Direct conversion of rodent fibroblasts to neural stem cells using the combinatory expression of Brn2, Ascl1, Myt1l and Bcl-xL

Abstract

Recent reports have demonstrated that direct conversion of somatic cells to other mature cell types can be achieved by expression of specifically defined factors. The first study to show direct conversion of fibroblasts to functional neurons was performed in the laboratory of Dr. Wernig, who used the neuronal lineage-inducing transcription factors Brn2 (B), Ascl1 (A), and Myt1L (M) as induced neuron (iN) factors. Subsequent studies have reported successful induction of neural lineages by various other factors. Together, the results of these studies suggest that fibroblasts do not pass through a neuronal stem cells (neural stem cells, NSCs) state on their way to becoming neurons. This observation is problematic in that neither differentiated fibroblasts nor mature neurons are capable of replicating into the number of cells required to be useful for cell therapies. Here, we investigated the possibility of generating an expandable population of NSCs during the fibroblast to neural stem cell conversion process using mouse fibroblasts and the NSC marker nestin-GFP. Co-expression of Bcl-xL in BAM-transduced fibroblasts enhanced the yield of GFP+ nestin+ cells. FACS-sorted GFP+ iNSCs could be expanded and differentiated into neuronal cells exhibiting midbrain dopamine neuron-specific marker expressions and functions. In addition to mouse fibroblasts, rat fibroblasts-derived iNSCs were expandable for multiple passages and were capable of differentiating to mature, functional dopaminergic neurons expressing midbrain dopamine neuron specific factors both in vitro and in vivo. Taken together, our results suggest that Bcl-xL elicits a positive effect on direct conversion toward neural stems and proliferation of induced neural stem cells.