July 2019
Volume 60, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2019
Generating lentoids from human iPSCs using an alternative substrate to Matrigel
Author Affiliations & Notes
  • Yvette Wormstone
    School of Biological Sciences, University of East Anglia, Norwich, United Kingdom
  • Michael Wormstone
    School of Biological Sciences, University of East Anglia, Norwich, United Kingdom
  • Footnotes
    Commercial Relationships   Yvette Wormstone, None; Michael Wormstone, None
  • Footnotes
    Support  The Humane Research Trust; Rosemary Rushbrook and Friends
Investigative Ophthalmology & Visual Science July 2019, Vol.60, 4296. doi:
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      Yvette Wormstone, Michael Wormstone; Generating lentoids from human iPSCs using an alternative substrate to Matrigel. Invest. Ophthalmol. Vis. Sci. 2019;60(9):4296.

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

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Abstract

Purpose : To date, all reported efforts to differentiate human induced pluripotent stem cells (iPSCs) into miniature ocular lenses (‘lentoids’) have relied upon the use of mouse sarcoma-derived Matrigel as a culture substrate. However, the composition of Matrigel varies from batch-to-batch and it contains undefined concentrations of growth factors. This inconsistency could influence the physiological outcomes of developmental experiments. The aim of this study is to generate lentoids from iPSCs using a defined replacement for Matrigel.

Methods : iPSCs (Cellartis ChiPSC22) were seeded onto iMatrix and subjected to the three-step differentiation scheme first detailed by Yang et al (2010). This involves treating the cells in mTesR with the addition of specific growth factors at different stages: 1) treatment of 100 ng/ml Noggin from d 0 to 6; 2) combination of 100 ng/ml bFGF, 20 ng/ml BMP4, and 20 ng/ml BMP7 from d 6 to 18; and 3) combination of 100 ng/ml FGF2 and 20 ng/ml Wnt-3a from d 18 onwards. Gene expression profiles were determined using quantitative Real-Time PCR (qRT-PCR) on RNA isolated from Days 6, 12 and 18, with naïve iPSC RNA as baseline control.

Results : Following stage 1 culture the cells formed a collection of single-layer clusters. Stage 2 culture induced progressive organisational changes that resulted in structures resembling ‘fried eggs’, as first described by Fu et al (2017). These matured into lentoid structures with defined outer boundaries and refractive properties. Addition of Stage 3 media didn’t appear to develop the lentoids further. All stages of culture were completed successfully on iMatrix. Gene expression profiling revealed – among other things - increased expression of the eye ‘master gene’ Pax6 from day 6 onwards and an enriched expression of aA crystallin was observed at Day 18, inferring initiation of lens fibre differentiation.

Conclusions : We have generated lentoid bodies from iPSCs using iMatrix as an alternative (fully defined) substrate to Matrigel. This, along with our observations and qRT-PCR data provides a strong base upon which to further refine and improve the ‘three stage’ schematic.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.

 

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