Keming Xu, Karthikeyan Narayanan, Fan Lee, Ki Hyun Bae, Shujun Gao, Motoichi Kurisawa, Enzyme-mediated hyaluronic acid–tyramine hydrogels for the propagation of human embryonic stem cells in 3D, Acta Biomaterialia, Volume 24, 2015, Pages 159-171, ISSN 1742-7061, https://doi.org/10.1016/j.actbio.2015.06.026.
Abstract:
The propagation of human embryonic stem cells (hESCs) in three-dimensional (3D) scaffolds facilitates the cell expansion process and supplies pluripotent cells of high quality for broad-spectrum applications in regenerative medicine. Herein, we report an enzyme-mediated hyaluronic acid–tyramine (HA–Tyr) hydrogel that encapsulated and propagated hESCs in 3D. HA–Tyr hydrogels were formed by crosslinking the tyramine moieties with horseradish peroxidase (HRP) and hydrogen peroxide (H2O2). By changing the HRP and H2O2 concentration, we prepared HA–Tyr hydrogels of different mechanical strength and studied the self-renewal properties of hESCs in these scaffolds. We observed that both the chemical composition and mechanical strength of substrates were important factors affecting cell proliferation and pluripotency. The HA–Tyr hydrogel with a compressive modulus of ∼350 Pa supported the proliferation of hESCs at the pluripotent state in both mTeSR1 medium and mouse embryonic fibroblast (MEF)-conditioned medium. Immunohistochemical analyses revealed that hESCs proliferated well and formed spheroid structures in 3D, without undergoing apoptosis. The hESCs cultured in HA–Tyr hydrogels showed high expression of CD44 and pluripotency markers. These cells exhibited the capability to form cell derivatives of all three embryonic germ layers in vitro and in vivo. In addition, the genetic integrity of the hESCs was unaffected in the 3D cultivation system.
License type:
http://creativecommons.org/licenses/by-nc-nd/4.0/
Funding Info:
This research is supported by the Institute of Bioengineering and Nanotechnology (Biomedical Research Council, Agency for Science, Technology and Research (A*STAR), Singapore).