title: Alginate Microfiber System for Expansion and Direct Differentiation of Human Embryonic Stem Cells.
abstract: Pluripotent human embryonic stem cells (hESCs) are a potential renewable cell source for regenerative medicine and drug testing. To obtain adequate cell numbers for these applications, there is a need to develop scalable cell culture platforms to propagate hESCs. In this study, we encapsulated hESCs in calcium alginate microfibers as single cells, for expansion and differentiation under chemically defined conditions. hESCs were suspended in 1% (w/v) alginate solution at high cell density (>10(7) cells/mL) and extruded at 5 m/min into a low calcium concentration bath (10 mM) for gelation. Mild citrate buffer (2.5 mM), which did not affect hESCs viability, was used to release the cells from the calcium alginate hydrogel. Encapsulation as single cells was critical, as this allowed the hESCs to grow in the form of relatively small and uniform aggregates. This alginate microfiber system allowed for expansion of an hESC line, HUES7, for up to five passages while maintaining pluripotency. Immunohistochemistry, polymerase chain reaction, and other analyses showed that passage 5 (P5) HUES7 cells expressed proteins and genes characteristic of pluripotent stem cells, possessed normal karyotype, and were able to form representative tissues of the three embryonic germ layers in vitro and in vivo. Encapsulated HUES7 cells at P5 could also be induced to directly differentiate into liver-like cells. Collectively, our experiments show that the alginate microfiber system can be used as a three-dimensional cell culture platform for long-term expansion and differentiation of hESCs under defined conditions.
authors.affiliation: Institute of Bioengineering and Nanotechnology , Singapore, Singapore .
authors.forename: Meng Fatt
authors.initials: MF
authors.lastname: Leong
authors.affiliation: Institute of Bioengineering and Nanotechnology , Singapore, Singapore .
authors.forename: Hong Fang
authors.initials: HF
authors.lastname: Lu
authors.affiliation: Institute of Bioengineering and Nanotechnology , Singapore, Singapore .
authors.forename: Tze Chiun
authors.initials: TC
authors.lastname: Lim
authors.affiliation: Institute of Bioengineering and Nanotechnology , Singapore, Singapore .
authors.forename: Karthikeyan
authors.initials: K
authors.lastname: Narayanan
authors.affiliation: Institute of Bioengineering and Nanotechnology , Singapore, Singapore .
authors.forename: Shujun
authors.initials: S
authors.lastname: Gao
authors.affiliation: Institute of Bioengineering and Nanotechnology , Singapore, Singapore .
authors.forename: Luna Yue
authors.initials: LY
authors.lastname: Wang
authors.affiliation: Institute of Bioengineering and Nanotechnology , Singapore, Singapore .
authors.forename: Rebecca P K
authors.initials: RP
authors.lastname: Toh
authors.affiliation: Institute of Bioengineering and Nanotechnology , Singapore, Singapore .
authors.forename: Henrik
authors.initials: H
authors.lastname: Funke
authors.affiliation: Institute of Bioengineering and Nanotechnology , Singapore, Singapore .
authors.forename: Muhammad Hamizan
authors.initials: MH
authors.lastname: Abdul Samad
authors.affiliation: Institute of Bioengineering and Nanotechnology , Singapore, Singapore .
authors.forename: Andrew C A
authors.initials: AC
authors.lastname: Wan
authors.affiliation: Institute of Bioengineering and Nanotechnology , Singapore, Singapore .
authors.forename: Jackie Y
authors.initials: JY
authors.lastname: Ying
mesh_terms: D000464:Alginates / Q000737:chemistry*; D002454:Cell Differentiation; D002478:Cells, Cultured; D018914:Cells, Immobilized; D020723:Glucuronic Acid / Q000737:chemistry; D006603:Hexuronic Acids / Q000737:chemistry; D000066449:Human Embryonic Stem Cells / Q000166:cytology*; D006801:Humans
chemical_list: D000464:Alginates; D006603:Hexuronic Acids; D020723:Glucuronic Acid