Introduction: Neurogenic transcription factors have been found to be instrumental in the formation of neurons, and their forced expression has been shown to direct neuronal conversion of somatic cells. We describe the generation of induced neuronal cells from adult mesenchymal stem cells from rat, equine and canine origin, using a combination of expression of neuronal transcription factors and a neuronal differentiation induction media.
Experimental Methods: Lentiviral vectors expressing the neuronal-specific transcription factors Ascl1, Brn2, Myt1l and NeuroD1 were produced, and doxycycline-induced expression was performed, in both the presence and absence of four pluripotency-inducing transcription factors; Oct4, Sox2, Klf4 and c-Myc (OSKM). Transduced cells were cultured in neuronal induction media supplemented with histone deacetylase inhibitors valproic acid or sodium butyrate.
Results: From day seven, neuronal-like cells were visible by light microscopy with rounded cell bodies and elongated axonal networks. Neuronal-specific βIII-tubulin antibody immunostaining demonstrated unipolar, bipolar and multipolar neurons, as well as long branching processes and extensive neurite outgrowth. βIII-tubulin positive cells were present in cultures transduced with both neurogenic factors alone or in combination with iPS cell factors. A lower conversion efficiency was noted in the presence of OSKM, with no significant differences seen in the presence of the histone deacetylase inhibitors. This method achieved a conversion efficiency of 8%, slightly higher than previously shown in studies using alternative cell types.
Conclusion: The forced expression of the neuronal-specific transcription factors Ascl1, Brn2, Myt1l and NeuroD1, in both the absence and presence of iPS cell factors induced the direct conversion of adult stem cells to neuronal cells, and may have applications in neurological disease modelling, drug screening, and regenerative medicine.