|
1
|
Sasano M, Hayashi H, Kawaji K, Usui E, Kodama EN. Establishing an accurate and sensitive in vitro drug screening system for human adenovirus infection with human corneal cells. Virology 2023; 581:34-38. [PMID: 36848735 DOI: 10.1016/j.virol.2023.02.005] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/10/2023] [Revised: 02/07/2023] [Accepted: 02/13/2023] [Indexed: 02/19/2023]
Abstract
Epidemic keratoconjunctivitis (EKC) is a hazardous and highly contagious disease, with the potential to cause epidemic outbreaks in hospitals and other community settings. There are currently no approved drugs for human adenovirus (HAdV), the causative agent of EKC. To establish a novel drug screening system for ocular HAdV infections, we employed CRL11516, a non-cancerous but immortalized human corneal epithelial cell line. Brincidoforvir and 3'-deoxy-3'-fluorothymidine inhibit replication of HAdV species C type 1 (C1), C2, E4, and C6 to the same extent. This alternative assay system may allow for the evaluation of anti-HAdV activity and cell cytotoxicity of compounds within 2 days and without the need of the rabbit eye infection model.
Collapse
Affiliation(s)
- Mina Sasano
- Division of Infectious Diseases, International Research Institute of Disaster Science, Tohoku University, 2-1, Seiryo-machi, Aoba-ku, Sendai, 980-0845, Japan
| | - Hironori Hayashi
- Division of Infectious Diseases, International Research Institute of Disaster Science, Tohoku University, 2-1, Seiryo-machi, Aoba-ku, Sendai, 980-0845, Japan; Department of Intelligent Network for Infection Control, Tohoku University Graduate School of Medicine, 2-1, Seiryo-machi, Aoba-ku, Sendai, Miyagi, 980-8575, Japan; Department of Refractory Viral Infection, National Center for Global Health and Medicine Research Institute, 1-21-1 Toyama Shinjuku-ku, Tokyo, 162-8655, Japan
| | - Kumi Kawaji
- Division of Infectious Diseases, International Research Institute of Disaster Science, Tohoku University, 2-1, Seiryo-machi, Aoba-ku, Sendai, 980-0845, Japan
| | - Emiko Usui
- Division of Infectious Diseases, International Research Institute of Disaster Science, Tohoku University, 2-1, Seiryo-machi, Aoba-ku, Sendai, 980-0845, Japan
| | - Eiich N Kodama
- Division of Infectious Diseases, International Research Institute of Disaster Science, Tohoku University, 2-1, Seiryo-machi, Aoba-ku, Sendai, 980-0845, Japan; Department of Intelligent Network for Infection Control, Tohoku University Graduate School of Medicine, 2-1, Seiryo-machi, Aoba-ku, Sendai, Miyagi, 980-8575, Japan; Department of Infectious Diseases, Graduate School of Medicine and Tohoku Medical Megabank Organization, Tohoku University, 2-1, Seiryo-machi, Aoba-ku, Sendai, Miyagi, 980-8575, Japan.
| |
Collapse
|
|
2
|
Luo Y, Kang KB, Sartaj R, Sun MG, Zhou Q, Guaiquil VH, Rosenblatt MI. Silk films with nanotopography and extracellular proteins enhance corneal epithelial wound healing. Sci Rep 2021; 11:8168. [PMID: 33854156 PMCID: PMC8046786 DOI: 10.1038/s41598-021-87658-1] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/20/2020] [Accepted: 03/30/2021] [Indexed: 02/08/2023] Open
Abstract
Corneal wound healing depends on extracellular matrix (ECM) and topographical cues that modulate migration and proliferation of regenerating cells. In our study, silk films with either flat or nanotopography patterned parallel ridge widths of 2000, 1000, 800 nm surfaces were combined with ECMs which include collagen type I (collagen I), fibronectin, laminin, and Poly-D-Lysine to accelerate corneal wound healing. Silk films with 800 nm ridge width provided better cell spreading and wound recovery than other size topographies. Coating 800 nm patterned silk films with collagen I proves to optimally further increased mouse and rabbit corneal epithelial cells growth and wound recovery. This enhanced cellular response correlated with redistribution and increase in size and total amount of focal adhesion. Transcriptomics and signaling pathway analysis suggested that silk topography regulates cell behaviors via actin nucleation ARP-WASP complex pathway, which regulate filopodia formation. This mechanism was further explored and inhibition of Cdc42, a key protein in this pathway, delayed wound healing and decreased the length, density, and alignment of filopodia. Inhibition of Cdc42 in vivo resulted in delayed re-epithelization of injured corneas. We conclude that silk film nanotopography in combination with collagen I constitutes a better substrate for corneal wound repair than either nanotopography or ECM alone.
Collapse
Affiliation(s)
- Yuncin Luo
- Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago, 1855 W. Taylor Street, MC648, Chicago, IL, 60612, USA
| | - Kai B Kang
- Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago, 1855 W. Taylor Street, MC648, Chicago, IL, 60612, USA
| | - Rachel Sartaj
- Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago, 1855 W. Taylor Street, MC648, Chicago, IL, 60612, USA
| | - Michael G Sun
- Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago, 1855 W. Taylor Street, MC648, Chicago, IL, 60612, USA
| | - Qiang Zhou
- Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago, 1855 W. Taylor Street, MC648, Chicago, IL, 60612, USA
| | - Victor H Guaiquil
- Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago, 1855 W. Taylor Street, MC648, Chicago, IL, 60612, USA
| | - Mark I Rosenblatt
- Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago, 1855 W. Taylor Street, MC648, Chicago, IL, 60612, USA.
| |
Collapse
|
|
3
|
Liu J, Yang L, Wang X, Wang S, Huang Z, Li C, Liu Y, Cheng Y, Liu C, Wang Z. Embryonic stem cell microenvironment enhances proliferation of human retinal pigment epithelium cells by activating the PI3K signaling pathway. Stem Cell Res Ther 2020; 11:411. [PMID: 32967731 PMCID: PMC7509927 DOI: 10.1186/s13287-020-01923-0] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/14/2020] [Revised: 08/05/2020] [Accepted: 09/03/2020] [Indexed: 12/27/2022] Open
Abstract
BACKGROUND Retinal pigment epithelium (RPE) replacement has been proposed as an efficacious treatment for age-related macular degeneration (AMD), which is the primary cause of vision loss in the elderly worldwide. The embryonic stem cell (ESC) microenvironment has been demonstrated to enable mature cells to gain a powerful proliferative ability and even enhance the stem/progenitor phenotype via activation of the phosphoinositide 3-kinase (PI3K) signaling pathway. As the PI3K signaling pathway plays a pivotal role in proliferation and homeostasis of RPE, we hypothesize that the stemness and proliferative capability of RPE can be enhanced by the ESC microenvironment via activation of the PI3K signaling pathway. METHODS To investigate whether the ESC microenvironment improves the stem cell phenotype and proliferation properties of human RPE (hRPE) cells by regulating the PI3K signaling pathway, primary hRPE cells were cocultured with either ESCs or human corneal epithelial cells (CECs) for 72 h, after which their proliferation, apoptosis, cell cycle progression, and colony formation were assayed to evaluate changes in their biological characteristics. Gene expression was detected by real-time PCR and protein levels were determined by western blotting or immunofluorescence. LY294002, an antagonist of the PI3K signaling pathway, was used to further confirm the mechanism involved. RESULTS In comparison to hRPE cells cultured alone, hRPE cells cocultured with ESCs had an increased proliferative capacity, reduced apoptotic rate, and higher colony-forming efficiency. The expression of the stem cell-associated marker KLF4 and the differentiation marker CRALBP increased and decreased, respectively, in hRPE cells isolated from the ESC coculture. Furthermore, PI3K pathway-related genes were significantly upregulated in hRPE cells after exposure to ESCs. LY294002 reversed the pro-proliferative effect of ESCs on hRPE cells. In contrast, CECs did not share the ability of ESCs to influence the biological behavior and gene expression of hRPE cells. CONCLUSIONS Our findings indicate that the ESC microenvironment enhances stemness and proliferation of hRPE cells, partially via activation of the PI3K signaling pathway. This study may have a significant impact and clinical implication on cell therapy in regenerative medicine, specifically for age-related macular degeneration.
Collapse
Affiliation(s)
- Jiahui Liu
- Affiliated Dongguan People's Hospital, Southern Medical University, Dongguan, China
| | - Liu Yang
- State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, 510060, China
| | - Xiaoran Wang
- State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, 510060, China
| | - Shoubi Wang
- State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, 510060, China
| | - Zheqian Huang
- State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, 510060, China
| | - Chaoyang Li
- State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, 510060, China
| | - Ying Liu
- State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, 510060, China
| | - Yaqi Cheng
- State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, 510060, China
| | - Chengxiu Liu
- Affiliated Hospital of Qingdao University Medical College, Qingdao, China
| | - Zhichong Wang
- State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, 510060, China.
| |
Collapse
|
|
4
|
NLRP12- and NLRC4-mediated corneal epithelial pyroptosis is driven by GSDMD cleavage accompanied by IL-33 processing in dry eye. Ocul Surf 2020; 18:783-794. [PMID: 32735949 DOI: 10.1016/j.jtos.2020.07.001] [Citation(s) in RCA: 59] [Impact Index Per Article: 11.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/30/2019] [Revised: 06/16/2020] [Accepted: 07/06/2020] [Indexed: 01/26/2023]
Abstract
PURPOSE Dry eye disease (DED) is a common and multifactor-induced autoimmune ocular surface disease. Environmental factors, such as desiccating stress (DS) and hyperosmolarity, affect the corneal epithelium to induce ocular surface inflammation in DED. We aimed to explore the potential mechanisms by which innate immunity and pyroptosis are initiated in the mucosal epithelium in response to environmental stress. METHODS Experimental dry eye was established in C57BL/6 J mice and genetic mice on the background of C57BL/6 J mice by subcutaneous injection of scopolamine and exposure to a desiccating environment. SDHCEC cell line was subjected to hyperosmolarity stress (450 mOsM). The phenol red thread tear test and corneal epithelial defects evaluation were used as assessments of severity of DED. RNA-sequencing, quantitative real-time PCR, western blotting and immunofluorescence staining were performed in this study. RESULTS Loss-of-function studies indicated that genetic deletion of GSDMD alleviates DS-induced corneal epithelium defects, and GSDMD is needed for IL-33 processing. We further found that NLRP12 collaborates with NLRC4 inflammasome to initiate GSDMD-dependent pyroptosis, which requires TLR4-induced caspase-8 (CASP8) activation in the mucosal corneal epithelium in response to DS. CONCLUSIONS These findings provide compelling evidence that GSDMD-dependent pyroptosis plays a pivotal role in DED. A novel mechanism involving NLRP12 and NLRC4 inflammasomes-induced GSDMD-dependent pyroptosis, accompanied by IL-33 processing is responsible for ocular surface epithelial defects in response to environmental stress. GSDMD is required for IL-33 processing and the subsequent amplification of inflammatory cascades. These findings reveal novel therapeutic targets for treating DED.
Collapse
|
|
5
|
Liu J, Huang Z, Yang L, Wang X, Wang S, Li C, Liu Y, Cheng Y, Wang B, Sang X, He X, Wang C, Liu T, Liu C, Jin L, Liu C, Zhang X, Wang L, Wang Z. Embryonic Stem Cells Modulate the Cancer-Permissive Microenvironment of Human Uveal Melanoma. Theranostics 2019; 9:4764-4778. [PMID: 31367256 PMCID: PMC6643444 DOI: 10.7150/thno.33139] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/14/2019] [Accepted: 05/14/2019] [Indexed: 02/03/2023] Open
Abstract
The currently used anti-cancer therapies work by killing cancer cells but result in adverse effects and resistance to treatment, which accelerates aging and causes damage to normal somatic cells. On one hand, chicken and zebrafish embryos can reprogram cancer cells towards a non-tumorigenic phenotype; however, they cannot be used in the clinical practice. On the other hand, embryonic stem cells (ESCs) mimic the early embryonic microenvironment and are easily available. We investigated the therapeutic efficacy of the ESC microenvironment (ESCMe) in human uveal melanoma in vitro and in vivo. Methods: Human uveal melanoma C918 cells co-cultured with ESCs were used to measure the levels of mRNA and protein of the phosphoinositide 3-kinase (PI3K) pathway. Cell proliferation, invasiveness, and tumorigenicity of C918 cells were also analyzed. To mimic the tumor microenvironment in vivo, we co-cultured C918 cells and normal somatic cells with ESCs in a co-culture system and evaluated the therapeutic potential of ESCMe in both cell types. For an in vivo study, a mouse tumor model was used to test the safety and efficacy of the transplanted ESC. Elimination of the transplanted ESCs in mice was carried out by using the ESC-transfected with a thymidine kinase suicidal gene followed by administration of ganciclovir to prevent the formation of teratomas by ESCs. Results: In vitro studies confirmed that ESCMe inhibits the proliferation, invasiveness, and tumorigenicity of C918 cells, and the PI3K agonist abolished these effects. ESCMe suppressed the various malignant behaviors of uveal melanoma cells but enhanced the proliferation of normal somatic cells both in vitro and in vivo. Further, we demonstrated that ESCMe suppressed the PI3K pathway in tumor cells but activated in somatic cells. Conclusions: The ESCMe can effectively suppress the malignant phenotype of uveal melanoma cells and modulate the tumor-promoting aging environment by preventing the senescence of normal cells through the bidirectional regulation of the PI3K signaling. Our results suggest that ESC transplantation can serve as an effective and safe approach for treating cancer without killing cells.
Collapse
|
|
6
|
Lai S, Wei Y, Wu Q, Zhou K, Liu T, Zhang Y, Jiang N, Xiao W, Chen J, Liu Q, Yu Y. Liposomes for effective drug delivery to the ocular posterior chamber. J Nanobiotechnology 2019; 17:64. [PMID: 31084611 PMCID: PMC6515668 DOI: 10.1186/s12951-019-0498-7] [Citation(s) in RCA: 65] [Impact Index Per Article: 10.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/23/2019] [Accepted: 05/04/2019] [Indexed: 01/03/2023] Open
Abstract
Background Age-related macular degeneration (AMD) is a leading cause of severe visual deficits and blindness. Meanwhile, there is convincing evidence implicating oxidative stress, inflammation, and neovascularization in the onset and progression of AMD. Several studies have identified berberine hydrochloride and chrysophanol as potential treatments for ocular diseases based on their antioxidative, antiangiogenic, and anti-inflammatory effects. Unfortunately, their poor stability and bioavailability have limited their application. In order to overcome these disadvantages, we prepared a compound liposome system that can entrap these drugs simultaneously using the third polyamidoamine dendrimer (PAMAM G3.0) as a carrier. Results PAMAM G3.0-coated compound liposomes exhibited appreciable cellular permeability in human corneal epithelial cells and enhanced bio-adhesion on rabbit corneal epithelium. Moreover, coated liposomes greatly improved BBH bioavailability. Further, coated liposomes exhibited obviously protective effects in human retinal pigment epithelial cells and rat retinas after photooxidative retinal injury. Finally, administration of P-CBLs showed no sign of side effects on ocular surface structure in rabbits model. Conclusions The PAMAM G3.0-liposome system thus displayed a potential use for treating various ocular diseases. Electronic supplementary material The online version of this article (10.1186/s12951-019-0498-7) contains supplementary material, which is available to authorized users.
Collapse
Affiliation(s)
- Sisi Lai
- School of Pharmaceutical Sciences, Guangzhou University of Chinese Medicine, Guangzhou, 510006, Guangdong, China
| | - Yanyan Wei
- School of Pharmaceutical Sciences, Guangzhou University of Chinese Medicine, Guangzhou, 510006, Guangdong, China
| | - Quanwu Wu
- School of Pharmaceutical Sciences, Guangzhou University of Chinese Medicine, Guangzhou, 510006, Guangdong, China
| | - Kang Zhou
- School of Pharmaceutical Sciences, Guangzhou University of Chinese Medicine, Guangzhou, 510006, Guangdong, China
| | - Tuo Liu
- School of Pharmaceutical Sciences, Guangzhou University of Chinese Medicine, Guangzhou, 510006, Guangdong, China
| | - Yingfeng Zhang
- School of Pharmaceutical Sciences, Guangzhou University of Chinese Medicine, Guangzhou, 510006, Guangdong, China
| | - Ning Jiang
- School of Pharmaceutical Sciences, Guangzhou University of Chinese Medicine, Guangzhou, 510006, Guangdong, China
| | - Wen Xiao
- School of Pharmaceutical Sciences, Guangzhou University of Chinese Medicine, Guangzhou, 510006, Guangdong, China
| | - Junjie Chen
- School of Pharmaceutical Sciences, Guangzhou University of Chinese Medicine, Guangzhou, 510006, Guangdong, China
| | - Qiuhong Liu
- The First Affiliated Hospital, Guangzhou University of Chinese Medicine, Guangzhou, 510006, Guangdong, China
| | - Yang Yu
- School of Pharmaceutical Sciences, Guangzhou University of Chinese Medicine, Guangzhou, 510006, Guangdong, China.
| |
Collapse
|
|
7
|
Qiu F, Shin Y, Chen D, Cheng R, Chen Q, Zhou K, Larrick JW, Mendelson AR, Ma JX. Anti-angiogenic effect of a humanized antibody blocking the Wnt/β-catenin signaling pathway. Microvasc Res 2018; 119:29-37. [PMID: 29630973 DOI: 10.1016/j.mvr.2018.03.011] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/23/2017] [Revised: 01/14/2018] [Accepted: 03/24/2018] [Indexed: 10/17/2022]
Abstract
PURPOSE Our previous study demonstrated that Mab2F1, a murine monoclonal antibody blocking the Wnt/β-catenin signaling pathway, has beneficial effects on experimental diabetic retinopathy and choroidal neovascularization (NV). The aforementioned antibody has been humanized. This study evaluated effects of the humanized antibody, H1L1, on NV. METHODS H1L1 was evaluated in the alkali burn-induced corneal NV rat model. Rats with corneal NV were injected subconjunctivally with Mab2F1 or H1L1 using non-specific mouse or human IgG as controls. Corneal NV and opacity were evaluated using corneal NV area and inflammatory index. Expression of angiogenic and inflammatory factors and components of the Wnt/β-catenin pathway in both the corneas of the animal model and human corneal epithelial (HCE) cells exposed to Wnt3a conditioned medium (WCM) were determined by Western blotting and a luciferase-based promoter assay. Cytotoxicities of these antibodies were evaluated by MTT assay. RESULTS H1L1 reduced the area of corneal NV and opacity, similar to Mab2F1. Both Mab2F1 and H1L1 down-regulated the overexpression of angiogenic and inflammatory factors including VEGF, TNF-α and ICAM-1, and blocked the aberrant activation of the Wnt/β-catenin pathway as shown by down-regulation of phosphorylated LRP6, total LRP6 and non-phosphorylated β-catenin in the cornea of the NV model and cultured HCE cells exposed to WCM. Both antibodies also inhibited the transcriptional activity of β-catenin induced by WCM in HCE cells. No toxic effects of the antibodies were observed in cultured HCE cells. CONCLUSIONS H1L1 exhibits anti-angiogenic activities through blocking the Wnt/β-catenin pathway.
Collapse
Affiliation(s)
- Fangfang Qiu
- Department of Physiology, The University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA.
| | - Younghwa Shin
- Department of Physiology, The University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA.
| | | | - Rui Cheng
- Department of Physiology, The University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA.
| | - Qian Chen
- Department of Physiology, The University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA
| | - Kelu Zhou
- Department of Physiology, The University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA.
| | | | | | - Jian-Xing Ma
- Department of Physiology, The University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA.
| |
Collapse
|
|
8
|
Morita M, Fujita N, Abe M, Hayashimoto K, Nakagawa T, Nishimura R, Tsuzuki K. Canine corneal epithelial cells possess a sustained proliferative capacity and generate a spontaneously derived cell line. Exp Eye Res 2018. [PMID: 29522723 DOI: 10.1016/j.exer.2018.03.003] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/17/2022]
Abstract
We have previously reported characteristics of canine corneal epithelial cells in vitro and found that canine corneal epithelial cells could maintain their proliferative capacity even after continuous culture without the use of feeder cells and growth promoting additives. The objective of this study was to elucidate proliferative characteristics of canine corneal epithelial cells independent of feeder cells and growth promoting additives, with the aim of developing a spontaneously derived corneal epithelial cell line. Canine and rabbit corneal epithelial cells were harvested from the limbus and cultured with, or without, feeder cells and growth promoting additives, and both were passaged continuously until growth arrest. Canine corneal epithelial cells could proliferate independently, and could be passaged more times than rabbit cells. A canine corneal epithelial cell line, cCEpi, which could be passaged more than 100 times without using feeder cells and growth promoting additives, was established. cCEpi cells maintained a cell morphology close to the primary culture and expressed p63, cytokeratin 15 (K15), and K3. Although changes in colony morphology, shortening of the population doubling time and a heteroploid karyotype were observed, cCEpi was not tumorigenic. Stratified cell sheets cultured from cCEpi were morphologically and immunohistologically similar to sheets cultivated from early passage cells. In conclusion, canine corneal epithelial cells can proliferate independent of feeder cells and growth promoting additives. cCEpi maintains properties similar to normal corneal epithelial cells and could be a useful source for studies in cellular biology and for developing novel therapies.
Collapse
Affiliation(s)
- Maresuke Morita
- Laboratory of Veterinary Surgery, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan
| | - Naoki Fujita
- Laboratory of Veterinary Surgery, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan.
| | - Momoko Abe
- Laboratory of Veterinary Surgery, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan
| | - Koji Hayashimoto
- Laboratory of Veterinary Surgery, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan
| | - Takayuki Nakagawa
- Laboratory of Veterinary Surgery, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan
| | - Ryohei Nishimura
- Laboratory of Veterinary Surgery, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan
| | - Keiko Tsuzuki
- Laboratory of Veterinary Surgery, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan
| |
Collapse
|
|
9
|
Chen Y, Song W, Zhao X, Han Q, Ren L. An antibacterial collagen membrane crosslinked by the inclusion complex of β-cyclodextrin dialdehyde and ofloxacin for bacterial keratitis. RSC Adv 2018; 8:18153-18162. [PMID: 35542096 PMCID: PMC9080568 DOI: 10.1039/c8ra02160k] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/12/2018] [Accepted: 05/04/2018] [Indexed: 11/27/2022] Open
Abstract
Infectious keratitis is one of the leading causes of blindness in the world, especially in developing countries. Corneal transplant surgery is a feasible treatment for bacterial keratitis when drug therapy cannot be effective. However, the amount of corneal donors is far from requirements of clinical treatment and thus, the prognosis of bacterial keratitis is not satisfactory. In this study, we developed a novel antibacterial corneal repair material (β-CD-DA/OFLX-Col) for bacterial keratitis. The inclusion complex of β-cyclodextrin dialdehyde (β-CD-DA) with ofloxacin formed by host–guest interaction was used as both a drug vector and a crosslinker for further reaction with the amino groups on lysine of collagen chains. Physical properties, cytocompatibility, and antibacterial property of β-CD-DA/OFLX-Col film were characterized. The results indicated that the film was mainly transparent and possessed superior mechanical properties. Moreover, human corneal epithelial cells could adhere to the film and proliferate normally, indicating that the β-CD-DA/OFLX-Col film was non-cytotoxic and had good biocompatibility. Most importantly, the β-CD-DA/OFLX-Col film exhibited prominent antibacterial effects against E. coli and S. aureus in vitro, which could minimize the risks of infection. The prepared β-CD-DA/OFLX-Col film could greatly increase bioavailability of drugs and reduce toxic side effects, thus displaying great potential in bacterial keratitis treatment. The synthesis of antibacterial collagen membrane crosslinked by the inclusion complex of β-cyclodextrin dialdehyde and ofloxacin.![]()
Collapse
Affiliation(s)
- Yawei Chen
- School of Materials Science and Engineering
- South China University of Technology
- Guangzhou 510641
- China
- National Engineering Research Center for Tissue Restoration and Reconstruction
| | - Wenjing Song
- School of Materials Science and Engineering
- South China University of Technology
- Guangzhou 510641
- China
- National Engineering Research Center for Tissue Restoration and Reconstruction
| | - Xuan Zhao
- School of Materials Science and Engineering
- South China University of Technology
- Guangzhou 510641
- China
- National Engineering Research Center for Tissue Restoration and Reconstruction
| | - Qianqian Han
- Department of Biomaterials
- National Institutes for Food and Drug Control
- Beijing 102629
- China
| | - Li Ren
- School of Materials Science and Engineering
- South China University of Technology
- Guangzhou 510641
- China
- National Engineering Research Center for Tissue Restoration and Reconstruction
| |
Collapse
|
|
10
|
Abstract
Corneal epithelial cells (CECs) play an important role in the function of the cornea, and are maintained by corneal epithelial stem cells (CESCs). Recent studies have shown that neuronal growth factors affect the proliferation and migration of CESCs. Neuregulin-1 (NR-1) is a neuronal growth factor that is expressed in the early stages of brain development. The aim of this study was to determine whether NR-1 activates corneal wound healing. We observed that NR-1 activated both proliferation and migration of CECs. In addition, the colony-forming efficacy of CESCs was enhanced. In mice, NR-1 treatment improved corneal wound healing. Furthermore, the expression of markers of corneal epithelium maintenance (ΔNp63) and CESC proliferation (Bmi-1 and Abcg2) was increased. These effects were mediated by intracellular signalling pathways (Stat3, Erk1/2 and p38). Taken together, our results suggest that NR-1 accelerates the recovery of corneal wounds, and may represent a novel treatment for corneal damage.
Collapse
Affiliation(s)
- Won-Yong Jeong
- a Department of Biotechnology, BK21 Plus Program, College of Life Sciences and Biotechnology , Korea University , Seoul , Korea
| | - Hye-Young Yoo
- a Department of Biotechnology, BK21 Plus Program, College of Life Sciences and Biotechnology , Korea University , Seoul , Korea
| | - Chan-Wha Kim
- a Department of Biotechnology, BK21 Plus Program, College of Life Sciences and Biotechnology , Korea University , Seoul , Korea
| |
Collapse
|
|
11
|
Liu Z, Zhan W, Zeng M, Chen J, Zou H, Min Z. Enhanced functional properties of human limbal stem cells by inhibition of the miR-31/FIH-1/P21 axis. Acta Ophthalmol 2017. [PMID: 28650568 DOI: 10.1111/aos.13503] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
OBJECTIVE On the basis of the functional roles of the embryonic stem cell niche (ESCN) in the human limbal stem cells (LSCs), we proposed to explore the potential roles of microRNAs in regulating the self-renewal and differentiation of LSCs cultured in the ESCN. METHODS The LSCs were cultured in different media, either in CnT-20 media or in CnT-20 + 20% ES culture supernatant (ESC-CM). The LSCs cultured in ESC-CM were then transfected with microRNA-31 (miR-31) mimic or antago-31. The colony-forming efficiency (CFE) was analysed. Cell cycle, apoptosis, mitochondrial potential and reactive oxygen species were analysed by flow cytometry, and quantitative real-time PCR was used to determine the expression levels of FIH-1, P21, P63, ABCG2, CK3, microRNA-31, microRNA-143, microRNA-145 and microRNA-184. Indirect immunostaining was employed to detect the expression of P63, ABCG2, survivin, connexin-43 and CK3. Western blot was employed to detect the expression of FIH-1, P63, P21, CK3, caspase 3, Tcf4, β-catenin, survivin, GSK3β and pGSK3β. RESULTS Compared with cells grown in CnT-20, the level of miR-31 in cells grown in ESC-CM was lower. We investigated the roles that miR-31 and FIH-1 play in regulating the functional properties of LSCs. We used antagomirs (antago) to reduce the level of miR-31 in LSCs. Antago-31 increased FIH-1 levels and significantly reduced P21 expressional level in LSCs compared to irrelevant-antago (Ir-antago) treatment. The downregulation of miR-31 in LSCs promotes the maintenance of stemness. CONCLUSION ES culture supernatant (ESC-CM) regulates the fate of LSCs in part by inhibiting the miR-31/FIH-1/P21 axis. This study may have a high impact on the expansion of LSCs in regenerative medicine, especially for ocular surface reconstruction.
Collapse
Affiliation(s)
- Zhiping Liu
- The Department of Ophthalmology; The Second Affiliated Hospital of Guangzhou Medical University; Guangzhou Guangdong China
| | - Weijiao Zhan
- The Department of Ophthalmology; Linyi People's Hospital; Linyi Shandong China
| | - Minzhi Zeng
- The Department of Ophthalmology; The Second Affiliated Hospital of Guangzhou Medical University; Guangzhou Guangdong China
| | - Jinghong Chen
- The Department of Hematology; The Second Affiliated Hospital of Guangzhou Medical University; Guangzhou Guangdong China
| | - Huyong Zou
- The Department of Ophthalmology; The Second Affiliated Hospital of Guangzhou Medical University; Guangzhou Guangdong China
| | - Zhiqun Min
- Molecular Biology Laboratory; The Second Affiliated Hospital of Guangzhou Medical University; Guangzhou Guangdong China
| |
Collapse
|
|
12
|
Chen LW, Chen YM, Lu CJ, Chen MY, Lin SY, Hu FR, Chen WL. Effect of air-lifting on the stemness, junctional protein formation, and cytokeratin expression of in vitro cultivated limbal epithelial cell sheets. Taiwan J Ophthalmol 2017; 7:205-212. [PMID: 29296553 PMCID: PMC5747231 DOI: 10.4103/tjo.tjo_101_17] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/25/2022] Open
Abstract
PURPOSE: The aim of this study is to evaluate the effects of air-lifting on the stemness, junctional protein formation, and cytokeratin expression of rabbit limbal stem cells cultivated in vitro, and to find out the proper timing of air-lifting before transplantation as limbal epithelial cell sheets for the treatment of limbal insufficiency. MATERIALS AND METHODS: Limbal epithelial cells were isolated from the limbus of New Zealand white rabbits and cultivated in vitro. After the cells became confluent, different durations of air-lifting (0, 1, 2, 4, and 7 days) were performed. At the end of cultivation, immunohistochemistry on cryosections was performed and observed by fluorescein microscopy and in vitro confocal microscopy for cytokeratins (K3, K10, K12, K13, and K14), junctional and structural proteins (ZO-1, p120, and actin) and stem cell markers (ABCG2 and P63). Scanning electron microscopy was used for observing the microstructure of superficial cells. Transepithelial electrical resistance (TEER) was used to measure the transepithelial permeability. RESULTS: The expression of K3, K10, K12, K13, K14, and ABCG2 showed no differences in pattern and location among different groups of airlifting. A time-dependent increase in corneal epithelial thickness was found after air-lifting. In vitro confocal microscopy demonstrated that K3, p120, and ZO-1 were expressed on the apical cell layer, whereas P63 and ABCG2 were expressed more on the basal epithelial layer. Scanning electron microscopy of the superficial layer demonstrated that airlifting induced time-dependent increase in the size of surface epithelial cells and triggered cellular differentiation. TEER results demonstrated a time-dependent increase of transepithelial electric resistance. CONCLUSIONS: During limbal epithelial cell expansion in vitro, air-lifting can increase cellular stratification, enlarge surface cells, trigger cellular differentiation, and increase the transepithelial barrier. However, the expression of cellular junctional, stem cell and cytokeratin markers seems to have no obvious differences in pattern and localization.
Collapse
Affiliation(s)
- Lily Wei Chen
- Department of Ophthalmology, National Taiwan University Hospital, Taipei, Taiwan
| | - Yan-Ming Chen
- Department of Ophthalmology, National Taiwan University Hospital, Taipei, Taiwan.,Department of Ophthalmology, E-Da Hospital, I-Shou University, Kaohsiung, Taiwan
| | - Chia-Ju Lu
- Department of Ophthalmology, National Taiwan University Hospital, Taipei, Taiwan
| | - Mei-Yun Chen
- Department of Ophthalmology, National Taiwan University Hospital, Taipei, Taiwan
| | - Szu-Yuan Lin
- Department of Ophthalmology, Cathay General Hospital, Taipei, Taiwan
| | - Fung-Rong Hu
- Department of Ophthalmology, National Taiwan University Hospital, Taipei, Taiwan.,Center of Corneal Tissue Engineering and Stem Cell Biology, National Taiwan University Hospital, Taipei, Taiwan
| | - Wei-Li Chen
- Department of Ophthalmology, National Taiwan University Hospital, Taipei, Taiwan.,Center of Corneal Tissue Engineering and Stem Cell Biology, National Taiwan University Hospital, Taipei, Taiwan
| |
Collapse
|
|
13
|
Characterization of Long-Term Cultured Murine Submandibular Gland Epithelial Cells. PLoS One 2016; 11:e0147407. [PMID: 26800086 PMCID: PMC4723076 DOI: 10.1371/journal.pone.0147407] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/12/2015] [Accepted: 01/04/2016] [Indexed: 12/31/2022] Open
Abstract
PURPOSE Human and rat salivary gland cell lines derived from tumors or genetic modification are currently available for research. Here, we attempted to culture and characterize long-term cultured cells spontaneously derived from wild type murine submandibular glands (SGs). METHODS SGs were removed from 3-week-old C57B/6J female mice and dissociated by collagenase type 1 and hyaluronidase digestion. Isolated SG epithelial cells were cultured in low calcium, serum-free growth media in the presence of cholera toxin (CT) during early passages. Single-cell colonies were isolated by limiting dilution culture after 25 passages. Early- and late-stage cell cultures were characterized for keratin 14, keratin 18, α-smooth muscle actin, and p63 by immunostaining and quantitative real-time PCR analysis. RESULTS SG epithelial cells cultured in optimized media maintained their proliferative ability and morphology for over 80 passages. Long-term cultured cells expressed keratin 14, keratin 18, and p63, indicative of an epithelial phenotype. CONCLUSIONS Epithelial cells originating from wild type murine SGs could be cultured for longer periods of time and remain phenotypically similar to ductal basal epithelium.
Collapse
|
|
14
|
1,25-dihydroxyvitamin D3 inhibits corneal wound healing in an ex-vivo mouse model. Graefes Arch Clin Exp Ophthalmol 2016; 254:717-24. [PMID: 26794222 DOI: 10.1007/s00417-016-3267-4] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/01/2015] [Revised: 12/07/2015] [Accepted: 01/07/2016] [Indexed: 10/22/2022] Open
Abstract
PURPOSE Impaired healing of corneal injuries can result in ulceration and complete loss of vision, especially in the elderly. Such patients frequently also exhibit vitamin D insufficiency. 1,25-dihydroxyvitamin D3 is the active vitamin D metabolite. As it affects cell proliferation and inflammation, we herein aimed at elucidating its influence on corneal wound healing after alkali burn by using in vitro and ex vivo techniques. METHODS mRNA abundance in human corneal epithelial cells in response to vitamin D3 was determined by RT-PCR. Corneal re-epithelialization after alkaline burn was analyzed using enucleated mouse eyes and fluorescein staining. RESULTS Human corneal epithelial cells (HCEC) expressed the vitamin D receptor (VDR) and retinoid x receptor (RXR) and were responsive to 1,25- dihydroxyvitamin D3, as shown by induction of the 1,25- dihydroxyvitamin D3 responsive gene cyp-24A1 and slightly reduced abundance of IL-6 mRNA. However, no effect on cell vitality and migration was observed. In contrast, re-epithelialization of mouse corneas ex vivo was dose dependently inhibited by 1,25- dihydroxyvitamin D3. CONCLUSIONS These data indicate that topically applied 1,25- dihydroxyvitamin D3 does not seem to be suitable for therapy of corneal lesions.
Collapse
|
|
15
|
van Essen TH, van Zijl L, Possemiers T, Mulder AA, Zwart SJ, Chou CH, Lin CC, Lai HJ, Luyten GPM, Tassignon MJ, Zakaria N, El Ghalbzouri A, Jager MJ. Biocompatibility of a fish scale-derived artificial cornea: Cytotoxicity, cellular adhesion and phenotype, and in vivo immunogenicity. Biomaterials 2015; 81:36-45. [PMID: 26717247 DOI: 10.1016/j.biomaterials.2015.11.015] [Citation(s) in RCA: 52] [Impact Index Per Article: 5.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/04/2015] [Accepted: 11/06/2015] [Indexed: 01/30/2023]
Abstract
PURPOSE To determine whether a fish scale-derived collagen matrix (FSCM) meets the basic criteria to serve as an artificial cornea, as determined with in vitro and in vivo tests. METHODS Primary corneal epithelial and stromal cells were obtained from human donor corneas and used to examine the (in)direct cytotoxicity effects of the scaffold. Cytotoxicity was assessed by an MTT assay, while cellular proliferation, corneal cell phenotype and adhesion markers were assessed using an EdU-assay and immunofluorescence. For in vivo-testing, FSCMs were implanted subcutaneously in rats. Ologen(®) Collagen Matrices were used as controls. A second implant was implanted as an immunological challenge. The FSCM was implanted in a corneal pocket of seven New Zealand White rabbits, and compared to sham surgery. RESULTS The FSCM was used as a scaffold to grow corneal epithelial and stromal cells, and displayed no cytotoxicity to these cells. Corneal epithelial cells displayed their normal phenotypical markers (CK3/12 and E-cadherin), as well as cell-matrix adhesion molecules: integrin-α6 and β4, laminin 332, and hemi-desmosomes. Corneal stromal cells similarly expressed adhesion molecules (integrin-α6 and β1). A subcutaneous implant of the FSCM in rats did not induce inflammation or sensitization; the response was comparable to the response against the Ologen(®) Collagen Matrix. Implantation of the FSCM in a corneal stromal pocket in rabbits led to a transparent cornea, healthy epithelium, and, on histology, hardly any infiltrating immune cells. CONCLUSION The FSCM allows excellent cell growth, is not immunogenic and is well-tolerated in the cornea, and thus meets the basic criteria to serve as a scaffold to reconstitute the cornea.
Collapse
Affiliation(s)
- T H van Essen
- Department of Ophthalmology, J3-S, Leiden University Medical Center, PO Box 9600, 2300 RC Leiden, The Netherlands.
| | - L van Zijl
- Department of Research, Aeon Astron Europe B.V., J.H. Oortweg 19, 2333 CH, Leiden, The Netherlands.
| | - T Possemiers
- Department of Ophthalmology, Antwerp University Hospital, Wilrijkstraat 10, 2650 Edegem, Belgium.
| | - A A Mulder
- Department of Molecular Cell-biology, Leiden University Medical Center, PO Box 9600, 2300 RC Leiden, The Netherlands.
| | - S J Zwart
- Department of Research, Aeon Astron Europe B.V., J.H. Oortweg 19, 2333 CH, Leiden, The Netherlands.
| | - C-H Chou
- Department of Research, Body Organ Biomedical Corporation, 5F, No. 153, Section 3, Xinyi Road, Da'an District, Taipei City 106, Taiwan, ROC.
| | - C C Lin
- Department of Research, Body Organ Biomedical Corporation, 5F, No. 153, Section 3, Xinyi Road, Da'an District, Taipei City 106, Taiwan, ROC.
| | - H J Lai
- Department of Research, Aeon Astron Europe B.V., J.H. Oortweg 19, 2333 CH, Leiden, The Netherlands.
| | - G P M Luyten
- Department of Ophthalmology, J3-S, Leiden University Medical Center, PO Box 9600, 2300 RC Leiden, The Netherlands.
| | - M J Tassignon
- Department of Ophthalmology, Antwerp University Hospital, Wilrijkstraat 10, 2650 Edegem, Belgium.
| | - N Zakaria
- Department of Ophthalmology, Antwerp University Hospital, Wilrijkstraat 10, 2650 Edegem, Belgium; University of Antwerp, Prinsstraat 13, 2000 Antwerpen, Belgium.
| | - A El Ghalbzouri
- Department of Molecular Cell-biology, Leiden University Medical Center, PO Box 9600, 2300 RC Leiden, The Netherlands.
| | - M J Jager
- Department of Ophthalmology, J3-S, Leiden University Medical Center, PO Box 9600, 2300 RC Leiden, The Netherlands.
| |
Collapse
|
|
16
|
Zhou Q, Liu Z, Wu Z, Wang X, Wang B, Li C, Liu Y, Li L, Wan P, Huang Z, Wang Z. Reconstruction of Highly Proliferative Auto-Tissue-Engineered Lamellar Cornea Enhanced by Embryonic Stem Cell. Tissue Eng Part C Methods 2015; 21:639-48. [DOI: 10.1089/ten.tec.2014.0481] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/28/2022] Open
Affiliation(s)
- Qiang Zhou
- State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China
| | - Zhao Liu
- State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China
| | - Zheng Wu
- State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China
| | - Xiaoran Wang
- State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China
| | - Bowen Wang
- State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China
| | - Chaoyang Li
- State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China
| | - Ying Liu
- State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China
| | - Liangliang Li
- State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China
| | - Pengxia Wan
- State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China
| | - Zheqian Huang
- State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China
| | - Zhichong Wang
- State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China
| |
Collapse
|
|
17
|
Collagen based film with well epithelial and stromal regeneration as corneal repair materials: Improving mechanical property by crosslinking with citric acid. MATERIALS SCIENCE & ENGINEERING. C, MATERIALS FOR BIOLOGICAL APPLICATIONS 2015; 55:201-8. [PMID: 26117756 DOI: 10.1016/j.msec.2015.05.030] [Citation(s) in RCA: 34] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 01/29/2015] [Revised: 04/15/2015] [Accepted: 05/08/2015] [Indexed: 10/23/2022]
Abstract
Corneal disease can lead to vision loss. It has become the second greatest cause of blindness in the world, and keratoplasty is considered as an effective treatment method. This paper presents the crosslinked collagen (Col)-citric acid (CA) films developed by making use of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS). The results showed that the Col-CA films had necessary optical performance, water content. The collagenase resistance of CA crosslinked films was superior to that of EDC crosslinked films. And CA5 film (Col:CA:EDC:NHS=60:3:10:10) had the best mechanical properties. Cell experiments showed that CA5 film was non-cytotoxic and human corneal epithelial cells could proliferate well on the films. Lamellar keratoplasty showed that the CA5 film could be sutured in the rabbit eyes and was epithelialized completely in about 10 days, and the transparency was restored quickly in 30±5 days. No inflammation and corneal neovascularization were observed at 6 months. Corneal stroma had been repaired; stromal cells and neo-stroma could be seen in the area of operation from the hematoxylin-eosin stained histologic sections and anterior segment optical coherence tomography images. These results indicated that Col-CA films were highly promising biomaterials that could be used in corneal tissue engineering and a variety of other tissue engineering applications.
Collapse
|
|
18
|
Wan PX, Wang BW, Wang ZC. Importance of the stem cell microenvironment for ophthalmological cell-based therapy. World J Stem Cells 2015; 7:448-460. [PMID: 25815128 PMCID: PMC4369500 DOI: 10.4252/wjsc.v7.i2.448] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/27/2014] [Revised: 09/17/2014] [Accepted: 10/29/2014] [Indexed: 02/06/2023] Open
Abstract
Cell therapy is a promising treatment for diseases that are caused by cell degeneration or death. The cells for clinical transplantation are usually obtained by culturing healthy allogeneic or exogenous tissue in vitro. However, for diseases of the eye, obtaining the adequate number of cells for clinical transplantation is difficult due to the small size of tissue donors and the frequent needs of long-term amplification of cells in vitro, which results in low cell viability after transplantation. In addition, the transplanted cells often develop fibrosis or degrade and have very low survival. Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPS) are also promising candidates for cell therapy. Unfortunately, the differentiation of ESCs can bring immune rejection, tumorigenicity and undesired differentiated cells, limiting its clinical application. Although iPS cells can avoid the risk of immune rejection caused by ES cell differentiation post-transplantation, the low conversion rate, the risk of tumor formation and the potentially unpredictable biological changes that could occur through genetic manipulation hinder its clinical application. Thus, the desired clinical effect of cell therapy is impaired by these factors. Recent research findings recognize that the reason for low survival of the implanted cells not only depends on the seeded cells, but also on the cell microenvironment, which determines the cell survival, proliferation and even reverse differentiation. When used for cell therapy, the transplanted cells need a specific three-dimensional structure to anchor and specific extra cellular matrix components in addition to relevant cytokine signaling to transfer the required information to support their growth. These structures present in the matrix in which the stem cells reside are known as the stem cell microenvironment. The microenvironment interaction with the stem cells provides the necessary homeostasis for cell maintenance and growth. A large number of studies suggest that to explore how to reconstruct the stem cell microenvironment and strengthen its combination with the transplanted cells are key steps to successful cell therapy. In this review, we will describe the interactions of the stem cell microenvironment with the stem cells, discuss the importance of the stem cell microenvironment for cell-based therapy in ocular diseases, and introduce the progress of stem cell-based therapy for ocular diseases.
Collapse
|
|
19
|
Reconstruction of auto-tissue-engineered lamellar cornea by dynamic culture for transplantation: a rabbit model. PLoS One 2014; 9:e93012. [PMID: 24705327 PMCID: PMC3976280 DOI: 10.1371/journal.pone.0093012] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/13/2014] [Accepted: 02/27/2014] [Indexed: 12/13/2022] Open
Abstract
To construct an auto-tissue-engineered lamellar cornea (ATELC) for transplantation, based on acellular porcine corneal stroma and autologous corneal limbal explants, a dynamic culture process, which composed of a submersion culture, a perfusion culture and a dynamic air-liquid interface culture, was performed using appropriate parameters. The results showed that the ATELC-Dynamic possessed histological structure and DNA content that were similar to native lamellar cornea (NLC, p>0.05). Compared to NLC, the protein contents of zonula occludens-1, desmocollin-2 and integrin β4 in ATELC-Dynamic reached 93%, 89% and 73%, respectively. The basal cells of ATELC-Dynamic showed a better differentiation phenotype (K3−, P63+, ABCG2+) compared with that of ATELC in static air-lift culture (ATELC-Static, K3+, P63−, ABCG2−). Accordingly, the cell-cloning efficiency of ATELC-Dynamic (9.72±3.5%) was significantly higher than that of ATELC-Static (2.13±1.46%, p<0.05). The levels of trans-epithelial electrical resistance, light transmittance and areal modulus variation in ATELC-Dynamic all reached those of NLC (p>0.05). Rabbit lamellar keratoplasty showed that the barrier function of ATELC-Dynamic was intact, and there were no signs of epithelial shedding or neovascularization. Furthermore, the ATELC-Dynamic group had similar optical properties and wound healing processes compared with the NLC group. Thus, the sequential dynamic culture process that was designed according to corneal physiological characteristics could successfully reconstruct an auto-lamellar cornea with favorable morphological characteristics and satisfactory physiological function.
Collapse
|
|
20
|
Liu Y, Ren L, Long K, Wang L, Wang Y. Preparation and characterization of a novel tobramycin-containing antibacterial collagen film for corneal tissue engineering. Acta Biomater 2014; 10:289-99. [PMID: 24013030 DOI: 10.1016/j.actbio.2013.08.033] [Citation(s) in RCA: 52] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/25/2013] [Revised: 08/13/2013] [Accepted: 08/26/2013] [Indexed: 11/29/2022]
Abstract
Corneal disease is a major cause of blindness and keratoplasty is an effective treatment method. However, clinical treatment is limited due to a severe shortage of high-quality allogeneic corneal tissues and the bacterial infection after corneal transplantation. In this study, we develop a novel artificial and antibacterial collagen film (called Col-Tob) for corneal repair. In the Col-Tob film, the tobramycin, which is an aminoglycoside antibiotic to treat various types of bacterial infections, was cross-linked by 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide and N-hydroxysuccinimide onto the collagen. Physical properties, antibacterial property and biocompatibility of the films were characterized. The results indicate that the film is basically transparent and has appropriate mechanical properties. Cell experiments show that human corneal epithelial cells could adhere to and proliferate well on the film. Most importantly, the film exhibits excellent antibacterial effect in vitro. Lamellar keratoplasty shows that the Col-Tob film can be sutured in rabbit eyes and are epithelialized completely in 15 ± 5 days, and their transparency is restored quickly in the first month. Corneal rejection reaction, neovascularization and keratoconus are not observed within 3 months. This film, which can be prepared in large quantities and at low cost,should have potential application in corneal repair.
Collapse
Affiliation(s)
- Yang Liu
- School of Materials Science and Engineering, South China University of Technology, Guangzhou 510641, China; National Engineering Research Center for Tissue Restoration and Reconstruction, Guangzhou 510006, China; Guangdong Province Key Laboratory of Biomedical Engineering, South China University of Technology, Guangzhou 510006, China
| | | | | | | | | |
Collapse
|
|
21
|
Sha X, Liu Z, Song L, Wang Z, Liang X. Human amniotic epithelial cell niche enhances the functional properties of human corneal endothelial cells via inhibiting P53-survivin-mitochondria axis. Exp Eye Res 2013; 116:36-46. [DOI: 10.1016/j.exer.2013.08.008] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/18/2013] [Revised: 07/11/2013] [Accepted: 08/13/2013] [Indexed: 12/17/2022]
|
|
22
|
Yao M, Chen J, Yang XX, Zhang XL, Ji QS, Zhou Q, Xu JT. Differentiation of human amniotic epithelial cells into corneal epithelial-like cells in vitro. Int J Ophthalmol 2013; 6:564-72. [PMID: 24195026 DOI: 10.3980/j.issn.2222-3959.2013.05.02] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/03/2013] [Accepted: 09/02/2013] [Indexed: 12/21/2022] Open
Abstract
AIM To explore the feasibility that human amniotic epithelial cells (hAECs) have the potential to differentiate into corneal epithelial-like cells under the microenvironment replicated by spontaneously immortalized human corneal epithelial cells (S-ihCECs). METHODS hAECs were isolated by enzyme digestion, and flow cytometry was used to analysis the expression of CD29/90/166/73/34 and HLA-DR. Recovered and cultured S-ihCECs, immunocytochemistry was used to detect the expression of CK3/12. The proliferation of S-ihCECs handled by different concentrations of mitomycin was detected by CCK-8. The proliferation of hAECs cultured by S-ihCECs culture media collected at different time was analyzed by CCK-8. After filtered out the optimal conditions, we collected S-ihCECs culture media for 5 days, then prepared conditioned medium to incubate hAECs, inverted phase contrast microscope and scanning electron microscope were used to observe the change of morphology in hAECs. Quantitative real-time reverse transcription-polymerase chain reaction (QRT-PCR) was carried out to evaluate the expression of Oct-4, NANOG, PAX6, and CK12 in the differentiation period. Immunocytochemistry and western bloting were used to detect the expression of CK3/12. RESULTS The culture media collected every 12h, from 20µg/mL mitomycin pretreatment S-ihCECs could significantly promote the proliferation of hAECs. In the period of differentiation, the morphology of differentiated hAECs was obviously different compared with the control group, and the distinctive CK3/12 for corneal epithelial cells was detected. CONCLUSION This study showed that hAECs can differentiate into corneal epithelial-like cells by in vitro replication of the corneal epithelial microenvironment, using the culture media collected from S-ihCECs, and it is possible that S-ihCECs culture media could be used in corneal tissue engineering.
Collapse
Affiliation(s)
- Min Yao
- Department of Ophthalmology, the First Affiliated Hospital of Jinan University, Guangzhou 510632, Guangdong Province, China
| | | | | | | | | | | | | |
Collapse
|
|
23
|
Shi Y, Lv H, Fu Y, Lu Q, Zhong J, Ma D, Huang Y, Xue W. Preparation and characterization of a hydrogel carrier to deliver gatifloxacin and its application as a therapeutic contact lens for bacterial keratitis therapy. Biomed Mater 2013; 8:055007. [DOI: 10.1088/1748-6041/8/5/055007] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022]
|
|
24
|
Dai Y, Zhou R, Liu L, Lu Y, Qi J, Wu W. Liposomes containing bile salts as novel ocular delivery systems for tacrolimus (FK506): in vitro characterization and improved corneal permeation. Int J Nanomedicine 2013; 8:1921-33. [PMID: 23690687 PMCID: PMC3656938 DOI: 10.2147/ijn.s44487] [Citation(s) in RCA: 51] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022] Open
Abstract
The objective of this study was to investigate the potential of liposomes containing bile salts as an ophthalmic delivery system for tacrolimus to improve corneal permeability. Liposomes containing bile salts, including sodium taurocholate, sodium deoxycholate, and sodium glycocholate, were produced by the thin-film dispersion method with a particle size of approximately 100 nm and an entrapment efficiency of more than 90%. Less than 5% tacrolimus was released from conventional liposomes and from liposomes containing sodium taurocholate, sodium deoxycholate, or sodium glycocholate over 12 hours. The cellular uptake of conventional liposomes was significantly higher than that of liposomes containing bile salts. However, liposomes containing bile salts exerted a 3–4-fold increase of tacrolimus in ex vivo corneal transport of tacrolimus compared with conventional liposomes. When rabbit eyes were treated with a DiI perchlorate-loaded liposome suspension, liposomes containing bile salts showed fast and sustained penetration across the cornea. Unfortunately, liposomes containing sodium deoxycholate caused toxicity or irritation to both spontaneously derived human corneal epithelial cells and the rabbit cornea. Therefore, liposomes containing sodium taurocholate and sodium glycocholate are potential carriers in ocular drug delivery systems, given their low toxicity and vastly improved permeability.
Collapse
Affiliation(s)
- Yikang Dai
- Department of Ophthalmology, Renji Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai, People's Republic of China
| | | | | | | | | | | |
Collapse
|
|
25
|
Chakraborty A, Dutta J, Das S, Datta H. Comparison of ex vivo cultivated human limbal epithelial stem cell viability and proliferation on different substrates. Int Ophthalmol 2013; 33:665-70. [PMID: 23529791 DOI: 10.1007/s10792-013-9765-z] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/09/2012] [Accepted: 03/16/2013] [Indexed: 11/26/2022]
Abstract
Ocular surface injury causes serious vision-related problems especially when limbal stem cells are affected. Treatment lies in the transplantation of viable donor cells. Various substrates are used for the cultivation of limbal epithelial stem cells. In the present study, viability and proliferation of ex vivo cultured limbal epithelial stem cells were examined on a variety of substrates like collagen type IV, direct plastic Petri plate, intact amniotic membrane and denuded amniotic membrane. Viability and proliferation of cells were examined by colorimetric assay and [(3)H]-thymidine incorporation study. Furthermore, matrix metalloproteinase is known to be a key regulator in stem cell migration and proliferation. This enzyme activity was studied by gelatinolytic zymography. It was found from this study that although human limbal epithelial stem cells could be cultivated on different substrates such as collagen type IV, direct plastic Petri plate, intact amniotic membrane and denuded amniotic membrane, maximum growth and proliferation was observed when cultured on intact amniotic membrane. The number of patients suffering from limbal epithelial stem cell deficiency is large compared to donor tissues available for transplantation. Hence, increased cell viability and proliferation is required to serve more patients.
Collapse
Affiliation(s)
- Anindita Chakraborty
- Neurobiology Division, Indian Institute of Chemical Biology, 4 Raja S. C. Mullick Road, Jadavpur, Kolkata, 700032, India
| | | | | | | |
Collapse
|
|
26
|
Defensin production by human limbo-corneal fibroblasts infected with mycobacteria. Pathogens 2013; 2:13-32. [PMID: 25436879 PMCID: PMC4235707 DOI: 10.3390/pathogens2010013] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/09/2012] [Revised: 12/24/2012] [Accepted: 01/24/2013] [Indexed: 11/22/2022] Open
Abstract
Epithelial cells of the cornea and the conjunctiva constitutively produce antimicrobial peptides; however, the production of defensins by other cell types located around the eye has not been investigated. We analyzed the production of beta-defensins (hBD) and cathelicidin LL-37 during the infection of primary limbo-corneal fibroblasts with M. tuberculosis (MTB), M. abscessus (MAB), and M. smegmatis (MSM). The intracellular survival of each mycobacterium, the production of cytokines and the changes on the distribution of the actin filaments during the infection were also analyzed. Fibroblasts produce basal levels of hBD1 and LL-37 and under PMA stimulation they produce hBD2, hBD3 and overexpress hBD1 and LL-37. MAB induced the highest levels of hBD1 and LL-37 and intermediate levels of IL-6; however, MAB was not eliminated. In addition, MAB induced the greatest change to the distribution of the actin filaments. MTB also produced changes in the structure of the cytoskeleton and induced low levels of hBD1 and IL-6, and intermediate levels of LL-37. The balance of these molecules induced by MTB appeared to contribute to the non-replicative state observed in the limbo-corneal cells. MSM induced the lowest levels of hBD1 and LL-37 but the highest levels of IL-6; MSM was eliminated. The results suggest that mycobacterial infections regulate the production of antimicrobial peptides and cytokines, which in conjunction can contribute to the control of the bacilli.
Collapse
|
|
27
|
Liu Z, Wan P, Duan H, Zhou J, Tan B, Liu Y, Zhou Q, Zhou C, Huang Z, Tian B, Li C, Wang Z. ES micro-environment enhances stemness and inhibits apoptosis in human limbal stem cells via the maintenance of telomerase activity. PLoS One 2013; 8:e53576. [PMID: 23326460 PMCID: PMC3543452 DOI: 10.1371/journal.pone.0053576] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/14/2012] [Accepted: 11/30/2012] [Indexed: 12/17/2022] Open
Abstract
Our previous work had found that telomerase rejuvenated in the cytoplasm of corneal epithelial cells cultured in embryonic stem cell-conditioned medium, the functional properties of stem-like corneal epithelial cells can be enhanced by co-culturing with embryonic stem cells (ESCs) via activation of the integrinβ1-FAK-PI3K/Akt signaling pathway. The goal of this study was to explore the potential molecular mechanisms of the ES micro-environment that enhance the stem cell-like phenotype and inhibit apoptosis in human limbal stem cells (LSC). The LSC were cultured in different media, either CnT-20 medium or CnT-20 +20% ES culture supernatant (ESC-CM). We observed that LSC cultured in ESC-CM had an increased proliferative capacity, greater serial passage capacity, higher colony-forming efficiency (CFE) and higher levels of stem cell-associated marker than those cultured in CnT-20. Compared with CnT-20, ESC-CM enhanced the undifferentiated status and inhibited apoptosis in the LSC by promoting the maintenance of telomerase activity, which could reduce the generation of reactive oxygen species (ROS), maintain the membrane potential (Δψm) at higher levels and reduce the expression of the p21 protein. Our findings indicated that ESC-CM system induced LSC to maintain a stem cell phenotype and inhibit the process of apoptosis. These effects might partially be achieved via the telomerase-p21-mitochondrial axis and the activation of the FAK/Wnt signaling pathways. This study may have high impact and clinic implication on the expansion of LSC in regenerative medicine, especially for ocular surface reconstruction.
Collapse
Affiliation(s)
- Zhiping Liu
- State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, Guangdong, China
| | - Pengxia Wan
- State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, Guangdong, China
| | - Hucheng Duan
- Ophthalmic Center of the Second People's Hospital of Foshan, Foshan, Guangdong, China
| | - Jin Zhou
- State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, Guangdong, China
| | - Bowei Tan
- State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, Guangdong, China
| | - Ying Liu
- State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, Guangdong, China
| | - Qiang Zhou
- State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, Guangdong, China
| | - Chenjing Zhou
- State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, Guangdong, China
| | - Zheqian Huang
- State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, Guangdong, China
| | - Bishan Tian
- State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, Guangdong, China
| | - Chaoyang Li
- State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, Guangdong, China
- * E-mail: (CYL); (ZCW)
| | - Zhichong Wang
- State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, Guangdong, China
- * E-mail: (CYL); (ZCW)
| |
Collapse
|
|
28
|
Yan L, Wu W, Wang Z, Li C, Lu X, Duan H, Zhou J, Wang X, Wan P, Song Y, Tang J, Han Y. Comparative study of the effects of recombinant human epidermal growth factor and basic fibroblast growth factor on corneal epithelial wound healing and neovascularization in vivo and in vitro. Ophthalmic Res 2012; 49:150-160. [PMID: 23258255 DOI: 10.1159/000343775] [Citation(s) in RCA: 39] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/13/2012] [Accepted: 09/06/2012] [Indexed: 01/18/2023]
Abstract
PURPOSE This study was undertaken to investigate the effects of recombinant human epidermal growth factor (rhEGF) and basic fibroblast growth factor (bFGF) on corneal wound healing and neovascularization (CNV). METHODS The positive effects of 10 ng/ml rhEGF and bFGF on the proliferation of corneal epithelial cells (SD-HCEC1s), rabbit keratocyte cells (RKCs) and human umbilical vein endothelial cells (HUVECs) as well as the effects on the migration capacity on HUVECs were observed. An animal central corneal wound and CNV model was established in rabbits. One eye of each group was chosen randomly for topical administration of rhEGF, bFGF or normal saline, and variability in the area of corneal epithelial wound healing and CNV was observed. RESULTS The optimal concentration of rhEGF and bFGF for the proliferation of corneal epithelial cells was 10 ng/ml. The promotive effect of 10 ng/ml rhEGF on the proliferation of RKCs and HUVECs was less than that of 10 ng/ml bFGF. In the animal experiment, the healing rate of the corneal epithelium in the rhEGF group was better than in the other groups on day 1. On day 3, the healing rates of the 3 groups were nearly equal. The CNV area in the rhEGF group was less than that of the bFGF group. CONCLUSIONS rhEGF and bFGF both had promotive effects on corneal epithelial wound healing, but rhEGF had a weaker promotive effect on CNV than bFGF. With long-term application of growth factor drugs, rhEGF is suggested for lessening the growth of CNV.
Collapse
Affiliation(s)
- Limeng Yan
- Department of Ophthalmology, Zhujiang Hospital, Southern Medical University, Guangzhou, PR China
| | | | | | | | | | | | | | | | | | | | | | | |
Collapse
|
|
29
|
Xu B, Fan TJ, Zhao J, Sun A, Wang RX, Hu XZ, Yu HZ, Fan XY, Xu XH. Transplantation of tissue-engineered human corneal epithelium in limbal stem cell deficiency rabbit models. Int J Ophthalmol 2012; 5:424-9. [PMID: 22937499 DOI: 10.3980/j.issn.2222-3959.2012.04.04] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/02/2012] [Accepted: 07/05/2012] [Indexed: 11/02/2022] Open
Abstract
AIM To evaluate the biological functions of tissue-engineered human corneal epithelium (TE-HCEP) by corneal transplantation in limbal stem cell deficiency (LSCD) rabbit models. METHODS TE-HCEPs were reconstructed with DiI-labeled untransfected HCEP cells and denuded amniotic membrane (dAM) in air-liquid interface culture, and their morphology and structure were characterized by hematoxylin-eosin (HE) staining of paraffin-sections, immunohistochemistry and electron microscopy. LSCD models were established by mechanical and alcohol treatment of the left eyes of New Zealand white rabbits, and their eyes were transplanted with TE-HCEPs with dAM surface outside by lamellar keratoplasty (LKP). Corneal transparency, neovascularization, thickness, and epithelial integrality of both traumatic and post transplantation eyes were checked once a week by slit-lamp corneal microscopy, a corneal pachymeter, and periodic acid-Schiff (PAS) staining. At day 120 post surgery, the rabbits in each group were sacrificed and their corneas were examined by DiI label observation, HE staining, immunohistochemistry and electron microscopy. RESULTS After cultured for 5 days on dAM, HCEP cells, maintaining keratin 3 expression, reconstructed a 6-7 layer TE-HCEP with normal morphology and structure. The traumatic rabbit corneas, entirely opaque, conjunctivalized and with invaded blood vessels, were used as LSCD models for TE-HCEP transplantation. After transplantation, obvious edema was not found in TE-HCEP-transplanted corneas which became more and more transparent, the invaded blood vessels reduced gradually throughout the monitoring period. The corneas decreased to normal thickness on day 25, while those of dAM eyes were over 575µm in thickness during the monitoring period. A 4-5 layer of epithelium consisting of TE-HCEP originated cells attached tightly to the anterior surface of stroma was reconstructed 120 days after TE-HCEP transplantation, which was similar to the normal control eye in morphology and structure. In contrast, intense corneal edema, turbid, invaded blood vessels were found in dAM eyes, and no multilayer epithelium was found but only a few scattered conjunctiva-like cells appeared. CONCLUSION The TE-HCEP, with similar morphology and structure to those of innate HCEP, could reconstruct a multilayer corneal epithelium with normal functions in restoring corneal transparency and thickness of LSCD rabbits after transplantation. It may be a promising HCEP equivalent for clinical therapy of corneal epithelial disorders.
Collapse
Affiliation(s)
- Bin Xu
- Key Laboratory for Corneal Tissue Engineering, College of Marine Life Sciences, Ocean University of China, Qingdao 266003, Shandong Province, China
| | | | | | | | | | | | | | | | | |
Collapse
|
|
30
|
Xu B, Fan TJ, Yang HS, Sun A, Zhao J, Ma XY, Hu XZ. In vitro reconstruction and characterization of tissue-engineered human corneal epithelium with seeder cells from an untransfected human corneal epithelial cell line. Int J Ophthalmol 2012; 5:281-5. [PMID: 22773973 DOI: 10.3980/j.issn.2222-3959.2012.03.06] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/09/2012] [Accepted: 05/10/2012] [Indexed: 02/04/2023] Open
Abstract
AIM To demonstrate the morphology and structure of in vitro reconstructed tissue-engineered human corneal epithelium (TE-HCEP) with seeder cells from an untransfected HCEP cell line. METHODS The TE-HCEPs were reconstructed in vitro with seeder cells from an untransfected HCEP cell line, and scaffold carriers of denuded amniotic membrane (dAM) in air-liquid interface culture for 3, 5, 7 and 9 days, respectively. The specimens were examined with hematoxylin-eosin (HE) staining of paraffin-section, immunocytochemical staining, scanning and transmission electron microscopy. RESULTS During in vitro reconstruction of TE-HCEP, HCEP cells formed a 3-4, 6-7 and 8-10 layers of an HCEP-like structure on dAMs in air-liquid interface culture for 3, 5 and 7 days, respectively. But the cells deceased to 5-6 layers and the structure of straified epithelium became loose at day 9. And the cells maintained positive expression of marker proteins (keratin 3 and keratin 12), cell-junction proteins (zonula occludens-1, E-cadherin, connexin 43 and integrin β1) and membrane transport protein of Na(+)-K(+) ATPase. The HCEP cells in TE-HCEP were rich in microvilli on apical surface and established numerous cell-cell and cell-dAM junctions at day 5. CONCLUSION The morphology and structure of the reconstructed TE-HCEP were similar to those of HCEP in vivo. The HCEP cells in the reconstructed TE-HCEP maintained the properties of HCEP cells, including abilities of forming intercellular and cell-extracellular matrix junctions and abilities of performing membrane transportation. The untransfected HCEP cells and dAMs could promisingly be used in reconstruction HCEP equivalent for clinical corneal epithelium transplantation.
Collapse
Affiliation(s)
- Bin Xu
- Key Laboratory for Corneal Tissue Engineering, Ocean University of China, Qingdao 266003, Shandong Province, China
| | | | | | | | | | | | | |
Collapse
|
|
31
|
Liu Z, Zhuang J, Li C, Wan P, Li N, Zhou Q, Zhou C, Huang Z, Wang Z. Long-term cultivation of human corneal endothelial cells by telomerase expression. Exp Eye Res 2012; 100:40-51. [PMID: 22575565 DOI: 10.1016/j.exer.2012.04.013] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/18/2012] [Revised: 04/18/2012] [Accepted: 04/23/2012] [Indexed: 12/13/2022]
Abstract
The objective of this study was to explore the potential role of human telomerase reverse transcriptase (TERT) in extending the proliferative lifespan of human corneal endothelial cells (HCECs) under long-term cultivation. A primary culture was initiated with a pure population of HCECs in DMEM/F12 media containing 10% fetal bovine serum and other various supplements. TERT gene was successfully transfected into normal HCECs. A stable HCECs cell line (TERT-HCECs) that expressed TERT was established. The cells could be subcultured for 36 passages. Within this line of cells, TERT not only extended proliferative lifespan and inhibited apoptosis but also enhanced the cell line remaining the normal characteristics similar to HCECs. There were no significantly differences in the expression of the pump function related proteins voltage dependent anion channel 3 (VDAC3), sodium bicarbonate cotransporter member 4 (SLC4A4), chloride channel protein 3 (CLCN3), Na(+)/K(+)-ATPase α1, and ZO-1 in the cell line TERT-HCECs and primary HCECs. TERT-HCECs formed a monolayer cell sheet, maintained similar cell junction formation and pump function with primary HCECs. Karyotype analysis exhibited normal chromosomal numbers. The soft agar colony assay and tumor formation in nude mice assay showed no malignant alterations in TERT-HCECs. Our findings indicated that we had established a cell line with its similar phenotype and properties to primary HCECs. Further study of the TERT-HCECs may be valuable in studying the function of the cells in vivo.
Collapse
Affiliation(s)
- Zhiping Liu
- State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou 510060, China
| | | | | | | | | | | | | | | | | |
Collapse
|
|
32
|
Saichanma S, Bunyaratvej A, Sila-Asna M. In vitro transdifferentiation of corneal epithelial-like cells from human skin-derived precursor cells. Int J Ophthalmol 2012; 5:158-63. [PMID: 22762041 DOI: 10.3980/j.issn.2222-3959.2012.02.08] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/14/2011] [Accepted: 03/15/2012] [Indexed: 12/28/2022] Open
Abstract
The damage of human corneal cells encounter with the problem of availability of corneal cells for replacement. Limitation of the source of corneal cells has been realized. An attempt of development of corneal epithelial-like cells from the human skin-derived precursor (hSKPs) has been made in this study. Combination of three essential growth factors: epidermal growth factor (EGF), keratinocyte growth factor (KGF) and hepatocyte growth factor (HGF) could demonstrate successfully induction of hSKPs to differentiation into corneal cells.The induced cells expressed the appearance of markers of corneal epithelial cells as shown by the presence of keratin 3 (K3) by antibody label and Western blot assay. The K3 gene expression of induced hSKPs cells as shown by reverse transcription-polymerase chain reaction (RT-PCR) technology was also demonstrated. The presence of these markers at both gene and protein levels could lead to our conclusion that the directional transdifferentiation of hSKPs cells into corneal epithelial cells was successfully done under this cell induction protocol. The finding shows a newly available stem cell source can be obtained from easily available skin. Cells from autologous human skin might be used for corneal disorder treatment in future clinical application.
Collapse
Affiliation(s)
- Sarawut Saichanma
- Department of Pathology, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok 14140, Thailand
| | | | | |
Collapse
|
|
33
|
HSV-1 miR-H6 inhibits HSV-1 replication and IL-6 expression in human corneal epithelial cells in vitro. Clin Dev Immunol 2012; 2012:192791. [PMID: 22550533 PMCID: PMC3329371 DOI: 10.1155/2012/192791] [Citation(s) in RCA: 39] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/18/2011] [Accepted: 02/01/2012] [Indexed: 11/17/2022]
Abstract
HSV-1 infection in the cornea could lead to blindness. The infected cell polypeptide 4 (ICP4) of herpes simplex virus 1 (HSV-1) is a regulator of viral transcription that is required for productive infection. It has been previously demonstrated that miR-H6 encoded from HSV-1 genome targets ICP4 to help maintain latency. In this study, synthesized miR-H6 mimics were transfected into HSV-1-infected human cornea epithelial (HCE) cells. The inhibition of HSV-1 replication and viral ICP4 expression in miR-H6-transfected HCE was confirmed by plaque assay, immunofluorescence, and Western blot. Compared to nontransfection or mock, miR-H6 produced a low-titer HSV-1 and weak ICP4 expression. In addition, miR-H6 can decrease the interleukin 6 released into the medium, which was determined by ELISA. Taken together, the data suggests that miR-H6 targeting of ICP4 inhibits HSV-1 productive infection and decreases interleukin 6 production in HCE, and this may provide an approach to prevent HSV-1 lytic infection and inhibit corneal inflammation.
Collapse
|
|
34
|
Niu JY, Liu J, Liu L, Lü YY, Chen JS, Xu JT, Zhong JX. Construction of eukaryotic plasmid expressing human TGFBI and its influence on human corneal epithelial cells. Int J Ophthalmol 2012; 5:38-44. [PMID: 22553752 DOI: 10.3980/j.issn.2222-3959.2012.01.08] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/17/2011] [Accepted: 02/03/2012] [Indexed: 11/02/2022] Open
Abstract
AIM To detect the expression of transforming growth factor beta-induced gene (TGFBI) protein in human corneal tissue and overexpress it in the human corneal epithelial cells in order to discuss the function of TGFBI in the pathogenesis of corneal dystrophy. METHODS Immunohistochemistry (IHC) was used to detect the expression of TGFBI in the human cornea tissue. TGFBI cDNA was obtained by reverse transcription-PCR from human corneal total RNA extracted from cornea transplant donor and cloned into pCMV-N-HA vector. The recombinant pCMV-N-HA-TGFBI plasmid transfected human corneal epithelial cells. Forty-eight hours later, mRNA and proteins were harvested from cells for real-time PCR analysis and western blot assay respectively. RESULTS IHC indicated TGFBI mainly exist below the human corneal epithelium layer. Transfection of recombinant pCMV-N-HA-TGFBI into human corneal epithelial cells resulted in effective expression of TGFBI, as shown by increased mRNA level detected by real-time PCR as well as increased protein level detected by Western blot. Meanwhile the result of real-time PCR and Western blot shown the expression of MMP1, MMP3 (matrix metalloproteinases MMP) increased while the expressin of TIMP1 (tissue inhibitors of matrix metalloproteinases TIMP) decreased. CONCLUSION TGFBI mainly exists below the corneal epithelial layer, recombinant eukaryotic expression vector harboring human TGFBI cDNA was obtained and efficiently overexpressed in human corneal epithelial cells. Meanwhile the TGFBI overexpression in human corneal epithelial cells result in MMP1, MMP3 increasing and TIMP1 decreasing. The result might be helpful for studying the function and role of TGFBI in pathogenesis of corneal dystrophy.
Collapse
Affiliation(s)
- Jing-Yi Niu
- Department of Ophthalmology, the First Affiliated Hospital of Jinan University, Guangzhou 510630, Guangdong Province, China
| | | | | | | | | | | | | |
Collapse
|
|
35
|
Miyake T, Ito N, Tajima K, Goto H, Furukawa T. Comparison of moxifloxacin and levofloxacin in an epithelial disorder model using cultured rabbit corneal epithelial cell sheets. Graefes Arch Clin Exp Ophthalmol 2012; 250:1035-41. [PMID: 22282216 DOI: 10.1007/s00417-011-1916-1] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/08/2011] [Revised: 12/14/2011] [Accepted: 12/19/2011] [Indexed: 10/14/2022] Open
Abstract
BACKGROUND When ophthalmic drug solutions are developed and clinically applied, their influence on corneal epithelium is an important issue. In the past, cells obtained by monolayer culture in vitro were used for evaluation of such influence. We recently created an experimental model of cell damage repair closer to the live body than conventional models by using layered sheets of cultured corneal epithelium. The present study was undertaken to evaluate the influence of ophthalmic moxifloxacin hydrochloride (MFLX) solution in comparison to that of ophthalmic levofloxacin (LVFX) solution using this model. METHODS Corneal epithelium cells were collected from corneal tissue specimens of white rabbits and subjected to air-lift culture to induce layering. Epithelial cell defects were created by a sponge soaked in 1 N aqueous sodium hydroxide. After removal of the sponge, either ophthalmic MFLX solution or ophthalmic LVFX solution was dropped onto the specimens three times daily (washed 1 min after each dose, followed by continuation of air-lifting culture). The percentage of the defective area repaired (percent defect repair) was evaluated. Each of the ophthalmic MFLX solution and the ophthalmic LVFX solution was used after the stock solution was diluted fourfold (1:4). Drug-free culture medium served as the negative control. Benzalconium chloride solution (BAC) 0.01% served as the positive control. RESULTS In the negative control group, complete repair of the defect with epithelial cells was seen 4 days after the start of treatment. In the positive control group, repair was suppressed. In the MFLX group and the LVFX group, the defect was repaired at each drug concentration, showing no significant difference from the negative control group. Thus, in this study using layered sheets of cultured corneal epithelium (a model closer to the living body than conventional models), the corneal epithelial defect was repaired in the ophthalmic MFLX solution treatment group and the ophthalmic LVFX solution treatment group to a degree similar to that in the negative control group. CONCLUSIONS These results suggest that neither MLFX nor LVFX suppresses repair of corneal epithelial damage.
Collapse
Affiliation(s)
- Taku Miyake
- Department of Ophthalmology, Tokyo Medical University, MD, 6-7-1 Nishi-shinjuku, Shinjyuku-ku, Tokyo, Japan
| | | | | | | | | |
Collapse
|
|
36
|
Exogenous MD-2 confers lipopolysaccharide responsiveness to human corneal epithelial cells with intracellular expression of TLR4 and CD14. Inflammation 2012; 34:371-8. [PMID: 20700758 DOI: 10.1007/s10753-010-9244-x] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/19/2022]
Abstract
In the present study, we aimed to investigate the responsiveness of human corneal epithelial cells (HCECs) to lipopolysaccharide (LPS) in vitro and to elucidate the underlying molecular mechanism(s) controlling the LPS responsiveness. The expression and subcellular localization of toll-like receptor 4 (TLR4) and CD14 and the expression of myeloid differentiation (MD)-2 were studied in SDHCEC1 cells, one HCEC cell line. Upon exposure to different concentrations of LPS, cell responses were evaluated by examining nuclear factor-kappaB (NF-κB) activation and the production of interleukin (IL)-8. The influence of soluble MD-2 on LPS responsiveness were assessed in SDHCEC1 cells pretreated with MD-2-containing conditioned medium before LPS challenge. SDHCEC1 cells expressed both TLR4 and CD14 intracellularly and had no detectable expression of MD-2 transcripts. Unresponsiveness to LPS at doses of up to 1,000 ng/ml was observed in SDHCEC1 cells, which was evidenced by no evident NF-κB activation and IL-8 production. The addition of MD-2 conditioned medium significantly induced NF-κB activation and enhanced the production of IL-8 as compared with the treatment with the control medium (p < 0.05). Meanwhile, the total mRNA amounts of TLR4 and CD14 and the surface expression of the two proteins were significantly (p < 0.05) increased by the pretreatment with MD-2 conditioned medium. LPS hyporesponsiveness of HCECs is largely due to deficient LPS receptor complex formation caused by lack of MD-2 expression. Exogenous MD-2 is capable of restoring the LPS responsiveness, at least partially, through promoting the surface expression of TLR4 and CD14.
Collapse
|
|
37
|
Duan F, Ni S, Nie Y, Huang Q, Wu K. Small interfering RNA targeting for infected-cell polypeptide 4 inhibits herpes simplex virus type 1 replication in retinal pigment epithelial cells. Clin Exp Ophthalmol 2011; 40:195-204. [PMID: 21883773 PMCID: PMC7162062 DOI: 10.1111/j.1442-9071.2011.02668.x] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
Background: This study sought to inhibit herpes simplex virus type 1 replication using small interfering RNA which targeting infected‐cell polypeptide 4 genes to mediate transcription of early and late viral genes in herpes simplex virus type 1 lytic (productive) infection in retina epithelial cells. Methods: After pre‐ or post‐infecting with herpes simplex virus type 1, small interfering RNAs were transfected into retina epithelial cells. The antiviral effects of small interfering RNA were evaluated by Western blot, plaque assays, indirect immunofluorescence and reverse transcription polymerase chain reaction. The viral titre was detected by the 50% tissue culture infective dose method. Results: Small interfering RNA decreased infected‐cell polypeptide 4 expression in retina epithelial cells that were infected with herpes simplex virus type 1 before or after small interfering RNA transfection. Compared with herpes simplex virus type 1 infection alone or transfection with negative control small interfering RNA, the viral titre and the retina epithelial cell cytopathic effect were significantly decreased in retina epithelial cells transfected with infected‐cell polypeptide 4‐targeting small interfering RNA (50 and 100 nM) (P < 0.05). The small interfering RNA effectively silenced herpes simplex virus type 1 infected‐cell polypeptide 4 expression on both mRNA and the protein levels (P < 0.05). The inhibition of infected‐cell polypeptide 4‐targeting small interfering RNA on infected‐cell polypeptide 4 protein expression was also verified by Western blot in herpes simplex virus type 1 infected human cornea epithelial cell, human trabecular meshwork cells and Vero cells. Conclusions: Infected‐cell polypeptide 4‐targeting small interfering RNA can inhibit herpes simplex virus type 1 replication in retina epithelial cells, providing a foundation for development of RNA interference as an antiviral therapy.
Collapse
Affiliation(s)
- Fang Duan
- Zhongshan Ophthalmic Center, State Key Laboratory of Ophthalmology, Sun Yat-sen University, Guangzhou, Guangdong, China
| | | | | | | | | |
Collapse
|
|
38
|
Zhou J, Chen F, Xiao J, Li C, Liu Y, Ding Y, Wan P, Wang X, Huang J, Wang Z. Enhanced functional properties of corneal epithelial cells by coculture with embryonic stem cells via the integrin β1-FAK-PI3K/Akt pathway. Int J Biochem Cell Biol 2011; 43:1168-1177. [PMID: 21550417 DOI: 10.1016/j.biocel.2011.04.010] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/03/2011] [Revised: 04/08/2011] [Accepted: 04/18/2011] [Indexed: 12/16/2022]
Abstract
Adult stem cells are important cell sources in regenerative medicine, but isolating them is technically challenging. This study employed a novel strategy to generate stem-like corneal epithelial cells and promote the functional properties of these cells by coculture with embryonic stem cells. The primary corneal epithelial cells were labelled with GFP and cocultured with embryonic stem cells in a transwell or by direct cell-cell contact. The embryonic stem cells were pre-transfected with HSV-tk-puro plasmids and became sensitive to ganciclovir. After 10 days of coculture, the corneal epithelial cells were isolated by treating the cultures with ganciclovir to kill the embryonic stem cells. The expression of stem cell-associated markers (ABCG2, p63) increased whereas the differentiation mark (Keratin 3) decreased in corneal epithelial cells isolated from the cocultures as evaluated by RT-PCR and flow cytometry. Their functional properties of corneal epithelial cells, including cell adhesion, migration and proliferation, were also enhanced. These cells could regenerate a functional stratified corneal epithelial equivalent but did not form tumors. Integrin β1, phosphorylated focal adhesion kinase and Akt were significantly upregulated in corneal epithelial cells. FAK Inhibitor 14 that suppressed the expression of phosphorylated focal adhesion kinase and Akt inhibited cell adhesion, migration and proliferation. LY294002 that suppressed phosphorylated Akt but not phosphorylated focal adhesion kinase inhibited cell proliferation but had no effect on cell adhesion or migration. These findings demonstrated that the functional properties of stem-like corneal epithelial cells were enhanced by cocultured embryonic stem cells via activation of the integrin β1-FAK-PI3K/Akt signalling pathway.
Collapse
Affiliation(s)
- Jin Zhou
- State Key Laboratory of Ophthalmology, Sun yet-sen University, Guangzhou, Guangdong, PR China
| | | | | | | | | | | | | | | | | | | |
Collapse
|
|
39
|
Fan TJ, Xu B, Zhao J, Yang HS, Wang RX, Hu XZ. Establishment of an untransfected human corneal epithelial cell line and its biocompatibility with denuded amniotic membrane. Int J Ophthalmol 2011; 4:228-34. [PMID: 22553650 DOI: 10.3980/j.issn.2222-3959.2011.03.02] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/01/2011] [Accepted: 05/20/2011] [Indexed: 11/02/2022] Open
Abstract
AIM To establish an untransfected human corneal epithelial (HCEP) cell line and characterize its biocompatibility with denuded amniotic membrane (dAM). METHODS The torn HCEP pieces were primarily cultured in DMEM/F12 media (pH 7.2) supplemented with 20% fetal bovine serum and other necessary factors, yielding an HCEP cell line which was its growth performance, chromosome morphology, tumorigenicity and expression of marker proteins analyzed. In addition, the biocompatibility of HCEP cells with dAM was evaluated through histological and immunocytochemistry analyses and with light, electron and slit-lamp microscopies. RESULTS HCEP cells proliferated to confluence in 3 weeks, which have been subcultured to passage 160. A continuous untransfected HCEP cell line, designated as utHCEPC01, was established with a population doubling time of 45.42 hours as was determined at passage 100. The cells retained HCEP cell properties as were approved by chromosomal morphology and the expression of keratin 3. They, with no tumorigenicity, formed a multilayer epithelium-like structure on dAMs through proliferation and differentiation during air-liquid interface culture, maintained expression of marker proteins including keratin 3 and integrin β1 and attached tightly to dAMs. The reconstructed HCEP was highly transparent and morphologically and structurally similar to the original. CONCLUSION An untransfected and non-tumorigenic HCEP cell line was established in this study. The cells maintained expression of marker proteins. The cell line was biocompatible with dAM. It holds the potential of being used for in vitro reconstruction of tissue-engineered HCEP, promising for the treatment of diseases caused by corneal epithelial disorders.
Collapse
Affiliation(s)
- Ting-Jun Fan
- Key Laboratory for Corneal Tissue Engineering, Ocean University of China, Qingdao 266003, Shandong Province, China
| | | | | | | | | | | |
Collapse
|
|
40
|
Ke Q, Wang X, Gao Q, Wu Z, Wan P, Zhan W, Ge J, Wang Z. Carrier-free epithelial cell sheets prepared by enzymatic degradation of collagen gel. J Tissue Eng Regen Med 2011; 5:138-45. [PMID: 20603893 DOI: 10.1002/term.298] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/30/2023]
Abstract
Limbal stem-cell deficiency by ocular trauma or disease causes corneal opacification and vision loss. Conventional tissue engineering using biodegradable scaffolds has met with limited success. In this study, we developed a novel method for preparing carrier-free epithelial cell sheets, which have potential for use in repairing defects of the ocular surface. Stratified corneal epithelial cell sheets were prepared in culture dishes coated with biodegradable type I collagen. Haematoxylin and eosin staining, electron microscopy and immunohistochemistry were performed to characterize the cell sheets. Then, carrier-free epithelial sheets were successfully engineered using specific collagenase to degrade the collagen gel. Cell sheets of four to six cell layers after culture for 14 days were similar to natural rabbit corneal epithelia, as shown by pathological examination. Microvillus, tight cell-cell junctions and desmosome junctions were observed via electron microscopy. K3 and basement membrane components, such as type IV collagen and laminin, were expressed in the cells sheets and integrin β1 was maintained in basal cells. This novel method of using collagenase to degrade collagen gel is both simple and effective in preparing intact carrier-free epithelial cell sheets. Such sheets have great potential for application during in vivo corneal regeneration.
Collapse
Affiliation(s)
- Qicheng Ke
- State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Centre, Sun Yat-Sen University, Guangzhou, People's Republic of China
| | | | | | | | | | | | | | | |
Collapse
|
|
41
|
Zhang W, Xiao J, Li C, Wan P, Liu Y, Wu Z, Huang M, Wang X, Wang Z. Rapidly constructed scaffold-free cornea epithelial sheets for ocular surface reconstruction. Tissue Eng Part C Methods 2011; 17:569-77. [PMID: 21214400 DOI: 10.1089/ten.tec.2010.0529] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022] Open
Abstract
PURPOSE To develop a centrifugal cell seeding method for rapid and efficient reconstruction of ocular surface with limbal stem cell deficiency (LSCD) in rabbits. METHODS The orthogonal design method was used to optimize centrifugation parameters for cell seeding. Methylthiazol tetrazolium proliferation assay, colony-forming efficiency, and flow cytometry were used to study cell viability. Histology, electron microscopy, and immunocytochemistry were evaluated for centrifugation-constructed cornea epithelial sheets (CCCESs). The rabbit eyes with LSCD were treated with or without CCCES for in vivo evaluation. RESULTS The 80.04% attached cells with 98.04% viability were achieved using optimal cell seeding density at 9 × 10(5) cm(-2) with centrifugation at 1800 rpm for 4 min. The 0.4% glycerin was added in the medium to increase the surface tension and osmotic pressure to optimal condition for obtaining higher cell density. The three-layer epithelial sheets were rapid constructed, which displayed the characteristics of normal corneal epithelium. In vivo transplantation, labeled cells of CCCES were detected at 30 days. CCCES reconstructed the LSCD corneal epithelia without conjunctivalization and neovascularation, evidenced by positive K3 and negative K4, Muc5AC. CONCLUSION The scaffold-free corneal epithelial sheets were rapidly constructed using optimal centrifugation procedure, which was demonstrated to reconstruct ocular surface with LSCD.
Collapse
Affiliation(s)
- Wei Zhang
- State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, 510060, PR China
| | | | | | | | | | | | | | | | | |
Collapse
|
|
42
|
Liu Y, Ding Y, Ma P, Wu Z, Duan H, Liu Z, Wan P, Lu X, Xiang P, Ge J, Wang Z. Enhancement of long-term proliferative capacity of rabbit corneal epithelial cells by embryonic stem cell conditioned medium. Tissue Eng Part C Methods 2010; 16:793-802. [PMID: 19842914 DOI: 10.1089/ten.tec.2009.0380] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/26/2022] Open
Abstract
Induction of autologous stem cells for directed differentiation has become a predominant method to obtain autologous cells for tissue reconstruction. However, the low inducing efficiency and contamination with other type of cells hinder its clinical utilization. Here we report a novel phenomenon that the corneal epithelial cells maintain long-term proliferative capacity and tissue-specific cell phenotype by factors secreted from murine embryonic stem cells (ESCs). The rabbit corneal epithelial cells grew very well in culture medium with addition of 40% ESC conditioned medium (ESC-CM). These corneal epithelial cells have been serially subcultured for more than 20 passages and maintained high cell purity, cobble-stone-like morphology, enhanced colony forming efficiency, normal diploid, and capacity to regenerate a functional stratified corneal epithelial equivalent. More importantly, these cells did not form tumor, and the cells lost their proliferative capacity after withdrawal of ESC-CM. The long-term proliferative capacity of corneal epithelial cells is partly resulted from enhancement of cell survival and colony formation, and mediated by ectopic expression of telomerase. Our findings indicate that this new ESC-CM culture system can generate low-immunogenic autologous cells sufficiently for use in regenerative medicine.
Collapse
Affiliation(s)
- Ying Liu
- State Key Laboratory of Ophthalmology, Sun Yat-sen University, Guangzhou, P.R. China
| | | | | | | | | | | | | | | | | | | | | |
Collapse
|
|
43
|
Cultured human corneal epithelial stem/progenitor cells derived from the corneal limbus. In Vitro Cell Dev Biol Anim 2010; 46:774-80. [DOI: 10.1007/s11626-010-9344-9] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/26/2010] [Accepted: 08/19/2010] [Indexed: 12/15/2022]
|
|
44
|
Song J, Li Y, Ge J, Duan Y, Sze SCW, Tong Y, Shaw PC, Ng TB, Tsui KC, Zhuo Y, Zhang KY. Protective effect of bilberry (Vaccinium myrtillus L.) extracts on cultured human corneal limbal epithelial cells (HCLEC). Phytother Res 2010; 24:520-4. [PMID: 20077406 DOI: 10.1002/ptr.2974] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/07/2022]
Abstract
The use of bilberry (Vaccinium myrtillus L.) as a food and medicine for improving human vision has a long history all over the world. However, there is lack of convincing evidence from rigorous clinical trials or scientific research. This study investigated the effects of different concentrations of bilberry extracts on the cell viability, cell cycle and the expression of hyaluronic acid and glycosaminoglycans of cultured human corneal limbal epithelial cells. The data showed that bilberry extracts had no cytotoxicity to the corneal limbal epithelial cells at a wide range of concentrations (10(-9)-10(-4) M, equalized to the content of cyanidin-3-O-glucoside). Bilberry extract (10(-6), 10(-5) and 10(-4) M) increased cell viability after 48 h incubation. The number of cells decreased in G(0)/G(1) phase and increased prominently in S and G(2)/M phases after treatment with bilberry extracts at a high concentration (10(-4) M). The expression of glycosaminoglycans increased prominently after incubation with bilberry extracts (10(-7) and 10(-4) M) for 48 h while no significant changes were observed for the expression of hyaluronic acid. The results indicated that bilberry extract may be beneficial for the physiological renewal and homeostasis of corneal epithelial cells.
Collapse
Affiliation(s)
- Juxian Song
- The School of Chinese Medicine, LKS Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong SAR, China
| | | | | | | | | | | | | | | | | | | | | |
Collapse
|
|
45
|
Lu X, Chen D, Liu Z, Li C, Liu Y, Zhou J, Wan P, Mou YG, Wang Z. Enhanced survival in vitro of human corneal endothelial cells using mouse embryonic stem cell conditioned medium. Mol Vis 2010; 16:611-22. [PMID: 20383337 PMCID: PMC2850933 DOI: 10.1167/2.7.611] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/28/2010] [Accepted: 04/02/2010] [Indexed: 12/13/2022] Open
Abstract
Purpose To determine whether mouse embryonic stem cell conditioned medium (ESC-CM) increases the proliferative capacity of human corneal endothelial cells (HCECs) in vitro. Methods Primary cultures of HCECs were established from explants of the endothelial cell layer, including the Descemet’s membrane. Cells were cultured in human corneal endothelium medium (CEM) containing 25% ESC-CM for the experimental group and CEM alone for the control group. Phase-contrast microscopy and reverse-transcription polymerase chain reaction (RT–PCR) were used to identify HCECs. The eruption time and HCEC morphology were observed under phase-contrast microscopy. We detected the protein expression of zona occludens protein-1 (ZO-1; a tight junction protein) and the Na+-K+-ATPase by western blot analysis and immunocytochemistry. The mRNA expression of the Na+-K+-ATPase, voltage-dependent anion channel 3 (VDAC3), solute carrier family 4, sodium bicarbonate cotransporter member 4 (SLC4A4), and chloride channel protein 3 (CLCN3) were detected by RT–PCR. To explore the proliferation capacity of HCECs, the colony forming efficiency (CFE) was determined by Giemsa staining and the cellular proliferation marker of Ki-67 protein (Ki-67) positive cells were detected by immunocytochemistry and flow cytometry. Progression of the cell cycle and apoptosis were analyzed by flow cytometry. Negative regulation of the cell cycle, as measured by cyclin-dependent kinase inhibitor p21 (p21) levels, was detected by western blot analysis and immunocytochemistry. Results In primary culture, HCECs in the 25%ESC-CM group erupted with polygonal appearance on day 2, while those in the CEM group erupted with slightly larger cells on day 3–4. HCECs in the 25%ESC-CM group could be subcultured until passage 6 without enlargement of cell volume, while those in the CEM group were enlarged and lost their polygonal appearance by passage 2. HCECs in both the 25%ESC-CM and CEM groups expressed ZO-1, Na+-K+-ATPase, VDAC3, SLC4A4, and CLCN3. The number of Ki67 positive cells, CFE, and percentage of cells entering the S and G2 phases were higher in the 25%ESC-CM group than in the CEM group. The number of apoptotic cells and p21 protein expression both decreased in the 25%ESC-CM group. Conclusions Use of 25%ESC-CM significantly increased the number of proliferating cells. These effects may be achieved through inhibition of p21 expression and apoptosis. These results suggested that 25%ESC-CM may be a new tool for cultivating HCECs for transplantation.
Collapse
Affiliation(s)
- Xiaoyan Lu
- State Key laboratory of Ophthalmology, Sun Yat-sen University, Guangzhou, PR China
| | | | | | | | | | | | | | | | | |
Collapse
|
|
46
|
Davies SB, Chui J, Madigan MC, Provis JM, Wakefield D, Di Girolamo N. Stem cell activity in the developing human cornea. Stem Cells 2010; 27:2781-92. [PMID: 19711455 DOI: 10.1002/stem.209] [Citation(s) in RCA: 35] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
The adult cornea harbors stem cells (SCs) in its periphery, in a niche known as the limbus. Over the past 2 decades there has been substantial research into these adult corneal SCs, their limbal niche, and their therapeutic applications. However, few studies have investigated how this niche and its SCs develop in humans. To better characterize this development, human fetal corneas from 8.5- to 22-weeks'-gestation (n = 173), neonatal (n = 2), and adult (n = 10) specimens were obtained. Histological and immunohistochemical assessments were conducted to determine embryological changes and expression of developmental and SC-related genes. Fresh fetal corneas were explanted to propagate corneal progenitors and cells characterized using reverse transcription-polymerase chain reaction, immunohistochemistry, flow cytometry, and colony-forming assays. A novel "ridge-like" structure was identified, circumscribing the fetal cornea, which we hypothesize represents the rudimentary SC niche. Immunohistochemistry disclosed "stem-like" cells across the cornea, becoming confined to this ridge with increasing gestational age. In addition, for the first time, pure long-term cultures of fetal corneal epithelium, which displayed phenotypical and functional properties similar to those of adult limbal SCs, were established. Optimization of culture techniques and purification of this SC population will allow for further investigation of their proliferative ability, with potential research and clinical applications. This study expands our understanding of limbal niche development and opens new avenues for investigation.
Collapse
Affiliation(s)
- Sarah B Davies
- Department of Pathology, School of Medical Sciences, Vision Science, University of New South Wales, Sydney, New South Wales 2052, Australia
| | | | | | | | | | | |
Collapse
|
|
47
|
Notara M, Daniels JT. Characterisation and functional features of a spontaneously immortalised human corneal epithelial cell line with progenitor-like characteristics. Brain Res Bull 2009; 81:279-86. [PMID: 19699783 DOI: 10.1016/j.brainresbull.2009.08.009] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/27/2009] [Revised: 07/24/2009] [Accepted: 08/16/2009] [Indexed: 10/20/2022]
Abstract
In this study a spontaneously formed corneal epithelial cell line, namely HCE-S, was established and characterised. The cell line was karyotyped and corneal epithelial maker expression of the cell line was assessed by immunostaining and semi-quantitative RT-PCR. The morphological characteristics were investigated using SEM and TEM analyses. The functional response to EGF in terms of cell proliferation, wound healing and cell migration was tested using Alamar Blue, scratch wound and colony dispersion assays, respectively. The cells were maintained in culture for more than 100 divisions and 35 passages suggesting that an immortalised cell line had been established. HCE-S, has maintained an epithelial morphology and has not phenotypicaly changed through passages. SEM and TEM microscopy showed morphological similarities to primary corneal epithelial cells. HCE-S expressed a battery of characteristic markers of primary corneal epithelial cells including cytokeratin 3 and PAX 6 as well as the basal cell integrins beta1 and alpha9 and the putative corneal stem cell marker ABCG2. HCE-S cells were responsive to exogenous EGF as shown by proliferation, migration and scratch wound assays. HCE-S can be cultured in a simple DMEM and only serum-based media which gives them an advantage against available corneal epithelial cell lines. This fact, along with the often limited availability and variability of primary corneal epithelial cells and the similarities of the cell line with primary cell characteristics suggest that HCE-S could be a useful tool for the study of corneal epithelial cell biology, ocular surface toxicity studies and pharmacological testing.
Collapse
Affiliation(s)
- Maria Notara
- Cells for Sight Transplantation and Research Programme, Ocular Repair and Regeneration Biology Unit, Institute of Ophthalmology, 11-43 Bath Street, London, EC1V 9EL, UK.
| | | |
Collapse
|
|
48
|
Wu Z, Zhou Y, Li N, Huang M, Duan H, Ge J, Xiang P, Wang Z. The use of phospholipase A(2) to prepare acellular porcine corneal stroma as a tissue engineering scaffold. Biomaterials 2009; 30:3513-22. [PMID: 19321202 DOI: 10.1016/j.biomaterials.2009.03.003] [Citation(s) in RCA: 82] [Impact Index Per Article: 5.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/13/2008] [Accepted: 03/04/2009] [Indexed: 12/17/2022]
Abstract
This study was to develop a method using phospholipase A(2) (PLA(2)) to prepare acellular porcine corneal stroma (APCS) for tissue engineering. The APCS was prepared from native porcine cornea (NPC) that was treated with 200 U/ml PLA(2) and 0.5% sodium deoxycholate (SD). The removal of DNA content, representing decellularization efficiency, reached to 91%, while all hydroxyproline and 80% of glycosaminoglycan were retained in the APCS when compared with NPC. The residual PLA(2) and SD were 0.35+/-0.04 U/mg dry weight and 4.3+/-0.8 ng/mg dry weight respectively. The extracts of APCS had no inhibitory effects on proliferation of corneal epithelial and endothelial cells as well as keratocytes. There was no sign of infiltration of neutrophilic leukocytes or leukomonocytes at 2 weeks after subcutaneous implantation of APCS. The prepared APCS displayed similar light transmittance to NPC. There were no significant differences in the areal modulus and curvature variation between APCS and NPC. Rabbit lamellar keratoplasty showed that the grafts of APCS were epithelialized completely in 8+/-2 days, and their transparency was restored in 84+/-11 days when the light transmittance of APCS-transplanted corneas displayed no significant difference compared with native corneas. Corneal neovascularization, corneal deformation, and graft degradation were not observed within 12 months.
Collapse
Affiliation(s)
- Zheng Wu
- State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou 510060, PR China
| | | | | | | | | | | | | | | |
Collapse
|
|
49
|
Jin HJ, Joo CK. The Effects of Wnt Protein on Proliferation and Stemness Maintenance of Corneal Limbal Stem Cells (CLSCs). JOURNAL OF THE KOREAN OPHTHALMOLOGICAL SOCIETY 2009. [DOI: 10.3341/jkos.2009.50.4.588] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/23/2022]
Affiliation(s)
- Hyun-Jin Jin
- Department of Ophthalmology and Visual Science, College of Medicine and Laboratory of Ophthalmology and Visual Science, Catholic Research Institutes of Medical Science, The Catholic University of Korea, Seoul, Korea
| | - Choun-Ki Joo
- Department of Ophthalmology and Visual Science, College of Medicine and Laboratory of Ophthalmology and Visual Science, Catholic Research Institutes of Medical Science, The Catholic University of Korea, Seoul, Korea
| |
Collapse
|
|
50
|
Castro-Muñozledo F. Corneal epithelial cell cultures as a tool for research, drug screening and testing. Exp Eye Res 2007; 86:459-69. [PMID: 18191836 DOI: 10.1016/j.exer.2007.11.017] [Citation(s) in RCA: 29] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/07/2007] [Revised: 11/27/2007] [Accepted: 11/28/2007] [Indexed: 11/29/2022]
Abstract
Understanding of visual system function and the development of new therapies for corneal diseases and damages depend upon comprehension of the biological roles of the tissue. The in vitro cultivation of corneal epithelial cells and cell lines derived from them has become a powerful tool to analyze and understand such issues. Currently, researchers have developed well-defined and precisely described culture protocols and a collection of corneal epithelial cell lines. These cell lines have been obtained through different experimental approaches: (1) the ectopic expression of oncogenes, (2) the inactivation of p16 and p53 pathways and hTERT expression, and (3) the spontaneous establishment after serial cultivation of cells. The advantages or disadvantages for these approaches are discussed. In conclusion, the availability of several culture protocols and immortalized cell lines that express corneal epithelial phenotype will be useful for investigating issues such as gene regulation and tissue development, or for validating alternative methods in toxicology.
Collapse
Affiliation(s)
- Federico Castro-Muñozledo
- Department of Cell Biology, Centro de Investigación y de Estudios Avanzados del IPN, México City, Mexico.
| |
Collapse
|