Studies On Biological And Electrophysiological Properties Of Retinal Stem Cells From Embryonic Rat

Retinitis Pigmentosa(RP) is one of the severe hereditary diseases resulting in blindness.So far,no effective treatment method to prevent progressive retinal degenerations or to restore their visual function has been discovered.In recent years,retinal stem cells (RSCs) transplantation has become one of the most promising treatments for retinal degeneration diseases.However,while the key goal is to supply functional RSCs,few studies of RSCs have examined their functional development.Meantime,specific differentiation ability of RSCs should also be focused on so as to make proper connections with retinal neurons.Thus,in this study,to investigate how specific differentiation of RSCs can be influenced by the extracellular microenvironment of special stages rats retina,embryonic 17 day(E 17) rat RSCs were isolated and co-cultured by probing with the special stages retinal cells in a Transwell co-culture system.The electrophysiological characteristics of cells at various stages of differentiation were characterised and relation between physiolgical and morphological differentiation were studied.In addition the research investigated whether endogenous retinal stem cells exist with the onset and development of RP in the pigmented Royal College of Surgeons(RCS) rats.The findings will be useful in the search for new treatments for RP.Thus this thesis research includes four parts:1.Culture and cryopreservation of RSCs.The isolated retina of timed-pregnancy embryonic day 17(E17) Long Evans rats was dissociated into single cells and retinal stem cells were cultured in the medium.The third passage retinal stem cells were stored in liquid nitrogen.After varying durations of freezing,these cells were thawed.Indirect immunofluorescence staining and patch-clamp whole-cell recording technique were used to identify the viability,differentiation,morphology and electrophysiological characters of retinal stem cells after cryopreservation. Following cryopreservation,the RSCs from E17d rats were isolated and cultured successfully in serum-free media.Immunofluorescence techniques identified specific antigens of the cells before and after differentiation.The isolated cells derived from E17d rats cultured in serum-free medium could gave rise to neural spheres in the presence of bFGF. These cells could proliferate successively and generate secondary neural spheres,thus displaying potential to self-renew.The neural spheres could be expanded for up to 5 passages and constantly expressed the neuroectodermal marker Nestin and retinal stem cell marker Chx-10,indicating their undifferentiated status.After addition of fetal bovine serum(FBS) in the culture medium,cells expressing specific antigens for retinal neurons,Müller cells, retinal ganglion cells and photoreceptors such as NSE,GFAP,Thy1.1 and opsin were found.2.Specific differentiation of RSCs probing with normal and RP retina.This investigation aimed to find how differentiation of RSCs can be influenced by the extracellular microenvironment.The RSCs were induced to differentiate by probing with retinal cells from aged postnatal 1 day rat(PN1) and the adult rat retinal cells in a Transwell co-culture system.The morphological changes of the RSCs were observed under phase contrast microscopy.The effects on RSC differentiation by co-culture were studied by using immunohistochmistry methods.RSCs were also co-cultured with the RP retinas in a Transwell co-culture system.The morphological changes and migration of the RSCs were observed under a scanning electromicroscope.Differentiation of cells in retinal spheres can be influenced by the extracellular microenvironment.It is likely that the retinal cells of rat neonates secrete a substance which promotes RSCs differentiation to photoreceptors,while adult do not.Staged release of various compounds may be crucial to the differentiation process.Co-culturing with RP retinal cells affects the ability of co-cultured RSCs to target RP tissues.In addition,the RSCs can be induced to differentiate into neuron-like cells.3.The electrophysiological characteristics of RSCs at various stages of differentiation. Whole-cell patch clamp was employed to record the resting membrane potential(RMP), input resistance(IR),presence of action potentials(AP) and the developmental features of sodium and potassium ion channels at various stages of differentiation.Relations with morphological and immunological development were also measured.For comparison,the electrophysiological characteristics of differentiated retinal cells from PN1 rats in a Transwell co-culture system were recorded by the whole cell patch clamp technique.RSCs and differentiated cells were stained by Lucifer Yellow which was injected into cells during whole cell recordings for the purpose of morphological study.The research confirmed the feasibility of applying whole-cell patch clamp techniques to analysing electrophysiological characteristics of RSCs.Also,cells stained by Lucifer Yellow showed excellent cell morphology resulting in benefit for morphological and functional studies.During the process of stem cell differentiation,voltage-dependent ion channels gradually develop and ultimately support repeated action potential firing.Distinct voltage-dependent currents emerged during different stages of stem cell development.This can be used to identify cells of the retinal stem sphere in undifferentiated and differentiated state,and to improve the functional criteria for determining cell differentiation.Ion channels may be involved in the adjustment mechanism of the neuron's differentiation and development with respect to its extracellular environment.Cultured retinal stem cells differentiated to the stage of fully functional neurons which generate APs.Some relationships between the cells of different electrophysiological properties and morphology were found.However,an overall correspondence mechanism is not yet clear.4.Endogenous retinal stem cells exist in RCS rats retina with the onset and development of RP.The pigmented RCS rats were divided into 4 groups(RCS 15d,30d, 90d,120d) according to severity of RP,which were compared with those of rats without retinal degeneration.Immunohistochmistry and Western-blot methods were employed to determine whether endogenous RSCs exist during different stages of RP in the RCS rats so as to seek new ways of treatment to restore visual function in RP.Endogenous RSCs exist in the RCS rats retina coinciding with the onset and development of RP,possibly resulting from the release of a chronic stimulating factor from the degenerating retina.Conclusions:Cryopreservation and resuscitation did not change biological properties of rats RSCs, including their morphological capacity for proliferation and differentiation into different types of retinal neurons This could be of great benefit to disease treatment theough the establishment of a "human retinal stem cells bank".As the outcomes of differentiation were shown to depend on the extracellular invironment of the RSCs,it is likely that staged release of various compunds may be crucial for the differentiation process.The sodium and potassium channels expressed on differentiating cell membranes showed a graudal development to the point of producing signatures suggesting action potentials.It appears likely that degenerating retina releases some sort of chronic stimulating factor which generates retinal stem cells in models of RP.It will be important for the study of the specific differentiation mechanism to combine both immunohistochemical and electrophysiological measures of cell functionality.