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D. M. Gamm, IV, R. L. Shearer, K. A. Wallace, E. E. Capowski, L. S. Wright, J. S. Meyer; Endogenous Mechanisms of Early Retinal Specification in Human Embryonic Stem Cells. Invest. Ophthalmol. Vis. Sci. 2009;50(13):3214.
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© ARVO (1962-2015); The Authors (2016-present)
To investigate endogenous signaling molecules involved in the differentiation of human embryonic stem (hES) cells toward a primitive eye field and early retinal phenotype.
hES cells were differentiated as embryoid bodies and then plated to allow for neural rosette formation. Rosettes were subsequently lifted and grown as neurospheres in minimal medium supplemented with B27. The appearance of cells belonging to the retinal lineage was monitored over time by PCR, immunocytochemistry and FACS. In addition, endogenous expression of signaling molecules involved in anterior neuralization and retinal specification, including FGF, Wnt and BMP family members, was examined at multiple developmental timepoints using RT-PCR and immunocytochemistry. Lastly, the effect of exogenous signaling molecules sonic hedgehog, IGF-1 and retinoic acid on gene expression of early eye field and retinal markers was investigated by qPCR.
Human ES cells were nearly uniformly differentiated to a primitive anterior neuroepithelial and eye field (Pax6+/Rx+) fate using established methods in the absence of exogenous BMP, Wnt or Nodal antagonists. Thereafter, up to 30% of these cells spontaneously assumed a definitive retinal progenitor (Chx10+/Pax6+) or RPE (MITF+/Pax6+) identity. The initial neural specification stage was partially regulated by endogenously produced FGFs. In addition, establishment of the primitive eye field was associated with the innate generation of the Wnt and BMP antagonists Dkk1 and Noggin, respectively. The efficiency of primitive retinal lineage specification was found to be enhanced by the addition of sonic hedgehog and IGF-1, while the addition of caudalizing factors such as retinoic acid largely eliminated the capacity for retinal specification.
Several known neural and retinal developmental events are endogenously regulated within differentiating hES cells. Thus, hES cells have the potential to be used as a model system to study the underlying mechanisms of early human retinal development.
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