New frontiers in retinal transplantation

New frontiers about retinal cell transplantation for retinal degenerative diseases start from the idea that acting on stem cells can help regenerate retinal layers and establish new synapses among retinal cells.Deficiency or alterations of synaptic input and neurotrophic factors result in trans-neuronal degeneration of the inner retinal cells.Thus,the disruption of photoreceptors takes place.However,even in advanced forms of retinal degeneration,a good percentage of the ganglion cells and the inner nuclear layer neurons remain intact.This phenomenon provides evidence for obtaining retinal circuitry through the transplantation of photoreceptors into the subretinal region.The eye is regarded as an optimal organ for cell transplantation because of its immunological privilege and the relatively small number of cells collaborating to carry out visual activities.The eyeball's immunological privilege,characterized by the suppression of delayed-type hypersensitivity responses in ocular tissues,is responsible for the low rate of graft rejection in transplant patients.The main discoveries highlight the capacity of embryonic stem cells(ESCs)and induced pluripotent stem cells to regenerate damaged retinal regions.Recent progress has shown significant enhancements in transplant procedures and results.The research also explores the ethical ramifications linked to the utilization of stem cells,emphasizing the ongoing issue surrounding ESCs.The analysis centers on recent breakthroughs,including the fabrication of three-dimensional retinal organoids and the innovation of scaffolding for cell transportation.Moreover,researchers are currently assessing the possibility of CRISPR and other advanced gene editing technologies to enhance the outcomes of retinal transplantation.The widespread use of universally recognized safe surgical and imaging methods enables retinal transplantation and monitoring of transplanted cell growth toward the correct location.Currently,most therapy approaches are in the first phases of development and necessitate further research,including both pre-clinical and clinical trials,to attain favorable visual results for individuals suffering from retinal degenerative illnesses.

Application of stem cell-derived retinal pigmented epithelium in retinal degenerative diseases： present and future

As a constituent of blood-retinal barrier and retinal outer segment（ROS） scavenger, retinal pigmented epithelium（RPE） is fundamental to normal function of retina. Malfunctioning of RPE contributes to the onset and advance of retinal degenerative diseases. Up to date, RPE replacement therapy is the only possible method to completely reverse retinal degeneration. Transplantation of human RPE stem cell-derived RPE（h RPESC-RPE） has shown some good results in animal models. With promising results in terms of safety and visual improvement, human embryonic stem cell-derived RPE（h ESC-RPE） can be expected in clinical settings in the near future. Despite twists and turns, induced pluripotent stem cell-derived RPE（i PSC-RPE） is now being intensely investigated to overcome genetic and epigenetic instability. By far, only one patient has received i PSCRPE transplant, which is a hallmark of i PSC technology development. During follow-up, no major complications such as immunogenicity or tumorigenesis have been observed. Future trials should keep focusing on the safety of stem cell-derived RPE（SC-RPE） especially in long period, and better understanding of the nature of stem cell and the molecular events in the process to generate SC-RPE is necessary to the prosperity of SC-RPE clinical application.

OPTIMAL CONDITIONS FOR DETECTING 5-BROMO-DEOXYURIDINE USED AS A MARKER OF TRANSPLANTED RETINAL PIGMENT EPITHELIAL CELLS

Objective To detemine optimal conditions by using 5 bromodeoxyuridine (BrdU) as a marker of transplanted retinal pigment epithelial (RPE) cells in the subretinal space of albino rabbits Methods Pigmented rabbit RPE cells at second to fifth passage were fed with 20 μmol/L BrdU in Eagle’s minimal essential medium (MEM) for five days After extensive wash with phosphate buffered saline (PBS),the cells were detached by trypsin and used for transplantation onto Bruch’s membrane of albino rabbits Eyes were enucleated at various times post transplantation Acetone, 4% paraformaldehyde, periodate lysine paraformaldehyde (PLP),or half strength Karnovsky’s fixatives were individually used to fix the tissue in order to find optimal condition for detecting BrdU marker The fixation was followed by embedding in OCT compound, glycol plastic, or paraffin The transplanted area was then sectioned, pepsin digested, and used for immunohistochemical staining with monoclonal antibody against BrdU and avidin biotin alkaline phosphatase complex (ABC AP) Results Frozen sections of acetone or paraformaldehyde fixed tissue gave strong immunostaining of BrdU but the overall morphology was poor Karnovsky’s fixed tissue offered strong staining but this was buried by strong background When using PLP as a fixative, we obtained strongly positive blue staining with very low background, and also excellent morphologic preservation Conclusion In combination with immunohistochemical detection method, BrdU labeling is an excellent long term marker for RPE transplantation one year after surgery To use BrdU as a marker necessitates the use of pepsin digestion to make the BrdU antigen in the nuclei accessible to antibody But the pepsin digestion may damage other tissues and result in overall poor morphology Among the fixatives tested in this study, PLP fixed tissues offered both strong BrdU staining and good preservation of structural integrity, particularly the fine structure of photoreceptor and RPE cells