May 2007
Volume 48, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2007
Isolation, Growth Kinetics and Differentiation of Human Retinal Progenitor Cells in Culture
Author Affiliations & Notes
  • U. Aftab
    Schepens Eye Research Institute, Boston, Massachusetts
  • C. Jiang
    Schepens Eye Research Institute, Boston, Massachusetts
  • H. Klassen
    Department of Ophthalmology, University of California, Irvine, Orange, California
  • E. Miljan
    ReNeuron Ltd., Guildford, United Kingdom
  • J. D. Sinden
    ReNeuron Ltd., Guildford, United Kingdom
  • M. Young
    Schepens Eye Research Institute, Boston, Massachusetts
  • Footnotes
    Commercial Relationships U. Aftab, ReNeuron, Ltd., Guildford, UK, F; C. Jiang, ReNeuron, Ltd., Guildford, UK, F; H. Klassen, None; E. Miljan, ReNeuron Ltd., Guildford, UK, E; J.D. Sinden, ReNeuron Ltd., Guildford, UK, E; M. Young, ReNeuron, Ltd., Guildford, UK, F.
  • Footnotes
    Support ReNeuron Ltd., Guildford, UK
Investigative Ophthalmology & Visual Science May 2007, Vol.48, 4076. doi:
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      U. Aftab, C. Jiang, H. Klassen, E. Miljan, J. D. Sinden, M. Young; Isolation, Growth Kinetics and Differentiation of Human Retinal Progenitor Cells in Culture. Invest. Ophthalmol. Vis. Sci. 2007;48(13):4076.

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

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Abstract

Purpose:: To study and compare the growth kinetics of human retinal progenitor cells (hRPCs) isolated from donor tissue of different developmental ages, to assess the number of passages over which these cells can be propagated, and to determine whether hRPCs can be differentiated into mature retinal cells.

Methods:: Eyes (11 to 18 weeks gestational age, G.A.) were obtained from an approved source with informed consent. Retinas were dissected and dissociated into single cells using 0.1% collagenase. The cells were suspended in media containing DMEM-F12 and 5% fetal bovine serum (FBS) and plated into culture flasks. FBS-containing media was removed the next day and replaced with fresh media without FBS. Cells were fed twice weekly and passaged when they reached 70-85% confluence. Chamber slides were plated at each passage, with and without FBS, and antibodies against Ki-67, nestin, rhodopsin, recoverin, red/green opsin, blue opsin, and Crx were added. Cells were counted at the time of isolation and at each passage using a hemocytometer.

Results:: hRPCs from donor tissue of less than 15 weeks G.A. did not survive well beyond the 2nd passage, whereas cells from donors above this age continued to proliferate in culture up to the 4th-5th passage. The yield of cells from retinal tissue of 15 weeks or more G.A. was on average 9.04 times more than that from tissue aged less than 15 weeks G.A. The maximum level of cell growth was observed at the level of passage 2. Neuron-like morphology was observed more often in the earlier passages. Cells stained positively for Ki-67, nestin, recoverin, Crx, red/green opsin and blue opsin, and weakly for rhodopsin.

Conclusions:: We conclude that tissue of 15-18 weeks gestational age results in a better yield, growth and survival of hRPCs than does tissue obtained from early ages under standard proliferation conditions. These cells can be readily passaged 5 times, after which increasing apoptosis is observed. These cells stain positively for photoreceptor markers when subjected to differentiation conditions.

Keywords: retinal culture • retinal degenerations: cell biology 
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