April 2014
Volume 55, Issue 13
Free
ARVO Annual Meeting Abstract  |   April 2014
Simple generation of self-forming neural retina and RPE cells from confluent human iPS cells
Author Affiliations & Notes
  • Olivier Goureau
    Institut de la Vision, UPMC Univ Paris 06, UMR_S 968; INSERM U968; CNRS UMR_7210, Paris, France
  • Sacha Reichman
    Institut de la Vision, UPMC Univ Paris 06, UMR_S 968; INSERM U968; CNRS UMR_7210, Paris, France
  • Angélique Terray
    Institut de la Vision, UPMC Univ Paris 06, UMR_S 968; INSERM U968; CNRS UMR_7210, Paris, France
  • Amélie Slembrouck
    Institut de la Vision, UPMC Univ Paris 06, UMR_S 968; INSERM U968; CNRS UMR_7210, Paris, France
  • Celine Nanteau
    Institut de la Vision, UPMC Univ Paris 06, UMR_S 968; INSERM U968; CNRS UMR_7210, Paris, France
  • Gael Orieux
    Institut de la Vision, UPMC Univ Paris 06, UMR_S 968; INSERM U968; CNRS UMR_7210, Paris, France
  • Christelle Monville
    I-STEM, INSERM; UEVE U861, Evry, France
  • Jose Alain Sahel
    Institut de la Vision, UPMC Univ Paris 06, UMR_S 968; INSERM U968; CNRS UMR_7210, Paris, France
    Centre Hospitalier National d’Ophtalmologie des Quinze-Vingts, INSERM-DHOS CIC 503, Paris, France
  • Footnotes
    Commercial Relationships Olivier Goureau, None; Sacha Reichman, None; Angélique Terray, None; Amélie Slembrouck, None; Celine Nanteau, None; Gael Orieux, None; Christelle Monville, None; Jose Sahel, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science April 2014, Vol.55, 3993. doi:
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      Olivier Goureau, Sacha Reichman, Angélique Terray, Amélie Slembrouck, Celine Nanteau, Gael Orieux, Christelle Monville, Jose Alain Sahel; Simple generation of self-forming neural retina and RPE cells from confluent human iPS cells. Invest. Ophthalmol. Vis. Sci. 2014;55(13):3993.

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

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Abstract

Purpose: For retinal cell therapy using human induced pluripotent stem cells (hiPSCs), the current challenge is to improve the generation of their therapeutic derivatives by eliminating time- and labor-consuming manual steps to allow large-scale production complying with certain criteria such as safety, efficiency, reproducibility and low production cost. Here, we developed a new retinal differentiation method using confluent hiPSCs bypassing embryoid body EB formation and selection and the use of exogenous molecules, coating or Matrigel.

Methods: Integration-free hiPSCs were generated via nucleofection of adult human dermal fibroblasts, with plasmids coding for the transcription factors OCT4, NANOG, SOX2, LIN28, KLF4 and C-MYC. Confluent hiPSCs were directed toward a retinal lineage in a serum free proneural medium containing N2 supplement. Emergent neural retina (NR)-like structures were isolated and cultured in floating conditions for their maturation. Capacity for retinal differentiation was determined by immunohistochemistry and qRT-PCR time course analysis triggering specific developmental and mature retinal markers.

Results: In two weeks, confluent hiPSCs are able to generate simultaneously RPE cells and self-forming NR-like structures containing multipotent retinal progenitor cells (RPCs). Floating cultures of isolated neuroretinal tissue enabled the differentiation of RPCs into all types of retinal cells in a sequential manner consistent with in vivo vertebrate retinogenesis. Indeed, early-born retinal cells were identified as early as 21 days in culture (i.e. ganglion, amacrine and horizontal cells), and late-born retinal cells appeared after 35 days (i.e. photoreceptor, Muller glial and bipolar cells). Interestingly, early inhibition of the Notch pathway led to massive differentiation of RPCs into photoreceptor precursors. hiPSC-derived RPE cells can be amplified while retaining their phenotype close to their in vivo state.

Conclusions: We thus propose an innovative process providing an easy and scalable approach to generate large numbers of mitotic RPCs and of both RPE cells and precursors of photoreceptors needed for regenerative medicine or drug screening purposes. Our retinal differentiation also provides an accessible in vitro model to investigate mechanisms involved in human retinogenesis and retinal diseases.

Keywords: 721 stem cells • 698 retinal development • 500 differentiation  
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