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J. S. Meyer, L. S. Wright, R. L. Shearer, E. E. Capowski, T. Lavaute, M. Pankratz, S.-C. Zhang, D. M. Gamm; Stepwise Generation of Early Retinal Cell Types From Human Embryonic Stem Cells Mimics Normal Retinal Development. Invest. Ophthalmol. Vis. Sci. 2008;49(13):3547.
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© ARVO (1962-2015); The Authors (2016-present)
Human embryonic stem (hES) cells have the potential to serve as a model system to unravel the molecular and developmental events that guide the development of an undifferentiated cell towards a retinal fate. We present a hES cell differentiation protocol that closely parallels the time-dependent sequence of molecular events that occurs during normal early human retinogenesis.
hES cells were differentiated toward a primitive anterior neuroepithelial fate following established methods, and then induced to differentiate along a retinal lineage. The acquisition of a primitive retinal fate was determined by gene and protein expression profiles at multiple developmental timepoints using RT-PCR and immunocytochemistry. Profiles were quantified using qRT-PCR and FACS analysis. Patterned cells were maintained long term as neurospheres or adherent cultures to assess the ability of these cells to generate retinal progenitors and RPE cells.
Using a specific patterning protocol, hES cell-derived anterior neuroepithelial cells begin to coexpress the transcription factors Pax6, Rx and Otx2 within the first week of differentiation. Traditionally, Pax6 and Rx have been used to identify cells of the early eye-forming field, from which the optic vesicles are derived. Within ten days, approximately 95% of differentiated hES cells coexpress Pax6 and Rx, as determined by FACS analysis, yielding a highly enriched population of potential primitive retinal cells. A full complement of early retina-associated genes is expressed within two weeks of differentiation, including Pax6, Rx, Otx2, Six3 and Six6. Prolonged differentiation of these cells as neurospheres or adherent cultures ultimately led to the expression of characteristics indicative of neural retinal progenitor cells or RPE, respectively.
In the current study, we explored the ability of hESCs to adopt a primitive retinal fate in vitro, and demonstrated that this differentiation process mimics the timeline of events that occur in vivo. Thus, hES cell cultures can be manipulated to produce the two components of early retina (retinal progenitor cells and RPE) in a manner reminiscent of normal human retinal development.
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