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P. G. Layer, M. Rieke, M. Araki; Cryptic Retinal Potencies Revealed in Reaggregated Spheres From Neonatal Rat Pineals. Invest. Ophthalmol. Vis. Sci. 2008;49(13):1318.
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The pineal organ represents a primitive "third eye" structure in vertebrates (Eakin, 1973). In mammals, however, the pineal gland is an endocrine organ that does not exhibit neuronal characteristics. Under monolayer-culture conditions, cells from neonatal rat pineals can differentiate into rod-like cells, as has been shown by rhodopsin immunoreactivity (Araki et al. 1992). To further reveal cryptic retinal potencies of pineal cells in a 3-dimensional tissue environment, we have reaggregated neonatal rat pineal cells to analyse their degree of retinal histotypic differentiation.
Dissociated cells of the pineals from postnatal day 1 rats were cultivated on a gyratory shaker at 68 rpm for reaggregation to form three-dimensional spheres. Reaggregation was performed in 3,5 cm Petri dishes in 2 ml full-medium. After 10 days, reaggregates were collected, fixed by 4% paraformaldehyde and cryosectioned. Differentiation and cellular reorganisation were investigated immunohistochemically with antibodies against Nestin, Pax6, , Islet-1, calretinin, CRALBP, rhodopsin and PKC-α and enzymehistological for AChE and BChE
Rat pineal cells reaggregated well and spheres grew continuously during 10 days of cultivation to form smoothly shaped spheres; only occasionally spheres with a rough surface were found. Immunostainings showed a large number of Nestin-positive cells, demonstrating the existence of neuroepithelial precursor cells. Many cells were calretinin-positive showing a typical neuronal morphology with particularly long processes. Fewer cells expressed rhodopsin, some of them were co-stained by Calretinin. Pax6-positive cells could be found mainly in the outer parts of spheres, whereby some of them were arranged in a rosette-like pattern. Many BChE- and AChE-positive cells appeared in coherent and mutually exclusive clusters. Noticeably, many glia-like cells were identified by the marker CRALBP.
Dissociated pineal cells from neonatal rats were able to form reaggregates, whereby pronounced cell sorting indicated a histotypic patterning. AChE-, BChE- and Pax6- positive cells became arranged in rosettes, reminiscent of follicles as typically detected in early pineals. Interestingly, the pineal reaggregates were dominated by both glial and neuronal cells, which appeared to form networks. Most remarkably, the postnatal rat pineal gland consists of a large source of neuroepithelial stem cells. Further work has to show whether pineal cells possess a capacity to differentiate into a complete retina.
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