title: Rapid and efficient generation of functional motor neurons from human pluripotent stem cells using gene delivered transcription factor codes.
abstract: Stem cell-derived motor neurons (MNs) are increasingly utilized for modeling disease in vitro and for developing cellular replacement strategies for spinal cord injury and diseases such as spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS). Human embryonic stem cell (hESC) differentiation into MNs, which involves retinoic acid (RA) and activation of the sonic hedgehog (SHH) pathway is inefficient and requires up to 60 days to develop MNs with electrophysiological properties. This prolonged differentiation process has hampered the use of hESCs, in particular for high-throughput screening. We evaluated the MN gene expression profile of RA/SHH-differentiated hESCs to identify rate-limiting factors involved in MN development. Based on this analysis, we developed an adenoviral gene delivery system encoding for MN inducing transcription factors: neurogenin 2 (Ngn2), islet-1 (Isl-1), and LIM/homeobox protein 3 (Lhx3). Strikingly, delivery of these factors induced functional MNs with mature electrophysiological properties, 11-days after gene delivery, with >60-70% efficiency from hESCs and human induced pluripotent stem cells (hiPSCs). This directed programming approach significantly reduces the time required to generate electrophysiologically-active MNs by approximately 30 days in comparison to conventional differentiation techniques. Our results further exemplify the potential to use transcriptional coding for rapid and efficient production of defined cell types from hESCs and hiPSCs.
authors.affiliation: Center for Gene Therapy, The Research Institute at Nationwide Children's Research Institute, Columbus, Ohio, USA.
authors.forename: Mark E
authors.initials: ME
authors.lastname: Hester
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Murtha
authors.forename: SungWon
authors.initials: S
authors.lastname: Song
authors.forename: Meghan
authors.initials: M
authors.lastname: Rao
authors.forename: Carlos J
authors.initials: CJ
authors.lastname: Miranda
authors.forename: Kathrin
authors.initials: K
authors.lastname: Meyer
authors.forename: Jinbin
authors.initials: J
authors.lastname: Tian
authors.forename: Gabriella
authors.initials: G
authors.lastname: Boulting
authors.forename: David V
authors.initials: DV
authors.lastname: Schaffer
authors.forename: Michael X
authors.initials: MX
authors.lastname: Zhu
authors.forename: Samuel L
authors.initials: SL
authors.lastname: Pfaff
authors.forename: Fred H
authors.initials: FH
authors.lastname: Gage
authors.forename: Brian K
authors.initials: BK
authors.lastname: Kaspar
mesh_terms: D002454:Cell Differentiation; D002460:Cell Line; D053595:Embryonic Stem Cells / Q000166:cytology; D020869:Gene Expression Profiling; D006801:Humans; D009046:Motor Neurons / Q000166:cytology* / Q000378:metabolism; D039904:Pluripotent Stem Cells / Q000473:pathology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
chemical_list: D014157:Transcription Factors