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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)
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).
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.
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.
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.
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