Feeder-free self-renewal of human embryonic stem cells in 3D porous natural polymer scaffolds

Human embryonic stem cells (hESCs) are routinely cultured on fibroblast feeder layers or in fibroblast-conditioned medium, which requires continued supply of feeder cells and poses the risks of xenogenic contamination and other complications such as feeder-dependent outcome. Here, we demonstrate a strategy that supports sustained self-renewal of hESCs in a three-dimensional porous natural polymer scaffold, comprised of chitosan and alginate, without the support of feeder cells or conditioned medium. We assessed the pluripotency of the renewed hESCs both in vitro by evaluation of cellular proliferation, functionality, and gene activities for 21 days, and in vivo by implantation of the stem cell populated scaffolds in an immunodeficient mouse model to induce teratoma formation. The self-renewed stem cells can be easily recovered for subculture by decomposing the scaffold under a mild condition. We further subcultured recovered hESCs for 14 days and verified their pluripotency. In addition to providing a clean environment for stem cell renewal, this strategy, with the demonstrated biocompatibility and biodegradability of chitosan and alginate, may potentially allow for the direct implantation of stem cell populated scaffolds for a broad spectrum of applications in tissue engineering and regenerative medicine.