Purchase this article with an account.
V. Marigo, A. De Marzo, C. G. Aruta, G. C. Demontis; Molecular and Electrophysiological Properties of Photoreceptors Derived From Retinal Neurospheres. Invest. Ophthalmol. Vis. Sci. 2009;50(13):3215.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Stem cells have been proposed as a prospective source of cells for retinal transplantation. Nevertheless, turning retinal progenitor cells into transplantable photoreceptors is a complex process. The application of retinal stem cells for regenerative medicine will require an enriched and homogeneous population of cells able to become functional rods in the retina. To this purpose we have developed a protocol for in vitro differentiation of rod-like photoreceptors and tested their molecular and physiological properties.
We derived retinal stem cells from the ciliary margin of adult mice and grew them as neurospheres in minimal medium. Cells were then allowed to differentiate on an extracellular matrix substrate in the absence of serum and in the presence of differentiating factors: retinoic acid and taurine. Gene expression was analyzed by Real-time PCR; protein localization by immunofluorescence; electrophysiology by patch-clamping.
During the first weeks of in vitro differentiation, cells expressed progenitor markers. Co-expression of several rod marker proteins, such as rhodopsin transducin and Pde6b, was detectable after at least two weeks of differentiation in 70% of the cells. Electrophysiological analysis demonstrates cGMP-gated currents after at least 3 weeks in vitro, suggesting that expression of cGMP-gated channels starts later than rhodopsin. Even if cGMP-gated channels were expressed at this time, cells did not show yet voltage-dependent currents typical of mature photoreceptors.
Expression, subcellular localization and electrophysiology suggest that our differentiation protocol allows a high percentage of neurosphere derived cells to acquire rod-like properties. Our data show that rhodopsin expression turns on early during the differentiation protocol and precedes functional maturation of the neurons. Long differentiation times are required to achieve high degree of maturation.
This PDF is available to Subscribers Only