Direct conversion of fibroblasts to functional neurons - iN cells

This week in Nature, Marius Wernig and colleagues demonstrate expression of three factors, Ascl1, Brn2, and Myt1l, can convert fibroblasts to functional neurons in vitro (iN cells), bypassing the pluripotent stage.

Vierbuchen et al. cloned 19 genes that are expressed in neural tissues, have roles in neural development or implicated in epigenetic reprogramming and expressed them in purified mouse embryonic fibroblasts (MEFs). After infection, they observed neuronal morphology, indicating some combination of these factors could convert MEFs to iNs. The group was able to narrow down the factors to five: Brn2, Myt1l, Zic1, Olig2, and Ascl1.

iN cells were generated using these 5 factors from both MEFs and post-natal tail-tip fibroblasts. These "5-factor" iNs expressed neuronal markers MAP2, NeuN, and synapsin, and demonstrated functional membrane properties similar to neurons. In both cases, the iN cells were able to produce action potentials and contained functional voltage-dependent and ligand-gated ion channels. The authors were able to detect vGLUT1-positive and GABA-positive cells, though they could not detect tyrosine hydroxylase, choline acetyltransferase or serotonin expression. Additionally, the iN cells were shown to form functional synapses. The induced neurons could integrate into pre-existing neural networks and form independent synapses with each other.

To further investigate which of the 5 genes were necessary to induce neuron-like cells in the MEFs, Vierbuchen and colleagues began removing each factor from the mix. The most efficient combinations of transcription factors were Ascl1, Brn2 and Myt1l or Zic1. Both combinations were able to induce neuron-like cells at 2 to 3 fold higher efficiency than the 5 factors; however, the Ascl1, Brn2 and Myt1l  iN cells showed a more complex morphology, leading the investigators to focus on that combination. Finally, the group showed that while Ascl1 alone or Ascl1 and Brn2 or Myt1l were sufficient to generate cells with neuronal traits, Ascl1, Brn2 and Myt1l together could generate iN cells that produced repetitive action potentials with mature characteristics and displayed more complex neuronal morphologies.

The cells generated in this study correspond mostly to excitatory cortical cells. Some interesting future directions for this work will be to elucidate which factors are necessary to produce dopaminergic neurons and to determine which cell type is optimal for conversion to neurons. This system bypasses production of tumorigenic pluripotent cells, a main barrier to using iPSCs in regenerative medicine, and may provide a platform for more efficient disease modeling and drug discovery.

Thomas Vierbuchen, Austin Ostermeier, Zhiping P. Pang, Yuko Kokubu, Thomas C. Sudhof & Marius Wernig. Direct conversion of fibroblasts to functional neurons by defined factors. Nature. Published online January 27, 2010.

doi:10.1038/nature08797

Image:Neurons derived from mouse embryonic fibroblasts infected with the three gene cocktail of Brn2, Myt1l, and Ascl1 and cultured for twelve days. The cells are labeled with Tuj1 antibody, which specifically recognizes neurons.
Credit: Thomas Vierbuchen/Marius Wernig