Conjunctival Reconstruction with Progenitor Cell-Derived Autologous Epidermal Sheets in Rhesus Monkey

et al. 2019

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This study was conducted to determine the dynamic Islet1 and Brn3 (POU4F) expression pattern in the human fetal retina and human-induced pluripotent stem cell- (hiPSC-) derived retinal organoid. Human fetal eyes from 8 to 27 fetal weeks (Fwks), human adult retina, hiPSC-derived retinal organoid from 7 to 31 differentiation weeks (Dwks), and rhesus adult retina were collected for cyrosectioning. Immunofluorescence analysis showed that Islet1 was expressed in retinal ganglion cells in the fetal retina, human adult retina, and retinal organoids. Unexpectedly, after Fwk 20, Brn3 expression gradually decreased in the fetal retina. In the midstage of development, Islet1 was detected in bipolar and developing horizontal cells. As the photoreceptor developed, the Islet1-positive cone precursors gradually became Islet1-negative/S-opsin-positive cones. This study highlights the distinguishing characteristics of Islet1 dynamic expression in human fetal retina development and proposes more concerns which should be taken regarding Brn3 as a cell-identifying marker in mature primate retina.

“…Subsequently, all samples were rinsed in PBS and mounted in Vectashield and coverslipped. H&E staining was performed as described previously [14, 15].…”

Section: Methodsmentioning

confidence: 99%

Islet1 and Brn3 Expression Pattern Study in Human Retina and hiPSC-Derived Retinal Organoid

et al. 2019

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“…The eyeball tissues were dissected to obtain 10 mm × 10 mm sections containing the transplants, which were frozen and sectioned via cryotomy (Leica). The cryosections and coverslips with retinal organoids were analyzed using immunofluorescence and hematoxylin and eosin staining as per the procedure described by Lu et al [17]. Antibodies against the following markers were used as primary antibodies for immunofluorescence microscopy at the dilutions indicated in parentheses: Ki67 (1 : 100, A11390, ABclonal, China), CHX10 (1 : 200, AB9016, Millipore, Germany), Brn3 (1 : 200, SC-6026X, Santa Cruz, USA), Islet1 (1 : 20, BM44446, Boster, China), HuD (1 : 100, SC-48421, Santa Cruz), RBPMS (1 : 100, ab152101, Abcam), β III-tubulin (1 : 200, ab7751, Abcam), neurofilament light polypeptide (NEFL; 1 : 100, A0257, ABclonal), microtubule-associated protein 2 (MAP2, 1 : 100, BM1243, Boster), vimentin (1 : 50, BM4029, Boster), glial fibrillary acidic protein (GFAP; 1 : 500, 3670S, Cell Signaling Technology, USA), CD68 (1 : 200, ab201340, Abcam), Iba1 (1 : 200, ab5076, Abcam), and SC121 (1 : 500, Y40410, Takara Bio).…”

Section: Methodsmentioning

confidence: 99%

Dexamethasone Provides Effective Immunosuppression for Improved Survival of Retinal Organoids after Epiretinal Transplantation

Xian

et al. 2019

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We investigated the efficacy of the immunosuppressants rapamycin (RAP) and dexamethasone (DEX) in improving the survival of retinal organoids after epiretinal transplantation. We first compared the immunosuppressive abilities of DEX and RAP in activated microglia in an in vitro setting. Following this, we used immunofluorescence, real-time polymerase chain reaction, and flow cytometry to investigate the effects of DEX and RAP on cells in the retinal organoids. Retinal organoids were then seeded onto poly(lactic-co-glycolic) acid (PLGA) scaffolds and implanted into rhesus monkey eyes (including a healthy individual and three monkeys with chronic ocular hypertension (OHT) induction) and subjected to different post-operative immunosuppressant treatments; 8 weeks after the experiment, histological examinations were carried out to assess the success of the different treatments. Our in vitro experiments indicated that both DEX and RAP treatments were equally effective in suppressing microglial activity. Although both immunosuppressants altered the morphologies of cells in the retinal organoids and caused a slight decrease in the differentiation of cells into retinal ganglion cells, the organoid cells retained their capacity to grow and differentiate into retinal tissues. Our in vivo experiments indicate that the retinal organoid can survive and differentiate into retinal tissues in a healthy rhesus monkey eye without immunosuppressive treatment. However, the survival and differentiation of these organoids in OHT eyes was successful only with the DEX treatment. RAP treatment was ineffective in preventing immunological rejection, and the retinal organoid failed to survive until the end of 8 weeks. DEX is likely a promising immunosuppressant to enhance the survival of epiretinal implants.

“…Subsequently, the tack was gently removed by forceps. Immunofluorescence and HE staining were performed as previously described [ 11 , 12 ]. Antibodies against the following proteins were used at the indicated dilutions: GFAP (mouse, 1 : 300, Cell Signaling Technology, 3670) and iba-1 (rabbit, 1 : 200, Abcam, Ab178680).…”

Section: Methodsmentioning

confidence: 99%

Establishing a Surgical Procedure for Rhesus Epiretinal Scaffold Implantation with HiPSC-Derived Retinal Progenitors

et al. 2018

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Background To develop an effective surgical procedure for cellular scaffold epiretinal implantation in rhesus, facilitating subsequent epiretinal stem cell transplantation. Methods Retinal progenitors were seeded onto a poly(lactic-co-glycolic) acid (PLGA) scaffold. First, the cellular scaffolds were delivered by 18G catheter or retinal forceps into rabbit epiretinal space (n = 50). Then, the cell survival rate was evaluated by Cell Counting Kit-8 (CCK-8). Second, three methods of scaffold fixation, including adhesion after gas-liquid exchange (n = 1), tamponade by hydrogel (n = 1), and fixation by retinal tacks (n = 4), were performed in rhesus monkeys. After one month, fundus photography and SD-OCT were performed to assess the outcomes, and histological examination was performed to evaluate proliferation. Results The cell survival rate was significantly higher in the catheter group. Follow-up examination showed that retinal tack fixation was the only method to maintain the scaffolds attached to host retina for at least 3 weeks, which is the minimal time required for cell integration. Histological staining demonstrated slight glial fibrillary acidic protein (GFAP) accumulation in the retinal tack insertion area. Conclusions The established surgical procedure offers a new insight into research of epiretinal cell replacement therapy in rhesus eyes. The successful delivery and long-term fixation provide a prerequisite for cell migration and integration.