Despite the fact that porcine embryonic stem cells (ESCs) are a practical study tool, in vitro long-term maintenance of these cells is difficult in a two-dimensional (2D) microenvironment using cellular niche or extracellular matrix proteins. However, a three-dimensional (3D) microenvironment, similar to that enclosing the inner cell mass of the blastocyst, may improve in vitro maintenance of self-renewal. Accordingly, as a first step toward constructing a 3D microenvironment optimized to maintain porcine ESC self-renewal, we investigated different culture dimensions for porcine ICM-derived cells to enhance the maintenance of self-renewal. Porcine ICM-derived cells were cultured in agarose-based 3D hydrogel with self-renewal-friendly mechanics and in 2D culture plates with or without feeder cells. Subsequently, the effects of the 3D microenvironment on maintenance of self-renewal were identified by analyzing colony formation and morphology, alkaline phosphatase (AP) activity, and transcriptional and translational regulation of self-renewal-related genes. The 3D microenvironment using a 1.5% (w/v) agarose-based 3D hydrogel resulted in significantly more colonies with stereoscopic morphology, significantly improved AP activity, and increased protein expression of self-renewal-related genes compared to those in the 2D microenvironment. These results demonstrate that self-renewal of porcine ICM-derived cells can be maintained more effectively in a 3D microenvironment than in a 2D microenvironment. These results will help develop novel culture systems for ICM-derived cells derived from diverse species, which will contribute to stimulating basic and applicable studies related to ESCs.
Keywords: agarose; culture dimension; embryonic stem cells; pig; self-renewal.