July 2018
Volume 59, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2018
Objective quantification of photoreceptor ribbon synapses in iPS cell-derived retina after transplantation to retinal degeneration model mice
Author Affiliations & Notes
  • Ryutaro Akiba
    Laboratory for Retinal Regeneration, Riken Center for Developmental Biology, Kobe, Hyogo, Japan
    Ophthalmology and Visual Science, Chiba University, Chiba, Chiba, Japan
  • Takesi Matsuyama Hoyos
    Laboratory for Retinal Regeneration, Riken Center for Developmental Biology, Kobe, Hyogo, Japan
  • Michiko Mandai
    Laboratory for Retinal Regeneration, Riken Center for Developmental Biology, Kobe, Hyogo, Japan
  • Shuichi Yamamoto
    Ophthalmology and Visual Science, Chiba University, Chiba, Chiba, Japan
  • Masayo Takahashi
    Laboratory for Retinal Regeneration, Riken Center for Developmental Biology, Kobe, Hyogo, Japan
Investigative Ophthalmology & Visual Science July 2018, Vol.59, 553. doi:
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      Ryutaro Akiba, Takesi Matsuyama Hoyos, Michiko Mandai, Shuichi Yamamoto, Masayo Takahashi; Objective quantification of photoreceptor ribbon synapses in iPS cell-derived retina after transplantation to retinal degeneration model mice. Invest. Ophthalmol. Vis. Sci. 2018;59(9):553.

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

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Abstract

Purpose : Photoreceptors in iPS cell-derived retina (iPSC-retina) has previously been shown to form synapses with host retina bipolar cells after transplantation to the end stage retinal degeneration mice (rd1). Quantification of host-graft synapses is one major parameter to estimate functional graft integration. However, Immunohistochemical characterization of synapse in the degenerated retina or of grafted retina is often challenging, as traits are not as clear as in the wild type retina. Therefore, using postnatal wild type mouse retina as a training data, we developed a new method to objectively count synapses. Using this method, we evaluated the synapses of iPSC-retina after transplantation to rd1 mice.

Methods : [Developmental synapse analysis]
Immunohistochemistry (IHC) of postnatal day (P) 7-35 wildtype B6J mice retina was carried out to visualize pre- and post-synaptic markers of photoreceptor-bipolar ribbon synapse. Regions Of Interest (ROI) were extracted from the images using ImageJ. The likeliness of synapse for each marker pair was then evaluated using a naïve Bayes classifier algorithm. Image properties of synapses were extracted and analyzed from each postnatal day retinas.
[Transplanted synapse analysis]
3D retinas differentiated from Mouse iPS cells, were transplanted into the subretinal space of end stage retinal degeneration model rd1 mice retina. The mice were sampled for IHC on 9, 14 and 30 days after transplantation (DAT). rd1 mice without transplantation were also sampled for IHC at the same time points as negative controls. Synapse formation of each group was evaluated using the method described above.

Results : [Developmental synapse analysis]
The numbers of synapses started to rise quickly around P14, and reached a peak on P28. The expression pattern of both pre- and post- synaptic markers were diffuse on P7, and gradually became more prominent in the outer plexiform layer around P10 to P14, consistent with previous reports.
[Transplanted synapse analysis]
The number of synapses increased on 30 DAT in rd1 mice with iPSC-retina transplantation when compared with 9 and 14 DAT and negative controls.

Conclusions : Objective quantification of photoreceptor synapses has been achieved. Synaptogenesis in iPSC-retina takes place around 14 to 30 DAT.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.

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