The generation of definitive neural stem cells from PiggyBac transposon-induced pluripotent stem cells can be enhanced by induction of the NOTCH signaling pathway.
Cell-based therapies using neural stem cells (NSCs) have shown positive outcomes in various models of neurological injury and disease. Induced pluripotent stem cells (iPSCs) address many problems associated with NSCs from various sources, including the immune response and cell availability. However, due to inherent differences between embryonic stem cells (ESCs) and iPSCs, detailed characterization of the iPS-derived NSCs will be required before translational experiments can be performed. Murine piggyBac transposon iPSCs were clonally expanded in floating sphere colonies to generate primitive NSCs initially with serum-free media (SFM) containing the leukemia inhibitory factor and followed by SFM with the fibroblast growth factor-2 (FGF2) to form colonies of definitive NSCs (dNSCs). Primitive and definitive clonally derived neurospheres were successfully generated using the default conditions from iPSCs and ESCs. However, the iPSC-dNSCs expressed significantly higher levels of pluripotency and nonectoderm lineage genes compared to equivalent ESC-dNSCs. The addition of the bone morphogenetic proteins antagonist, Noggin, to the media significantly increased primary neurosphere generation from the iPSC lines, but did not affect the dNSC sphere colonies generated. The induction of the NOTCH pathway by the Delta-like ligand 4 (DLL4) improved the generation and quality of dNSCs, as demonstrated by a reduction in pluripotency and nonectodermal markers, while maintaining NSC-specific gene expression. The iPS-dNSCs (+DLL4) showed functional neural differentiation by immuncytochemical staining and electrophysiology. This study suggests the intrinsic differences between ESCs and iPSCs in their ability to acquire a dNSC fate that can be overcome by inducing the NOTCH pathway.
Division of Genetics and Development, Toronto Western Research Institute, Toronto, Canada.
Ryan P
RP
Salewski
Josef
J
Buttigieg
Robert A
RA
Mitchell
Derek
D
van der Kooy
Andras
A
Nagy
Michael G
MG
Fehlings
D000818:Animals; D051792:Basic Helix-Loop-Helix Proteins / Q000235:genetics / Q000378:metabolism; D002352:Carrier Proteins / Q000502:physiology; D002454:Cell Differentiation; D002478:Cells, Cultured; D004251:DNA Transposable Elements / Q000235:genetics*; D004151:Dipeptides / Q000494:pharmacology; D053595:Embryonic Stem Cells / Q000187:drug effects / Q000502:physiology; D057026:Induced Pluripotent Stem Cells / Q000187:drug effects / Q000502:physiology*; D047908:Intracellular Signaling Peptides and Proteins / Q000502:physiology; D008564:Membrane Potentials; D008565:Membrane Proteins / Q000502:physiology; D051379:Mice; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism; D058953:Neural Stem Cells / Q000378:metabolism* / Q000502:physiology; D051881:Receptor, Notch1 / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins; D015398:Signal Transduction; D018874:Spheroids, Cellular / Q000187:drug effects / Q000502:physiology; D059467:Transcriptome; D000099176:Noggin Protein
D051792:Basic Helix-Loop-Helix Proteins; D002352:Carrier Proteins; D004251:DNA Transposable Elements; D004151:Dipeptides; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D051881:Receptor, Notch1; D012097:Repressor Proteins; D000099176:Noggin Protein; C411886:Hes3 protein, mouse; C419410:N-(N-(3,5-difluorophenacetyl)alanyl)phenylglycine tert-butyl ester; C497762:Notch1 protein, mouse; C064045:delta protein