June 2017
Volume 58, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2017
Recapitulation of the Human Fetal Crumbs Complex in human iPSCs-derived Retinas and Retinal Pigment Epithelium
Author Affiliations & Notes
  • Peter M Quinn
    Ophthalmology, Leiden University Medical Center, Leiden, Netherlands
  • Thilo Matthias Buck
    Ophthalmology, Leiden University Medical Center, Leiden, Netherlands
  • Celso Alves
    Ophthalmology, Leiden University Medical Center, Leiden, Netherlands
  • Charlotte Ohonin
    Ophthalmology, Leiden University Medical Center, Leiden, Netherlands
  • Susana M Chuva de Sousa Lopes
    Anatomy and Embryology, Leiden University Medical Center , Leiden, Netherlands
  • Harald Mikkers
    Molecular Cell Biology, Leiden University Medical Center , Leiden, Netherlands
  • Jan Wijnholds
    Ophthalmology, Leiden University Medical Center, Leiden, Netherlands
  • Footnotes
    Commercial Relationships   Peter Quinn, None; Thilo Buck, None; Celso Alves, None; Charlotte Ohonin, None; Susana Chuva de Sousa Lopes, None; Harald Mikkers, None; Jan Wijnholds, None
  • Footnotes
    Support  FFB project nr TA-GT-0715-0665-LUMC, Curing Retinal Blindness Foundation, Stichting Retina Nederland Fonds, Landelijke St. Blinden en Slechtzienden, Rotterdamse Stichting, Blindenbelangen, St. Blindenhulp, St. Blinden-Penning, Algemene Nederlandse Vereniging ter Voorkoming van Blindheid (ANVVB), Gelderse Blinden Stichting, MaculaFonds
Investigative Ophthalmology & Visual Science June 2017, Vol.58, 3758. doi:
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      Peter M Quinn, Thilo Matthias Buck, Celso Alves, Charlotte Ohonin, Susana M Chuva de Sousa Lopes, Harald Mikkers, Jan Wijnholds; Recapitulation of the Human Fetal Crumbs Complex in human iPSCs-derived Retinas and Retinal Pigment Epithelium. Invest. Ophthalmol. Vis. Sci. 2017;58(8):3758.

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

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Abstract

Purpose : Mutations in the CRB1 gene cause severe inherited retinal dystrophies from birth or early childhood. The early developmental processes of normal and diseased human retinas are still not well understood. Having in vitro human retinas which mimic these developmental processes is highly desirable allowing a bridge in treatment development between animal models and the clinic. Here, we compare the developing Crumbs complex in 1st and 2nd trimester human fetal retina and retinal pigment epithelium (RPE) and how well this is recapitulated in retinal organoids and RPE differentiated from human induced pluripotent stem cells (iPSCs).

Methods : Human retinal organoids and RPE were differentiated from the LUMC0004iCTRL10 iPSC line following Zhong et al, 2014. Material was analysed on a range of differentiation day (DD) iPSCs-derived retinas and RPE and 1st and 2nd trimester human fetal eyes. Immunohistochemistry was carried out to allude to positioning of proteins of interest at the subapical region and adherens junctions.

Results : Human fetal retina from the 1st trimester expressed CRB2 at the subapical region above the adherens junction protein P120-catenin however CRB1 was not visibly present. During the 2nd trimester however both CRB1 and CRB2 along with other Crumbs complex members PALS1 and PAR3 are present. In both the 1st and 2nd trimester CRB2 is present apically in the RPE, while p120-catenin localized basolateral. Early DD28 iPSCs-derived retinas did not express CRB1 but CRB2 was located at the subapical region. Later DD160 iPSCs-derived retinas did express CRB1 at the subapical region along with CRB2, PALS1 and MUPP1. Human iPSCs-derived RPE also apically expressed CRB2 but not CRB1, while β-catenin localized basolateral.

Conclusions : We show for the first time that CRB2, but not CRB1, is present at the subapical region in 1st trimester human fetal retina. But CRB1 localises as rod and Müller glial cell birth takes place during the 2nd trimester. Suggesting a role for CRB2 but not CRB1 in the earliest human retinal radial glial progenitors. Also we show that human fetal RPE expresses CRB2 but not CRB1. Finally, human iPSCs-derived retinas and RPE recapitulated the developing Crumbs complex as seen in 1st and 2nd trimester fetal eyes. Development of knockout CRB1 and/or CRB2 retinal organoids may therefore provide robust in vitro systems for potency assays for gene therapy vectors.

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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