title: Efficient neuronal differentiation and maturation of human pluripotent stem cells encapsulated in 3D microfibrous scaffolds.
abstract: Developing an efficient culture system for controlled human pluripotent stem cell (hPSC) differentiation into selected lineages is a major challenge in realizing stem cell-based clinical applications. Here, we report the use of chitin-alginate 3D microfibrous scaffolds, previously developed for hPSC propagation, to support efficient neuronal differentiation and maturation under chemically defined culture conditions. When treated with neural induction medium containing Noggin/retinoic acid, the encapsulated cells expressed much higher levels of neural progenitor markers SOX1 and PAX6 than those in other treatment conditions. Immunocytochemisty analysis confirmed that the majority of the differentiated cells were nestin-positive cells. Subsequently transferring the scaffolds to neuronal differentiation medium efficiently directed these encapsulated neural progenitors into mature neurons, as detected by RT-PCR and positive immunostaining for neuron markers βIII tubulin and MAP2. Furthermore, flow cytometry confirmed that >90% βIII tubulin-positive neurons was achieved for three independent iPSC and hESC lines, a differentiation efficiency much higher than previously reported. Implantation of these terminally differentiated neurons into SCID mice yielded successful neural grafts comprising MAP2 positive neurons, without tumorigenesis, suggesting a potential safe cell source for regenerative medicine. These results bring us one step closer toward realizing large-scale production of stem cell derivatives for clinical and translational applications.
authors.affiliation: Institute of Bioengineering and Nanotechnology, 31 Biopolis Way, The Nanos, Singapore 138669, Singapore. hflu@ibn.a-star.edu.sg
authors.forename: Hong Fang
authors.initials: HF
authors.lastname: Lu
authors.forename: Sze-Xian
authors.initials: SX
authors.lastname: Lim
authors.forename: Meng Fatt
authors.initials: MF
authors.lastname: Leong
authors.forename: Karthikeyan
authors.initials: K
authors.lastname: Narayanan
authors.forename: Rebecca P K
authors.initials: RP
authors.lastname: Toh
authors.forename: Shujun
authors.initials: S
authors.lastname: Gao
authors.forename: Andrew C A
authors.initials: AC
authors.lastname: Wan
mesh_terms: D002454:Cell Differentiation / Q000502:physiology; D002460:Cell Line; D002470:Cell Survival / Q000235:genetics / Q000502:physiology; D005136:Eye Proteins / Q000235:genetics / Q000378:metabolism; D005434:Flow Cytometry; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D009416:Nerve Regeneration / Q000502:physiology; D000071841:PAX6 Transcription Factor; D051761:Paired Box Transcription Factors / Q000235:genetics / Q000378:metabolism; D039904:Pluripotent Stem Cells / Q000166:cytology*; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D055748:SOXB1 Transcription Factors / Q000235:genetics / Q000378:metabolism; D054457:Tissue Scaffolds / Q000737:chemistry*
chemical_list: D005136:Eye Proteins; D018398:Homeodomain Proteins; D000071841:PAX6 Transcription Factor; C000605730:PAX6 protein, human; D051761:Paired Box Transcription Factors; C000605732:Pax6 protein, mouse; D012097:Repressor Proteins; C490906:SOX1 protein, human; D055748:SOXB1 Transcription Factors