Abstract
Purpose:
Retinal diseases are often characterized by the degeneration of the photoreceptors. A new approach to restore the lost cells could be the utilization of stem or progenitor cells. Since in zebrafish Müller cells (MC) can transdifferentiate and regenerate photoreceptors these cells might also serve that purpose. Therefore, we wanted to investigate the differentiation potential of human MC in vitro.
Methods:
In our experiment the spontaneously immortalized human MC line MIO-M1 has been used. The cells were cultured with different soluble factors [hEGF + bFGF, PEDF, retinoic acid (RA) and hRPE-conditioned medium (RPE-CM)]. At different time points (3, 7, 10, 14 or 21 days) the expression of cell-specific markers [nestin, SOX-2, glutamine synthetase (GS), GFAP, MAP-2, RPE65, βIII-tubulin] was quantified. Thereby, quantitative real time PCR (qRT-PCR), Western blot and immunohistochemistry have been employed.
Results:
Whereas no increase of progenitor markers could be detected at the molecular level an increase of GFAP and βIII-tubulin after treatment with the soluble factors was found. On the other hand, protein expression of the progenitor markers was found in all treatment groups with a maximum at day 10. Furthermore, GFAP protein was significantly upregulated after treatment (hEGF + bFGF, RA) as was βIII-tubulin (hEGF + bFGF, RPE-CM). Furthermore, expression of the neuronal markers MAP-2 and βIII-Tubulin increased up to day 14. The MC marker GS remained unchanged at all time points.
Conclusions:
The results indicate that human MC can be induced to dedifferentiate into a progenitor state and directed further along the neuronal lineage in vitro. The activation (GFAP) by soluble factors is regulated on the translational level whereas the differentiation (MAP-2, βIII-tubulin) was initiated on the transcriptional level. In summary, pre-differentiated MC might be a potential source for retinal regeneration in humans.