(587f) Engineering of Embryonic Stem Cell Neurogenic Differentiation for Neurodegenerative Diseases

Embryonic stem cells (ESCs) have the potential to become a limitless source of tissue for cell therapies and drug testing. Specifically, efficient generation of neuronal cells from human ESCs (hESCs) will have a major impact in transplantation therapies to replace aged or diseased neural tissue. This study focuses on better understanding the specific signals directing the differentiation of ESCs to neural progeny. We further examine the differentiation of hESCs in a bioreactor as a way of producing neuronal cells in quantities suitable for clinical therapies. The integrative nuclear fibroblast growth factor (FGF) signaling (INFS) has been show to mediate the activation of genes thereby coaxing the differentiation of umbilical cord blood cells and human neuronal precursors to neuronal cells. Here, we investigated if a similar mechanism can be utilized for the neurogenic commitment of mouse ESCs (mESCs). We found that treatment of mESCs with agents such as retinoic acid (RA) and cyclic adenosine monophosphate (cAMP) activates INFS, in part by nuclear translocation of an FGF receptor. Immunopositivity for glial fibrillary acidic protein (GFAP) revealed an increased propensity of mESCs for astrocyte differentiation upon stimulation with cAMP. In contrast, RA prompted the differentiation towards neurons as shown by enhanced expression of tyrosine hydroxylase and β3-tubulin. To that end, the localization of the RA receptor in differentiating stem cells in various stages of neuronal differentiation was also studied. These findings may lead to the development of methods for directed differentiation of ESCs to neuronal cells with high efficiency. For such methods to be utilized in clinical therapies, ESC-derived neurons must be generated in high quantities. Hence, we also examine if neuronal differentiation of hESCs can be performed in stirred suspension bioreactors. Cells formed neurosphere-like aggregates in the bioreactor and in a serum-free medium. A transition was noted to neuroepithelial cells and then radial glial cells. Finally, neuron-like cells emerged upon plating of the neurospheres. Differentiated cells displayed increased gene/protein levels of Pax6, Nestin, β3-tubulin, MAP2 and GABA (Fig. 1) compared to undifferentiated hESCs, and similar levels of ectoderm genes when compared to cells differentiated exclusively in static culture. This scalable system has the ability to produce mass quantities of neurospheres. In conjunction with an INFS-based method for directing the differentiation of these cells further to neuronal cells, this stem cell culture modality may facilitate the production of cellular material suitable for therapies against Parkinson and Alzheimer diseases.