Lentoid

Abstract: Dissociated cells of the lens epithelium of newly hatched chickens were cultured in vitro to investigate whether cells actively grown in culture retain their own differentive entiative traits to form lens fibers. After an exponential growth phase of the flattened epithelial cells, a number of “islets” of smaller epithelial cells with polygonal shape appeared. Along the periphery of these islets, the characteristic morphological change which leads to the formation of spherical bodies was observed. Electron microscopic observation showed the differentiation of lens fibers in these spherical bodies comparable to those in the lens in situ. Accumulation of δ-chrystallin was confirmed in such “lentoid” bodies. Outgrowth of the lens epithelial cells was maintained in in vitro culture up to about 50 days with several subculturings. The formation of lentoid bodies occurred in each subculture generation, which started from a homogeneous population of flattened epithelial cells. The present culture conditions permit the maintenance of such a population of cells that have a high growth potential and stably retains their differentiative trait to form lens fiber, even after repeated replication under in vitro conditions.

Abstract: The eye lens is an encapsulated avascular organ whose function is to focus light on the retina. Lens comprises a single progenitor cell lineage in multiple states of differentiation. Disruption of lens function leading to protein aggregation and opacity results in age-onset cataract. Cataract is a complex disease involving genetic and environmental factors. Here, we report the development of a new 3-stage system that differentiates human embryonic stem cells (hESCs) into large quantities of lens progenitor-like cells and differentiated 3-dimensional lentoid bodies. Inhibition of BMP signaling by noggin triggered differentiation of hESCs toward neuroectoderm. Subsequent reactivation of BMP and activation of FGF signaling stimulated formation of lens progenitor cells marked by the expression of PAX6 and alpha-crystallins. The formation of lentoid bodies was most efficient in the presence of FGF2 and Wnt-3a, yielding approximately 1000 lentoid bodies/30-mm well. Lentoid bodies expressed and accumulated lens-specific markers including alphaA-, alphaB-, beta-, and gamma-crystallins, filensin, CP49, and MIP/aquaporin 0. Collectively, these studies identify a novel procedure to generate lens cells from hESCs that can be applied for studies of lens differentiation and cataractogenesis using induced pluripotent stem (iPS) cells derived from various cataract patients.