A novel technique for studying the growth properties of human embryonic corneal endothelial and stromal cells in organ culture was devised. Human embryonic eye globes were microdissected so that a passage was opened between the outer environment and the anterior chamber, which rendered free access of tissue culture medium to the endothelial cell monolayer. The dissected eye globes were maintained in organ culture for 24 h in the continuous presence of tritiated thymidine. Cross-sections were cut through the whole eye globes and subjected to autoradiographic analysis in order to estimate the mitogenic response of each corneal cell type to externally supplied growth factors and hormones. It was found that the corneal endothelial cells could be stimulated to initiate DNA synthesis by exposure to epidermal growth factor (EGF). The stimulatory effect of this growth factor could be enhanced if either insulin-like growth factor I (IGF I) or a combination of insulin, transferrin and high density lipoprotein (HDL) was simultaneously added. Similar results were obtained by adding growth factors and hormones to primary cell cultures from human embryonic corneas. It was also found that the stromal cell could be stimulated to initiate DNA synthesis by the addition of EGF and IGF I or a combination of insulin, transferrin and HDL. Taken together, these results suggest that the proliferation of corneal endothelial cells and stromal cells is dependent on EGF-like factors as well as on some insulin-like substance during embryogenesis.

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