Three-dimensional-cultured MSC-derived exosome with hydrogel for cerebral ischemia repair.

Microglia-mediated neuroinflammatory response, one of the most essential pathological processes of cerebral ischemia-reperfusion (I/R) injury, is acknowledged as the main factors leading to poor prognosis of cerebral ischemia. Exosome derived from mesenchymal stem cell (MSC-Exo) exhibits neuroprotective functions by reducing cerebral ischemia-induced neuroinflammatory response and promoting angiogenesis. However, MSC-Exo has disadvantages such as insufficient targeting capability and low production, which limits their clinical applications. Here, we fabricated gelatin methacryloyl (GelMA) hydrogel for three-dimensional (3D) culture of MSCs. It is indicated that 3D environment could simulate the biological niches of MSCs, thereby significantly increasing the cell stemness of MSCs and improving the yield of MSCs-derived exosomes (3D-Exo). In this study, we utilized the modified Longa method to induce middle cerebral artery occlusion (MCAO) model. Additionally, in vitro and in vivo studies were conducted to interrogate the mechanism of the stronger neuroprotective effect of 3D-Exo. Furthermore, the administration of 3D-Exo in MCAO model could promote neovascularization in infarct region and result in a significant suppression of inflammatory response. This study proposed an exosome-based targeting delivery system for cerebral ischemia and provided a promising strategy for efficient and large-scale production of MSC-Exo.

Healing Ability of Central Corneal Epithelium in Rabbit Ocular Surface Injury Models.

Purpose: Wound healing of the corneal epithelium mainly involves two types of cells: limbal stem/progenitor cells (LSCs) and differentiated central corneal epithelial cells (CECs). The healing ability of CECs is still debatable, and its correlated transcriptomic alterations during wound healing are yet to be elucidated. This study aimed to determine the healing ability and mechanisms underlying the actions of CECs using rabbit ocular surface injury models. Methods: A central corneal ring-like residual epithelium model was used to investigate the healing ability of CECs. Uninjured and injury-stimulated LSCs and CECs were collected for transcriptomic analysis. The analysis results were verified by quantitative reverse transcriptase polymerase chain reaction, immunofluorescence staining, and two types of rabbit corneal injury models. Results: During wound healing, the upregulated genes in LSCs were mostly enriched in the mitotic cell cycle-related processes, but those in CECs were mostly enriched in cell adhesion and migration. CECs could repair the epithelial defects successfully at one-time injuries. However, after repetitive injuries, the CECs repaired notably slower and failed to completely heal the defect, but the LSCs repaired even faster than the one-time injury. Conclusions: Our results indicated rabbit CECs repair the epithelial defect mainly depending on migration and its proliferative ability is limited, and LSCs are the main source of regenerative epithelial cells. Translational Relevance: This study provides information on gene expression in the corneal epithelium during wound healing, indicating that regulation of the cell cycle, cell adhesion, and migration may be the basis for future treatment strategies for corneal wound healing.

Generation of a DKK2 homozygous knockout human embryonic stem cell line using the CRISPR/Cas9 system.

Dickkopf-2 (DKK2) is an antagonist of canonical Wnt signaling, which is involved invarious biological processes of development, such as epidermal appendage formation andeye development. To identify underlying effects of DKK2 during embryonic development, we generated a DKK2 homozygous knockout human embryonic stem cell (hESC) line through the CRISPR/Cas9 genome-editing technology. This cell line, which maintains normal stem cell morphology and stably expresses pluripotent markers, could provide an ideal platform for exploring the role of DKK2 in embryonic development. In addition, Zeocin selection combined with tiny clone picking might be a highly efficient way to generate gene-knockout hESC lines.

Generating an MEIS1 homozygous knockout human embryonic stem cell line using the CRISPR/Cas9 system.

Myeloid ecotropic viral integration site 1 (MEIS1) plays an essential role in the development of several embryonic organs, such as the central nervous system and eyes. To further investigate the role of MEIS1 in embryonic development, herein, we generated a MEIS1 homozygous knockout human embryonic stem cell (hESC) line using the CRISPR/Cas9 genome-editing technology. We believe that this cell line will be a good resource for exploring the function of the MEIS1 gene in embryonic development in vitro. Furthermore, the gene-knockout method reported in this study is efficient and labor-saving, which may provide an effective strategy for hESC gene deletion.

Bilateral congenital macular coloboma and cataract: A case report.

RATIONALE: The case with congenital macular coloboma and cataract was rarely reported, and the pathogenic gene of the disease is still not clear. Moreover, it is difficult to improve the visual acuity of the eye with this disease. PATIENT CONCERNS: An 11-year-old boy presented low visual acuity and horizontal nystagmus in both eyes. Ophthalmologic examination showed the patient with bilateral congenital coloboma and cataract. The visual acuity of the patient improved slightly after cataract surgery. Heterozygous mutations of frizzled-4 (FZD4) and nucleotide-binding oligomerization domain-containing protein 2 (NOD2) were identified by next-generation sequencing in this case. DIAGNOSIS: Congenital macular coloboma and cataract of both eyes. INTERVENTIONS: We performed the standard phacoemulsification and intraocular lens implantation on both eyes of the patient for the treatment of congenital cataract, and then followed up the fundus lesions regularly. OUTCOMES: Cataract surgery may improve the visual acuity of the eyes with congenital macular coloboma and cataract at some degree, but the vision of this patient was still very poor postoperatively. Furthermore, the heterozygous mutations of FZD4 and NOD2 were found in this patient. LESSONS: Cataract surgery may improve the visual acuity of the eyes with congenital macular coloboma and cataract at some degree, and heterozygous mutations of FZD4 and NOD2 may be involved in the occurrence of congenital macular coloboma and cataract.

Expression of matrix Metalloproteinases-2 and aquaporin-1 in corneoscleral junction after angle-closure in rabbits.

BACKGROUND: To investigate the expression of Matrix Metalloproteinases 2 and aquaporin-1 in corneoscleral junction and explore the mechanism of trabecular damageafter angle-closure. METHODS: Thirty New Zealand white rabbits were randomly assigned into 2 groups, theexperimental group (Group 1) including twenty five rabbits and the control group (Group 2) including 5 rabbits. The rabbits in the experimental group were used to establish angle-closure models, and the rabbits in the control group were not subjected to any operation. All the rabbits were followed by slit lamp microscopy, Tonopen tonometer, and anterior segment optical coherent tomography (AS-OCT). The expressions of metalloproteinase MMP-2, aquaporin-1, and tissue inhibitors of metalloproteinase-2 in corneoscleral junctionwere evaluatedin both groups byimmunofluorescence, quantitative reverse-transcription polymerase chain reaction (qRT-PCR), and enzyme-linked immunosorbent assay (ELISA). RESULTS: Slit-lamp examination showed that angle-closure model was successfully established in twenty rabbits. The extent of angle-closure was about 2 to 4 clock hours in all the rabbit models, but the intraocular pressure (IOP) of the rabbits distributed from 8.57 to 15.25 mmHg and no significant high IOP was found in the follow-up period. The AQP-1-positive cells mainly located in Schlemm's canal, the inner surface of trabecular meshwork (TM), and the surface of iris, which began to decline on 1 month after angle-closure. MMP2 staining was diffuse in trabecular meshwork and iris. Immunofluorescence signal of MMP2 was strong within 1 month after angle-closure, and subsequently became weak. qRT-PCR and ELISA showed that the expression of MMP-2 and TIMP-2 increased within 1 month after angle-closure and then declined gradually. The AQP-1 levels showed slightly declined on 1 month after angle-closure. CONCLUSIONS: Altered levels of MMPs, TIMPs, and AQP-1 were found in the area of angle-closure, which may be involved in the damage of TM and Schlemm's canal after angle-closure.

Clinical effectiveness of ranibizumab and conbercept for neovascular age-related macular degeneration: a meta-analysis.

INTRODUCTION: To assess the ocular efficacy of intravitreal ranibizumab and conbercept injection in patients with neovascular age-related macular degeneration. MATERIALS AND METHODS: We 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. RESULTS: Eight 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. CONCLUSION: Pooled 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.

Promoting the expansion and function of human corneal endothelial cells with an orbital adipose-derived stem cell-conditioned medium.

BACKGROUND: Corneal 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. METHODS: The 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. RESULTS: CD29, 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. CONCLUSIONS: The 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.

Therapy of corneal endothelial dysfunction with corneal endothelial cell-like cells derived from skin-derived precursors.

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.

Differentiation of human embryonic stem cells into corneal epithelial progenitor cells under defined conditions.

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 ß3 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.

Construction of tissue-engineered full-thickness cornea substitute using limbal epithelial cell-like and corneal endothelial cell-like cells derived from human embryonic stem cells.

The aim of this study was to construct a full-thickness artificial cornea substitute in vitro by coculturing limbal epithelial cell-like (LEC-like) cells and corneal endothelial cell-like (CEC-like) cells derived from human embryonic stem cells (hESCs) on APCM scaffold. A 400 µm thickness, 11 mm diameter APCM lamella containing Bowman's membrane was prepared as the scaffold using trephine and a special apparatus made by ourselves. LEC-like cells and CEC-like cells, derived from hESCs as our previously described, were cocultured on the scaffold using a special insert of 24-well plates that enabled seeding both sides of the scaffold. Three or four layers of epithelium-like cells and a uniform monolayer of CEC-like cells could be observed by H&E staining. The thickness, endothelial cell density, and mechanical properties of the construct were similar to that of native rabbit corneas. Immunofluorescence analysis showed expression of ABCG2 and CK3 in the epithelium-like cell layers and expression of N-cadherin, ZO-1 and Na+/K + ATPase in the CEC-like cells. The corneal substitutes were well integrated within the host corneas, and the transparency increased gradually in 8-week follow-up after transplantation in the rabbits. These results suggest that the strategy we developed is feasible and effective for construction of tissue-engineered full-thickness cornea substitute with critical properties of native cornea.

Acellular porcine corneal matrix as a carrier scaffold for cultivating human corneal epithelial cells and fibroblasts in vitro.

AIM: To investigate the feasibility of corneal anterior lamellar reconstruction with human corneal epithelial cells and fibroblasts, and an acellular porcine cornea matrix (APCM) in vitro. METHODS: The scaffold was prepared from fresh porcine corneas which were treated with 0.5% sodium dodecyl sulfate (SDS) solution and the complete removal of corneal cells was confirmed by hematoxylin-eosin (HE) staining and 4', 6-diamidino-2-phenylindole (DAPI) staining. Human corneal fibroblasts and epithelial cells were cultured with leaching liquid extracted from APCM, and then cell proliferative ability was evaluated by MTT assay. To construct a human corneal anterior lamellar replacement, corneal fibroblasts were injected into the APCM and cultured for 3d, followed by culturing corneal epithelial cells on the stroma construction surface for another 10d. The corneal replacement was analyzed by HE staining, and immunofluorescence staining. RESULTS: Histological examination indicated that there were no cells in the APCM by HE staining, and DAPI staining did not detect any residual DNA. The leaching liquid from APCM had little influence on the proliferation ability of human corneal fibroblasts and epithelial cells. At 10d, a continuous 3 to 5 layers of human corneal epithelial cells covering the surface of the APCM was observed, and the injected corneal fibroblasts distributed within the scaffold. The phenotype of the construction was similar to normal human corneas, with high expression of cytokeratin 12 in the epithelial cell layer and high expression of vimentin in the stroma. CONCLUSION: Corneal anterior lamellar replacement can be reconstructed in vitro by cultivating human corneal epithelial cells and fibroblasts with an acellular porcine cornea matrix. This laid the foundation for the further transplantation in vivo.