Therapeutic Effect And Mechanism Of HEROs-derived Retinal Progenitor Cells In The Treatment Of Retinitis Pigmentosa

Background:Retinitis pigmentosa(RP)is a group of severe blinding eye diseases characterized by the progressive loss of photoreceptors with no effective therapy currently.Stem cell therapy is considered to have the most potential.However,how to obtain the appropriate donor cells is a key problem to be solved before application of stem cells in treating RP.One donor cell source with potential is retinal progenitor cells(RPCs)—retina-specific stem cells,which have been shown to improve retinal functions after transplantation.In retinal degeneration(RD)animal models.Presently,several clinical studies using human fetal retina-derived RPCs(hfRPCs)to treat patients with RP are under way with some preliminary confirmation of the safety and tolerance.However,the limited source and ethical limitations of hfRPCs prevent their widespread clinical application.Alternatively,human embryonic stem cells(hESCs)have pluripotency and can proliferate in vitro to acquire large number of cells.In addition,several groups have established clinical grade hESC lines.Donor cells differentiated from such hESC lines have been approved by health administration in China for clinical trials,paving the way for the therapeutic application of hESCs.Nonetheless,direct application of hESCs has a high risk of tumorigenesis.In recen t years,3D culture techniques enable the formation of human embryonic stem cell derived retinal organoids(hEROs)that are highly reminiscent to the retina developed in vivo.This provides a promising source of alternative donor cells for RP treatment.However,previous reports indicated that ESC-originated cells may still contain early stage embryonic cells that could cause tumor formation after transplantation.Thus,how to enrich RPCs from hEROs while eliminating tumorigenic early embryonic cells remains a great challenge.To this end,cell surface markers can be used to screen target cells from hEROs with complex cellular components.Cell surface antigen C-Kit,also known as CD117,is a type III tyrosine kinase receptor expressed on the cell surface of various types of stem cells.C-Kit was previously shown to mark a group of RPCs in mouse and human retinal development,thus can be used to screen RPCs.Stage-specific embryonic antigen-4(SSEA4)is expressed in hESCs and cells of early developmental stages,and by negative screening,it can be used to identify and eliminate those cells,therefore greatly reducing the risk of tumorigenesis.Previously,our research group has isolated C-Kit~+/SSEA4~-RPCs from hfRPCs by using fluorescence-activated cell sorting(FACS),and confirmed the safety and efficacy of its therapeutic effect.However,it is unknown whether a similar approach can be applied to hEROs.Therefore,this study set out to explore the feasibility of enriching C-Kit~+/SSEA4~-RPCs from hEROs and study the biological characteristics,which will provide critical information for the potential clinical application.The mechanisms of stem cell therapy mainly include two categories:1)Cell replacement through regeneration of various types of retinal cells;2)Protective effect by delaying degeneration process through nutritional support,immune regulation,and neural plasticity regulation.Recently,it has been proposed that stem cells transplanted into the subretinal space can rescue damaged photoreceptor cells by inter-cellular material exchange.This new mechanism has led to a re-evaluation of the method and outcome of the previous retinal cell transplantation therapy.As a new therapeutic mechanism,the cellular exchange(including forms,contents,and triggers)between stem cells and host retinal photoreceptor cells after transplantation needs to be further understood and studied.The long-term efficacy of stem cell transplantation depends not only on the ability of the transplanted stem cells to differentiate into retinal photoreceptor cells,but also on the host retinal microenvironment.The microglial cell activation,neuroinflammatory reaction and gliosis often occur during retinal degeneration,which do harm to long-term survival,migration and integration after stem cell transplantation.Meanwhile,excessive activation of microglia is one of the pathogenic mechanisms of photoreceptor cell death during retinal degeneration.In recent years,researchers have been exploring strategies to improve the microenvironment of retinal degeneration in order to achieve better therapeutic effects,including drug pretreatment and combined stem cell transplantation.However,there has been any report on possible donor cells with both RPC features and the capability to regulate microenvironment.In the CNS,C-Kit/SCF signaling pathway has been implicated in modulating local immune response of microglial cells,and C-Kit~+cells in other system have also been reported to participate in immune regulation.Nonetheless,it remains elusive whether C-Kit~+/SSEA4~-RPCs from hEROs can regulate retinal microglia and improve retinal microenvironment.Based on the research background,we hypothesized that using a combination of cell surface markers,we will be able to isolate C-Kit~+/SSEA4~-cells from hEROs that have RPC features but with low risk of tumorigenesis;C-Kit~+/SSEA4~-RPCs are safe and effective for the treatment of retinal degeneration in rodent models;The therapeutic effects may be achieved through cell replacement,inter-cellular material exchange,and improvement of the retinal degenerative microenvironment.This study includes three parts:Part One:Biological characteristics of hEROs-derived C-Kit~+/SSEA4~-RPCs1.Isolation of C-Kit~+/SSEA4~-RPCs from hEROsNeural retinal organoids were derived from hESCs through three-dimensional culture.The expression pattern of C-Kit and SSEA4 on hEROs was analyzed at different time points(30D,45D,and 60D).Transcriptome comparison was performed using RNAseq data from hfRPCs and C-Kit~+/SSEA4~-RPCs from hEROs.Results:At 30D,retinal organoids mainly composed of neural retina can be acquired.In 30D hEROs,C-Kit and SSEA4 expressions were high and gradually reduced afterwards.C-Kit was co-expressed with many RPCs markers,suggesting that it marked a group of RPCs.C-Kit~+/SSEA4~-RPCs derived from30D,45D and 60D were similar to hfRPCs in gene expression patterns,with 30D ones being the closest.Thus,enrichment of C-Kit~+/SSEA4~-RPCs from hEROs is feasible,and30D is the optimal time point for sorting.2.Biological features of C-Kit~+/SSEA4~-RPCs from hEROsC-Kit~+/SSEA4~-RPCs were isolated and cultured from 30D hEROs and RPC-specific markers were surveyed by immunocytochemical labeling.Clone formation and induced differentiation experiments were performed to verify the self-proliferation and pluripotency features of the RPCs respectively.Embryonic antigen expression was examined using flowcytometry.Results:C-Kit~+/SSEA4~-RPCs at passage 3(P3)can maintain RPC-specific marker expression,form clones,and self-proliferate.With specific culture medium,these cells can be induced to express markers for photoreceptors and other retinal neurons,suggesting pluripotency.No embryonic antigens such as OCT4 and Nanog can be detected through flowcytometry,indicating effective elimination of ESCs in C-Kit~+/SSEA4~-RPCs.In sum,C-Kit~+/SSEA4~-RPCs enriched from hEROs are self-renewable,pluripotent,and with little contamination of ESCs,hence are suitable donor cells for preclinical study treating retinal degenerations.Part Two:Preclinical study of hEROs-derived C-Kit~+/SSEA4~-RPCs for transplantation therapy of RP1.Survival,migration,and differentiation of C-Kit~+/SSEA4~-RPCs from hEROs aftersubretinal transplantation into RD ratsC-Kit~+/SSEA4~-RPCs from hEROs were transfected with EGFP by lentivirus and subretinally transplanted into RCS rats.Immunocytochemical analysis was performed to assess survival,migration,and differentiation of transplanted cells.Results:C-Kit~+/SSEA4~-RPCs from hEROs survived up to 24 weeks post-transplantation(PO 24W).At PO 2W,C-Kit~+/SSEA4~-RPCs mostly distributed within the subretinal space.At PO 4W,they spread laterally and vertically across the outer nuclear layer and reached as far as retinal ganglion cell layer,demonstrating excellent migration capability.Moreover,at PO 4W,transplanted cells can differentiate into rods and cones,with mature morphology,photoreceptor-specific protein expression,and even synaptic proteins at their terminals.In addition to photoreceptors,C-Kit~+/SSEA4~-RPCs also expressed specific cellular markers corresponding as they migrated to different layers of the retina.These results indicate that C-Kit~+/SSEA4~-RPCs from hEROs,when transplanted subretinally into RD rats,can survive,migrate,differentiate,and even form putative synapses with the host retinal neurons.2.Safety of C-Kit~+/SSEA4~-RPCs from hEROs after subretinal transplantation intoRD ratsC-Kit~+/SSEA4~-RPCs and unsorted cells from hEROs,transfected with EGFP,by lentivirus and subretinally transplanted into RCS rats.Immunocytochemical analysis was performed to evaluate tumor formation or abnormal cell proliferation.Results:Up to 24weeks post-transplantation,no tumor formation and no Ki67(a proliferation marker)–positive cells,suggesting no abnormal proliferation.In contrast,at PO 4W,66.7%of rats transplanted with unsorted cells form tumor-like structures in the eye,which contained abundant SSEA4~+and Ki67~+poorly differentiated cells mixed with differentiated GFAP~+glial cell and Tuj1~+neurons.Flowcytometry analysis showed that unsorted cells contain as high as 24%SSEA4~+cells.These results indicate that C-Kit~+/SSEA4~-RPCs are safe for transplantation,whereas unsorted cells,due to high SSEA4~+cell fraction,are likely to cause tumor formation after transplantation,further highlighting the importance of SSEA4negative screening.3.Therapeutic effects of subretinal transplantation of C-Kit~+/SSEA4~-RPCs fromhEROs into RD animal modelsC-Kit~+/SSEA4~-RPCs from hEROs were subretinally transplanted into a fast degeneration model,RD1 mice,and a slow degeneration model,RCS rats.Therapeutic outcomes were evaluated by comparing to the sham surgery group,the untreated control group,and the C-Kit~-/SSEA4~-control group(in RCS rat model).Retinal structural protection was assessed by measuring the thickness of the outer nuclear layer,while fERG and optomotor responses were used to examine visual function changes.Results:Retinal ONL thickness was better maintained with subretinal transplantation of C-Kit~+/SSEA4~-RPCs in both RD1 mice(PO 14D)and RCS rats(PO 24W).fERG showed significant improvement of retinal function in transplanted RD1 mice(PO 14D)and RCS rats(PO12W).In RCS rats,C-Kit~+/SSEA4~-RPCs transplantations had better effects than that of C-Kit~-/SSEA4~-cells both at PO 4W and 8W.Optomotor responses revealed slowing down of the visual sensitivity degradation up to PO 24W.Thus,by treating both the fast and slow RD animal models,it was clear that subretinal transplantation of C-Kit~+/SSEA4~-RPCs from hEROs led to protection of retinal structure and function for prolonged period of time.Part Three:Mechanisms of hEROs-derived C-Kit~+/SSEA4~-RPCs transplantation for the treatment of retinal pigmentosa1.Subretinal transplantation of C-Kit~+/SSEA4~-RPCs from hEROs rendered cell replacement in the host retina and material exchange with host photoreceptors.Human specific cell markers were used to tracing hEROs-derived C-Kit~+/SSEA4~-RPCs after transplantation.Results:Using human mitochondria specific antibody MTCO2and human cell nuclear antibody HuNu were used to demarcate human cells,it was found that some EGFP~+photoreceptor-like cells in the ONL also expressed MTCO2 or HuNu along with mature photoreceptor markers,suggesting that C-Kit~+/SSEA4~-RPCs can differentiate and replace degenerated photoreceptors after transplantation.In addition,EGFP~+/HuNu~-cells have been identified in the ONL,likely due to material exchange(EGFP)between transplanted cells and the host photoreceptor.Thus,C-Kit~+/SSEA4~-RPCs from hEROs,after transplantation,may both involved in cell replacement of and material exchange with the host photoreceptors.2.Subretinal transplantation of C-Kit~+/SSEA4~-RPCs from hEROs improve micro environment of the degenerating retina.2.1 hEROs-derived C-Kit~+/SSEA4~-RPCs transplantation can improve microenvironment in RD animal models.C-Kit~+/SSEA4~-RPCs from hEROs were subretinally transplanted into RD1 mice,and RCS rats to study their impacts to degenerative microenvironment featured by microglia activation and Müller cell gliosis.Immunocytopchemistry,western blot,and real time PCR were used to study the impact of transplanted C-Kit~+/SSEA4~-RPCs to cell number,morphology,and inflammatory cytokine expression of microglia as well as Müller cell gliosis.Results:C-Kit~+/SSEA4~-RPC transplantation significantly reduced retinal microglia number and Iba1 protein expression in both RD1 mice(PO 14D)and RCS rats(PO4W&8W).In RCS rats,the number of activated,phagocytotic microglia decreased drastically,along with reduced TSPO protein expression and more ramified,inactivate morphology.Real time PCR revealed a reduced inflammatory cytokine level after transplantation.In addition,in both RD1 and RCS model,C-Kit~+/SSEA4~-RPCs transplantation significantly inhibited Müller cell gliosis.These results suggest that C-Kit~+/SSEA4~-RPCs transplantation can improve retinal microenvironment through inhibition of microglial activation,the associated inflammation response,as well as Müller cell gliosis.2.2 Mechanisms and pathways of microglia regulation by hEROs-derived C-Kit~+/SSEA4~-RPCs.C-Kit~+/SSEA4~-RPCs from hEROs and LPS treated human or mouse microglial cell lines were co-cultured.Real-Time PCR was used to analyze the expression level of cytokines.Transcriptional comparison of C-Kit~+/SSEA4~-RPCs and hfRPCs revealed DEGs and signaling pathways related with immune and inflammation responses,which were then verified by Real-Time PCR.Results:hEROs-derived C-Kit~+/SSEA4~-RPCs can suppress inflammatory cytokine expression by human/mouse microglial cells triggered by LPS.They demonstrated superior anti-inflammatory capability than hfRPCs and C-Kit~-/SSEA4~-cells.Transcriptional comparison of C-Kit~+/SSEA4~-RPCs and hfRPCs revealed over 700 DEGs related to immune response and inflammation.KEGG signaling pathway analysis indicated that cytokine and cytokine receptor interactions are important pathways related to regulation of microglial responses by C-Kit~+/SSEA4~-RPCs.Real-Time PCR results confirmed that immune-regulatory effects of C-Kit~+/SSEA4~-RPCs may be achieved by upregulation of microglia inhibitory genes and downregulation of microglia recruitment genes.These results indicate that hEROs-derived C-Kit~+/SSEA4~-RPCs have unique microglia-regulatory function thus are superior donor cells for RD treatment.The mechanisms of immune-regulation shed light on our understanding of the role of stem cells in regulating degenerative microenvironment.In summary,we draw the following conclusion:1.We first isolated the C-Kit~+/SSEA4~-RPCs from hESCs derived retinal organoids(hEROs),and confirmed that those cells are a subgroup of RPCs with self-renewal,multi-directional differentiation potential and low risk of tumorigenesis,which meanwhile shares the similar gene expression pattern with hfRPCs.2.hEROs derived C-Kit~+/SSEA4~-RPCs showed a low risk of tumor formation after transplantation,which confirmed the importance of SSEA4 negative screening.3.After transplantation,hEROs derived C-Kit~+/SSEA4~-RPCs showed superior survival,migration,and multidirectional differentiation potential,and demonstrated long-term efficacy in treating both fast and slow retinal degeneration models(RD1 mice and RCS rats),providing new donor cells for clinical treatment of RP4.It showed that both cell replacement and cellular material exchange between donor and host photoreceptor cells were involved after the transplantation of hEROs derived C-Kit~+/SSEA4~-RPCs.5.Using both fast and slow retinal degeneration models,it has been illustrated that hEROs derived C-Kit~+/SSEA4~-RPCs play a unique role in improving retinal degenerative microenvironment,by inhibiting microglia and Müller cell gliosis after transplantation.The underlying mechanism of microglial regulation may involve upregulation of microglia inhibitory genes and downregulation of microglia recruitment genes.