Abstract
Purpose::
Adult stem cells may provide an accessible and autologous source for cell-based therapy on a wide range of degenerative disease and damaged tissues such as retinal degeneration. Thus, better understanding of adult stem cell capacity may lay the groundwork for acquiring cells highly capable of regeneration. For this purpose, we isolated adult retinal stem cells (RSCs) and studied their proliferation and differentiation potentials.
Methods::
Adult RSCs were isolated from the ciliary margin zone of 11-12 week-old NMRI mouse eyes. Isolated cells were grown under the stimulation of exogenous EGF and FGF-2. After one week these cells proliferate as spheres. The spheres were individually picked and grown as monolayer with addition of serum. RT-PCR was used to study the expressions of stem, progenitor, and differentiated markers.
Results::
We found that these cells posses a high proliferative capacity. Starting with single cell, we were able to generate more than 1040 cells in 190 days and these cells are still growing in a linear manner. RT-PCR analysis shows that these cells expressed several markers characteristic of stem cells (c-kit, Bmi1, Sox2) and Mash1, a bHLH pro-neural protein. Immunocytochemistry analysis showed a strong expression of nestin, a neural precursor marker. We also detected the expression of Pax6 and Chx10, indicating a retinal fate of these cells. In addition, we observed the expression of FGF-2 and Bmp4 which are known to play important roles on eye development. Karyotype analysis shows that these cells are mostly polyploid. Interestingly, in comparison to the adult RSCs, newborn RSCs show a significantly lower frequency of polyploidy.
Conclusions::
Together, our finding shows a range of possibilities on the differentiation potential of the adult RSCs. However, polyploidy of the cells raises a concern over the cell genomic stability and thus the safety of using adult RSCs grown in such culture condition for transplantation. It is tempting to speculate that the higher genomic instability is related to cellular aging in adult RSCs. We are also interested to find whether the concentration of exogenous mitogens in the culture medium plays a role in polyploidisation. In addition, knowing that not all polyploid cells are tumorigenic, it would be important to test this. Data on these points will be presented together with a continuing assessment of the cell capacity and potentiality.
Keywords: retina • proliferation • retinal development