Long term, feeder free maintenance of human embryonic stem cells by mussel inspired adhesive heparin and collagen type I.
UNLABELLED For practical applications of human embryonic stem cells (hESCs) in regenerative medicine, hESCs should be cultured on a large scale, and at the same time their properties have to be maintained in a controllable manner. Here, we report a chemically defined, scalable culture platform involving co immobilization of heparin catechol (HepC) and collagen type 1 (Col) for the long term maintenance >18 passages) of hESCs in a feeder free condition. This platform utilizes a wet adhesive, mussel inspired heparin catechol conjugate as a key component. We hypothesized that the heparin's affinity toward a wide range of proteins, might support undifferentiated in vitro growth of hESC. In fact, on the HepC coated substrate, most hESC clumps were adhered ~78% at passage 2 (P2) and expressed pluripotency markers (Fig. 2) Although HepC alone wasn't able to support long term maintenance of hESCs in a feeder free system due to decrease in the adhesion rate of hESCs on HepC coating 44% at P4) during the repeated passaging processes, we found that when collagen type I was co immobilized in the process of HepC coating, the long term maintenance (passage 18 or more) of hESCs could be achieved with 100% adhesion efficiency (Fig. 4) One remarkable observation is that hESCs on collagen type I underwent spontaneous differentiation after P6 (Fig. 3) which implied co immobilized HepC played a role to suppress differentiation of hESCs. This study suggests that unlike the previous studies using proteins, peptides, or synthetic polymers, a polysaccharide, heparin, can be used as a cost effective component for chemically defined, feeder free culture of hESC. STATEMENT OF SIGNIFICANCE Towards practical applications of human embryonic stem cells (hESCs) in regenerative medicine, hESCs should be cultured on a large scale, and their pluripotent property has to be maintained in a controllable manner. To address these issues, studies that develop chemically defined culture substrates have been explored to replace the widely used, complex, and undefined culture materials represented by Matrigel. Most reports have focused on utilizing proteins, peptides and/or synthetic polymers. However, there have not yet been studies on using polysaccharides as two dimensional coating materials to potentially replace Matrigel coating. Here, we report that heparin is an effective polysaccharide for the feeder free, two dimensional culture of hESCs. Our study implies that use of polysaccharides or a polysaccharide/ECM combination can be a new, alternative design principle for hESC culture systems.
Acta biomaterialia