title: A method for efficiently generating neurospheres from human-induced pluripotent stem cells using microsphere arrays.
abstract: In vitro, human neural stem cells can be selectively expanded from fetal or adult neural tissues as neurospheres consisting of immature neural progenitor cells. Access to human neural tissues is limited, making it difficult to propagate and use primary neural stem or progenitor cells (NSPCs) from human neural tissues (hN-NSPCs). It was recently demonstrated that hN-NSPCs can be differentiated from either human embryonic stem cells (hESC-NSPCs) or human-induced pluripotent stem cells (hiPSC-NSPCs), and that hESC-NSPCs and hiPSC-NSPCs are adaptable, powerful substitutes for hN-NSPCs in both regenerative medicine and pharmacological or neurotoxicological assays. We here describe a new protocol to generate neurospheres consisting of hiPSC-NSPCs using microsphere arrays, the surface of which is modified with polyethylene glycol to render it nonadhesive to cells. Primary hiPSCs treated with noggin formed neurospheres on the microsphere arrays and could be stably propagated as free-floating spheroids. The hiPSC-NSPCs proliferating in these neurospheres were almost identical in phenotype to hN-NSPCs, in both cell-surface marker expression and their ability to differentiate into neuronal cells, although gene expression profiles showed that the hiPSC-NSPCs had higher neural and lower glial gene expression, along with mid-hindbrain-like regional specificity. This convenient propagation protocol can be used to evaluate the neurosphere-forming efficiency of hiPSC clones. This method will support the generation of neurospheres from hESCs and hiPSCs and contribute to the use of hESC-NSPCs and hiPSC-NSPCs in research.
authors.affiliation: Division of Stem Cell Research, Institute for Clinical Research, Osaka National Hospital, National Hospital Organization, Osaka, Japan.
authors.forename: Tomoko
authors.initials: T
authors.lastname: Shofuda
authors.forename: Hayato
authors.initials: H
authors.lastname: Fukusumi
authors.forename: Daisuke
authors.initials: D
authors.lastname: Kanematsu
authors.forename: Atsuyo
authors.initials: A
authors.lastname: Yamamoto
authors.forename: Mami
authors.initials: M
authors.lastname: Yamasaki
authors.forename: Norio
authors.initials: N
authors.lastname: Arita
authors.forename: Yonehiro
authors.initials: Y
authors.lastname: Kanemura
mesh_terms: D018929:Cell Culture Techniques / Q000379:methods; D002454:Cell Differentiation; D002460:Cell Line; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000378:metabolism; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology*; D008863:Microspheres; D058953:Neural Stem Cells / Q000166:cytology*; D009474:Neurons / Q000166:cytology; D044968:Regenerative Medicine / Q000379:methods