Abstract
Human embryonic stem cells (hESCs) can potentially differentiate into any cell type, including dopaminergic neurons to treat Parkinson's disease (PD), but hyperproliferation and tumor formation must be avoided. Accordingly, we use myocyte enhancer factor 2C (MEF2C) as a neurogenic and anti-apoptotic transcription factor to generate neurons from hESC-derived neural stem/progenitor cells (NPCs), thus avoiding hyperproliferation. Here, we report that forced expression of constitutively active MEF2C (MEF2CA) generates significantly greater numbers of neurons with dopaminergic properties in vitro. Conversely, RNAi knockdown of MEF2C in NPCs decreases neuronal differentiation and dendritic length. When we inject MEF2CA-programmed NPCs into 6-hydroxydopamine—lesioned Parkinsonian rats in vivo, the transplanted cells survive well, differentiate into tyrosine hydroxylase-positive neurons, and improve behavioral deficits to a significantly greater degree than non-programmed cells. The enriched generation of dopaminergic neuronal lineages from hESCs by forced expression of MEF2CA in the proper context may prove valuable in cell-based therapy for CNS disorders such as PD.
Highlights
Human embryonic stem cells are an important model system for human neurogenesis [1,2] but are a potential source of therapeutic cells to treat neurodegenerative disorders such as Parkinson’s disease (PD) [3,4,5,6,7], stroke [8,9], and spinal cord injury [10,11,12]
H9 Human embryonic stem cells (hESCs) were passaged by manual microdissection and monitored for normal karyotype [33]. hESCs were maintained on Hs27 human fibroblasts but changed to feeder-free conditions when differentiated to facilitate cell-based therapies in humans [34]
MEF2CA Increases DA Neuron-Specific Markers In Vitro In the absence of exogenous MEF2CA, we found that our protocol for differentiating rosette-neural stem cells (R-NSCs)/neural stem/progenitor cells (NPCs) resulted in increased expression of endogenous myocyte enhancer factor 2C (MEF2C) (Figure 2A) in addition to some degree of expression of TH+ dopaminergic neuronal markers in vitro (Figure 5A)
Summary
Human embryonic stem cells (hESC) are an important model system for human neurogenesis [1,2] but are a potential source of therapeutic cells to treat neurodegenerative disorders such as Parkinson’s disease (PD) [3,4,5,6,7], stroke [8,9], and spinal cord injury [10,11,12] Such cell-based therapies require protocols for differentiating hESCs into neural precursors and further directing them towards a specialized regional neurotransmitter identity, such as midbrain dopaminergic neurons. A method of directed differentiation into neurons would facilitate transplantation therapies
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