Preclinical Study Of Photoreceptor Progenitor Cells Transplantation On Retinitis Pigmentosa

Retinitis pigmentosa (RP) is one of the most common etiological factors for hereditary blind. As the reason of unremitting decrease of visual perception in RP is degeneration and apoptosis of photoreceptor cells, which could not be regenerated, transplantation of healthy retina tissue or cells, has become the most perspective treatment strategy currently. In reported clinical trails, the sources of donor tissue or cells were quite different and ultimate outcomes were incongruent, which had no substantial breakthrough. Since 2006, research from several centers of retina transplantation suggested, retinal photoreceptor progenitors(RPP) of mouse at a definite period could establish contact with synapse of host retina,accordingly, the vision of host improved. This creative research showed a guide for clinical trail for retina transplantation. According to the result of this research, if we could find RPP with the characteristics mentioned above from human embryo, it perhaps would do great help to clinical application of retina transplantation.Another major reason of incongruent clinical outcome of retina transplantation is the absence of objective evaluation method of visual function, as the function of photoreceptor cells of patients with RP is so weak that routine examination of visual electro-physiology could not discriminate the subtile changes of visual function. Function evaluation of photoreceptor cells includes examine of cone and rod. Till now, the most sensitive method, multifocal electroretinogram (mERG),could only detected local functional changes of cone in partial patients. To distinguish the electric activity of cone and rod, and try to evaluate the function of survived rod in patients with RP, perhaps will do help in analysing the rule of functional changes of cone and rod in patients with RP. While RP developed further, mERG could not detected local electric activity of retina. According to non-imaging visual transmission system of mammal(including human being), the transient pupil constriction occurs while beams of light shine into the eye. With the enhancement of intensity of light, extent of pupil constriction will augment. extent of pupil constriction is an important and sensitive objective parameter for functional evaluation of retina and Optic nerve,but whether extent of pupil constriction could quantify the function of photoreceptor cells in patients with RP, need systematic research.Animal experiments suggested that RPP of mouse has a window phase lasting for about a week, in which the cells have plasticity. It is the precondition of clinical trails on retina transplantation that acquiring the retinal photoreceptor progenitor at the definite phase in human embryo. How to separate the photoreceptor cells from embryo has been researched in animal experiments, but the developmental bionomics of photoreceptor cells in human embryo was seldom reported. It is so important to study the development of photoreceptor cells in human embryo, and to separate, collect highly purified retinal photoreceptor progenitor.We planned a research project about"preclinical study of photoreceptor progenitor cells transplantation on retinitis pigmentosa". This project was approved by medical ethic committee of Southwest Hospital, Third Military Medical University, and registered at the Registration center of Chinese clinical trails affiliated to World Health Organization. (registration No ChiCTR-TNC-00000193)。The content and result of our research is described as follow:1. We established an mERG recording technique for rods in normal persons, and developed the full-field pupillometry independently. The results showed that in RP patients, when fERG showed non-recordable, the cone mERG can still detect effective waves, the detection rate was 65.79%, and the rod mERG detection rate was 10.51%. The cone mERG wave tracing array of RP patients can be categorized into center, eccenter and nor-recordable types. In center type, amplitude density of ring 1 and 2 was in positive correlation with central vision. According to the light stimulation properties of rods and cones, we used short term and progressive increased light to stimulate retina of RP patients, finding that pupil contraction extent was generally lower than normal. The lower the threshold was, the higher the pupil contraction extent at all light stimulation levels were. Under stimulation of blue lights with low intensity, the relative pupillary construction (RPC) value of pupil was in positive correlation with fundus autofluorescence; under stimulation of white lights with middle intensity, RPC was in positive correlation with visual field. It implied that pupil contraction under full field pupillometry under dark adaptation can be taken as an objective and quantitive index to assess photoreceptor function of RP patients.2. We applied immunohistochemistry to observe expression of opsin, a marker of mature photoreceptors in progress of human retinal development. The results showed that opsin was expressed in outer nuclear layer since pregnancy 16 week, and the expression increased with retinal development, implying that photoreceptors before 16 week were at the stage of precursor. We acquired RPP cells with 85% purity from fetal retina at 12-16 week. The immunostain of cultured RPP cells showed that CRX,NRL,Tuj-1 was positive, while opsin,Chx-10,GFAP was negative. By induction of retinoic acid. the expression of opsin in RPP cells increased significantly.3. We performed 6 cases of allograft fetal RPP cells transplantation and 4 cases of fetal neuroretina cells transplantation in RP patients. The results showed that the surgery succeeded 100%, no complications were found in the preoperative period. In RPP transplantation group, the grafts can merge with host retina morphologically well in 2~9 months follow-up. The examinations of visual acuity, visual field and full-field pupillometry were coincident, showing a light improvement of the host visual function in 2~6 month follow-up. In human fetal neuroretina cells transplantation group, the rejection rate was 75%, manifesting delayed hypersensitivity at 1month after surgery, and the visual function was impaired. Combination of cyclosporine A and prednisone can ease but not terminate rejection reaction. It implied that allotransplantation of human fetal RPP cells is safe, the host visual function can be improved mildly in 6 months follow-up. Meanwhile, the neuroretinal cells allotransplantation resulted in high rejection.We concluded that:1. mERG can distinguish of rods and cones function respectively and contribute to evaluation of the impared degree in most of RP patients.2. pupil contraction extent under full field stimulation after dark adaptation can be taken as an objective and quantitive index to assess photoreceptor function of RP patients. Full-field pupillometry provides a new objective assessment for RP patients after therapy.3. Photoreceptors of human foetus before 16 week are at the stage of precursor. High purity of RPP cells can be acquired from fetal retina at P12-P16 weeks using suitable methods.4. Allotransplantation of fetal RPP cells is safe, the visual function of host can improved in 6 months follow-up, it will promotes us to carry out efficacy Study in the future.