Our goals for this grant were to determine the role of the transcription factor MEF2C in neurogenesis, including all of the targets of this factor in the genome, use this knowledge to direct differentiation of human embryonic stem cells (hESC) into specific types of neurons, and investigate the transplantation of these cells into rodent models of Parkinson’s disease (PD) and stroke. During the tenure of this grant, we accomplished these goals to a very significant degree. Our investigations into the role of MEF2C in neurogenesis produced a large body of knowledge pertinent to its essential role in this process. This knowledge base was achieved through both monitoring expression levels of MEF2C during the entire process of neurogenesis and by knocking down its expression by use of siRNA. We now have a very detailed view of the temporal contribution of MEF2C as stem cells differentiate into neurons. Using this knowledge, we optimized a differentiation protocol for directing hESC into neuronal precursor cells and then initiated expression of a constitutively active MEF2 transcription factor (MEF2CA) via lentiviral technology. We discovered that the forced expression of MEF2CA provided a strong bias to neurons to differentiate along a dopaminergic (DA) lineage. Our network analysis for MEF2C confirmed that many of the known effector proteins for DA neurons are indeed targets for this transcription factor. Histological and electrophysiological investigations into the nature of these cells grown in vitro showed that they are indeed functional neurons displaying the anticipated qualities during the various stages of differentiation.
Our in vivo transplantation studies have been equally productive. Owing to the strong tendency of the MEF2CA-expressing cells to differentiate into DA neurons, we first investigated their effects on a rat PD model where the dopaminergic cells of the substantia nigra are ablated on one side of the brain by injection of 6-hydroxydopamine. In response to an injection of the dopamine analog apomorphine, these rats will turn in a circle and the readout is the number of turns in a 30 minute period measured on a rotometer. Fewer turns indicate that the rat has less pathology, i.e., is getting better. We transplanted hESC-derived neural progenitor cells (hESC-NPC) either expressing MEF2CA or not and monitored recovery of the rats. While rats receiving both preparations of stem cells showed considerable improvement, the ones receiving MEF2C-expressing cells did significantly better on the rotometer. Also, histologically the MEF2CA-expressing cells could all be seen to differentiate, whereas those that did not express MEF2CA were often found in an undifferentiated state, which potentially posses a problem of continuing proliferation in the brain and tumor formation. Thus, the forced expression of MEF2CA forced the cells to differentiate and prevented uncontrolled cell division. An additional advantage was that the remaining endogenous DA neurons showed much greater density of fibers in the vicinity of the transplanted cells, suggesting that there was an additional benefit of factor secretion. Thus, the MEF2CA genetically modified cells appear to have significant advantages for transplantation for PD.
We are also investigating the use of the MEF2CA-expressing hESC-NPC in rat and mouse models of stroke. Preliminary data shows that in both systems we see behavioral improvements following the transplantations with these cells. In the period of the no cost extension, we will complete these studies and characterize the types of neurons these transplanted cells become and their role in reversing the pathology caused by the brain ischemia from stroke. Our hypothesis is that there is a strong bias toward the DA neuron phenotype produced by the expression of MEF2CA, but that this is overridden by the context within the brain. Therefore, in a stroke model, the context of damage to the cortex provides signals to the newly transplanted cells that they should migrate to the damaged area and become cells appropriate to that region, not DA neurons. We will test this hypothesis in the remaining months of the grant.