The direct conversion of mouse fibroblasts to neurons 1 was developed while many scientists were interested in neuronal differentiation using induced pluripotent stem cells (iPSCs) 2. This direct reprogramming skips a pluripotent state, making patient- and/or disease-specific cell therapy much faster and more feasible. Recent reports have indicated that mouse or human fibroblasts could be directly converted to a number of different cell types, such as cardiomyocytes, blood progenitor cells, hepatocyte-like cells, neural progenitors and specific dopaminergic neurons 3, 4, 5, 6, 7, 8, 9. However, all of the studies in this field have employed the retroviral or lentiviral vector delivery system. These genome-integration viruses may cause insert mutations and activation of tumor-related genes; therefore, it is necessary to generate neurons with a non-integrating system. Concerning transcription factors, the reported combination of three neural-specific transcription factors, Ascl1, Brn2 and Myt1l (ABM), or the three combined with NeuroD1, can convert mouse and human fibroblasts to functional neurons 1, 4, 5. It has also been reported that Ngn2 alone could direct astrocytes into neurons 10, 11. Therefore, it is possible that a different combination of transcription factors could induce fibroblasts to neurons as well. In this study, we used non-integrating adenoviruses carrying a different combination of transcription factors of Ascl1, Brn2 and Ngn2 (ABN) for the direct conversion of mouse embryonic and adult fibroblasts to functional neurons.

The GFP expression level helped us observe changes of MEFs during the whole process of transdifferentiation. At 1 day post infection (dpi), the infection efficiency was estimated to be approximately 80% - 90% by fluorescence, and the MEFs infected displayed typical fibroblast morphology (Supplementary information, Figure S3A). At 3-5 dpi, the infected MEFs began to show the morphological appearance of neurons with short and slender projections (Supplementary information, Figure S3B). At 7-10 dpi, more cells with complex processes were observed (Supplementary information, Figure S3C). At 13-18 dpi, neuronal-like cells with long processes forming a complex network with each other were readily observed (Supplementary information, Figure S3D).

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