Three-dimensional early retinal progenitor 3D tissue constructs derived from human embryonic stem cells
Gabriel Nistor, Magdalene J. Seiler, Fengrong Yan, David Ferguson, Hans S. Keirstead
Journal of Neuroscience Methods Article in Press, Corrected Proof doi:10.1016/j.jneumeth.2010.04.025
Purpose: To develop three-dimensional (3D) constructs of retinal pigment epithelium (RPE) and early retina progenitor cells from human embryonic stem cells (hESCs).
Methods: 3D tissue constructs were developed by culturing hESC-derived neural retinal progenitors in a matrix on top of hESC-derived RPE cells in a cell culture insert. An osmolarity gradient maintained the nutrition of the 3D cell constructs. Cross-sections through hESC-derived tissue constructs were characterized by immunohistochemistry for various transcription factors and cell markers.
Results: hESC-derived tissue constructs expressed transcription factors characteristic of retinal development, such as pax6, Otx2, Chx10, retinal RAX; Brn3b (necessary for differentiation of retinal ganglion cells); and crx and nrl (role in photoreceptor development). Many cells expressed neuronal markers including nestin, beta-tubulin and microtubule-associated proteins.