Therapeutic angiogenesis via solar cell facilitated electrical

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1 Supporting information Therapeutic angiogenesis via solar cell facilitated electrical stimulation Gun-Jae Jeong 1,, Jin Young Oh 2,, Yeon-Ju Kim 3,, Suk Ho Bhang 4, Hyeon-Ki Jang 5, Jin Han 1, Jeong-Kee Yoon 1, Sang-Mo Kwon 3,*, Tae Il Lee 6,*, Byung-Soo Kim 1,7,*. 1 School of Chemical and Biological Engineering, Seoul National University, Seoul, Republic of Korea 2 Department of Chemical Engineering, Stanford University, California, USA. 3 Department of Physiology, School of Medicine, Pusan National University, Yangsan, Republic of Korea 4 School of Chemical Engineering, Sungkyunkwan University, Suwon, Republic of Korea 5 Interdisciplinary Program for Bioengineering, Seoul National University, Seoul, Republic of Korea 6 Department of BioNano Technology, Gachon University, Seongnam, Republic of Korea 7 Institute of Chemical Processes, Seoul National University, Seoul, Republic of Korea These authors contributed equally to this work. *Authors to whom the correspondence should be addressed: Sang-Mo Kwon, Ph.D., smkwon323@hotmail.com, Tel.: , Fax: Tae Il Lee, Ph.D., t2.lee77@gachon.ac.kr, Tel.: , Fax: Byung-Soo Kim, Ph.D., byungskim@snu.ac.kr, Tel.: , Fax:

2 Materials and Methods Apoptosis assay Apoptosis in each cells was measured after 24hr of electric stimulation by using a FITC Annexin V apoptosis detection kit (BD Pharmingen) according to the manufacturer s instructions. After staining, the % of viable, apoptotic and necrotic cells was quantified by flow cytometry (BD accuri C6). Cell cycle analysis For cell cycle analysis, Cells were collected after 24hr of electric stimulation and then washed with PBS, fixed in cold 70% ethanol at 4 C for 1hr, and incubated in 0.2mg/ml RNase A (Sigma-Aldrich, St. Louis, MO, USA) for 1hr at 37 C. Prior to analysis, cells were stained with 10µg/ml propidium iodide (PI; Sigma-Aldrich). Distribution of the cell cycle phase was determined with flow cytometry (BD FACS Canto 2). Data for cell cycle distributions were analyzed using BD FACSDiva software (BD Biosciences, Bedford, MA). Histology and Immunohistochemistry Ischemic limb muscles were embedded in optimal cutting temperature compound (O.C.T. compound, Tissue-Tek 4583; Sakura Finetek USA, Torrance, CA), frozen, and cut into 10- µm sections at 22 C. To examine histological abnormality, the sections were stained with hematoxylin and eosin (H&E) to assess muscle degeneration and tissue inflammation. To examine macrophages distribution in the electrode implantation region, the sections were subjected to immunofluorescent staining by anti-cd68 antibodies (Abcam, Cambridge, UK). Fluorescein isothiocyanate conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA) were used to visualize the signals. The sections were counterstained with 4',6-diamidino-2-phenylindole (DAPI) and examined under a S1

3 fluorescence microscope (Nikon TE2000, Tokyo, Japan). Surface marker expression analysis Cells were subjected to flow cytometry analysis using the following: the labeled endothelial progenitor cell (EPC) markers CD34 (BD Pharmingen, San Jose, CA, USA), c-kit (BD Pharmingen), Flk-1 (BD Pharmingen, San Jose, CA, USA), VE-cadherin (BD Pharmingen), and mesenchymal stem cell (MSC) markers CD90 (BioLegend, San Diego, CA, USA), CD105 (BD Pharmingen), Sca-1 (Thermo Fisher Scientific, Waltham, MA, USA), and skeletal muscle markers Integrin α1 (BioLegend), Integrin α7 (Miltenyi Biotec, Bergisch Gladbach, Germany). S2

4 Figure S1. Evaluation of cell apoptosis using annexin V/PI staining. Cells were treated with 24hr of electrical stimulation (ES). Dot plot profile and the percentage of apoptotic and dead cells were analyzed by FACS. (a) mmsc, (b) m-myoblasts, (c) mepcs, and (d) mecs. Cells cultured without ES served as the controls (Con = control). *P < 0.05 compared with the ES S3

5 group. **P < 0.01 compared with the ES group. PI = propidium iodide. Figure S2. Evaluation of cell cycle using PI staining. Cells were treated with 24hr of S4

6 electrical stimulation (ES). After fixation, the cells were stained with PI and then analyzed using a flow cytometer. (a) mmsc, (b) m-myoblasts, (c) mepcs, and (d) mecs. Cells cultured without ES served as the controls (Con = control). PI = propidium iodide. Figure S3. Histological evaluations of the electrode implantation region. Tissues in the implantation regions were stained with H&E and CD68 immunohistochemistry (green) at 3 days after implantation. Scale bars = 100 µm. Blue indicates nuclei stained with 4,6- diamidino-2-phenylindole (DAPI). E = electrode. S5

7 Figure S4. Phenotypic characterization by FACS analysis of primary cultured cells. (a) The primarily-derived mmscs were CD90 negative, CD105 positive and Sca-1 positive. (b) The primarily-derived mepcs were CD34 negative, c-kit negative, Flk-1 positive and VEcadherin negative. (c) The primarily-derived mmyoblasts were Integrin α1 and Integrin α7 negative. S6