Parkinson's disease (PD) is currently treated with dopamine agonists, although fetal cell-based therapy has also been attempted 
. Pharmacological agents treat symptoms but do not restore DA neurons in PD patients. In addition, long-term treatments with dopamine agonists such as l
-DOPA cause dyskinesias and eventually become ineffective. To overcome these disadvantages, cell therapy using fetal mesencephalic brain tissue has been employed, but the results have been mixed and largely unsatisfactory. One reason for the failure of such transplants is graft-induced dyskinesias; in fact, many of these grafts contain more serotonin and GABAergic neurons than DA neurons. Both pulsatile delivery of DA agonists and upregulation of GABAA
receptors have been shown to contribute to dyskinesias in primates 
, so we reasoned that improved production of DA neurons might improve this situation. An additional problem has been extensive cell death in the grafts 
. Therefore, strategies to enrich for DA neurons, especially of the A9 subtype rather than other neuronal types, and to prolong cell survival are likely to result in improved cell-based therapies for PD.
hESC-derived DA neuronal precursors have been considered as an alternative source for cell-based therapies in PD. Although this approach holds promise, hESC-based therapies face several hurdles, including death of engrafted cells, failure of migration/incorporation into host brain, lack of differentiation into appropriate neuronal cell types, and formation of tumors after transplantation. One technical advance that we propose here to overcome these problems is the forced expression of a transcription factor that drives more restricted neuronal lineage choices from ESCs, while also fostering survival of the differentiated cells. In this study, we present evidence that MEF2C represents a transcription factor that can accomplish these goals. Our feeder-free/neurosphere-based protocol for isolating rosettes followed by transduction with MEF2CA addresses many of the issues associated with the production of human stem cells for therapeutic use in the brain. This approach is particularly well suited for PD since, unlike previous methods 
, a substantial number of dopaminergic neurons can be generated in the absence of hyperproliferating cell types. Previously, we and others have shown that the MEF2 transcription factors, with MEF2C the predominant isoform during early brain development, represent a family of activity-dependent neurogenic effectors that enhance survival and neuronal differentiation 
. Moreover, we have recently shown that conditional knockout of Mef2c
in mice during very early brain development – at the NPC stage – impairs neurogenesis, migration, and synaptogenesis in vivo
, resulting in a behavioral phenotype resembling Autism-spectrum disorders 
Here, we initially investigated the function of MEF2C during early neurogenesis of hESCs in vitro
by knocking down endogenous MEF2C using shRNAs at the R-NSC/NPC stage. At this stage, MEF2C expression was maximal, and cells homogenously expressed nestin and musashi1. As a result, we found a reduction in the number of hESC-derived neurons and dendritic/synaptic spines on those neurons, fitting well with our previous in vivo
results in mice when we knocked out MEF2C at the NPC stage 
Conversely, when we overexpressed MEF2CA in vivo
, we drove neurogenesis from hESC-derived R-NSCs/NPCs, specifically producing an enrichment of the dopaminergic phenotype. We found that MEF2C-mediated neurogenesis is not simply due to neuronal survival. For example, to distinguish neurogenic vs. survival effects of MEF2CA, we included Bcl-xL as a control and found that it did not significantly increase neuronal differentiation. Previously, both growth factors and transcription factors have been used to increase the percentage of NSCs that express neuronal and dopaminergic phenotypes 
, but no method has proven to be totally effective. In this regard, a critical consideration is that if 100% of the hESCs do not become terminally differentiated as neuronal cells, then hyperproliferation can occur with possible tumorigenic potential. In contrast, with our approach, the great majority of cells receiving MEF2CA became neuronal, which can overcome this conundrum. Thus, our findings suggest that MEF2C is an effective driver of neurogenesis and in the proper context its expression prevents hyperproliferation. Additionally, our results show that under our conditions expression of MEF2C produces substantial enrichment in the dopaminergic phenotype. We show that the mechanism for this dopaminergic effect includes up-regulation by MEF2C of two additional transcription factors, nurr1 and the ETS family member Etv1. In the future, with more efficient transduction of MEF2CA or sorting of MEF2CA-positive cells, coupled with transient expression of MEF2CA only during the critical neurogenesis stage, this approach may provide an even more valuable source of human neural progenitor cells that are programmed to become dopaminergic neurons after transplantation in PD patients.
Our in vivo
experiments using 6-OHDA-lesioned Parkinsonian rats and implanted lenti-MEF2CA/R-NSCs revealed good survivability for at least 6 months after transplantation, possibly because of the anti-apoptotic properties in addition to the neurogenic effect of MEF2CA 
. Most importantly, we observed significant neurobehavioral/motoric improvement after transplantation of MEF2CA/R-NSCs in this PD animal model compared to control/R-NSCs. By comparing control/R-NSCs vs. MEF2CA/R-NSCs in the same experimental model, we controlled for nonspecific transplantation-related effects on behavior. Prior studies had observed comparable improvement in motor function in the same Parkinsonian rat model after transplanting hESCs that had been co-cultured with telomerase-immortalized human mesencephalic astrocytes to enhance differentiation of the stem cells into dopaminergic neurons; however, this improvement was accompanied by hyperproliferation, heralding potential tumor formation 
. Our approach using MEF2CA-transduced stem cells can avoid this major deterrent to transplantation.
In conclusion, our results demonstrate that MEF2C restricts hESCs to the neuronal lineage and that this attribute can be used to generate neurons and avoid tumor formation when used for cell-based therapies. Furthermore, hESC/NPCs programmed to become neurons via MEF2C activity were protected from apoptosis and enriched for the dopaminergic phenotype. This approach could potentially provide a limitless supply of stem cells for therapeutic application in PD. Our technique represents a unique approach for the production of cells for regenerative medicine, while at the same time avoiding apoptosis and tumorigenesis by promoting directed differentiation with a pro-survival/neurogenic transcription factor.