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The generation of definitive neural stem cells from PiggyBac transposon-induced pluripotent stem cells can be enhanced by induction of the NOTCH signaling pathway.

ABSTRACT: 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.

SUBMITTER: Salewski RP

PROVIDER: S-EPMC3549637 | biostudies-literature | 2013 Feb

REPOSITORIES: biostudies-literature

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The generation of definitive neural stem cells from PiggyBac transposon-induced pluripotent stem cells can be enhanced by induction of the NOTCH signaling pathway.

Salewski Ryan P RP Buttigieg Josef J Mitchell Robert A RA van der Kooy Derek D Nagy Andras A Fehlings Michael G MG

Stem cells and development 20120917 3

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 ...[more]

PMID: 22889305

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Project description:BackgroundDecades of efforts have attempted to differentiate the pluripotent stem cells (PSCs) into truly functional hematopoietic stem cells (HSCs), yet the problems of low differentiation efficiency in vitro and poor hematopoiesis reconstitution in vivo still exist, mainly attributing to the lack of solid, reproduced, or pursued differentiation system.MethodsIn this study, we established an in vitro differentiation system yielding in vivo hematopoietic reconstitution hematopoietic cells from mouse PSCs through a 3D induction system followed by coculture with OP9 stromal cells. The in vivo hematopoietic reconstitution potential of c-kit+ cells derived from the mouse PSCs was evaluated via m-NSG transplantation assay. Flow cytometry analysis, RNA-seq, and cell cycle analysis were used to detect the in vitro hematopoietic ability of endothelial protein C receptor (EPCR, CD201) cells generated in our induction system.ResultsThe c-kit+ cells from 3D self-assembling peptide induction system followed by the OP9 coculture system possessed apparently superiority in terms of in vivo repopulating activity than that of 3D induction system followed by the 0.1% gelatin culture. We interestingly found that our 3D+OP9 system enriched a higher percentage of CD201+c-kit+cells that showed more similar HSC-like features such as transcriptome level and CFU formation ability than CD201-c-kit+cells, which have not been reported in the field of mouse PSCs hematopoietic differentiation. Moreover, CD201+ hematopoietic cells remained in a relatively slow cycling state, consistent with high expression levels of P57 and Ccng2. Further, we innovatively demonstrated that notch signaling pathway is responsible for in vitro CD201+ hematopoietic cell induction from mouse PSCs.ConclusionsAltogether, our findings lay a foundation for improving the efficiency of hematopoietic differentiation and generating in vivo functional HSC-like cells from mouse PSCs for clinical application.

Dataset Information

The generation of definitive neural stem cells from PiggyBac transposon-induced pluripotent stem cells can be enhanced by induction of the NOTCH signaling pathway.

Publications

The generation of definitive neural stem cells from PiggyBac transposon-induced pluripotent stem cells can be enhanced by induction of the NOTCH signaling pathway.

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