Supporting Information: Enhanced Therapeutic Effects of MSC-derived Exosomes with an Injectable Hydrogel for Hindlimb Ischemia Treatment

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1 Supporting Information: Enhanced Therapeutic Effects of MSC-derived Exosomes with an Injectable Hydrogel for Hindlimb Ischemia Treatment Kaiyue Zhang 1,2, Xiangnan Zhao 1, Xiaoniao Chen 3, Yongzhen Wei 2, Wei Du 1, Yuebing Wang 1, Linan Liu 4, Weian Zhao 4, Zhibo Han 5,6, Deling Kong 2, Qiang Zhao 2, Zhikun Guo 7, Zhongchao Han 5, Na Liu 1, Fengxia Ma 6* 1,,2, 8*, Zongjin Li 1 Nankai University School of Medicine, Tianjin, China; 2 The Key Laboratory of Bioactive Materials, Ministry of Education, Nankai University, the College of Life Science, Tianjin, China; 3 Department of Ophthalmology, Chinese PLA General Hospital, Beijing, China; 4 Department of Pharmaceutical Sciences, Department of Biomedical Engineering, Sue and Bill Gross Stem Cell Research Center, Chao Family Comprehensive Cancer Center & Edwards Lifesciences Center for Advanced Cardiovascular Technology, and Department of Biological Chemistry University of California, Irvine, USA; 5 Beijing Engineering Laboratory of Perinatal Stem Cells, Beijing Institute of Health and Stem Cells, Health & Biotech Co., Beijing, China; 6 State Key Lab of Experimental Hematology, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin, China; 7 Henan Key Laboratory of Medical Tissue Regeneration, Xinxiang Medical University, Xinxiang, China; 8 State Key Laboratory of Kidney Diseases, Chinese PLA General Hospital, Beijing, China Corresponding Author * Zongjin Li, zongjinli@nankai.edu.cn * Fengxia Ma, mafengxia1978@163.com S-1

2 Table S-1. Primers used in the qpcr assay Gene Forward primer sequence, 5-3 Reverse primer sequence, 5-3 VEGFA TGTCTAATGCCCTGGAGCCT GTCACATCTGCAAGTACGTTCG VEGFR2 CAAGTGGCTAAGGGCATGGA ATTTCAAAGGGAGGCGAGCA ANG1 CGCCGAAGTCCAGAAAACAG GGGAAGAGAAATCCGGTTCCA ANG2 GCTCGAATACGATGACTCGGT GTTTGCTCCGCTGTTTGGTT enos ATCTTCAGCCCCAAACGGAG CTGGAACATCTTCCGCCTGT BCL2 GGTCATGTGTGTGGAGAGCG GGTGCCGGTTCAGGTACTCA BAX GATGCGTCCACCAAGAAGCT CGGCCCCAGTTGAAGTTG CASP3 TGGTTCATCCAGTCGCTTTG CATTCTGTTGCCACCTTTCG CASP8 CTGCTGGGGATGGCCACTGTG TCGCCTCGAGGACACGCTCTC CASP9 CGAACTAACAGGCAAGCAGC ACCTCACCAAATCCTCCAGAAC GAPDH AGGGCTGCTTTTAACTCTGGT CCCCACTTGATTTTGGAGGGA Shown is the sequences of primers in 5-3 orientation for real-time qpcr used in this study. S-2

3 Supporting Figures and Captions Figure S-1 Figure S-1. Identification of exosomes labeled with Gluc-lactadherin fusion proteins. (A) Schematic representation of plv-gluc and plv-gluc-lactadherin plasmids. (B) BLI images of exosomes and exosome-free proteins isolated from hp-mscs transfected with plv-gluc or plv-gluc-lactadherin plasmid. (C) Internalization of exosomes and exosome-free proteins detected by Gluc signals. Data were expressed as the mean ± SEM. All experiments were performed in three independent experiments triplicate and were shown as the mean ± SEM. (n=3; ** P < 0.01) S-3

4 Figure S-2 Figure S-2. Characterization of thermo-sensitive chitosan hydrogel. (A) Dynamic rheological properties of chitosan hydrogel including frequency sweep and strain sweep at 37 C. (B) Swelling and degradation ratio of chitosan hydrogel at 37 C. All experiments were performed in three independent experiments triplicate and were shown as the mean ± SEM. S-4

5 Figure S-3 Figure S-3. The expression of apoptosis-related genes following administrating exosomes to HUVECs under challenge from H 2 O 2 -induced stress. Real-time qpcr analysis of apoptosis relative genes in HUVECs treated with 100 μg/ml exosomes upon exposure to 400 μm H 2 O 2 for 12 h. Relative gene expression was normalized to GAPDH, and data were analyzed via the 2 - ΔΔ Ct method. All experiments were performed in three independent experiments and were shown as the mean ± SEM. (n=3; * P < 0.05). S-5

6 Figure S-4 Figure S-4. The expression of apoptosis-related genes following administrating exosomes or CS-Exo to HUVECs under challenge from H 2 O 2 -induced stress. All experiments were performed in three independent experiments and were shown as the mean ± SEM. (n=3; * P < 0.05). S-6

7 Figure S-5 Figure S-5. The total length and meshes number of capillary-like tubes structure in tube formation assay. The data were measured in 3 random fields in each group. All experiments were performed in three independent experiments triplicate and were shown as the mean ± SEM. (n=3; * P < 0.05; ** P < 0.01 versus PBS; # P < 0.05 versus Exo). S-7

8 Figure S-6 Figure S-6. Pathological status of ischemic hindlimbs. Pathological status of ischemic hindlimbs 28 days after treatments. S-8