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Philip Nigel Lewis, Keith Meek; Cell-directed stromal morphogenesis in the embryonic human cornea. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):1943. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Keratocytes play a pivotal role in the synthesis and assembly of the corneal stroma during embryonic development, but the mechanisms by which the cells carry out this process remain unresolved in the human cornea. We examined developing human embryonic corneal stroma by serial block face scanning electron microscopy to visualise cell matrix interactions in 3-D, and thus to gain insight into the underlying cellular mechanisms involved in the assembly of the tissue. <br /> <br />
Corneas from 8, 13 and 15 week human embryos were fixed in modified Karnovsky fixative, then processed through a series of staining solutions, including tannic acid, osmium tetroxide, uranyl acetate and lead acetate, prior to dehydration and embedding in CY212 epoxy resin. The surfaces of polymerised resin blocks were then trimmed and the blocks were attached to aluminium specimen pins and transferred to a Zeiss Sigma VP FEG SEM equipped with a Gatan 3View®2 system. Data sets of up to 2000 images of the block surface were acquired every 25 or 50nm through automated sectioning. Selected serial image sequences were extracted from the image data and 3-D reconstructions were generated using Amira 5.6 software.
3-D analysis revealed that the 8 week embryonic corneal stroma was characterised by quasi-orthogonally arranged, stratified layers of densely packed prospective keratocyte cells. Thin strands of collagen were present orientated in each layer in parallel with the axes of elongated cells and their nuclei. At later 13 and 15 week developmental stages, the stroma comprised numerous thin superimposed lamellae made up of highly aligned collagen fibrils and prospective keratocytes. Many of the keratocytes exhibited highly complex cellular extensions which appeared to be aligned with the collagen fibrils in each lamella.
In the development of the human cornea, the corneal embryonic stroma would appear to form from an initial cellular template, without the formation of a primary stroma as seen in chick. Once formed the template facilitates the formation of superimposed lamellae from which keratocytes synthesise and organise the collagen in the stroma.
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