels as the microgels were hard to handle for the measurements and expected to show
similar characteristics to bulk gels. Bulk hydrogel samples were fabricated as our group
previously studied [1].
Figure S5. hMSCs viability after encapsulation in r(GO/alginate) microgels. Fluorescence
images after live/dead staining. Scale bar = 100 µm.
Figure S6. Viability of MSCs cultured on TCPs in 2 mg/mL ascorbic acid solution at 37°C
for different reduction time. (A) Live/dead staining. Scale bar = 50 µm. (B) Relative
metabolic acvitites of the MSCs by WST assay.
Figure S7. Viability of MSCs during incubation at 400 μM H2O2 concentrations up to 7 days.
Figure S8. Effect of pre-treatment with ascorbic acid solution on MSC viability during
incubation at various H2O2 concentrations. Scale bar = 50 μm.
Figure S9. Representative electrocardiography of the treated rats two weeks after MI.
Treatment with mesenchymal stem cells encapsulated in reduced graphene alginate beads
(MSCs in r(GO/alginate)) improved contractility after MI compared to mesenchymal stem
cells encapsulated in graphene alginate beads (MSCs in GO/alginate).
Figure S10. Immunostaining for hMSCs in the heart tissues after 2 weeks of transplantation.
Samples included hMSCs only, and hMSCs in GO/algiante, and hMSCs encapsulated in
r(GO/alginate). Scale bars are 50 µm. * indicates significant difference between the
individual sample groups (p<0.05). Immunofluorescence was performed to identify the
remaining transplanted human cells in the heart tissue. First, the sectioned sample was
immersed in acetone for 15 min and washed twice with 1X PBS for 5 min. The samples were
incubated in 0.1 % triton-X solution for 10 min and washing twice with 1X PBS for 5 min.
The samples were then placed in 1 X citrate buffer (sodium citrate dihydrate 12.044g, citric
acid 11.341 g, pH 6.0) and boiled for 40 sec in a microwave oven, placed at room
temperature for 20 min. Blocking was performed by incubating the samples in horse serum at
room temperature for 1 h. The samples were incubated in the primary antibody solution
(Anti-CD44 monoclonal antibody, diluted at 1:100 with 0.05% triton-X) overnight at 4 °C.
The samples were incubated in the secondary antibody solution (Alexa 488/555 goat anti-
mouse IgG, 1:200 in 1X PBS) at 37°C for 1 h. The sample was then treated for 30 min with a
solution of sudan black B (0.1 g of sudan black B, EtOH 100 mL). Subsequently, the samples
were immersed twice in a 1X MHB solution (for 1L of 10X MHB solution; NaCl 80 g, KCl 4
g, KH2PO4 0.6 g, glucose 10 g, Na2HPO4 0.479 g, EGTA 2 mM, MES 5 mM) for 15 min.
The samples were finally stained with DAPI (1:1000 in 1 X PBS) for 3 min. Fluorescence
images were acquired randomly with a fluorescence microscope (Leica DMI 3000B).
Figure S11. Immunostaining for sarcomeric α-actinin and Ki67 in the heart tissues after 2
weeks post-transplantation. Representative images from the PBS, hMSCs only, hMSCs
encapsulated in GO/algiante, and hMSCs encapsulated in r(GO/alginate) treatment groups.
(A) Representative images of the samples. Scale bars are 50 µm. (B) Numbers of sarcomeric
α-actinin stained cells per image. An asterik (*) indicates significant difference between the
individual sample groups (p<0.05).
Table S1. Potential functions of the cytokines mentioned in this paper for cardiac regenration.
Group Cytokines Function References
I
bFGF, β-NGF, G-CSF,
GDNF, HB-EGF, HGF,
M-CSF, NT-3, TGF-α
CM survival and neovascularization
(proangiogenic and proarteriogenic
factors)[2–7]
TGF-beta family Heart restoration and left ventricular
remodeling[8]
FGF-7, TGF- α Wound healing [9]
IGF-2 Promote pluripotency and self-renewal
of MSCs[10]
c-kit Enhance migration, survival and
growth of CPCs through PI3K and
MAPK pathway
[11]
II
FGF-4 Proliferation of MSCs [12]
FGF, IGF-1, IGF-1R,
PDGFRβ, PLGF, SCF
Cardiac regeneration including
neovascularization, attenuation of
ventricular wall thinning and increased
angiogenesis
[13–16]
PDGF-AA, PDGFRα Recruitment of cardiac atrial
appendage stem cells to infarcted sites[17]
VEGF/VEGFR CSC migration through
SDF-1a/CXCR4 cardiac regeneration[18,19]
Table S2. Cardiac output parameters in the treated rats as measured by echocardiography (n/s=not significant, * p<0.05, ** p<0.01, and *** p<0.001).
References
[1] S. Kim, Y. Yoo, H. Kim, E. Lee, J.Y. Lee, Reduction of graphene oxide/alginate composite hydrogels for enhanced adsorption of hydrophobic compounds, Nanotechnology. 26 (2015) 405602.
[2] M. Mirotsou, T.M. Jayawardena, J. Schmeckpeper, M. Gnecchi, V.J. Dzau, Paracrine mechanisms of stem cell reparative and regenerative actions in the heart, J. Mol. Cell. Cardiol. 50 (2011) 280–289.
[3] T. Okazaki, S. Ebihara, M. Asada, S. Yamanda, Y. Saijo, Y. Shiraishi, T. Ebihara, K. Niu, H. Mei, H. Arai, Macrophage colony-stimulating factor improves cardiac function after ischemic injury by inducing vascular endothelial growth factor production and survival of cardiomyocytes, Am. J. Pathol. 171 (2007) 1093–1103.
[4] J. Yang, J. Xia, Y. He, J. Zhao, G. Zhang, MSCs transplantation with application of G-CSF reduces apoptosis or increases VEGF in rabbit model of myocardial infarction, Cytotechnology. 67 (2015) 27–37.
[5] S.M. Watt, F. Gullo, M. van der Garde, D. Markeson, R. Camicia, C.P. Khoo, J.J. Zwaginga, The angiogenic properties of mesenchymal stem/stromal cells and their therapeutic potential, Br. Med. Bull. 108 (2013) 25–53.
[6] M. Blais, P. Lévesque, S. Bellenfant, F. Berthod, Nerve growth factor, brain-derived neurotrophic factor, neurotrophin-3 and glial-derived neurotrophic factor enhance angiogenesis in a tissue-engineered in vitro model, Tissue Eng. Part A. 19 (2013) 1655–1664.
[7] A. Singh, A. Singh, D. Sen, Mesenchymal stem cells in cardiac regeneration: a detailed progress report of the last 6 years (2010–2015), Stem Cell Res. Ther. 7 (2016) 82. doi:10.1186/s13287-016-0341-0.
[8] M. Bujak, N.G. Frangogiannis, The role of TGF-β signaling in myocardial infarction and cardiac remodeling, Cardiovasc. Res. 74 (2007) 184–195.
[9] L. Lin, L. Du, The role of secreted factors in stem cells-mediated immune regulation, Cell. Immunol. 326 (2018) 24–32.
[10] A. Youssef, D. Aboalola, V.K. Han, The roles of insulin-like growth factors in mesenchymal stem cell niche, Stem Cells Int. 2017 (2017).
[11] B.N. Vajravelu, K.U. Hong, T. Al-Maqtari, P. Cao, M.C. Keith, M. Wysoczynski, J. Zhao, J.B. Moore IV, R. Bolli, C-Kit promotes growth and migration of human cardiac progenitor cells via the PI3K-AKT and MEK-ERK pathways, PloS One. 10 (2015) e0140798.
[12] S.-C. Choi, S.-J. Kim, J.-H. Choi, C.-Y. Park, W.-J. Shim, D.-S. Lim, Fibroblast growth factor-2 and-4 promote the proliferation of bone marrow mesenchymal stem cells by the activation of the PI3K-Akt and ERK1/2 signaling pathways, Stem Cells Dev. 17 (2008) 725–736.
[13] Z. Ma, H. Yang, H. Liu, M. Xu, R.B. Runyan, C.A. Eisenberg, R.R. Markwald, T.K. Borg, B.Z. Gao, Mesenchymal stem cell-cardiomyocyte interactions under defined contact modes on laser-patterned biochips, PLoS One. 8 (2013) e56554.
[14] C. Sesti, S.L. Hale, C. Lutzko, R.A. Kloner, Granulocyte colony-stimulating factor and stem cell factor improve contractile reserve of the infarcted left ventricle independent of restoring muscle mass, J. Am. Coll. Cardiol. 46 (2005) 1662–1669.
[15] J. Zhang, A. Chen, Y. Wu, Q. Zhao, Placental growth factor promotes cardiac muscle repair via enhanced neovascularization, Cell. Physiol. Biochem. 36 (2015) 947–955.
[16] S. Wang, M. Mo, J. Wang, S. Sadia, B. Shi, X. Fu, L. Yu, E.E. Tredget, Y. Wu, Platelet-derived growth factor receptor beta identifies mesenchymal stem cells with enhanced
engraftment to tissue injury and pro-angiogenic property, Cell. Mol. Life Sci. 75 (2018) 547–561.
[17] S. Windmolders, A. De Boeck, R. Koninckx, A. Daniëls, O. De Wever, M. Bracke, M. Hendrikx, K. Hensen, J.-L. Rummens, Mesenchymal stem cell secreted platelet derived growth factor exerts a pro-migratory effect on resident cardiac atrial appendage stem cells, J. Mol. Cell. Cardiol. 66 (2014) 177–188.
[18] A.D. Berendsen, B.R. Olsen, How vascular endothelial growth factor-A (VEGF) regulates differentiation of mesenchymal stem cells, J. Histochem. Cytochem. 62 (2014) 103–108.
[19] J.-M. Tang, J.-N. Wang, L. Zhang, F. Zheng, J.-Y. Yang, X. Kong, L.-Y. Guo, L. Chen, Y.-Z. Huang, Y. Wan, VEGF/SDF-1 promotes cardiac stem cell mobilization and myocardial repair in the infarcted heart, Cardiovasc. Res. 91 (2011) 402–411.
