Transfection With Pax6 Gene of Mouse ES Cells and Subsequent Cell Cloning Induced Retinal Neuron Progenitors, in vitro

Abstract

Purpose: : Neural cell transplantation is considered to be a potential therapeutic approach which may repair or sustain the functions of damaged retinal neurons. Differentiation from embryonic stem (ES) cells into Retinal neuron progenitors becomes promising a new strategy.Here, we attempted to establish selective retinal neuron progenitors from ES cells transfected with the pax6 gene in vitro.

Methods: : Mouse undifferentiated ES cells were transfected with pax6 gene. Pax6 gene is known as an essential transcription factor for early eye development. The transfected cells were cultured in the G418 containing media and limiting dilution culture was conducted to select cloned neural progenitors. We only expanded the cloned cells which expressed nestin and musashi-1 by RT-PCR. To induce fully differentiation, we cultured these cells in our original differentiation media. The growing neural like cells was characterized using RT-PCR and immunostaining.

Results: : After selection of the clones for nestin and musashi-1 mRNAs as neural progenitors, we obtained the cells which begin to express retinal progenitor transcripts, six3 and chx10 on the next day after culture. The cells developed axon-like processes that gradually extended over time. After 4 days in culture, the cells also expressed immature neuronal and RGC-related markers, such as betaIII tubulin (Tuj-1), neurofilament middle chain (NFM), Islet1, brn3a, brn3b, Thy1 and melanopsin. Cells showing neuron-like feature appeared at day 7. Immunostaining for the cells were Nestin⁺ (14.8 ± 1.8 %), NFM⁺ (9.5 ± 1.2 %), Tuj1⁺ (16.6 ± 1.8 %), Brn3⁺ (2.2 ± 0.01 %) and Thy1⁺ (1.3 ± 0.01 %) at that point. After 14 days in culture, the cells were Tuj1⁺ (84.3 ± 1.6 %), NFM⁺ (91.8 ± 1.9 %), Brn3⁺ (91.8 ± 0.6 %), PKC⁺ (bipolar cell marker; 2.0 ± 0.2 %), Crx⁺ (photoreceptor-related marker; 8.1 ± 0.8 %) and, Opsin⁺ (photoreceptor-related marker; 0.5 ± 0.1 %). The number of the neuron-like feature increased with time and similar of retinal neuron progenitors including RGCs. Without pax6 transfection, these phenomena were not observed.

Conclusions: : Our results demonstrate that ES cells with pax6 transduction can differentiate into retinal neuron progenitor cells, which have the potential of RGC like cells in vitro.

Keywords: retina: proximal (bipolar, amacrine, and ganglion cells) • gene/expression • transcription factors