Cathay General Hospital 1 article published in JoVE Bioengineering Human Pluripotent Stem Cell Culture on Polyvinyl Alcohol-Co-Itaconic Acid Hydrogels with Varying Stiffness Under Xeno-Free Conditions Tzu-Cheng Sung*1, Hsing-Fen Li*1, Akon Higuchi1,2, Qing-Dong Ling3,4, Jia-Sin Yang1, Yeh-Chia Tseng1, Chih-Hsien Pan Pan1, Abdullah A. Alarfaj2, Murugan A. Munusamy2, Suresh Kumar5, Shih-Tien Hsu6, Kadarkarai Murugan7,8 1Department of Chemical and Materials Engineering, National Central University, 2Department of Botany and Microbiology, King Saud University, 3Cathay Medical Research Institute, Cathay General Hospital, 4Graduate Institute of Systems Biology and Bioinformatics, National Central University, 5Department of Medical Microbiology and Parasitology, Universiti Putra Malaysia, 6Department of Internal Medicine, Taiwan Landseed Hospital, 7Department of Zoology, Bharathiar University, 8Thiruvalluvar University A protocol is presented to prepare polyvinyl alcohol-co-itaconic acid hydrogels with varying stiffness, which were grafted with and without oligopeptides, to investigate the effect of the stiffness of biomaterials on the differentiation and proliferation of stem cells. The stiffness of the hydrogels was controlled by the crosslinking time.