| Retinal degenerative diseases(RDD)are a group of diseases that can cause visual impairment and even blindness,seriously affecting the quality of life of patients.Retinitis pigmentosa(RP)is one of the most common diseases of RDD,in which the progressive loss of photoreceptors is a typical pathological process.Due to the gradual loss of rod and cone cells,RP patients often lose their night vision ability at an early stage,leading to loss of peripheral vision and ultimately central vision.The exact pathogenesis of RP is still unclear.The incidence rate of RP in the world is about 1/4000,which is recognized as one of the most common serious blinding eye diseases in the world,and there is no effective treatment.Stem cell transplantation has brought new hope for the treatment of RP due to its ability to replace damaged neurons and promote the recovery of visual function.Currently,several preclinical and clinical trials have shown that stem cell transplantation is safe and effective for the treatment of RP.However,the potential mechanism of stem cell therapy for RP remains to be elucidated.Müller glia are the main macroglia in the retina,which run through the entire retina and account for 90%of all retinal glia.As a supporting cell of the retina,Müller glia have important biological effects.In mammals,Müller glia respond to retinal damage in a variety of ways.Reactive Müller glia can protect tissue function by releasing neurotrophic factors and antioxidants.However,excessive gliosis of Müller glia can also promote retinal degeneration by forming glial scars,impeding the regeneration process of retinal tissue.In contrast to mammals,the retina of lower vertebrates can undergo complete spontaneous repair through the reprogramming process of Müller glia after injury.Müller glia are also considered an important source of endogenous stem cells,which provides insights for the treatment of RP.For example,when zebrafish undergo Müller glial reprogramming under pathological conditions,Müller glia change from static to active state,dedifferentiate into precursor cells and transdifferentiate into retinal neurons,thereby repairing the damaged retina and achieving the recovery of visual function.However,in higher mammals,Müller glia rapidly undergo gliosis after retinal damage,and their ability to repair the retina is even more minimal.Therefore,finding strategies to promote reprogramming of mammalian Müller glia to achieve retinal regeneration is a novel approach to the treatment of RP.Under certain conditions,the reprogramming process of Müller glia in higher mammals can be initiated,and Müller glia can undergo dedifferentiation and transdifferentiation into retinal neurons.Reprogramming of Müller glia in higher mammals can be initiated by genetic methods,such as specific overexpression of neural transcription factor Ascl1 in Müller glia or introduction of neural stem cell transformation factorβ-catenin and photoreceptor differentiation factors(Otx2,Crx and Nrl).In addition,the injection of exogenous factors,such as epithelial growth factor(EGF),fibroblast growth factor 1(FGF1),FGF2 and ciliary neurotrophic factor(CNTF),confirmed the promotion of dedifferentiation of Müller glia and transdifferentiation into retinal neurons.It is worth noting that our team and other teams have previously proved that exogenous stem cell transplantation could also stimulate the retinal endogenous stem cell potential of Müller glia,such as retinal stem cells(r SCs),bone marrow mesenchymal stem cells(BMSCs),hematopoietic stem cells and progenitor cells(HSPCs).However,the role of stem cell transplantation in the reprogramming of Müller glia needs further clarification.Retinal organoids technology provides a new source of therapeutic cells for stem cell transplantation.In our laboratory,human embryonic stem cells(h ESCs)were previously differentiated into retinal organoids(hERO)through three-dimensional culture,and then seed cells hERO-C-Kit~+/SSEA4~-RPCs(later referred to as retinal progenitor cells from human embryonic stem cells derived retinal organoids,hERO-RPCs)were obtained through flow cytometry(C-Kit~+/SSEA4~-).Previous studies have confirmed that hERO-RPCs have the ability to self-renewal,multidirectional differentiation potential and low risk of tumor formation.After transplantation,hERO-RPCs could protect the visual function and retinal structure of the grafted area in degenerative mice,regulate the microenvironment of retinal degeneration,and inhibit excessive activation of microglia and reactive gliosis of Müller glia.Reactive gliosis and reprogramming of Müller glia are different responses of Müller glia to injury.Whether hERO-RPCs could promote Müller glia reprogramming is unclear,and there have been no reports of organoid derived retinal progenitor cells promoting Müller glial reprogramming.Based on literature reports and previous research foundation in our laboratory,this study proposed the following hypothesis:Transplantation of hERO-RPCs into the subretinal space of RCS rats could inhibit reprogramming events such as reactive gliosis,promoting dedifferentiation and transdifferentiation of retinal Müller glia,thereby exerting a protective effect on retinal function and structure and delaying the progression of retinal degeneration.In view of the above assumptions,the main research contents and results of this paper are as follows:Part One:Effect of subretinal space transplantation of hERO-RPCs on retinal function and structure in RCS rats1.Retinal organoids were obtained by three-dimensional culture of h ESCs and used for subsequent isolation of hERO-RPCs at the 30th day of culture.C-Kit~+/SSEA4~-RPCs were sorted from hERO cultured for 30 days by flow cytometry and cultured until passage 3,where retinal progenitors and cell proliferation markers were identified by cell immunofluorescence.This study observed that hERO-RPCs in passage 3 were various in size and shape,most of them were spindle shaped,and hERO-RPCs expressed markers of retinal progenitor cells and cell proliferation.It showed that hERO-RPCs have been successfully isolated hERO-RPCs from retinal organoids through combined screening of cell surface markers in this study,which could be used for subsequent transplantation and in vitro experiments.2.In this study,hERO-RPCs were transfected with lentivirus to label EGFP,followed by subretinal transplantation in RCS rats.The blank group and PBS group were set as control groups,and the improvement of visual function in RCS rats after transplantation was detected by flash electroretinogram(f ERG).The results showed that the amplitude of f ERG a in the hERO-RPCs group significantly increased 4 weeks after transplantation compared to the control group.At 4,8 weeks after transplantation,the amplitude of the b wave of f ERG was also significantly increased compared to the control group,indicating that hERO-RPCs transplantation has a protective effect on the visual function of RCS rats.3.In this study,immunofluorescence techniques were used to identify the human characteristics of transplanted cells and evaluate their survival and migration.The results showed that 8 weeks after transplantation,EGFP~+cells coexpressed with human specific antibodies MTCO2 and Hu Nu,proving that EGFP~+cells were of human origin,and hERO-RPCs could survive well in the host retina and subretinal space.At 2 weeks after transplantation,it was observed that the transplanted cells were located in the subretinal space and the outer nuclear layer of the retina.At 4 weeks after transplantation,the transplanted cells migrated longitudinally to the inner nuclear layer of the retina,reaching as far as the ganglion cell layer.At 8 weeks after transplantation,the transplanted cells migrated longitudinally and evenly to all layers of the retina,indicating their good migration ability.This study analyzed the protection of retinal structures after transplantation through the thickness of the outer nuclear layer.The results showed that the outer nuclear layer of the retina of RCS rats continued to become thinner as the degeneration progressed.At 2,4,8weeks after transplantation,the thickness of the outer nuclear layer in the grafted area group was significantly greater than that in the control group.In addition,the thickness of the outer nuclear layer in the contralateral area group was analyzed.It showed that the thickness of the outer nuclear layer in the contralateral area group was also significantly increased compared to the control group at 2,4 weeks after transplantation,but the protective effect was weaker than that in the grafted area group.The results showed that hERO-RPCs in the early and middle late stages could promote the protection of retinal structures in the transplanted and contralateral areas and delay retinal degeneration.Part Two:Study on reprogramming of Müller glia in RCS rats by subretinal transplantation of hERO-RPCs1.Immunofluorescence assay was used to analyze the changes in Müller glial gliosis after hERO-RPCs transplantation.In this study,it was found that the average fluorescence intensity of the gliosis marker GFAP in the grafted area group and PBS group was significantly higher than that in the blank group and contralateral area group at 2 weeks after transplantation.At 4,8 weeks after transplantation,the average fluorescence intensity of GFAP in the grafted area group was significantly lower than that in the control group,and the protrusions of GFAP~+cells became thinner and shorter,but the expression of GFAP in the contralateral area group was not inhibited.The results suggested that hERO-RPCs could inhibit the gliosis of Müller glia in the grafted area during the middle and late stages of transplantation.2.The effect of hERO-RPCs transplantation on dedifferentiation of Müller glia was analyzed by immunofluorescence assay.The study found that at 2,4,8 weeks after transplantation,the number of Sox9~+Müller glia in the grafted area group and the contralateral area group significantly increased compared to the control group.In the control group,most of the Sox9~+cells were located in the upper part of the inner nuclear layer,but in the grafted area group,the Sox9~+cells migrated towards the outer nuclear layer,while a small number of cells entered the outer nuclear layer.At 2,4,8 weeks after transplantation,the vertical distribution distance of Sox9~+cells in the grafted area group was significantly increased compared to the control group.At 2,4 weeks after transplantation,the vertical distribution distance of Sox9~+cells in the contralateral area group was also significantly increased compared to the control group.In addition,immunofluorescence recognized dedifferentiation related transcription factors Chx10,Müller glia markers Vimentin and Sox9,at 2,4,8 weeks after transplantation,the number of Chx10~+cells,Vimentin~+/Chx10~+cells,and Sox9~+/Chx10~+cells in the grafted area group significantly increased compared to the control group.At 4,8 weeks after transplantation,the number of Chx10~+cells in the contralateral area group increased significantly compared to the control group.Only at 8weeks after transplantation,the number of Vimentin~+/Chx10~+cells in the contralateral area group increased significantly compared to the control group.The above results suggested that hERO-RPCs transplantation promotes the increase in the number and migration and the expression of transcription factors related to dedifferentiation of Müller glia in the grafted area and the contralateral area,suggesting that Müller glia might undergo early dedifferentiation.3.The effect of hERO-RPCs transplantation on Müller glial transdifferentiation was analyzed by immunofluorescence assay.At 8 weeks after transplantation,no colabeling of Sox9~+Müller glia with retinal photoreceptor marker Rhodopsin,ganglion cell marker Brn3a,ganglion/amacrine cell marker Hu C/D,and photoreceptor precursor marker Crx was found in this study.It suggested that hERO-RPCs transplantation for 8 weeks did not promote Müller glial transdifferentiation.Part Three:Preliminary study on the mechanism of reprogramming Müller glia by hERO-RPCs1.In vitro Transwell indirect coculture experiments were performed by hERO-RPCs with primary LE rat Müller glia,and Müller glia cultured alone were set as the control group,and cell immunofluorescence was used to analyze the changes of Müller glial proliferation ability after coculture.Primary cells were passaged to P3,and their morphology was stable,showing large cell bodies and multiple protrusions,with protrusions intertwined into networks,high purity,and good proliferative activity.In this study,we observed that the number of Müller glia and the proportion of Ki67~+cells in the experimental group were significantly higher than those in the control group at 1,3,5 days after coculture,suggesting that hERO-RPCs could promote Müller glial proliferation in vitro.2.Cell migration experiments were conducted with hERO-RPCs and primary Müller glia,and the effect of hERO-RPCs on the migration ability of Müller glia in vitro was analyzed,with Müller glia cultured alone as a control group.The results showed that at 24 hours after culture,crystal violet staining increased in the experimental group compared to the control group.Statistics showed that the number of Müller glia migrated in the experimental group was significantly increased compared to the control group,suggesting that hERO-RPCs had a promoting effect on the migration of Müller glia in vitro.3.Cell immunofluorescence technique was used to analyze the changes in glial characteristics and precursor characteristic related proteins of Müller glia after coculture,and Real-Time PCR was used to analyze the changes in glial characteristics and precursor characteristic m RNA expression of Müller glia after co culture.In this study,it was found that there was no significant difference in the expression of glial characteristic and precursor characteristic related proteins in Müller glia after coculture of hERO-RPCs with Müller glia.However,the expression of glial characteristic m RNA in Müller glia was down regulated and the expression of precursor characteristic m RNA was up regulated in experimental groups at 1and 3 days after coculture with hERO-RPCs,suggesting that hERO-RPCs could inhibit the gliosis of Müller glia at the transcriptional level and promote dedifferentiation events in their reprogramming in vitro.In summary,the following research conclusions were drawn in this topic:1.This study found that hERO-RPCs survived and migrated well in RCS rats after transplantation,which could protect the visual function of RCS rats,promote the protection of retinal structures in the grafted and contralateral areas in the early and middle late stages,and delay retinal degeneration.2.This study was the first to find that hERO-RPCs transplantation only inhibited the gliosis of Müller glia in the grafted area in the middle late stages,promoted the increase in the number and migration and the expression of transcription factors related to dedifferentiation of Müller glia in the grafted area and the contralateral area,but no expression of transdifferentiation indicators was observed,suggesting that hERO-RPCs transplantation promoted early dedifferentiation of Müller glia,but did not promote the occurrence of transdifferentiation of Müller glia.3.Taking the lead in conducting a preliminary study on the mechanism of hERO-RPCs affecting the reprogramming of Müller glia,it was suggested that hERO-RPCs could promote the proliferation and migration of Müller glia in vitro,and could downregulate the expression of glial characteristic m RNA and upregulate the expression of precursor characteristic m RNA,indicating that hERO-RPCs could inhibit gliosis and promote reprogramming of Müller glia at the transcriptional level in vitro. |
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