Int J Stem Cells.  2015 Nov;8(2):228-234. 10.15283/ijsc.2015.8.2.228.

FGF8 is Essential for Functionality of Induced Neural Precursor Cell-derived Dopaminergic Neurons

• Lim MS
• Lee SY
• Park CH

• KMID: 2380817
• DOI: http://doi.org/10.15283/ijsc.2015.8.2.228

Abstract

Induced neural precursor cells (iNPCs) are one source of transplantable dopaminergic neurons used in cell therapy for Parkinson's disease. In the present study, we demonstrate that iNPCs can be generated by transducing Brn2, Ascl1, Myt1L and Bcl-xL in a culture supplemented with several mitogens and subsequently can be differentiated to dopaminergic neurons (DA). However, studies have shown that iDA and/or iNPC-derived DA neurons using various conversion protocols have low efficiency. Here, we show that early exposure of FGF8 to fibroblasts efficiently improves differentiation of DA neurons. So our study demonstrates that FGF8 is a critical factor for generation of iNPC-derived DA neurons.

Keyword

Direct conversion; FGF8; Dopamine neuron; iNPC

MeSH Terms

Cell- and Tissue-Based Therapy
Dopaminergic Neurons*
Fibroblasts
Mitogens
Neurons
Parkinson Disease
Mitogens

Figure

• 

  Fig. 1 Generation of iNPCs from rat embryonic fibroblasts in two culture conditions; (A) Schematic diagram depicting the timeline of iNPCs during the conversion process; (B, C) Morphology of iNPCs in the absence of FGF8 (B) and presence of FGF8 (C); Four weeks after the conversion period, cluster-forming cells were observed (B, C, white dotted circle). Scale bar=100 μm.

• 

  Fig. 2 Characterization of iNPCs between two culture conditions: (A, B) Cultured iNPCs in the presence of FGF8 were stably expandable without loss of self-renewing potentials. iNPCs were cultured for more than six passages and were analyzed using immunocytochemistry analyses with the proliferating marker, ki67, neural precursor marker nestin, and sox2 on the last day of each passage. (C, D) Cultured iNPCs in the absence of FGF8 were also not associated with significant changes in the percentage of iNPCs compared to FGF8-supplemented culture. Error bars denote the standard error of the mean (SEM, n=3). Scale bar=20 μm.

• 

  Fig. 3 Functional DA neurons enhanced by FGF8 treatment during the iNPC generation period: (A, B) Cultured DA neurons in the presence of FGF8 were more functional than TuJ1+ and TH+ neurons after short- and long-term differentiation (5 and 18 days) without loss of TuJ1 or TH+ cells. (C, D) Cultured DA neurons in the absence of FGF8 were observed to include TuJ1+ and TH+ cells; however, most of cells had an immature morphology, and the numbers of TuJ1+ and TH+ cells decreased over long-term differentiation compared to those supplemented with FGF8. Error bars denote the standard error of the mean (SEM, n=3). Scale bar=20 μm. *p<0.05, **p<0.001 compared to the FGF8 condition.

Reference

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