Corneal endothelial dysfunction occurs when corneal endothelial cells (CECs) are dramatically lost and eventually results in vision loss. Corneal transplantation is the only solution at present. However, corneal transplantation requires a fresh human cornea and there is a worldwide shortage of donors. Therefore, finding new functional CECs to replace human CECs is urgent. Skin-derived precursors (SKPs) can be easily acquired and have multiple differential potential. We co-cultured human SKPs with B4G12 cells in serum-free medium and obtained abundant CEC-like cells which had similar morphology and characteristic to human CECs. CEC-like cells exerted excellent therapeutic effect when they were transplanted into rabbit and monkey corneal endothelial dysfunction models by injection method. This protocol enables efficient production of CEC-like cells from SKPs. The renewable cell source, novel derivation method and simple treatment strategy may lead to potential applications in cell replacement therapy for corneal endothelial dysfunction.Corneal endothelial cells (CECs) are a monolayer of hexagonal cells covering the posterior surface of the cornea, and serve as a barrier between the corneal stroma and the aqueous humor. The tight junction as well as the ionic "pump" functions of CECs are important factors that maintain corneal transparency 1 . Many pathological factors such as trauma, surgery, and inflammation may cause dramatic loss of endothelial cell density, resulting in corneal endothelial dysfunction which is characterized by corneal edema, bullous keratopathy, and loss of visual acuity 2 . Human CECs have limited proliferative capacity in vivo and cannot be subcultured for more than a few passages in vitro 3,4 . Corneal transplantation is the only solution at present. However, corneal transplantation requires a fresh human cornea and there is a worldwide shortage of donors 5 . Therefore, finding new functional CECs to replace human CECs is a new and potential direction for future clinical application.Skin-derived precursors (SKPs) were discovered by Tomas 6,7 , and they have been a research hotspot in recent years. SKPs can be isolated in large quantities from many parts of the skin in rodents and humans. As they are multipotent, they can be differentiated into several functional cell types 8 , and are able to suppress the allogeneic activation of T-lymphocytes resulting in an improved health status of animals suffering from a graft-versus-host reaction 9 . Moreover, SKPs were demonstrated to be embryonic neural-crest-related precursors and share many properties with neural crest stem cells 10

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BackgroundCorneal endothelial dysfunction causes severe impairment of vision. The only solution is corneal transplantation. However, this treatment is hampered by a worldwide shortage of donor corneas. New therapies may replace the conventional donor corneal transplantation alongside the developments in regenerative medicine and tissue engineering, but sufficient functional corneal endothelial cells (CECs) are essential. The aim of this study was to promote the expansion and function of human corneal endothelial cells (HCECs) in vitro and in vivo.MethodsThe phenotypes of human orbital adipose-derived stem cells (OASCs) were detected by flow cytometry and immunofluorescence. HCECs were isolated and cultured using a conditioned medium obtained from OASCs (OASC-CM) in vitro. Related cell markers of HCECs were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR), Western blot, and immunofluorescence. The cell counting kit-8 (CCK-8) assay and the wound healing assay were performed to evaluate the proliferation ability of the cells. The cultured HCECs were then transplanted into rabbit and monkey corneal endothelial dysfunction models by cell injection.ResultsCD29, CD105, CD49e, CD166, and vimentin were highly expressed in cultured human OASCs. The CEC-relative markers zonula occludens-1 (ZO-1), Na+/K+ ATPase, N-cadherin, Col8a2, and SLC4A4 were expressed in HCECs cultured by OASC-CM. The HCECs were able to maintain polygonal cell morphology and good proliferative capacity. In animal experiments, corneal transparency was achieved after the injection of HCECs, which demonstrated the good repair capacity of the cells.ConclusionsThe proliferation abilities of the cells were significantly enhanced, and related functional markers were strongly positive, while HCEC morphology was maintained using OASC-CM. HCECs obtained some stem cell-like properties. This preclinical study confirmed the therapeutic ability of the HCECs in vivo. Our findings demonstrated that cultured HCECs with OASC-CM might be a promising source for research and clinical treatment.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-017-0737-5) contains supplementary material, which is available to authorized users.

IntroductionTo assess the ocular efficacy of intravitreal ranibizumab and conbercept injection in patients with neovascular age-related macular degeneration.Materials and methodsWe searched PubMed, Wed of Science, Cochrane Library, EMBASE, Google Scholar, Medline, China National Knowledge Infrastructure, and WANFANG DATA databases, up to June 20, 2018. We also searched abstracts and clinical study presentations at meetings as well as trial registries; we contacted authors of included studies if questions arose. Eligibility criteria for selection of studies were randomized controlled trials and retrospective trials that compared ranibizumab with conbercept for treatment of neovascular age-related macular degeneration.ResultsEight randomized controlled trials and four retrospective studies were included with a total of 853 patients. Best-corrected visual acuity after loading dosage was improved in the conbercept group, compared with the ranibizumab group (weighted mean difference: -0.04; 95% CI: -0.07 to 0.00; P=0.04). There was a significant difference between conbercept and ranibizumab therapy with respect to unchanged or recurrent leakage of choroidal neovascularization (OR: 0.46; 95% CI: 0.24–0.88; P=0.02). No significant differences were observed in central macular thickness (weighted mean difference: -2.92; 95% CI: -9.00 to 3.17; P=0.35), complete and partial closure of leakage of choroidal neovascularization (complete closure, P=0.70; partial closure, P=0.35), or number of injections (weighted mean difference: 0.42; 95% CI: -0.46 to 1.29; P=0.35) between the conbercept and ranibizumab groups at the end of the follow-up periods.ConclusionPooled evidence confirmed that conbercept was superior to ranibizumab with respect to visual gain after treatment. Additional studies with long-term follow-up are needed to support our conclusion.

The development of cell-based therapies using stem cells represents a significant breakthrough in the treatment of limbal stem cell deficiency (LSCD). The aim of this study was to develop a novel protocol to differentiate human embryonic stem cells (hESCs) into corneal epithelial progenitor cells (CEPCs), with similar features to primary cultured human limbal stem cells (LSCs), using a medium composed of DMEM/F12 and defined keratinocyte serum-free medium (KSFM) (1:1) under different carbon dioxide (CO2) levels in culture. The differentiated cells exhibited a similar morphology to limbal stem cells under 5%, 7%, and 9% CO2 and expressed the LSC markers ABCG-2 and p63; however, CK14 was only expressed in the cells cultured under 7% and 9% CO2. Quantitative reverse-transcription polymerase chain reaction (RT-PCR) analysis indicated that the ABCG2, p63, and CK14 levels in the 7% CO2 and 9% CO2 groups were higher than those in the 5% CO2 group and in undifferentiated hESCs (p<0.05). The highest expression of ABCG2 and p63 was exhibited in the cells cultured under 7% CO2 at day 6 of differentiation. Western blotting indicated that the ABCG2 and p63 levels were higher at day 6 than the other time points in the 7% CO2 and 9% CO2 groups. The highest protein expression of ABCG2 and p63 was identified in the 7% CO2 group. The neural cell-specific marker tubulin β3 and the epidermal marker K1/10 were also detected in the differentiated cells via immunofluorescent staining; thus, cell sorting was performed via fluorescence-activated cell sorting (FACS), and ABCG2-positive cells were isolated as CEPCs. The sorted cells formed three to four layers of epithelioid cells by airlifting culture and expressed ABCG2, p63, CK14, and CK3. In conclusion, the novel induction system conditioned by 7% CO2 in this study may be an effective and feasible method for CEPC differentiation.