June 2017
Volume 58, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2017
3D Retinal Cups from Human iPS Cells Display Differentiated Subtypes of Amacrine Cells and Immature Synaptic Contacts
Author Affiliations & Notes
  • Dunja Lukovic
    National Stem Cell Bank , Research Center Principe Felipe , Valencia, Valencia, Spain
  • Ana Artero Castro
    National Stem Cell Bank , Research Center Principe Felipe , Valencia, Valencia, Spain
  • Laura Campello
    Department of Physiology, Genetics and Microbiology , University of Alicante, Alicante , Spain
  • Nicolás Cuenca
    Department of Physiology, Genetics and Microbiology , University of Alicante, Alicante , Spain
  • Slaven Erceg
    National Stem Cell Bank , Research Center Principe Felipe , Valencia, Valencia, Spain
  • Footnotes
    Commercial Relationships   Dunja Lukovic, None; Ana Artero Castro, None; Laura Campello, None; Nicolás Cuenca, None; Slaven Erceg, None
  • Footnotes
    Support  PRB2, Plataforma de Recursos Biomoleculares y Bioinformaticos, ISCIII, Spain; Ministerio de Economia, Industria y Competitividad, ISCIII, PI16/00409, Spain.
Investigative Ophthalmology & Visual Science June 2017, Vol.58, 1362. doi:
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      Dunja Lukovic, Ana Artero Castro, Laura Campello, Nicolás Cuenca, Slaven Erceg; 3D Retinal Cups from Human iPS Cells Display Differentiated Subtypes of Amacrine Cells and Immature Synaptic Contacts. Invest. Ophthalmol. Vis. Sci. 2017;58(8):1362.

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

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Abstract

Purpose : Self-organizing 3D retinas derived from patient–specific iPS cells offer unprecedented opportunities to study the human retinal biology, as well as serve to develop in vitro models of human retinal diseases allowing to study the molecular mechanisms of these pathologies and potential therapeutic treatments. Here, we performed a detailed cellular and ultrastructural analysis of human 3D retinas in order to define the maturation stages and organizational features of this tissue.

Methods :
iPS cells were generated by reprogramming human fibroblasts from a healthy individual and directed to differentiate toward neural retina in 3D cell culture. In the initial stage of photoreceptor outer segment formation, 3D retinal cups were analyzed by confocal immunohistochemistry and transmission electron microscopy.

Results : The iPS-derived 3D neural retina exhibited the complex laminar morphology of a mature retina. Rod and cone photoreceptors were recognized using antibodies against rhodopsin and cone arrestin and these cells presented an inner and outer segment under development. The existence of disks in the incipient outer segments was observed by electron microscopy. Horizontal cells were properly located in the inner nuclear layer, evidenced by the specific marker parvalbumin, and their dendrites were decorated with spots of ribeye immunoreactivity. The synaptic marker synaptophysin was expressed by photoreceptors. Small ribbon synapses were observed in the outer plexiform layer using electron microscopy, which indicates potential incipient synaptic contacts formation. Müller cells displayed their normal columnar retinal structure while astrocytes showed immature morphologies. Different types of amacrine cells, dopaminergic, starburst and AII cells, were detected indicating advanced stage of retinal differentiation. Collagen IV immunoreactivity was found filling the empty spaces of the tissue and in the circular structures of the center of 3D retinal cups.

Conclusions : The identification of advanced photoreceptor morphology as well as different subtypes of amacrine cells, glial cells and well defined retinal layering point out the complex degree of development that can be reached by 3D cell culture of patient-specific iPS cells. The presence of synaptic ribbons suggests that the photoreceptors are capable of signal neurotransmission to their postsynaptic neurons.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.

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