Development and clinical deployment of a smartphone-based visual field deep learning system for glaucoma detection.

By 2040, ~100 million people will have glaucoma. To date, there are a lack of high-efficiency glaucoma diagnostic tools based on visual fields (VFs). Herein, we develop and evaluate the performance of 'iGlaucoma', a smartphone application-based deep learning system (DLS) in detecting glaucomatous VF changes. A total of 1,614,808 data points of 10,784 VFs (5542 patients) from seven centers in China were included in this study, divided over two phases. In Phase I, 1,581,060 data points from 10,135 VFs of 5105 patients were included to train (8424 VFs), validate (598 VFs) and test (3 independent test sets-200, 406, 507 samples) the diagnostic performance of the DLS. In Phase II, using the same DLS, iGlaucoma cloud-based application further tested on 33,748 data points from 649 VFs of 437 patients from three glaucoma clinics. With reference to three experienced expert glaucomatologists, the diagnostic performance (area under curve [AUC], sensitivity and specificity) of the DLS and six ophthalmologists were evaluated in detecting glaucoma. In Phase I, the DLS outperformed all six ophthalmologists in the three test sets (AUC of 0.834-0.877, with a sensitivity of 0.831-0.922 and a specificity of 0.676-0.709). In Phase II, iGlaucoma had 0.99 accuracy in recognizing different patterns in pattern deviation probability plots region, with corresponding AUC, sensitivity and specificity of 0.966 (0.953-0.979), 0.954 (0.930-0.977), and 0.873 (0.838-0.908), respectively. The 'iGlaucoma' is a clinically effective glaucoma diagnostic tool to detect glaucoma from humphrey VFs, although the target population will need to be carefully identified with glaucoma expertise input.

Induction of pluripotent stem cells by reprogramming human ocular fibroblasts under xeno-free conditions / Indução de células-tronco pluripotentes pela reprogramação de fibroblastos oculares humanos sob condições xeno-livre

ABSTRACT Purposes: To develop an efficient and xeno-free standard eye-derived induced pluripotent stem cell reprogramming protocol for use during induced pluripotent stem cell-based cell therapies in treating retinal degenerative diseases and to compare the relative effectiveness of both animal- and non-animal-derived culture systems in the generation of induced pluripotent stem cells. Methods: Primary cultured human pterygium fibroblasts and human Tenon's capsule fibroblasts were induced to induced pluripotent stem cells using a non-in­tegrated virus under two xeno-free systems; as part of this study, a traditional non-xeno-free reprogramming system was also assessed. Induced pluripotent stem cell clones were selected and counted by live staining. Reprogramming efficiencies were evaluated between the fibroblasts and among different culture systems. In a series of experiments, such as PCR and immunofluorescence staining, the induced pluripotent stem cells were characterized. Results: Human pterygium fibroblast- and human Tenon's capsule fibroblast-derived induced pluripotent stem cells were successfully established using different reprogramming systems, under which they exhibited properties of induced pluripotent stem cells. Reprogramming efficiencies of induced pluripotent stem cells using the cell therapy system, the traditional system, and the E6/E8 system were 0.014%, 0.028%, and 0.001%, respectively, and those of human pterygium fibroblast- and human Tenon's capsule fibroblast-derived induced pluripotent stem cells-using the aforementioned systems-were 0.018% and 0.017%, respectively. Conclusions: Sendai virus facilitates induced pluripotent stem cell reprogramming of ocular fibroblasts-both human pterygium and human Tenon's capsule fibroblasts being safe and efficient for induced pluripotent stem cell reprogramming. Although the reprogramming efficiencies of ocular-derived induced pluripotent stem cells under xeno-free conditions were not superior to those observed using the traditional reprogramming system, the cell therapy system reprogramming system is a good option when induced pluripotent stem cells are to be induced under xeno-free conditions.

RESUMO Objetivos: Desenvolver um protocolo padrão, eficiente e xeno-livre, para a reprogramação de células-tronco pluripotentes induzidas, que possa ser usado durante as terapias de células-tronco pluripotentes induzidas para o tratamento de doenças degenerativas da retina, e comparar a eficácia relativa de sistemas de cultivo de origem animal e de origem não animal na geração de células-tronco pluripotentes induzidas. Métodos: Cultivos primários de fibroblastos de pterígio humano e de fibroblastos da cápsula de Tenon humanos foram induzidos a células-tronco pluripotentes induzidas usando um vírus não integrado sob dois sistemas xeno-livres; um sistema tradicional de reprogramação não xeno-livre também foi avaliado como parte deste estudo. Os clones de células-tronco pluripotentes induzidas foram selecionados e contados por coloração de células vivas. As eficiências de reprogramação foram avaliadas entre os diferentes fibroblastos e entre os diferentes sistemas de cultivo. Uma série de experimentos, como o PCR e a coloração por imunofluorescência, foram conduzidos para caracterizar as células-tronco pluripotentes induzidas. Resultados: Célu­las-tronco pluripotentes induzidas derivadas de fibroblastos de pterígio humano e fibroblastos da cápsula de Tenon humanos foram estabelecidas com sucesso sob diferentes sistemas de reprogramação e exibiram propriedades de células-tronco pluripotentes induzidas. As eficiências de reprogramação das células-tronco pluripotentes induzidas usando o sistema de terapia celular, o sistema tradicional e o sistema E6/E8 foram 0,014, 0,028% e 0,001%, respectivamente. Além disso, as efi­ciências de reprogramação de células-tronco pluripotentes induzidas derivadas de fibroblastos de pterígio humano e de fibroblastos da cápsula de Tenon humanos usando todos os sistemas acima foram de 0,018% e 0,017%, respectivamente. Conclusões: O vírus Sendai pode ser usado para facilitar a reprogramação de fibroblastos oculares pelas células-tronco pluripotentes induzidas. Tanto os fibroblastos de pterígio humano quanto os fibroblastos da cápsula de Tenon humanos são seguros e eficientes para a reprogramação de células-tronco pluripotentes induzidas. Embora as eficiências de reprogramação das células-tronco pluripotentes induzidas de origem ocular sob condições xeno-livres não tenham sido superiores às eficiências observadas para o sistema tradicional de reprogramação, o sistema de reprogramação sistema de terapia celular é uma boa opção para a indução de células-tronco pluripotentes induzidas sob condições xeno-livres.

Induction of pluripotent stem cells by reprogramming human ocular fibroblasts under xeno-free conditions.

PURPOSES: To develop an efficient and xeno-free standard eye-derived induced pluripotent stem cell reprogramming protocol for use during induced pluripotent stem cell-based cell therapies in treating retinal degenerative diseases and to compare the relative effectiveness of both animal- and non-animal-derived culture systems in the generation of induced pluripotent stem cells. METHODS: Primary cultured human pterygium fibroblasts and human Tenon's capsule fibroblasts were induced to induced pluripotent stem cells using a non-in-tegrated virus under two xeno-free systems; as part of this study, a traditional non-xeno-free reprogramming system was also assessed. Induced pluripotent stem cell clones were selected and counted by live staining. Reprogramming efficiencies were evaluated between the fibroblasts and among different culture systems. In a series of experiments, such as PCR and immunofluorescence staining, the induced pluripotent stem cells were characterized. RESULTS: Human pterygium fibroblast- and human Tenon's capsule fibroblast-derived induced pluripotent stem cells were successfully established using different reprogramming systems, under which they exhibited properties of induced pluripotent stem cells. Reprogramming efficiencies of induced pluripotent stem cells using the cell therapy system, the traditional system, and the E6/E8 system were 0.014%, 0.028%, and 0.001%, respectively, and those of human pterygium fibroblast- and human Tenon's capsule fibroblast-derived induced pluripotent stem cells-using the aforementioned systems-were 0.018% and 0.017%, respectively. CONCLUSIONS: Sendai virus facilitates induced pluripotent stem cell reprogramming of ocular fibroblasts-both human pterygium and human Tenon's capsule fibroblasts being safe and efficient for induced pluripotent stem cell reprogramming. Although the reprogramming efficiencies of ocular-derived induced pluripotent stem cells under xeno-free conditions were not superior to those observed using the traditional reprogramming system, the cell therapy system reprogramming system is a good option when induced pluripotent stem cells are to be induced under xeno-free conditions.

Regulated differentiation of WERI-Rb-1 cells into retinal neuron-like cells.

The encouraging response and improved survival of acute promyelocytic leukemia patients following retinoic acid treatment has rendered differentiation therapy an attractive option in cancer treatment. Given that terminal differentiation represents a considerable barrier in retinoblastoma tumorigenesis and that retinoblastoma has a significantly higher spontaneous degeneration rate compared with other tumors (1,000-fold change), differentiation therapy represents a promising alternative in the treatment of retinoblastoma. However, the full differentiation potential of retinoblastoma still unknown. The present study was designed to investigate the extend differentiation of the classical retinoblastoma cell line WERI-Rb-1 (W-RBCs). Several critical cell signaling pathways and key genes related to cell proliferation and differentiation were comprehensively regulated to control the fate of W-RBCs. Various strategies were applied to optimize simple and time-saving methods to induce W-RBCs into different types of retinal neuron-like cells (RNLCs) in vitro. Further, the tumorigenesis of these differentiated W-RBCs was tested in nude mice in vivo. W-RBCs were found to inherently express both retinal progenitor cell- and embryonic stem cell-related genes or proteins. Moreover, the addition of antagonists of critical cell signals (Wnt, Nodal, BMP4 and Notch), even without atonal bHLH transcription factor 7 gene transfection, could directly induce W-RBCs into RNLCs, and especially into photoreceptor-like and retinal ganglion-like cells. Interestingly, the differentiated cells showed remarkably poorer tumorigenesis in vivo. These findings may offer new insights on the oriented differentiation of W-RBCs into RNLCs with low tumorigenicity and provide potential targets for retinoblastoma differentiation therapy.

An optimized gene transfection system in WERI-Rb1 cells.

The pathogenesis of Rb1 gene inactivation indicates that gene therapy could be a promising treatment for retinoblastoma. An appropriate gene transfer system is the basis for successful gene therapy; however, little attention has been given to an effective gene transfer system for retinoblastoma therapy in previous studies. This study was designed to provide an optimized transgene system for WERI­Rb1 cells (W-RBCs). Green fluorescent protein (GFP) was adopted as a reporter. Four classic viral vectors based on retroviruses, recombinant adeno-associated viruses (rAAV2, rAAV2/1), lentiviruses (LVs) and a novel non-viral vector X-treme HP reagent were adopted for W-RBC gene transfection. The efficacy and cytotoxicity were comprehensively compared among the different vectors through GFP expression and the trypan blue exclusion test. Furthermore, the serum and cell culture status were also optimized for better transfection. Cells transfected by rAAV2/1 expressed more GFP protein and exhibited less staining with trypan blue, compared to the rAAV2 counterpart. However, in comparison to the retroviral group, both the rAAV2/1 and LV groups had considerably less GFP+ cells. Interestingly, the X-treme HP presented a similar GFP transfection capacity to the retroviral vector, but with a much lower cytotoxicity. Furthermore, there were more GFP+ cells in a suspended condition than that in an adherent culture. Moreover, cells in a serum-positive system expressed more GFP, while cells in a serum-free system showed lower GFP expression and higher cytotoxicity. In conclusion, the retroviral vector and the X-treme HP are effective for W-RBC gene transfection, while the X-treme HP is more preferable due to its lower cytotoxicity. Moreover, the suspended cell culture system is superior to the adherent system, and the serum protects cell viability and facilitates the gene transfection of W-RBCs. This study presents an effective, convenient, and low toxic transfection system for gene delivery in W-RBCs and provides a promising system for further gene therapy of retinoblastoma.

Stage-specific differentiation of iPSCs toward retinal ganglion cell lineage.

PURPOSE: As an alternative and desirable approach for regenerative medicine, human induced pluripotent stem cell (hiPSC) technology raises the possibility of developing patient-tailored cell therapies to treat intractable degenerative diseases in the future. This study was undertaken to guide human Tenon's capsule fibroblasts-derived iPSCs (TiPSCs) to differentiate along the retinal ganglion cell (RGC) lineage, aiming at producing appropriate cellular material for RGC regeneration. METHODS: By mimicking RGC genesis, we deliberately administered the whole differentiation process and directed the stage-specific differentiation of human TiPSCs toward an RGC fate via manipulation of the retinal inducers (DKK1+Noggin+Lefty A) alongside master gene (Atoh7) sequentially. Throughout this stepwise differentiation process, changes in primitive neuroectodermal, eye field, and RGC marker expression were monitored with quantitative real-time PCR (qRT-PCR), immunocytochemistry, and/or flow cytometry. RESULTS: Upon retinal differentiation, a large fraction of the cells developed characteristics of retinal progenitor cells (RPCs) in response to simulated environment signaling (DKK1+Noggin+Lefty A), which was selectively recovered with manual isolation approaches and then maintained in the presence of mitogen for multiple passages. Thereafter, overexpression of ATOH7 further promoted RGC specification in TiPSC-derived RPCs. A subset of transfected cells displayed RGC-specific expression patterns, including Brn3b, iSlet1, calretinin, and Tuj, and approximately 23% of Brn3b-positive RGC-like cells were obtained finally. CONCLUSIONS: Our DKK1+Noggin+Lefty A/Atoh7-based RGC-induction regime could efficiently direct TiPSCs to differentiate along RGC lineage in a stage-specific manner, which may provide a benefit to develop possible cell therapies to treat retinal degenerative diseases such as glaucoma.

[The study of laser-induced choroidal neovascularization in rhesus monkeys].

OBJECTIVE: To investigate the morphological and functional changes of the laser-induced choroidal neovascularization (CNV) in rhesus monkeys. METHODS: Experimental study. Eight adult rhesus monkeys weighted 4 to 7 kg were used in this study. CNVs were induced with small high-energy laser spots at short pulse duration by an argon green laser. Eyes were monitored weekly by color fundus photography, fluorescence fundus angiography (FFA) , and optical coherence tomography (OCT) . Fluorescein leaking intensities of grade 4 CNV lesions were analyzed by the method of ANOVA for repeated measures. Electroretinogram (ERG) was performed before laser photocoagulation and 56 days after laser photocoagulation and the data were analyzed with paired t-test. RESULTS: (1) FFA revealed that the mean intensities of grade 4 CNV lesions were 89.44 ± 26.28, 97.56 ± 26.47, 110.22 ± 29.76, 100.26 ± 29.24, 91.77 ± 28.11, 77.76 ± 24.85 and 63.23 ± 22.34 on day 14, day 21, day 28, day 35, day 42, day 49, and day 56 respectively and the differences were statistically significant (F = 39.715, P < 0.01) . The differences between any time-point and its previous time-point were also statistically significant (t14-21 = 4.824, P < 0.01; t21-28 = 5.225, P < 0.01; t28-35 = 7.378, P < 0.01;t35-42 = 2.954, P < 0.05; t42-49 = 5.386, P < 0.01; t49-56 = 6.138, P < 0.01). (2) OCT images showed retinal edema, subretinal fluid and hyper-reflective lesions of CNVs in the laser sites and histopathology showed that fibrovascular tissues together with proliferating retinal pigment epithelium cells were seen in the laser sites. (3) ERG data revealed that implicit time of dark-adapted b wave (t = 4.23, P < 0.01) increased while the amplitudes of dark-adapted a wave (t = 6.35, P < 0.01) , dark-adapted b wave (t = 3.12, P < 0.01) and light-adapted b wave (t = 3.93, P < 0.01) decreased 56 days after laser photocoagulation compared with those before laser photocoagulation. CONCLUSION: The laser-induced CNV in non-human primate model shows continuous leakage on FFA examination and reduced amplitudes as well as increased implicit time on ERG examination, suggesting that laser-induced CNV primate model not only could be used to study the pathogenesis of CNV formation but also could be used for screening of drug effectiveness.

Intravitreal injection of (99)Tc-MDP inhibits the development of laser-induced choroidal neovascularization in rhesus monkeys.

BACKGROUND: The aim of this study was to investigate the safety and efficacy of intravitreal injection of (99)Tc-MDP, a decay product of (99m)Tc-MDP, on the development of laser-induced choroidal neovascularization (CNV) in rhesus monkeys. METHODS: Experimental CNV was induced by argon laser with a small high-energy laser spot. Monkeys were given 50 µL of (99)Tc-MDP at a concentration of 0.005 µg/mL (n = 6) or 0.01 µg/mL (n = 6) by intravitreal injection once a week immediately after laser injury for a period of 56 days. Control animals were treated with the same volume of PBS (n = 6) in the same way. Eyes were monitored by ophthalmic examination, color fundus photography, fluorescence fundus angiography (FFA), optical coherence tomography (OCT) and histology. Incidences of grade 4 CNV lesions as well as the leakage areas of grade 4 CNVs on the late-phase of fluorescein angiograms were measured in a standardized, randomized and masked fashion fortnightly. The maximum widths and heights of grade 4 CNVs were also calculated by histology at the end of the experiment. Toxicity of (99)Tc-MDP on the retina was evaluated by electroretinogram (ERG) and histologic analysis. RESULTS: (99)Tc-MDP reduced the incidences of grade 4 CNVs by 33.33 % and 39.40 % in the 0.005 µg/mL and 0.01 µg/mL groups, respectively, compared with the PBS group on day 28 (P < 0.05; n = 6). The leakage areas of grade 4 CNVs were smaller in the 0.005 µg/mL (0.7136 ± 0.0283 mm(2), p <0.01; n = 6) and 0.01 µg/mL (0.4351 ± 0.0349 mm(2), p < 0.01; n = 6) groups than those in the PBS control group (0.9373 ± 0.0455 mm(2); n = 6) in a dose-dependent manner on day 28. OCT and histology also showed that the sizes of CNVs were smaller in the (99)Tc-MDP treated groups than those in the PBS group. Although intravitreal injection of (99)Tc-MDP led to mild inflammatory reaction in the anterior chamber, histology and ERG findings demonstrated that (99)Tc-MDP did not cause any change in histological structure or function of the retina (p>0.05). CONCLUSIONS: Intravitreal injection of (99)Tc-MDP can inhibit the development of laser-induced CNV without toxic effect on retina, suggesting that (99)Tc-MDP has therapeutic potential for CNV related diseases.