April 2014
Volume 55, Issue 13
Free
ARVO Annual Meeting Abstract  |   April 2014
Co-culture of stem cell derived retinal progenitors and retinal pigment epithelium promotes tissue maturity
Author Affiliations & Notes
  • Peter Yu Cheng Zhao
    Surgery/Ophthalmology, Yale University, New Haven, CT
  • Shaomin Peng
    Surgery/Ophthalmology, Yale University, New Haven, CT
  • Lilangi Ediriwickrema
    Surgery/Ophthalmology, Yale University, New Haven, CT
  • Caihong Qiu
    Cell Biology, Yale University, New Haven, CT
  • Katherine Jean Davis
    Surgery/Ophthalmology, Yale University, New Haven, CT
  • Ron A Adelman
    Ophthalmology & Visual Science, Yale University, New Haven, CT
  • Lawrence J Rizzolo
    Surgery/Ophthalmology, Yale University, New Haven, CT
  • Footnotes
    Commercial Relationships Peter Zhao, None; Shaomin Peng, None; Lilangi Ediriwickrema, None; Caihong Qiu, None; Katherine Davis, None; Ron Adelman, None; Lawrence Rizzolo, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science April 2014, Vol.55, 3995. doi:https://doi.org/
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      Peter Yu Cheng Zhao, Shaomin Peng, Lilangi Ediriwickrema, Caihong Qiu, Katherine Jean Davis, Ron A Adelman, Lawrence J Rizzolo; Co-culture of stem cell derived retinal progenitors and retinal pigment epithelium promotes tissue maturity. Invest. Ophthalmol. Vis. Sci. 2014;55(13):3995. doi: https://doi.org/.

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

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Abstract

Purpose: To restore vision, stem cell therapies for retinal degenerations must address the loss of both photoreceptors and retinal pigment epithelium. To explore the effects of each tissue layer on the other’s maturation, we co-cultured human embryonic stem cell derived retinal progenitor cells (hESC-RPC) with retinal pigment epithelium (hESC-RPE).

Methods: Cultures were derived from the H9 human embryonic stem cell line using modifications of previously published techniques. To generate hESC-RPC, H9 were seeded as clusters to polycaprolactone (PCL) scaffolds and cultured in retinal differentiation media. hESC-RPE were expanded as monolayers on laminin-coated Transwell filters in serum-free media, and adapted to the retinal differentiation media for co-culture. In the co-culture group, hESC-RPC cultures were placed on top of hESC-RPE monolayers during the retinal differentiation protocol. For controls, cultures were maintained separately in retinal differentiation media. Transepithelial electrical resistance (TER) was monitored over time to assess RPE integrity and function. After 2-4 weeks, co-cultured tissue layers were separated and compared to controls by RT-PCR and immunofluorescence.

Results: Neural retinal marker mRNAs were expressed by hESC-RPC in both monoculture and co-culture. Co-cultures expressed several of these markers at higher levels, including Crx and Rhodopsin. Immunofluorescence revealed multilayered clusters positive for markers including Otx2, Crx, and Recoverin. In co-cultures, these markers localized to the surface opposing the RPE. An RT-PCR array for monitoring RPE maturation showed that co-cultured hESC-RPE was more mature. In addition, co-cultured hESC-RPE maintained a high TER in retinal differentiation medium, while in controls the TER decreased after 2-4 weeks.

Conclusions: Co-culture increases the maturation of both hESC-RPC and hESC-RPE, underlining the interdependence of these tissues. Secretions of the RPE and contact with the RPE could supplement or replace media as promoters of RPC differentiation, facilitating the creation of a transplantable tissue.

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