May 2007
Volume 48, Issue 13
ARVO Annual Meeting Abstract  |   May 2007
Genetic Manipulation of Murine Retinal Stem Cells
Author Affiliations & Notes
  • V. Broccoli
    Stem Cell Research Department, San Raffaele Scientific Institute, Milan, Italy
  • S. G. Giannelli
    Stem Cell Research Department, San Raffaele Scientific Institute, Milan, Italy
  • M. Andreazzoli
    Department of Biological Sciences, Pisa University, Pisa, Italy
  • P. Rama
    Ophthalmology Unit, San Raffaele Hospital, Milan, Italy
  • M. Codenotti
    Ophthalmology Unit, San Raffaele Hospital, Milan, Italy
  • Footnotes
    Commercial Relationships V. Broccoli, None; S.G. Giannelli, None; M. Andreazzoli, None; P. Rama, None; M. Codenotti, None.
  • Footnotes
    Support None.
Investigative Ophthalmology & Visual Science May 2007, Vol.48, 4069. doi:
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      V. Broccoli, S. G. Giannelli, M. Andreazzoli, P. Rama, M. Codenotti; Genetic Manipulation of Murine Retinal Stem Cells. Invest. Ophthalmol. Vis. Sci. 2007;48(13):4069.

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      © ARVO (1962-2015); The Authors (2016-present)

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Purpose:: For long time, mammalian retina has been considered a tissue with low or absent regeneration potential during its entire life. However, recent findings have challenged this view. In fact, the isolation of proliferative cells from the adult ciliary margin and the assessment of retinal endogenous regenerative capability upon damage have revealed unexpected features of the retina. We have isolated mouse retinas from several different stages and looked for lasting cell growth in vitro.

Methods:: We have been successful in isolating cells from embryonic and postnatal mouse retina able to constantly grow in vitro using dedicated cell culture conditions.

Results:: These cells are endowed with all the cardinal properties of stem cells, showing high and stable growth ability in vitro without any signs of senescence or growth-factor independent proliferation. Indeed, cells have been maintained in vitro for more than 6 months up to passage 65. Interestingly, upon switch in cell culture conditions all cells differentiated in mature retinal cell types and in particular in Muller glia and photoreceptor progenitors. Interestingly, stem cell derived photoreceptors express a large fraction of molecules of the light cycle, indicating a highly correct and complete differentiation program. However, bipolar, ganglion, amacrine cells were not generally found in the differentiated cultures indicating a biased differentiation potential. This raises the question whether these cells can be induced toward these cell types. For this reason, we genetically manipulated the cell cultures forcing the expression of different master genes of the bipolar and ganglion cell fate. In particular, we used Mash1 and NeuroD genes to push bipolar cell fate differentiation and Math5 gene to direct ganglion cell formation. Interestingly, in these conditions retinal stem cells gave rise to a large fraction of neurons with few glial or photoreceptor cells. Molecular characterization indicated that all neurons were bipolar cells expressing G0alpha and PKCalpha. Notably, similar results were obtained with cell cultures at early or late in vitro passages.

Conclusions:: These results indicate that cultures of retinal stem cells maintain a stable differentiation potential in vitro and may be stimulated to differentiate toward many of the retinal cell types. This strategy will also allow to obtain pure populations of specific retina cell types useful for gene expression profiling and cell transplantation.

Keywords: retinal development • retina • genetics 

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