ABSTRACT
Fate-specific differentiation of neural progenitors attracts keen interest in modern medicine due to its application in cell replacement therapy. Though various signaling pathways are involved in maintenance and differentiation of neural progenitors, the mechanism of development of lineage-restricted progenitors from embryonic stem (ES) cells is not clearly understood. Here, we have demonstrated that neuronal vs. glial differentiation potential of ES cell-derived neural progenitors (ES-NPs) are governed by the growth factors, exposed during their proliferation/expansion phase and cannot be significantly altered during differentiation phase. Exposure of ES-NPs to fibroblast growth factor-2 (FGF2) during proliferation triggered the expression of pro-neural genes that are required for neuronal lineage commitment, and upon differentiation, predominantly generated neurons. On the other hand, epidermal growth factor (EGF)-exposed ES-NPs are not committed to neuronal fate due to decreased expression of pro-neural genes. These ES-NPs further generate more glial cells due to expression of glial-restricted factors. Exposure of ES-NPs to the same growth factors during proliferation/expansion and differentiation phase augments the robust differentiation of neurons or glial subtypes. We also demonstrate that, during differentiation, exposure to growth factors other than that in which the ES-NPs were expanded does not significantly alter the fate of ES-NPs. Thus, we conclude that FGF2 and EGF determine the neural vs. glial fate of ES-NPs during proliferation and augment it during differentiation. Further modification of these protocols would help in generating fate-specified neurons for various regenerative therapies.
Subject(s)
Cell Proliferation/drug effects , Embryonic Stem Cells/drug effects , Epidermal Growth Factor/pharmacology , Fibroblast Growth Factor 2/pharmacology , Neural Stem Cells/drug effects , Neuroglia/drug effects , Neurons/drug effects , Animals , Cell Differentiation/drug effects , Embryonic Stem Cells/cytology , Embryonic Stem Cells/physiology , Mice , Molecular Sequence Data , Neural Stem Cells/cytology , Neural Stem Cells/physiology , Neuroglia/cytology , Neuroglia/physiology , Neurons/cytology , Neurons/physiologyABSTRACT
Hes-1 and Hes-5 are downstream effectors of Notch signaling that are known to be involved in different aspects of neural stem cell proliferation and differentiation. Evidence has emerged that Hes-1 expression can be regulated by alternate signaling pathways independent of canonical Notch/CBF1 interaction. This context-dependent differential regulation of Hes-1 expression in neural progenitor gains a lot of importance as it would help in its exponential expansion without the requirement of interaction from neighboring cells during development. Here, we have clearly demonstrated the existence of a population of neural progenitors with Notch/CBF1-independent Hes-1 expression in vitro. Further analysis demonstrated the role of FGF2 in activating Hes-1 expression through the direct binding of ATF2, a JNK downstream target, on Hes-1 promoter. This raises the possibility for the existence of two distinct populations of neural progenitors - one maintained by Hes-1 expression exclusively through Notch-independent mechanism and the other mediating Hes-1 expression through both canonical Notch and FGF2-ATF2 pathway. This alternative pathway will insure a constant expression of Hes-1 even in the absence of canonical Notch intracellular domain-mediated signaling, thereby maintaining a pool of proliferating neural progenitors required during development.
Subject(s)
Activating Transcription Factor 2/metabolism , Basic Helix-Loop-Helix Transcription Factors/metabolism , Central Nervous System/embryology , Homeodomain Proteins/metabolism , Neurons/metabolism , Receptors, Notch/metabolism , Stem Cells/metabolism , Activating Transcription Factor 2/genetics , Animals , Basic Helix-Loop-Helix Transcription Factors/genetics , Cell Differentiation/physiology , Cell Line , Fibroblast Growth Factor 2/genetics , Fibroblast Growth Factor 2/metabolism , Gene Expression Regulation, Developmental/physiology , Homeodomain Proteins/genetics , Immunoglobulin J Recombination Signal Sequence-Binding Protein/genetics , Immunoglobulin J Recombination Signal Sequence-Binding Protein/metabolism , JNK Mitogen-Activated Protein Kinases/genetics , JNK Mitogen-Activated Protein Kinases/metabolism , Mice , Neurons/cytology , Promoter Regions, Genetic/genetics , Receptors, Notch/genetics , Signal Transduction/physiology , Stem Cells/cytology , Transcription Factor HES-1 , Transcriptional Activation/physiologyABSTRACT
ES cells have been reported to serve as an excellent source for obtaining various specialized cell types and could be used in cell replacement therapy. Here, we demonstrate the potential of ES cells to differentiate along retinal ganglion cell (RGC) lineage. FGF2-induced ES cell derived neural progenitors (ES-NPs) were able to generate RGC-like cells in vitro upon differentiation. These cells expressed RGC regulators and markers such as, Ath5, Brn3b, RPF-1, Thy-1 and Islet-1, confirming their potential to differentiate into RGCs. The generation of RGCs from ES-NPs was enhanced with the exposure of FGF2 and Sonic hedgehog (Shh), although Shh treatment alone did not affect RGC differentiation significantly. ES-NPs, after exposure to FGF2, were capable of integrating and differentiating into RGCs in vivo upon transplantation. Thus, our study suggests that ES cells can serve an excellent renewable source for generating RGCs that can be used to treat neurodegenerative diseases like glaucoma.
