title: Improved method of producing human neural progenitor cells of high purity and in large quantities from pluripotent stem cells for transplantation studies.
abstract: Transplantation of human neural progenitor cells (hNPCs) is a promising therapeutic approach for various diseases of the central nervous system (CNS). Reliable testing of hNPC transplantation in animal models of neurological diseases requires that these cells can be produced in sufficient amounts, show consistent homogeneity as a neural cell population, and be reliably labeled for in vivo tracking. In addition, the cells should be characterized as being at the optimal state of differentiation favoring successful engraftment. Here, we show that high numbers of purified hNPCs can be produced from human embryonic stem cells (hESCs) by manually selecting specifically sized and shaped spheres followed by fluorescence-activated cell sorting based on the relative cell size. In addition, we report that labeling of hNPCs with ultra-small superparamagnetic iron oxide (USPIO) particles does not affect the cellular morphology or growth. More importantly, we show that the transduction with lentiviral vector encoding green fluorescent protein (GFP) decreases the neurality of the cell population. We conclude that our cost-effective protocol of generating hNPCs is widely applicable for preclinical studies on CNS disorders. This improved method of producing large quantities of high-purity hNPCs maybe useful also when generating hNPCs from human induced pluripotent stem (hiPS) cell lines. However, caution should be used when lenti-GFP transduction is applied for hNPC labeling.
authors.affiliation: Department of Neurobiology, A. I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, Kuopio, Finland.
authors.forename: Katja A
authors.initials: KA
authors.lastname: Puttonen
authors.forename: Marika
authors.initials: M
authors.lastname: Ruponen
authors.forename: Riitta
authors.initials: R
authors.lastname: Kauppinen
authors.forename: Sara
authors.initials: S
authors.lastname: Wojciechowski
authors.forename: Outi
authors.initials: O
authors.lastname: Hovatta
authors.forename: Jari
authors.initials: J
authors.lastname: Koistinaho
mesh_terms: D002454:Cell Differentiation; D048429:Cell Size; D003470:Culture Media; D000089622:Doublecortin Domain Proteins; D053595:Embryonic Stem Cells / Q000166:cytology*; D005136:Eye Proteins / Q000235:genetics / Q000378:metabolism; D052203:Ferrosoferric Oxide / Q000737:chemistry; D005434:Flow Cytometry; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D016086:Lentivirus / Q000235:genetics; D058185:Magnetite Nanoparticles / Q000737:chemistry; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism; D000071317:Nanog Homeobox Protein; D058953:Neural Stem Cells / Q000166:cytology* / Q000378:metabolism / Q000637:transplantation; D009479:Neuropeptides / Q000235:genetics / Q000378:metabolism; D000071841:PAX6 Transcription Factor; D051761:Paired Box Transcription Factors / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D003470:Culture Media; D000089622:Doublecortin Domain Proteins; D005136:Eye Proteins; D018398:Homeodomain Proteins; C489788:MAP2 protein, human; D058185:Magnetite Nanoparticles; D008869:Microtubule-Associated Proteins; C486780:NANOG protein, human; D000071317:Nanog Homeobox Protein; D009479:Neuropeptides; D000071841:PAX6 Transcription Factor; C000605730:PAX6 protein, human; D051761:Paired Box Transcription Factors; D012097:Repressor Proteins; D049452:Green Fluorescent Proteins; D052203:Ferrosoferric Oxide