Generation of Dopamine Neurons from Rodent Fibroblasts through the Expandable Neural Precursor Cell Stage

• Published in: The Journal of biological chemistry 2015-07, Vol.290 (28), p.17401-17414
• Main Authors: Lim, Mi-Sun, Chang, Mi-Yoon, Kim, Sang-Mi, Yi, Sang-Hoon, Suh-Kim, Haeyoung, Jung, Sung Jun, Kim, Min Jung, Kim, Jin Hyuk, Lee, Yong-Sung, Lee, Soo Young, Kim, Dong-Wook, Lee, Sang-Hun, Park, Chang-Hwan
• Format: Article
• Language: English
• Subjects: Animals; Basic Helix-Loop-Helix Transcription Factors - genetics; cell therapy; Cell Transdifferentiation; Cellular Reprogramming; Developmental Biology; Dopamine - metabolism; Dopaminergic Neurons - cytology; Dopaminergic Neurons - metabolism; Fibroblasts - cytology; Fibroblasts - metabolism; Gene Expression; Hepatocyte Nuclear Factor 3-beta - genetics; Mesencephalon - cytology; Mesencephalon - metabolism; Mice; Mice, Inbred C57BL; Mice, Inbred ICR; Mice, Transgenic; Nerve Tissue Proteins - genetics; neural stem cell (NSC); Neural Stem Cells - cytology; Neural Stem Cells - metabolism; neurodegeneration; Nuclear Receptor Subfamily 4, Group A, Member 2 - genetics; Parkinson disease; Parkinsonian Disorders - metabolism; Parkinsonian Disorders - pathology; Parkinsonian Disorders - therapy; POU Domain Factors - genetics; Rats; Rats, Inbred Lew; Rats, Wistar; reprogramming; Transcription Factors - genetics
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M114.629808

Recent groundbreaking work has demonstrated that combined expression of the transcription factors Brn2, Ascl1, and Myt1L (BAM; also known as Wernig factors) convert mouse fibroblasts into postmitotic neuronal cells. However, questions remain regarding whether trans-conversion is achieved directly or involves an intermediary precursor stage. Trans-conversion toward expandable neural precursor cells (NPCs) is more useful than direct one-step neuron formation with respect to yielding a sufficient number of cells and the feasibility of manipulating NPC differentiation toward certain neuron subtypes. Here, we show that co-expression of Wernig factors and Bcl-xL induces fibroblast conversion into NPCs (induced NPCs (iNPCs)) that are highly expandable for >100 passages. Gene expression analyses showed that the iNPCs exhibited high expression of common NPC genes but not genes specific to defined embryonic brain regions. This finding indicated that a regional identity of iNPCs was not established. Upon induction, iNPCs predominantly differentiated into astrocytes. However, the differentiation potential was not fixed and could be efficiently manipulated into general or specific subtypes of neurons by expression of additional genes. Specifically, overexpression of Nurr1 and Foxa2, transcription factors specific for midbrain dopamine neuron development, drove iNPCs to yield mature midbrain dopamine neurons equipped with presynaptic DA neuronal functions. We further assessed the therapeutic potential of iNPCs in Parkinson disease model rats. Background: Fibroblasts can be converted into neurons by transduction with BAM. Results: Multiple lines of evidence were used to demonstrate that a significant percentage of BAM-transduced fibroblasts can be converted into iNPCs by co-expression of Bcl-xL. Conclusion: BAMX-derived iNPCs were expandable over multiple passages in vitro, and differentiation phenotypes of iNPCs were readily manipulated by specific developmental cues. Significance:Bcl-xL has a critical role in neural precursor cell conversion.