title: Long-term, feeder-free maintenance of human embryonic stem cells by mussel-inspired adhesive heparin and collagen type I.
abstract: 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Chemistry, KAIST, 291 University Rd., Yuseong-gu, Daejeon 305-701, Republic of Korea.
authors.forename: Mihyun
authors.initials: M
authors.lastname: Lee
authors.affiliation: Department of Biological Sciences, 291 University Rd., Yuseong-gu, Daejeon 305-701, Republic of Korea.
authors.forename: Youngjin
authors.initials: Y
authors.lastname: Kim
authors.affiliation: The Graduate School of Nanoscience and Technology, 291 University Rd., Yuseong-gu, Daejeon 305-701, Republic of Korea.
authors.forename: Ji Hyun
authors.initials: JH
authors.lastname: Ryu
authors.affiliation: The Graduate School of Nanoscience and Technology, 291 University Rd., Yuseong-gu, Daejeon 305-701, Republic of Korea.
authors.forename: Kyuri
authors.initials: K
authors.lastname: Kim
authors.affiliation: Department of Biological Sciences, 291 University Rd., Yuseong-gu, Daejeon 305-701, Republic of Korea. Electronic address: ymhan@kaist.ac.kr.
authors.forename: Yong-Mahn
authors.initials: YM
authors.lastname: Han
authors.affiliation: Department of Chemistry, KAIST, 291 University Rd., Yuseong-gu, Daejeon 305-701, Republic of Korea. Electronic address: haeshin@kaist.ac.kr.
authors.forename: Haeshin
authors.initials: H
authors.lastname: Lee
mesh_terms: D000269:Adhesives / Q000494:pharmacology*; D000818:Animals; D049872:Bivalvia / Q000737:chemistry*; D002460:Cell Line; D018914:Cells, Immobilized / Q000166:cytology / Q000187:drug effects; D020099:Coated Materials, Biocompatible / Q000494:pharmacology; D024042:Collagen Type I / Q000494:pharmacology*; D016326:Extracellular Matrix Proteins / Q000378:metabolism; D061252:Feeder Cells / Q000166:cytology*; D006493:Heparin / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D000066449:Human Embryonic Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D011485:Protein Binding / Q000187:drug effects; D051381:Rats; D013997:Time Factors
chemical_list: D000269:Adhesives; D020099:Coated Materials, Biocompatible; D024042:Collagen Type I; D016326:Extracellular Matrix Proteins; D006493:Heparin
keywords: Collagen type-I; Dopamine; Feeder-free culture; Heparin; Human embryonic stem cell