AimTo generate human embryonic stem cell derived corneal endothelial cells (hESC-CECs) for transplantation in patients with corneal endothelial dystrophies.Materials and MethodsFeeder-free hESC-CECs were generated by a directed differentiation protocol. hESC-CECs were characterized by morphology, expression of corneal endothelial markers, and microarray analysis of gene expression.ResultshESC-CECs were nearly identical morphologically to primary human corneal endothelial cells, expressed Zona Occludens 1 (ZO-1) and Na+/K+ATPaseα1 (ATPA1) on the apical surface in monolayer culture, and produced the key proteins of Descemet’s membrane, Collagen VIIIα1 and VIIIα2 (COL8A1 and 8A2). Quantitative PCR analysis revealed expression of all corneal endothelial pump transcripts. hESC-CECs were 96% similar to primary human adult CECs by microarray analysis.ConclusionhESC-CECs are morphologically similar, express corneal endothelial cell markers and express a nearly identical complement of genes compared to human adult corneal endothelial cells. hESC-CECs may be a suitable alternative to donor-derived corneal endothelium.
Much of the peripheral nervous system of the head is derived from ectodermal thickenings, called placodes, that delaminate or invaginate to form cranial ganglia and sense organs. The trigeminal ganglion, which arises lateral to the midbrain, forms via interactions between the neural tube and adjacent ectoderm. This induction triggers expression of Pax3, ingression of placode cells and their differentiation into neurons. However, the molecular nature of the underlying signals remains unknown. Here, we investigate the role of PDGF signaling in ophthalmic trigeminal placode induction. By in situ hybridization, PDGF receptor β is expressed in the cranial ectoderm at the time of trigeminal placode formation, with the ligand PDGFD expressed in the midbrain neural folds. Blocking PDGF signaling in vitro results in a dose-dependent abrogation of Pax3 expression in recombinants of quail ectoderm with chick neural tube that recapitulate placode induction. In ovo microinjection of PDGF inhibitor causes a similar loss of Pax3 as well as the later placodal marker, CD151, and failure of neuronal differentiation. Conversely, microinjection of exogenous PDGFD increases the number of Pax3+ cells in the trigeminal placode and neurons in the condensing ganglia. Our results provide the first evidence for a signaling pathway involved in ophthalmic (opV) trigeminal placode induction.
Whereas neural crest cells are the source of the peripheral nervous system in the trunk of vertebrates, the “ectodermal placodes,” together with neural crest, form the peripheral nervous system of the head. Cranial ectodermal placodes are thickenings in the ectoderm that subsequently ingress or invaginate to make important contributions to cranial ganglia, including epibranchial and trigeminal ganglia, and sensory structures, the ear, nose, lens, and adenohypophysis. Recent studies have uncovered a number of molecular signals mediating induction and differentiation of placodal cells. Here, we described recent advances in understanding the tissue interactions and signals underlying induction and neurogenesis of placodes, with emphasis on the trigeminal and epibranchial. Important roles of Fibroblast Growth Factors, Platelet Derived Growth Factors, Sonic Hedgehog, TGFβ superfamily members, and Wnts are discussed.
The largest of the cranial ganglia, the trigeminal ganglion relays cutaneous sensations of the head to the central nervous system. Its sensory neurons have a dual origin from both ectodermal placodes and neural crest. Here, we show that birth of neurons derived from the chick ophthalmic trigeminal placode begins prior to their ingression (HH11), as early as HH8, and considerably earlier than previously suspected (HH16). Furthermore, cells exiting the cell cycle shortly thereafter express the ophthalmic trigeminal placode marker Pax3 (HH9). At HH11, these post-mitotic Pax3+ placode cells begin to express the pan neuronal marker, neurofilament, while still in the ectoderm. Analysis of the ectodermal origin and distribution of these early post-mitotic neurons reveals that the ophthalmic placode extends further rostrally than anticipated, contributing to neurons that reside in and make a significant contribution to the ophthalmic trigeminal nerve. These data redefine the timing and extent of neuron formation from the ophthalmic trigeminal placode.
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