Pre-clinical toxicity analysis following subretinal transplantation of an iPSC-derived retinal cell graft in immune suppressed rats.
Laura R. BohrerIan C. HanJessica A. CookeErin R. BurnightKristin R. AnfinsonEmily KaalbergMallory J. UlfertsJeremy M. HoffmannKelsey L WielandLuke A. WileyKristan S. WorthingtonKatherine N. Gibson‐CorleyRobert F. MullinsEdwin M. StoneBudd A. Tucker
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Because immune rejection is likely to be a major barrier to successful retinal transplantation, it is important to determine whether immune privilege for allogeneic retinal grafts is a feature of the subretinal space and vitreous cavity.Newborn neural retinas of C57BL/6 mice were implanted into the subretinal space, vitreous cavity, or subconjunctival space of eyes of adult BALB/c (disparate from C57BL/6 at major and minor histocompatibility loci). At postimplantation day 12, the recipients were evaluated for donor-specific delayed hypersensitivity and examined clinically and histologically for evidence of rejection.Newborn neural retinal allografts in the subconjunctival space were destroyed by postimplantation day 12 and these recipients displayed intense donor-specific delayed hypersensitivity. By contrast, grafts in the subretinal space and vitreous cavity at postimplantation day 12 were found to be well differentiated and with no evidence of inflammation; these recipients failed to display donor-specific delayed hypersensitivity. Moreover, their spleens contained regulatory T cells that suppressed donor-specific delayed hypersensitivity in naive syngeneic recipients.Allogeneic newborn neural retinal grafts implanted in the subretinal space and vitreous cavity experience immune privilege and induce deviant immune responses resembling anterior chamber associated immune deviation.
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Purpose: The role of activated retinal pigment epithelium (RPE) cells was investigated in the rejection after subretinal transplantation. Methods: RPE cells from 7 pigmented rabbits were separated and evaluated regarding their MHC class II expression as the sign of activation. The activation of the RPE cells was augmented with a treatment of 1,000 U/ml interferon γ (IFN-γ) for 8 days. These cells were then transplanted into 7 albino rabbits. As control, RPE transplantations without a pretreatment were performed in 7 albino rabbits. Six weeks after the transplantation, the transplanted eyes were enucleated and histology was performed. Results: In culture, without IFN-γ addition, 11.38 ± 0.94% of the RPE cells presented MHC class II. After IFN-γ treatment, this quantity increased to 78.26 ± 1.46% of the RPE cells. These cells transplanted into the rabbits caused an obvious rejection in the transplantation area which was verified histologically. The control group presented a transplantation area without signs of rejection or inflammation. Conclusion: In culture, some of the adult RPE cells are activated. These cells may accelerate the rejection cascade after transplantation. An elimination of activated RPE cells from the transplant should be recommended before transplantation.
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Successful subretinal transplantation is limited by considerable early graft loss despite pharmacological suppression of adaptive immunity. We postulated that early innate immune activity is a dominant factor in determining graft survival and chose a nonimmunosuppressed mouse model of retinal pigment epithelial (RPE) cell transplantation to explore this. Expression of almost all measured cytokines by DH01 RPE cells increased significantly following graft preparation, and the neutrophil chemoattractant KC/GRO/CINC was most significantly increased. Subretinal allografts of DH01 cells (C57BL/10 origin) into healthy, nonimmunosuppressed C57BL/6 murine eyes were harvested and fixed at 1, 3, 7, and 28 days postoperatively and subsequently cryosectioned and stained. Graft cells were detected using SV40 large T antigen (SV40T) immunolabeling and apoptosis/necrosis by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL). Sections were also immunolabeled for macrophage (CD11b and F4/80), neutrophil (Gr1 Ly-6G), and T-lymphocyte (CD3-ɛ) infiltration. Images captured with an Olympus FV1000 confocal microscope were analyzed using the Imaris software. The proportion of the subretinal bolus comprising graft cells (SV40T+) was significantly (p < 0.001) reduced between postoperative day (POD) 3 (90 ± 4%) and POD 7 (20 ± 7%). CD11b+, F4/80+, and Gr1 Ly-6G+ cells increased significantly (p < 0.05) from POD 1 and predominated over SV40T+ cells by POD 7. Colabeling confocal microscopic analysis demonstrated graft engulfment by neutrophils and macrophages at POD 7, and reconstruction of z-stacked confocal images confirmed SV40T inside Gr1 Ly-6G+ cells. Expression of CD3-ɛ was low and did not differ significantly between time points. By POD 28, no graft cells were detectable and few inflammatory cells remained. These studies reveal, for the first time, a critical role for innate immune mechanisms early in subretinal graft rejection. The future success of subretinal transplantation will require more emphasis on techniques to limit innate immune-mediated graft loss, rather than focusing exclusively on suppression of the adaptive immune response.
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Whether differentiation of induced pluripotent stem cells (iPSCs) in ischemic myocardium enhances their immunogenicity, thereby increasing their chance for rejection, is unclear. Here, we dynamically demonstrated the immunogenicity and rejection of iPSCs in ischemic myocardium using bioluminescent imaging (BLI). Murine iPSCs were transduced with a tri-fusion (TF) reporter gene consisting of firefly luciferase-red fluorescent protein-truncated thymidine kinase (fluc-mrfp-tTK). Ascorbic acid (Vc) were used to induce iPSCs to differentiate into cardiomyocytes (CM). iPSCs and iPS-CMs were intramyocardially injected into immunocompetent or immunosuppressed allogenic murine with myocardial infarction. BLI was performed to track transplanted cells. Immune cell infiltration was evaluated by immunohistochemistry. Syngeneic iPSCs were also injected and evaluated. The results demonstrated that undifferentiated iPSCs survived and proliferated in allogenic immunocompetent recipients early post-transplantation, accompanying with mild immune cell infiltration. With in vivo differentiation, a progressive immune cell infiltration could be detected. While transplantation of allogenic iPSC-CMs were observed an acute rejection from receipts. In immune-suppressed recipients, the proliferation of iPSCs could be maintained and intramyocardial teratomas were formed. Transplantation of syngeneic iPSCs and iPSC-CMs were also observed progressive immune cell infiltration. This study demonstrated that iPSC immunogenicity increases with in vivo differentiation, which will increase their chance for rejection in iPSC-based therapy.
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Stem cell strategies focused on replacement of RPE cells for the treatment of geographic atrophy are under intense investigation. Although the eye has long been considered immune privileged, there is limited information about the immune response to transplanted cells in the subretinal space of large animals. The purpose of this study was to evaluate the survival of allogenic induced pluripotent stem cell-derived RPE cells (iPSC-RPE) delivered to the subretinal space of the pig as well as determine whether these cells induce an immune response in non-diseased eyes. GFP positive iPSC-RPE, generated from outbred domestic swine, were injected into the subretinal space of vitrectomized miniature swine. Control eyes received vehicle only. GFP positive iPSC-RPE cells were identified in the subretinal space 3 weeks after injection in 5 of 6 eyes. Accompanying GFP-negative cells positive for IgG, CD45 and macrophage markers were also identified in close proximity to the injected iPSC-RPE cells. All subretinal cells were negative for GFAP as well as cell cycle markers. We found that subretinal injection of allogenic iPSC-RPE cells into wild-type mini-pigs can induce the innate immune response. These findings suggest that immunologically matched or autologous donor cells should be considered for clinical RPE cell replacement.
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We studied and developed a gene-based intraocular erythropoietin (EPO) therapy for diabetic retinopathy (DR), by which the applicability of neuroprotective therapy with favorable safety profile is attempted.Hematocrit (Hct) was measured in C57BL/6 mice after intramuscular injection of AAV2-CMV-hEPO virus. Diabetes was induced by intraperitoneal injection of streptozotocin in Sprague-Dawley (SD) rats. Subretinal or intravitreal injection was performed in SD rats and Dark Agouti (DA) rats. The human EPO (hEPO) concentration was measured with ELISA. Blood-retinal barrier (BRB) breakdown was measured with Evans blue permeation. Retinal function was evaluated with electroretinography (ERG). Retinal cell apoptosis was detected with TUNEL. Retinal thickness and cell counts were examined by light microscopy. Retinal vascular changes were evaluated with fundus fluorescein angiography (FFA) and confocal microscopy.The serum hEPO was elevated 2 weeks after AAV2-CMV-hEPO virus injection, and Hct began to increase after 4 weeks. After subretinal injection, hEPO expressions in aqueous humor, vitreous, and retina followed a dose- and time-dependent manner. In the AAV2-CMV-hEPO-treated diabetic group, BRB was maintained, and retinal cell apoptosis was significantly reduced. The ERG results showed that the retinal function remained unchanged for at least one year after subretinal injection of AAV2-CMV-hEPO virus. Long-term expression of hEPO following subretinal injection of AAV2-CMV-hEPO virus did not induce neovascularization in retina and choroid.The AAV2-CMV-hEPO gene therapy is safe, and it exerts long-term protective effects on diabetic retinas. Thus, the gene therapy by using AAV2-CMV-hEPO for DR is feasible.
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Age-related macular degeneration (AMD) is the leading cause of blindness in the elderly due in large part to age-dependent atrophy of retinal pigment epithelium (RPE) cells. RPE cells form a monolayer located between the choroid and the outer segments of photoreceptors, playing multifarious roles in maintenance of visual function. Allogeneically induced pluripotent stem cell-derived RPE (iPSC-RPE or iRPE) has become a potential approach for providing an abundant source of donors for clinical cell products. Transplantation of iRPE has been proven effective in rescuing impaired retinas in Royal College of Surgeons (RCS) rats after approximately 5 to 6 weeks. Here, we explore the long-term (19 weeks) safety and efficacy of human iRPE cell transplantation in pre-clinical animal models.The expression of human RPE-specific markers in iRPE cells was determined using immunofluorescence staining. For the proliferative test, Ki-67 expression was also verified by immunofluorescence and flow cytometric analysis. Then, iRPE cells were transplanted into the subretinal space of immune-deficient NOD/SCID/IL-2Rgcnull (NSG) mice to assess their safety. To evaluate whether the transplanted cells could survive and rescue visual function, we performed color fundus photography, focal electroretinogram and immunostaining after delivering iRPE cells into the subretinal space of RCS rats.Human iRPE cells expressed native RPE-specific markers, such as microphthalmia-associated transcription factor (MiTF), retinal pigment epithelium-specific 65-kDa protein (RPE65) and tight-junction associated structural protein (ZO-1), and their proliferative capacity (Ki-67 expression) was poor after 25 days of induction. A tumorigenicity test revealed no tumor formation or abnormal proliferation in the immunodeficient mice after subretinal injection of 5×105 iRPE cells. The transplanted iRPE cells survived for at least 19 weeks and maintained visual function for 15 weeks.In the present study, we provided further evidence for the use of human iRPE transplantation to treat retinal degenerative disease in pre-clinical animal models. Therefore, we consider human iRPE cells a promising source of cell replacement therapy for AMD.
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Whether physiologically induced pluripotent stem cell (iPSC)-derived organs are immunogenic and can be used for transplantation is unclear. Here, we generated iPSC-derived skin, islet, and heart representing three germ layers of the body through 4n complementation and evaluated their immunogenicity and therapeutic efficacy. Upon transplantation into recipient mice, iPSC-derived skin successfully survived and repaired local tissue wounds. In diabetic mouse models, explanted iPSC-derived islets effectively produced insulin and lowered blood glucose to basal levels. iPSC-derived heart grafts maintained normal beating for more than 3 months in syngeneic recipients. Importantly, no obvious immune rejection responses against iPSC-derived organs were detected long after transplantation. Our study not only demonstrates the fundamental immunogenicity and function of iPSC derivatives, but also provides preclinical evidence to support the feasibility of using iPSC-derived skin, islet, and heart for therapeutic use.
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