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Garg A, Goel K, Gour A, Sapra M, Sangwan VS, Tripathi R, Tiwari A. Unveiling the Molecular Mechanisms Underlying the Success of Simple Limbal Epithelial Transplantation (SLET). Cells 2025; 14:200. [PMID: 39936991 PMCID: PMC11817669 DOI: 10.3390/cells14030200] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/26/2024] [Revised: 01/17/2025] [Accepted: 01/19/2025] [Indexed: 02/13/2025] Open
Abstract
Simple limbal epithelial transplantation (SLET) has emerged as an effective treatment option for limbal stem cell deficiency (LSCD). However, the precise molecular mechanisms underlying its success remain incompletely understood. This review delves into the proposed mechanisms involving the donor limbus, host microenvironment, and the amniotic membrane as a scaffold in SLET. The donor limbus contributes to SLET efficacy through various factors secreted by limbal epithelial stem cells, including hepatocyte growth factor (HGF), soluble Fms-like tyrosine kinase-1 (sFLT-1), and pigment epithelium-derived factor (PEDF), which support corneal healing and transparency. Additionally, the presence of melanocytes, immune cells, limbal fibroblasts, and adhesion molecules within the donor tissue helps preserve the integrity of the limbal niche. The host environment plays a critical role in supporting the transplanted stem cells, with mesenchymal stem cell-secreted factors promoting proliferation and differentiation. Although the amniotic membrane has traditionally been used as a scaffold, emerging evidence suggests that it may not always be necessary. Further studies are needed to validate this scaffold-free approach and to evaluate the vitality and functional contributions of individual components used in SLET. Understanding these complex interactions and molecular mechanisms sheds light on the importance of the donor tissue, host microenvironment, and scaffold in SLET, paving the way for the optimization of this technique for the effective treatment of LSCD.
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Affiliation(s)
- Aastha Garg
- Department of Cornea and Anterior Segment Services, Dr. Shroff’s Charity Eye Hospital, New Delhi 110002, India; (A.G.); (A.G.); (V.S.S.)
- Department of Stem Cells Research, Dr. Shroff’s Charity Eye Hospital, New Delhi 110002, India; (K.G.); (M.S.)
| | - Kartik Goel
- Department of Stem Cells Research, Dr. Shroff’s Charity Eye Hospital, New Delhi 110002, India; (K.G.); (M.S.)
| | - Abha Gour
- Department of Cornea and Anterior Segment Services, Dr. Shroff’s Charity Eye Hospital, New Delhi 110002, India; (A.G.); (A.G.); (V.S.S.)
- Department of Stem Cells Research, Dr. Shroff’s Charity Eye Hospital, New Delhi 110002, India; (K.G.); (M.S.)
| | - Mehak Sapra
- Department of Stem Cells Research, Dr. Shroff’s Charity Eye Hospital, New Delhi 110002, India; (K.G.); (M.S.)
| | - Virender Singh Sangwan
- Department of Cornea and Anterior Segment Services, Dr. Shroff’s Charity Eye Hospital, New Delhi 110002, India; (A.G.); (A.G.); (V.S.S.)
- Department of Stem Cells Research, Dr. Shroff’s Charity Eye Hospital, New Delhi 110002, India; (K.G.); (M.S.)
| | - Ratnakar Tripathi
- Department of Veterinary Medicine, University of Missouri, Columbia, MO 65201, USA
| | - Anil Tiwari
- Department of Stem Cells Research, Dr. Shroff’s Charity Eye Hospital, New Delhi 110002, India; (K.G.); (M.S.)
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Xiao Y, McGhee CNJ, Zhang J. Posterior Limbal Mesenchymal Stromal Cells Promote Proliferation and Stemness of Transition Zone Cells: A Novel Insight Into Corneal Endothelial Rejuvenation. Invest Ophthalmol Vis Sci 2025; 66:44. [PMID: 39820276 PMCID: PMC11753478 DOI: 10.1167/iovs.66.1.44] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/01/2024] [Accepted: 12/21/2024] [Indexed: 01/19/2025] Open
Abstract
Purpose Progenitors for the corneal endothelium have been identified in the transition zone (TZ), but their cellular interactions remain undefined. Posterior limbal mesenchymal stromal cells (P-LMSCs) may support TZ cells in the posterior limbus. This study aims to characterize P-LMSCs and investigate their effects on TZ cells. Methods Human P-LMSCs and TZ cells were isolated by explant culture. P-LMSCs were characterized by comparing with anterior limbal mesenchymal stromal cells (A-LMSCs) using immunocytochemistry. TZ cells were cocultured with P-LMSCs in a Transwell, with TZ cell and A-LMSC coculture and TZ cells only as the control groups. The proliferation and wound healing capacity of TZ cells were assessed by EdU assay and scratch wound assay. Colony forming assay, droplet digital PCR, Western blotting, and immunocytochemistry were used to compare the stemness of TZ cells. The effect of P-LMSC conditioned medium on endothelial wound healing was evaluated in organ-cultured mouse corneas. Endothelial regeneration was measured by trypan blue staining. Results P-LMSCs expressed similar proteins (vimentin, Nestin, TRA-1-60, Oct3/4) as A-LMSCs. TZ cells cocultured with P-LMSCs had significantly higher proliferation, wound healing speed, and colony-forming efficiency than TZ cells only. TZ cells supported by P-LMSCs expressed higher levels of stem/progenitor markers (Nestin, Sox9, AP-2α, Pitx2) than the control groups. P-LMSC conditioned medium stimulated regeneration of mouse corneal endothelium from the TZ region. Conclusions The proliferation and stemness of TZ cells were enhanced by P-LMSCs in both cell and organ culture models. Our study provides an innovative strategy for corneal endothelial rejuvenation.
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Affiliation(s)
- Yuting Xiao
- Department of Ophthalmology, New Zealand National Eye Centre, Faculty of Medical and Health Sciences, The University of Auckland, Auckland, New Zealand
| | - Charles N. J. McGhee
- Department of Ophthalmology, New Zealand National Eye Centre, Faculty of Medical and Health Sciences, The University of Auckland, Auckland, New Zealand
| | - Jie Zhang
- Department of Ophthalmology, New Zealand National Eye Centre, Faculty of Medical and Health Sciences, The University of Auckland, Auckland, New Zealand
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Li Y, Dong L, Chen Y, Cai W, Yang G, Wang Y. Epithelial differentiation of gingival mesenchymal stem cells enhances re-epithelialization for full-thickness cutaneous wound healing. Stem Cell Res Ther 2024; 15:455. [PMID: 39609719 PMCID: PMC11605919 DOI: 10.1186/s13287-024-04081-9] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/07/2024] [Accepted: 11/25/2024] [Indexed: 11/30/2024] Open
Abstract
BACKGROUND Increasing evidence suggests that mesenchymal stem cells (MSCs) repair traumatized tissues primarily through paracrine secretion and differentiation into specific cell types. However, the role of epithelial differentiation of MSCs in cutaneous wound healing is unclear. This study aimed to investigate the epithelial differentiation potential of gingival tissue-derived MSCs (GMSCs) in epithelial cell growth medium and the mechanisms underlying their differentiation into an epithelial-like cell phenotype. METHODS We used scanning electron microscopy to examine GMSCs for epithelial differentiation. Quantitative real-time polymerase chain reaction and Western blotting were respectively used to measure genes and proteins related to epithelial differentiation. Immunofluorescence was used to examine subcellular localization of KLF4, KRT19, and β-catenin proteins. Transcriptome sequencing was used to enrich the mechanisms underlying epithelial differentiation in GMSCs. An MSAB inhibitor was used to validate the Wnt signaling pathway further. The wound healing rate and re-epithelialization were assessed through macroscopical observation and hematoxylin and eosin staining. RESULTS GMSCs cultured in epithelial cell growth medium from days 3 to 15 exhibited decreased expression of mesenchymal-epithelial transition and stemness-related proteins (N-cadherin, Vimentin, KLF4, and SOX2), increased expression of epithelial-related proteins (KRT12, KRT15, KRT19, and E-cadherin), and exhibited epithelial-like morphology. Mechanistically, high-throughput sequencing revealed that the Wnt and TGF-beta signaling pathways were inhibited during epithelial differentiation of GMSCs (Epi-GMSCs). MSAB-induced Wnt signaling pathway inhibition promoted epithelial-related gene and protein expression. Furthermore, we demonstrated the ability of Epi-GMSCs to facilitate wound healing by improving re-epithelialization in a full-thickness skin defect model. CONCLUSIONS Collectively, this study uncovers that GMSCs have the ability to differentiate into epithelia and highlights a promising strategy for using Epi-GMSCs to improve cutaneous wound healing.
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Affiliation(s)
- Yongzheng Li
- Stomatology Hospital, School of Stomatology, Zhejiang University School of Medicine, Zhejiang Provincial Clinical Research Center for Oral Diseases, Key Laboratory of Oral Biomedical Research of Zhejiang Province, Cancer Center of Zhejiang University, Hangzhou, 310000, China
| | - Lingling Dong
- Stomatology Hospital, School of Stomatology, Zhejiang University School of Medicine, Zhejiang Provincial Clinical Research Center for Oral Diseases, Key Laboratory of Oral Biomedical Research of Zhejiang Province, Cancer Center of Zhejiang University, Hangzhou, 310000, China
| | - Yani Chen
- Stomatology Hospital, School of Stomatology, Zhejiang University School of Medicine, Zhejiang Provincial Clinical Research Center for Oral Diseases, Key Laboratory of Oral Biomedical Research of Zhejiang Province, Cancer Center of Zhejiang University, Hangzhou, 310000, China
| | - Wenjin Cai
- Stomatology Hospital, School of Stomatology, Zhejiang University School of Medicine, Zhejiang Provincial Clinical Research Center for Oral Diseases, Key Laboratory of Oral Biomedical Research of Zhejiang Province, Cancer Center of Zhejiang University, Hangzhou, 310000, China
| | - Guoli Yang
- Stomatology Hospital, School of Stomatology, Zhejiang University School of Medicine, Zhejiang Provincial Clinical Research Center for Oral Diseases, Key Laboratory of Oral Biomedical Research of Zhejiang Province, Cancer Center of Zhejiang University, Hangzhou, 310000, China.
| | - Ying Wang
- Stomatology Hospital, School of Stomatology, Zhejiang University School of Medicine, Zhejiang Provincial Clinical Research Center for Oral Diseases, Key Laboratory of Oral Biomedical Research of Zhejiang Province, Cancer Center of Zhejiang University, Hangzhou, 310000, China.
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Setiawan AM, Kamarudin TA. Differentiation of Human Mesenchymal Stem Cells into Corneal Epithelial Cells: Current Progress. Curr Issues Mol Biol 2024; 46:13281-13295. [PMID: 39727920 DOI: 10.3390/cimb46120792] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/17/2024] [Revised: 11/13/2024] [Accepted: 11/17/2024] [Indexed: 12/28/2024] Open
Abstract
The limited availability of corneal tissue grafts poses significant challenges in the treatment of corneal blindness. Novel treatment utilizes stem cell grafts transplanted from the healthy side of the cornea to the damaged side. However, this procedure is only possible for those who have one-sided corneal blindness. Human stem cells offer promising potential for corneal tissue engineering, providing an alternative solution. Among the different types of stem cells, mesenchymal stem cells (MSCs) stand out due to their abundance and ease of isolation. Human MSCs can be derived from bone marrow, adipose, and umbilical cord tissues. Differentiating MSC toward corneal tissue can be achieved through several methods including chemical induction and co-culture with adult corneal cells such as human limbal epithelial stem cells (LESCs) and human corneal epithelial cells (hTCEpi). Adipose-derived stem cells (ADSCs) are the most common type of MSC that has been studied for corneal differentiation. Corneal epithelial cells are the most common corneal cell type targeted by researchers for corneal differentiation. Chemical induction with small molecules, especially bone morphogenetic protein 4 (BMP4), all-trans retinoic acid (ATRA), and epidermal growth factor (EGF), has gained more popularity in corneal epithelial cell differentiation. This review highlights the current progress in utilizing MSCs for corneal differentiation studies, showcasing their potential to revolutionize treatments for corneal blindness.
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Affiliation(s)
- Abdul Malik Setiawan
- Department of Anatomy, Faculty of Medicine, Universiti Kebangsaan Malaysia, Cheras, Kuala Lumpur 56000, Malaysia
- Department of Anatomy, Maulana Malik Ibrahim State Islamic University, Malang 65144, Indonesia
| | - Taty Anna Kamarudin
- Department of Anatomy, Faculty of Medicine, Universiti Kebangsaan Malaysia, Cheras, Kuala Lumpur 56000, Malaysia
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Li Y, Ge L, Ren B, Zhang X, Yin Z, Liu H, Yang Y, Liu Y, Xu H. De-Differentiation of Corneal Epithelial Cells Into Functional Limbal Epithelial Stem Cells After the Ablation of Innate Stem Cells. Invest Ophthalmol Vis Sci 2024; 65:32. [PMID: 39546294 DOI: 10.1167/iovs.65.13.32] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/17/2024] Open
Abstract
Purpose Regeneration after tissue injury is often associated with cell fate plasticity, which restores damaged or lost cells. Here, we examined the de-differentiation of corneal epithelial cells (CECs) into functional limbal epithelial stem cells (LESCs) after the ablation of innate stem cells. Methods The regeneration of LESCs after the ablation of innate LESCs was identified by a set of markers: ApoE+/Cx43low/CK12-. CK14-CreERT2 or Slc1a3-CreERT mice were crossed with reporter mice to trace the fate of CECs. YAP-TEAD inhibitor verteporfin (VTP) and LATS inhibitor TRULI were used to examine the role of Hippo/YAP pathway in the de-differentiation of CECs. Results LESCs-ablation cornea showed to be functionally normal, including the maintenance of corneal transparency, prevention of conjunctivalization, and wound healing rate equivalent to that of normal cornea. ApoE+/Cx43low/CK12- LESCs regenerated at the limbus at 6 days after the ablation of innate stem cells, and maintained for at least 6 months. Corneal epithelial lineage tracing showed that CECs migrated back to the limbus after the ablation of innate stem cells, and de-differentiated into active and quiescent LESCs (aLESCs and qLESCs), which participated in corneal epithelial homeostasis and wound healing, respectively, like their innate counterparts. However, when the limbus niche was destroyed by NaOH (1 M, 5 seconds), CECs that occupied the limbus could not de-differentiate into ApoE+/Cx43low/CK12- LESCs and cornea developed into conjunctivalization. In addition, the protein level and activity of YAP increased at the early stage (1-2 days) after the ablation of limbal epithelium, and decreased when the de-differentiation occurred. The YAP-TEAD inhibitor VTP promoted the de-differentiation, whereas LATS inhibitor TRULI inhibited the de-differentiation of CECs. However, the persistent activation of YAP prevented the de-differentiation of CECs after an additional NaOH burn to the limbal stroma, and VTP could not rescue the capacity of CECs to de-differentiate into LESCs. Conclusions These results reveal the de-differentiation of CECs into functional LESCs after the ablation of innate stem cells, and suggest potential role of Hippo/YAP pathway in the de-differentiation of CECs in vivo.
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Affiliation(s)
- Yijian Li
- Southwest Hospital/Southwest Eye Hospital, Third Military Medical University (Army Medical University), Chongqing, China
- Key Lab of Visual Damage and Regeneration and Restoration of Chongqing, Chongqing, China
| | - Lingling Ge
- Southwest Hospital/Southwest Eye Hospital, Third Military Medical University (Army Medical University), Chongqing, China
- Key Lab of Visual Damage and Regeneration and Restoration of Chongqing, Chongqing, China
| | - Bangqi Ren
- Southwest Hospital/Southwest Eye Hospital, Third Military Medical University (Army Medical University), Chongqing, China
- Key Lab of Visual Damage and Regeneration and Restoration of Chongqing, Chongqing, China
| | - Xue Zhang
- Southwest Hospital/Southwest Eye Hospital, Third Military Medical University (Army Medical University), Chongqing, China
- Key Lab of Visual Damage and Regeneration and Restoration of Chongqing, Chongqing, China
| | - Zhiyuan Yin
- Southwest Hospital/Southwest Eye Hospital, Third Military Medical University (Army Medical University), Chongqing, China
- Key Lab of Visual Damage and Regeneration and Restoration of Chongqing, Chongqing, China
| | - Hongling Liu
- Southwest Hospital/Southwest Eye Hospital, Third Military Medical University (Army Medical University), Chongqing, China
- Key Lab of Visual Damage and Regeneration and Restoration of Chongqing, Chongqing, China
| | - Yuli Yang
- Southwest Hospital/Southwest Eye Hospital, Third Military Medical University (Army Medical University), Chongqing, China
- Key Lab of Visual Damage and Regeneration and Restoration of Chongqing, Chongqing, China
| | - Yong Liu
- Southwest Hospital/Southwest Eye Hospital, Third Military Medical University (Army Medical University), Chongqing, China
- Key Lab of Visual Damage and Regeneration and Restoration of Chongqing, Chongqing, China
- Jinfeng Laboratory, Chongqing, China
| | - Haiwei Xu
- Southwest Hospital/Southwest Eye Hospital, Third Military Medical University (Army Medical University), Chongqing, China
- Key Lab of Visual Damage and Regeneration and Restoration of Chongqing, Chongqing, China
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Pourjabbar B, Shams F, Heidari Keshel S, Biazar E. Proliferation and differentiation of Wharton's jelly-derived mesenchymal stem cells on prgf-treated hydrogel scaffold. Regen Med 2024; 19:549-560. [PMID: 39558722 PMCID: PMC11633401 DOI: 10.1080/17460751.2024.2427513] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/04/2024] [Accepted: 11/04/2024] [Indexed: 11/20/2024] Open
Abstract
BACKGROUND To address the limitations of Cultivated Limbal Epithelial Transplantation (CLET) and the use of amniotic membrane (AM) in treating Limbal Stem Cell Deficiency (LSCD), we aimed to develop a Collagen/Silk Fibroin (Co/SF) scaffold enriched with Platelet-Rich Growth Factor (PRGF) to support the proliferation, maintenance, and differentiation of Wharton's jelly-derived mesenchymal stem cells (WJMSCs) into corneal epithelial cells (CECs). METHOD Scaffolds loaded with PRGF were evaluated through release studies, cytotoxicity assays, and cell differentiation. The proliferation and differentiation of WJMSCs and Limbal Epithelial Stem Cells (LESCs) were investigated using MTT assays, real-time PCR and immunostaining. RESULTS The PRGF-loaded Co/SF scaffold significantly promoted the proliferation of both WJMSCs and LESCs in a concentration-dependent manner. Real-time PCR and immune staining revealed a significant increase in the expression of P63, ABCG2, and cytokeratin 3/12 markers in WJMSCs, a significant decrease in the expression of P63 and ABCG2, and a significant increase in the expression of cytokeratin 3/12 markers indicating successful differentiation into CECs. CONCLUSION The WJMSC cultured on PRGF-enriched Co/SF scaffold demonstrates potential as a viable alternative to conventional CLET, offering a promising strategy for corneal tissue regeneration.
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Affiliation(s)
- Bahareh Pourjabbar
- Department of Tissue Engineering and Applied Cell Sciences, School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran
| | - Forough Shams
- Department of Medical Biotechnology, School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran
| | - Saeed Heidari Keshel
- Department of Tissue Engineering and Applied Cell Sciences, School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran
- Medical Nanotechnology and Tissue Engineering Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran
| | - Esmaeil Biazar
- Tissue Engineering Group, Department of Biomedical Engineering, Tonekabon Branch, Islamic Azad University, Tonekabon, Iran
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Long Q, Huang C, Zhang L, Jiang H, Zhao S, Zhang L, Zheng X, Ou S, Gu H. A novel tissue-engineered corneal epithelium based on ultra-thin amniotic membrane and mesenchymal stem cells. Sci Rep 2024; 14:17407. [PMID: 39075142 PMCID: PMC11286932 DOI: 10.1038/s41598-024-68219-8] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/20/2024] [Accepted: 07/22/2024] [Indexed: 07/31/2024] Open
Abstract
Currently, in vitro cultured corneal epithelial transplantation is effective in treating limbal stem cell dysfunction (LSCD). Selecting carriers is crucial for constructing the corneal epithelium through tissue engineering. In this study, the traditional amniotic membrane (AM) was modified, and mesenchymal stem cells (MSCs) were inoculated into the ultra-thin amniotic membrane (UAM) stroma to construct a novel UAM-MSC tissue-engineered corneal epithelial carrier, that could effectively simulate the limbal stem cells (LSCs) microenvironment. The structure of different carriers cultured tissue-engineered corneal epithelium and the managed rabbit LSCD model corneas were observed through hematoxylin-eosin staining. Cell phenotypes were evaluated through fluorescence staining, Western blotting, and RT-qPCR. Additionally, cell junction genes and expression markers related to anti-neovascularization were evaluated using RT-qPCR. Corneal epithelium cell junctions were observed via an electron microscope. The tissue-engineered corneal epithelium culture medium was analyzed through mass spectrometry. Tissue-engineered corneal epithelial cells expanded by LSCs on UAM-MSCs had good transparency. Simultaneously, progenitor cell (K14, PNCA, p63) and corneal epithelial (PAX6) gene expression in tissue-engineered corneal epithelium constructed using UAM-MSCs was higher than that in corneal epithelial cells amplified by UAM and de-epithelialized amniotic membrane. Electron microscopy revealed that corneal epithelial cells grafted with UAM-MSCs were closely connected. In conclusion, the UAM-MSCs vector we constructed could better simulate the limbal microenvironment; the cultured tissue-engineered corneal epithelium had better transparency, anti-neovascularization properties, closer intercellular connections, and closer resemblance to the natural corneal epithelial tissue phenotype.
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Affiliation(s)
- Qiurong Long
- Guizhou Medical University, Guiyang, Guizhou, China
- The Affiliated Hospital of Guizhou Medical University, No.9 Beijing Road, Yunyan District, Guiyang, Guizhou, China
| | - Chao Huang
- Guizhou Medical University, Guiyang, Guizhou, China
| | - Liying Zhang
- Guizhou Medical University, Guiyang, Guizhou, China
- The Affiliated Hospital of Guizhou Medical University, No.9 Beijing Road, Yunyan District, Guiyang, Guizhou, China
| | - Hao Jiang
- Guizhou Medical University, Guiyang, Guizhou, China
- The Affiliated Hospital of Guizhou Medical University, No.9 Beijing Road, Yunyan District, Guiyang, Guizhou, China
| | - Su Zhao
- Guizhou Medical University, Guiyang, Guizhou, China
- The Affiliated Hospital of Guizhou Medical University, No.9 Beijing Road, Yunyan District, Guiyang, Guizhou, China
| | - Lingli Zhang
- Guizhou Medical University, Guiyang, Guizhou, China
| | - Xueer Zheng
- Guizhou Medical University, Guiyang, Guizhou, China
| | - Shangkun Ou
- Guizhou Medical University, Guiyang, Guizhou, China.
- The Affiliated Hospital of Guizhou Medical University, No.9 Beijing Road, Yunyan District, Guiyang, Guizhou, China.
| | - Hao Gu
- Guizhou Medical University, Guiyang, Guizhou, China.
- The Affiliated Hospital of Guizhou Medical University, No.9 Beijing Road, Yunyan District, Guiyang, Guizhou, China.
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Su G, Guo X, Xu L, Jin B, Tan Y, Zhou X, Wang W, Li X, Wang S, Li G. Isolation and characterization of rabbit limbal niche cells. Exp Eye Res 2024; 241:109838. [PMID: 38395213 DOI: 10.1016/j.exer.2024.109838] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/01/2024] [Revised: 02/19/2024] [Accepted: 02/20/2024] [Indexed: 02/25/2024]
Abstract
Limbal niche cells (LNCs) are one of the most important supporting cells for corneal epithelial stem cells (CES), however, research on LNCs has been mostly limited to humans and rats previously. To expand the research work into the rabbit animal model, one of the most often used animals in stem cell study, this study was carried out for the in vitro isolation and identification of rabbit LNCs. Rabbit LNCs were isolated by collagenase A digestion method and single cells were obtained, the cells were then seeded on 5% Matrigel-coated plastic surface and cultured in modified embryonic stem cell medium (MESCM). Three biological replicates of the isolating and characterization were recorded from New Zealand White rabbits aged from 2.5 months to 5 months. LNC markers (VIM/CD90/CD105/SCF/PDGFRβ) were analyzed using tyramide signal amplification (TSA) staining, immunohistochemical staining (IHC), western blotting (WB), and real-time reverse transcription polymerase chain reaction (qPCR). TSA staining suggested that VIM was highly expressed in rabbit limbus stroma, which was confirmed by WB, and P63α was expressed in the basal limbus epithelium. Pan-CK and CK12 were highly expressed in the central corneal epithelium but lightly expressed in the limbal epithelium. The WB result indicated that PDGFRβ and VIM expressions in rabbit-LNCs P4 were higher than in P1 and P7. In addition, rabbit corneal epithelium highly expressed Paired Box 6 (PAX6) and Epidermal growth factor-like domain 6(EGFL6). For the three repeat experiments, the cell expansion activity of rabbit-LNC was highest at P4. Rabbit-LNCs were passaged from P0 to P7, and the number of cell doublings (NCD) of P4 for the three repeat experiments was 2.816, 2.737, and 2.849. qPCR showed that high mRNA expression levels of VIM, CD90, CD105, SCF, and PDGFRβ in rabbit-LNCs P4. In conclusion, rabbit-LNCs could be successfully isolated by the collagenase A digestion method as used in human tissue. There were similar characteristics between rabbit and human LNCs (VIM+/CD90+/CD105+/SCF+/PAX6+/PDGFRβ+).
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Affiliation(s)
- Guanyu Su
- Department of Ophthalmology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei Province, 430030, China
| | - Xiaojie Guo
- Department of Integrative Medicine, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei Province, 430030, China
| | - Lingjuan Xu
- Department of Ophthalmology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei Province, 430030, China
| | - Bihui Jin
- Department of Ophthalmology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei Province, 430030, China
| | - Yongyao Tan
- Department of Ophthalmology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei Province, 430030, China
| | - Xiao Zhou
- Department of Ophthalmology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei Province, 430030, China
| | - Wei Wang
- Department of Ophthalmology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei Province, 430030, China
| | - Xinyu Li
- Department of Ophthalmology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei Province, 430030, China
| | - Shusheng Wang
- Department of Cell and Molecular Biology & Ophthalmology, 2000 Percival Stern Hall, Tulane University, New Orleans, LA, 70118-5698, USA
| | - Guigang Li
- Department of Ophthalmology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei Province, 430030, China.
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Zong Q, Bundkirchen K, Neunaber C, Noack S. Effect of High BMI on Human Bone Marrow-Derived Mesenchymal Stromal Cells. Cell Transplant 2024; 33:9636897241226546. [PMID: 38258516 PMCID: PMC10807335 DOI: 10.1177/09636897241226546] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/08/2023] [Revised: 12/22/2023] [Accepted: 01/02/2024] [Indexed: 01/24/2024] Open
Abstract
Bone marrow-derived mesenchymal stromal cells (BMSCs) are attractive candidates in tissue engineering and regenerative medicine. Growing evidence has suggested that a high body mass index (BMI) can affect the properties of BMSCs, resulting in a reduced quality of the cells. However, the results are not consistent. Therefore, this study aimed to investigate the influences of high BMI on human BMSCs (hBMSCs). To avoid gender bias, BMSCs from females and males were studied independently. Finally, hBMSCs from 89 females and 152 males were separately divided into the normal BMI group (18.5 kg/m2 ≤ BMI < 25 kg/m2) and the high BMI group (BMI > 25 kg/m2). The cells were analyzed for the colony-forming potential; proliferation capacity; in vitro adipogenic, osteogenic, and chondrogenic differentiation potentials; and the expression of 32 common surface antigens. The results showed that high BMI did not change the number of colonies at passage 1 in females and males. In contrast, significantly reduced colony numbers at passage 4 (P4) were found in both female and male donors with high BMI. The doubling time of hBMSCs was comparable between the normal and the high BMI groups of females and males. Furthermore, the results of trilineage differentiation did not differ between the different BMI groups of males. In females, the high and the normal BMI groups also showed similar adipogenic and chondrogenic differentiation, while osteogenic differentiation was significantly enhanced in the high-BMI group. Regarding the expression of surface antigens, the expressions of CD200 and SSEA4 on hBMSCs were reduced in the high-BMI group of females and males, respectively. In conclusion, high BMI suppressed the clonogenicity of female and male hBMSCs at P4, improved the in vitro osteogenesis of female hBMSCs, and decreased the expressions of CD200 on hBMSCs in females and SSEA4 in males.
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Affiliation(s)
- Qiang Zong
- Department of Trauma Surgery, Hannover Medical School, Hannover, Germany
| | - Katrin Bundkirchen
- Department of Trauma Surgery, Hannover Medical School, Hannover, Germany
| | - Claudia Neunaber
- Department of Trauma Surgery, Hannover Medical School, Hannover, Germany
| | - Sandra Noack
- Department of Trauma Surgery, Hannover Medical School, Hannover, Germany
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10
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Hopkinson A, Notara M, Cursiefen C, Sidney LE. Increased Anti-Inflammatory Therapeutic Potential and Progenitor Marker Expression of Corneal Mesenchymal Stem Cells Cultured in an Optimized Propagation Medium. Cell Transplant 2024; 33:9636897241241992. [PMID: 38602231 PMCID: PMC11010753 DOI: 10.1177/09636897241241992] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/03/2024] [Revised: 02/19/2024] [Accepted: 03/05/2024] [Indexed: 04/12/2024] Open
Abstract
There is a huge unmet need for new treatment modalities for ocular surface inflammatory disorders (OSIDs) such as dry eye disease and meibomian gland dysfunction. Mesenchymal stem cell therapies may hold the answer due to their potent immunomodulatory properties, low immunogenicity, and ability to modulate both the innate and adaptive immune response. MSC-like cells that can be isolated from the corneal stroma (C-MSCs) offer a potential new treatment strategy; however, an optimized culture medium needs to be developed to produce the ideal phenotype for use in a cell therapy to treat OSIDs. The effects of in vitro expansion of human C-MSC in a medium of M199 containing fetal bovine serum (FBS) was compared to a stem cell medium (SCM) containing knockout serum replacement (KSR) with basic fibroblast growth factor (bFGF) and human leukemia inhibitory factor (LIF), investigating viability, protein, and gene expression. Isolating populations expressing CD34 or using siRNA knockdown of CD34 were investigated. Finally, the potential of C-MSC as a cell therapy was assessed using co-culture with an in vitro corneal epithelial cell injury model and the angiogenic effects of C-MSC conditioned medium were evaluated with blood and lymph endothelial cells. Both media supported proliferation of C-MSC, with SCM increasing expression of CD34, ABCG2, PAX6, NANOG, REX1, SOX2, and THY1, supported by increased associated protein expression. Isolating cell populations expressing CD34 protein made little difference to gene expression, however, knockdown of the CD34 gene led to decreased expression of progenitor genes. C-MSC increased viability of injured corneal epithelial cells whilst decreasing levels of cytotoxicity and interleukins-6 and -8. No pro-angiogenic effect of C-MSC was seen. Culture medium can significantly influence C-MSC phenotype and culture in SCM produced a cell phenotype more suitable for further consideration as an anti-inflammatory cell therapy. C-MSC show considerable potential for development as therapies for OSIDs, acting through anti-inflammatory action.
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Affiliation(s)
- Andrew Hopkinson
- Academic Ophthalmology, Mental Health and Clinical Neurosciences, University of Nottingham, Nottingham, UK
| | - Maria Notara
- Department of Ophthalmology, Faculty of Medicine and University Hospital Cologne, University of Cologne, Koln, Germany
| | - Claus Cursiefen
- Department of Ophthalmology, Faculty of Medicine and University Hospital Cologne, University of Cologne, Koln, Germany
| | - Laura E. Sidney
- Academic Ophthalmology, Mental Health and Clinical Neurosciences, University of Nottingham, Nottingham, UK
- Regenerating and Modelling Tissues, Translational Medical Sciences, School of Medicine, University of Nottingham, Nottingham, UK
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11
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Ye P, Gu R, Zhu H, Chen J, Han F, Nie X. SOX family transcription factors as therapeutic targets in wound healing: A comprehensive review. Int J Biol Macromol 2023; 253:127243. [PMID: 37806414 DOI: 10.1016/j.ijbiomac.2023.127243] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/07/2023] [Revised: 10/02/2023] [Accepted: 10/02/2023] [Indexed: 10/10/2023]
Abstract
The SOX family plays a vital role in determining the fate of cells and has garnered attention in the fields of cancer research and regenerative medicine. It also shows promise in the study of wound healing, as it actively participates in the healing processes of various tissues such as skin, fractures, tendons, and the cornea. However, our understanding of the mechanisms behind the SOX family's involvement in wound healing is limited compared to its role in cancer. Gaining insight into its role, distribution, interaction with other factors, and modifications in traumatized tissues could provide valuable new knowledge about wound healing. Based on current research, SOX2, SOX7, and SOX9 are the most promising members of the SOX family for future interventions in wound healing. SOX2 and SOX9 promote the renewal of cells, while SOX7 enhances the microvascular environment. The SOX family holds significant potential for advancing wound healing research. This article provides a comprehensive review of the latest research advancements and therapeutic tools related to the SOX family in wound healing, as well as the potential benefits and challenges of targeting the SOX family for wound treatment.
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Affiliation(s)
- Penghui Ye
- Key Lab of the Basic Pharmacology of the Ministry of Education & Joint International Research Laboratory of Ethnomedicine of Ministry of Education, Zunyi Medical University, Zunyi 563006, China; College of Pharmacy, Zunyi Medical University, Zunyi 563006, China
| | - Rifang Gu
- Key Lab of the Basic Pharmacology of the Ministry of Education & Joint International Research Laboratory of Ethnomedicine of Ministry of Education, Zunyi Medical University, Zunyi 563006, China; School Medical Office, Zunyi Medical University, Zunyi 563006, China
| | - Huan Zhu
- Key Lab of the Basic Pharmacology of the Ministry of Education & Joint International Research Laboratory of Ethnomedicine of Ministry of Education, Zunyi Medical University, Zunyi 563006, China; College of Pharmacy, Zunyi Medical University, Zunyi 563006, China
| | - Jitao Chen
- Key Lab of the Basic Pharmacology of the Ministry of Education & Joint International Research Laboratory of Ethnomedicine of Ministry of Education, Zunyi Medical University, Zunyi 563006, China; College of Pharmacy, Zunyi Medical University, Zunyi 563006, China
| | - Felicity Han
- Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Brisbane, QLD 4072, Australia
| | - Xuqiang Nie
- Key Lab of the Basic Pharmacology of the Ministry of Education & Joint International Research Laboratory of Ethnomedicine of Ministry of Education, Zunyi Medical University, Zunyi 563006, China; College of Pharmacy, Zunyi Medical University, Zunyi 563006, China; Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Brisbane, QLD 4072, Australia.
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12
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Yam GHF, Pi S, Du Y, Mehta JS. Posterior corneoscleral limbus: Architecture, stem cells, and clinical implications. Prog Retin Eye Res 2023; 96:101192. [PMID: 37392960 DOI: 10.1016/j.preteyeres.2023.101192] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/19/2023] [Revised: 06/22/2023] [Accepted: 06/23/2023] [Indexed: 07/03/2023]
Abstract
The limbus is a transition from the cornea to conjunctiva and sclera. In human eyes, this thin strip has a rich variation of tissue structures and composition, typifying a change from scleral irregularity and opacity to corneal regularity and transparency; a variation from richly vascularized conjunctiva and sclera to avascular cornea; the neural passage and drainage of aqueous humor. The limbal stroma is enriched with circular fibres running parallel to the corneal circumference, giving its unique role in absorbing small pressure changes to maintain corneal curvature and refractivity. It contains specific niches housing different types of stem cells for the corneal epithelium, stromal keratocytes, corneal endothelium, and trabecular meshwork. This truly reflects the important roles of the limbus in ocular physiology, and the limbal functionality is crucial for corneal health and the entire visual system. Since the anterior limbus containing epithelial structures and limbal epithelial stem cells has been extensively reviewed, this article is focused on the posterior limbus. We have discussed the structural organization and cellular components of the region beneath the limbal epithelium, the characteristics of stem cell types: namely corneal stromal stem cells, endothelial progenitors and trabecular meshwork stem cells, and recent advances leading to the emergence of potential cell therapy options to replenish their respective mature cell types and to correct defects causing corneal abnormalities. We have reviewed different clinical disorders associated with defects of the posterior limbus and summarized the available preclinical and clinical evidence about the developing topic of cell-based therapy for corneal disorders.
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Affiliation(s)
- Gary Hin-Fai Yam
- Department of Ophthalmology, University of Pittsburgh, Pittsburgh, PA, USA; Tissue Engineering and Cell Therapy Group, Singapore Eye Research Institute, Singapore; McGowan Institute for Regenerative Medicine, Pittsburgh, PA, USA.
| | - Shaohua Pi
- Department of Ophthalmology, University of Pittsburgh, Pittsburgh, PA, USA
| | - Yiqin Du
- Department of Ophthalmology, University of South Florida, Tampa, FL, USA
| | - Jodhbir S Mehta
- Tissue Engineering and Cell Therapy Group, Singapore Eye Research Institute, Singapore; Department of Cornea and External Eye Disease, Singapore National Eye Centre, Singapore; Ophthalmology and Visual Sciences Academic Clinical Program, Duke-National University of Singapore (NUS) Medical School, Singapore.
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13
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Corneal Reconstruction with EGFP-Labelled Limbal Mesenchymal Stem Cells in a Rabbit Model of Limbal Stem Cell Deficiency. Int J Mol Sci 2023; 24:ijms24065431. [PMID: 36982507 PMCID: PMC10051408 DOI: 10.3390/ijms24065431] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/03/2023] [Revised: 03/07/2023] [Accepted: 03/10/2023] [Indexed: 03/14/2023] Open
Abstract
Ocular surface reconstruction is essential for treating corneal epithelial defects and vision recovery. Stem cell-based therapy demonstrates promising results but requires further research to elucidate stem cell survival, growth, and differentiation after transplantation in vivo. This study examined the corneal reconstruction promoted by EGFP-labeled limbal mesenchymal stem cells (L-MSCs-EGFP) and their fate after transplantation. EGFP labeling allowed us to evaluate the migration and survival rates of the transferred cells. L-MSCs-EGFP seeded onto decellularized human amniotic membrane (dHAM) were transplanted into rabbits with a modeled limbal stem cell deficiency. The localization and viability of the transplanted cells in animal tissue were analyzed using histology, immunohistochemistry, and confocal microscopy up to 3 months after transplantation. EGFP-labeled cells remained viable for the first 14 days after transplantation. By the 90th day, epithelialization of the rabbit corneas reached 90%, but the presence of viable labeled cells was not observed within the newly formed epithelium. Although labeled cells demonstrated low survivability in host tissue, the squamous corneal-like epithelium was partially restored by the 30th day after transplantation of the tissue-engineered graft. Overall, this study paves the way for further optimization of transplantation conditions and studying the mechanisms of corneal tissue restoration.
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14
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Kopecny LR, Lee BWH, Coroneo MT. A systematic review on the effects of ROCK inhibitors on proliferation and/or differentiation in human somatic stem cells: A hypothesis that ROCK inhibitors support corneal endothelial healing via acting on the limbal stem cell niche. Ocul Surf 2023; 27:16-29. [PMID: 36586668 DOI: 10.1016/j.jtos.2022.12.008] [Citation(s) in RCA: 5] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/09/2022] [Revised: 12/18/2022] [Accepted: 12/22/2022] [Indexed: 12/29/2022]
Abstract
Rho kinase inhibitors (ROCKi) have attracted growing multidisciplinary interest, particularly in Ophthalmology where the question as to how they promote corneal endothelial healing remains unresolved. Concurrently, stem cell biology has rapidly progressed in unravelling drivers of stem cell (SC) proliferation and differentiation, where mechanical niche factors and the actin cytoskeleton are increasingly recognized as key players. There is mounting evidence from the study of the peripheral corneal endothelium that supports the likelihood of an internal limbal stem cell niche. The possibility that ROCKi stimulate the endothelial SC niche has not been addressed. Furthermore, there is currently a paucity of data that directly evaluates whether ROCKi promotes corneal endothelial healing by acting on this limbal SC niche located near the transition zone. Therefore, we performed a systematic review examining the effects ROCKi on the proliferation and differentiation of human somatic SC, to provide insight into its effects on various human SC populations. An appraisal of electronic searches of four databases identified 1 in vivo and 58 in vitro studies (36 evaluated proliferation while 53 examined differentiation). Types of SC studied included mesenchymal (n = 32), epithelial (n = 11), epidermal (n = 8), hematopoietic and other (n = 8). The ROCK 1/2 selective inhibitor Y-27632 was used in almost all studies (n = 58), while several studies evaluated ≥2 ROCKi (n = 4) including fasudil, H-1152, and KD025. ROCKi significantly influenced human somatic SC proliferation in 81% of studies (29/36) and SC differentiation in 94% of studies (50/53). The present systemic review highlights that ROCKi are influential in regulating human SC proliferation and differentiation, and provides evidence to support the hypothesis that ROCKi promotes corneal endothelial division and maintenance via acting on the inner limbal SC niche.
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Affiliation(s)
- Lloyd R Kopecny
- School of Clinical Medicine, University of New South Wales, Sydney, Australia.
| | - Brendon W H Lee
- Department of Ophthalmology, School of Clinical Medicine, University of New South Wales, Level 2 South Wing, Edmund Blacket Building, Prince of Wales Hospital, Randwick, NSW, 2031, Australia
| | - Minas T Coroneo
- Department of Ophthalmology, Prince of Wales Hospital, Sydney, Australia
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15
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Di Girolamo N, Park M. Cell identity changes in ocular surface Epithelia. Prog Retin Eye Res 2022:101148. [DOI: 10.1016/j.preteyeres.2022.101148] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/31/2022] [Revised: 10/13/2022] [Accepted: 11/09/2022] [Indexed: 11/21/2022]
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16
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Cialdai F, Risaliti C, Monici M. Role of fibroblasts in wound healing and tissue remodeling on Earth and in space. Front Bioeng Biotechnol 2022; 10:958381. [PMID: 36267456 PMCID: PMC9578548 DOI: 10.3389/fbioe.2022.958381] [Citation(s) in RCA: 107] [Impact Index Per Article: 35.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/31/2022] [Accepted: 09/05/2022] [Indexed: 11/18/2022] Open
Abstract
Wound healing (WH) and the role fibroblasts play in the process, as well as healing impairment and fibroblast dysfunction, have been thoroughly reviewed by other authors. We treat these topics briefly, with the only aim of contextualizing the true focus of this review, namely, the microgravity-induced changes in fibroblast functions involved in WH. Microgravity is a condition typical of spaceflight. Studying its possible effects on fibroblasts and WH is useful not only for the safety of astronauts who will face future interplanetary space missions, but also to help improve the management of WH impairment on Earth. The interesting similarity between microgravity-induced alterations of fibroblast behavior and fibroblast dysfunction in WH impairment on Earth is highlighted. The possibility of using microgravity-exposed fibroblasts and WH in space as models of healing impairment on Earth is suggested. The gaps in knowledge on fibroblast functions in WH are analyzed. The contribution that studies on fibroblast behavior in weightlessness can make to fill these gaps and, consequently, improve therapeutic strategies is considered.
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17
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Trends in using mesenchymal stromal/stem cells (MSCs) in treating corneal diseases. Ocul Surf 2022; 26:255-267. [DOI: 10.1016/j.jtos.2022.10.003] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/28/2021] [Revised: 10/03/2022] [Accepted: 10/07/2022] [Indexed: 12/05/2022]
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18
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Tavakkoli F, Damala M, Koduri MA, Gangadharan A, Rai AK, Dash D, Basu S, Singh V. Transcriptomic Profiling of Human Limbus-Derived Stromal/Mesenchymal Stem Cells-Novel Mechanistic Insights into the Pathways Involved in Corneal Wound Healing. Int J Mol Sci 2022; 23:ijms23158226. [PMID: 35897793 PMCID: PMC9368612 DOI: 10.3390/ijms23158226] [Citation(s) in RCA: 7] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/21/2022] [Revised: 07/16/2022] [Accepted: 07/16/2022] [Indexed: 01/27/2023] Open
Abstract
Limbus-derived stromal/mesenchymal stem cells (LMSCs) are vital for corneal homeostasis and wound healing. However, despite multiple pre-clinical and clinical studies reporting the potency of LMSCs in avoiding inflammation and scarring during corneal wound healing, the molecular basis for the ability of LMSCs remains unknown. This study aimed to uncover the factors and pathways involved in LMSC-mediated corneal wound healing by employing RNA-Sequencing (RNA-Seq) in human LMSCs for the first time. We characterized the cultured LMSCs at the stages of initiation (LMSC−P0) and pure population (LMSC−P3) and subjected them to RNA-Seq to identify the differentially expressed genes (DEGs) in comparison to native limbus and cornea, and scleral tissues. Of the 28,000 genes detected, 7800 DEGs were subjected to pathway-specific enrichment Gene Ontology (GO) analysis. These DEGs were involved in Wnt, TGF-β signaling pathways, and 16 other biological processes, including apoptosis, cell motility, tissue remodeling, and stem cell maintenance, etc. Two hundred fifty-four genes were related to wound healing pathways. COL5A1 (11.81 ± 0.48) and TIMP1 (20.44 ± 0.94) genes were exclusively up-regulated in LMSC−P3. Our findings provide new insights involved in LMSC-mediated corneal wound healing.
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Affiliation(s)
- Fatemeh Tavakkoli
- Prof. Brien Holden Eye Research Center, LV Prasad Eye Institute, Hyderabad 500034, India; (F.T.); (M.D.); (M.A.K.); (S.B.)
- Center for Genetic Disorders, Banaras Hindu University, Varanasi 221005, India;
| | - Mukesh Damala
- Prof. Brien Holden Eye Research Center, LV Prasad Eye Institute, Hyderabad 500034, India; (F.T.); (M.D.); (M.A.K.); (S.B.)
- School of Life Sciences, University of Hyderabad, Hyderabad 500046, India
| | - Madhuri Amulya Koduri
- Prof. Brien Holden Eye Research Center, LV Prasad Eye Institute, Hyderabad 500034, India; (F.T.); (M.D.); (M.A.K.); (S.B.)
- Manipal Academy of Higher Education, Manipal 576104, India
| | - Abhilash Gangadharan
- CSIR-Institute of Genomics and Integrative Biology, Mathura Road Campus, New Delhi 110025, India; (A.G.); (D.D.)
| | - Amit K. Rai
- Center for Genetic Disorders, Banaras Hindu University, Varanasi 221005, India;
| | - Debasis Dash
- CSIR-Institute of Genomics and Integrative Biology, Mathura Road Campus, New Delhi 110025, India; (A.G.); (D.D.)
| | - Sayan Basu
- Prof. Brien Holden Eye Research Center, LV Prasad Eye Institute, Hyderabad 500034, India; (F.T.); (M.D.); (M.A.K.); (S.B.)
- Center for Ocular Regeneration (CORE), Prof. Brien Holden Eye Research Center, LV Prasad Eye Institute, Hyderabad 500034, India
| | - Vivek Singh
- Prof. Brien Holden Eye Research Center, LV Prasad Eye Institute, Hyderabad 500034, India; (F.T.); (M.D.); (M.A.K.); (S.B.)
- Center for Ocular Regeneration (CORE), Prof. Brien Holden Eye Research Center, LV Prasad Eye Institute, Hyderabad 500034, India
- Correspondence: ; Tel.: +91-40-6810-2286
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19
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Zhu H, Wang W, Tan Y, Su G, Xu L, Jiang ML, Li S, Meir YJJ, Wang Y, Li G, Zhou H. Limbal Niche Cells and Three-Dimensional Matrigel-Induced Dedifferentiation of Mature Corneal Epithelial Cells. Invest Ophthalmol Vis Sci 2022; 63:1. [PMID: 35499835 PMCID: PMC9078055 DOI: 10.1167/iovs.63.5.1] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/30/2021] [Accepted: 04/11/2022] [Indexed: 01/11/2023] Open
Abstract
PURPOSE To investigate the phenotypic changes of mature corneal epithelial cells (MCECs) that cocultured with limbal niche cells (LNCs) in three-dimensional Matrigel (3D Matrigel) in vitro. METHODS MCECs were isolated from central corneas, and limbal epithelial progenitor cells (LEPCs) were isolated from limbal segments with Dispase II. LNCs were isolated and cultured from limbal niche using the collagenase A digestion method and identified with PCK/VIM/CD90/CD105/SCF/PDGFRβ. MCECs were cultured on 3D Matrigel (50%, v/v) with or without LNCs for 10 days. Expression of CK12 and p63α and clone formation test were used to compare the progenitor phenotypic changes for MCECs before and after induction using LEPCs as control. RESULTS Homogeneous LNCs were isolated and identified as spindle shape and adherent to a plastic surface coated with 5% Matrigel. Double immunostaining of the fourth-passage LNCs was uniformly PCK-/VIM+/CD90+/CD105+/SCF+/PDGFRβ+. Reverse transcription and quantitative real-time polymerase chain reaction (RT-qPCR) revealed the decrease of PCK expression from the second passage and elevation of Vim, CD90, CD105, SCF, and PDGFRβ transcripts from the third passage, and the transcription level of Vim, CD90, CD105, SCF, and PDGFRβ was elevated statistically in the fourth passage compared to the first passage (P < 0.01). Both immunofluorescence (IF) staining for cross section and cytospin cells demonstrated that MCECs expressed higher CK12 while lower p63α than LEPCs (P < 0.01). Sphere growth formation was noticed as early as 24 hours in the MCEC + LNC group, 48 hours in the LEPC group, and 72 hours in the MCEC group. The diameters of the spheres were the biggest in the MCEC + LNC group (182.24 ± 57.91 µm), smaller in the LEPC group (125.71 ± 41.20 µm), and smallest in the MCEC group (109.39 ± 34.85 µm) by the end of the 10-day culture (P < 0.01). Double immunostaining with CK12/p63α showed that cells in the sphere formed from MCECs expressed CK12 but not p63α; in contrast, some cells in the MCEC + LNC group expressed CK12, but most of them expressed p63α. RT-qPCR revealed a significant reduction of CK12 transcript but elevation of p63α, Oct4, Nanog, Sox2, and SSEA4 (P < 0.05). Holoclone composed of cubic epithelial cells could be generated in the MCEC + LNC group but not in the other two groups. CONCLUSIONS The data shows that human MCEC cell phenotype could be induced to the dedifferentiation stage when cocultured with LNCs in 3D Matrigel that simulated the microenvironment of limbal stem cells in vitro.
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Affiliation(s)
- Hui Zhu
- Department of Ophthalmology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei Province, China
| | - Wei Wang
- Department of Ophthalmology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei Province, China
| | - Yongyao Tan
- Department of Ophthalmology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei Province, China
| | - Guanyu Su
- Department of Ophthalmology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei Province, China
| | - Lingjuan Xu
- Department of Ophthalmology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei Province, China
| | - Meng lin Jiang
- Department of Ophthalmology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei Province, China
| | - Shen Li
- Department of Ophthalmology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei Province, China
| | - Yaa-Jyuhn James Meir
- Department of Biomedical Sciences, College of Medicine, Chang Gung University, Linkou, Taiwan
| | - Yunming Wang
- State Key Laboratory of Material Processing and Die & Mould Technology, School of Materials Science and Engineering, Huazhong University of Science & Technology, Wuhan, Hubei Province, China
| | - Guigang Li
- Department of Ophthalmology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei Province, China
| | - Huamin Zhou
- State Key Laboratory of Material Processing and Die & Mould Technology, School of Materials Science and Engineering, Huazhong University of Science & Technology, Wuhan, Hubei Province, China
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20
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Nguyen HT, Theerakittayakorn K, Somredngan S, Ngernsoungnern A, Ngernsoungnern P, Sritangos P, Ketudat-Cairns M, Imsoonthornruksa S, Assawachananont J, Keeratibharat N, Wongsan R, Rungsiwiwut R, Laowtammathron C, Bui NX, Parnpai R. Signaling Pathways Impact on Induction of Corneal Epithelial-like Cells Derived from Human Wharton’s Jelly Mesenchymal Stem Cells. Int J Mol Sci 2022; 23:ijms23063078. [PMID: 35328499 PMCID: PMC8949174 DOI: 10.3390/ijms23063078] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/08/2022] [Revised: 03/07/2022] [Accepted: 03/10/2022] [Indexed: 02/05/2023] Open
Abstract
Corneal epithelium, the outmost layer of the cornea, comprises corneal epithelial cells (CECs) that are continuously renewed by limbal epithelial stem cells (LESCs). Loss or dysfunction of LESCs causes limbal stem cell deficiency (LSCD) which results in corneal epithelial integrity loss and visual impairment. To regenerate the ocular surface, transplantation of stem cell-derived CECs is necessary. Human Wharton’s jelly derived mesenchymal stem cells (WJ-MSCs) are a good candidate for cellular therapies in allogeneic transplantation. This study aimed to test the effects of treatments on three signaling pathways involved in CEC differentiation as well as examine the optimal protocol for inducing corneal epithelial differentiation of human WJ-MSCs. All-trans retinoic acid (RA, 5 or 10 µM) inhibited the Wnt signaling pathway via suppressing the translocation of β-catenin from the cytoplasm into the nucleus. SB505124 downregulated the TGF-β signaling pathway via reducing phosphorylation of Smad2. BMP4 did not increase phosphorylation of Smad1/5/8 that is involved in BMP signaling. The combination of RA, SB505124, BMP4, and EGF for the first 3 days of differentiation followed by supplementing hormonal epidermal medium for an additional 6 days could generate corneal epithelial-like cells that expressed a CEC specific marker CK12. This study reveals that WJ-MSCs have the potential to transdifferentiate into CECs which would be beneficial for further applications in LSCD treatment therapy.
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Affiliation(s)
- Hong Thi Nguyen
- Embryo Technology and Stem Cell Research Center, School of Biotechnology, Institute of Agricultural Technology, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand; (H.T.N.); (K.T.); (S.S.)
- Laboratory of Embryo Technology, Institute of Biotechnology, Vietnam Academy of Science and Technology, Hanoi 100000, Vietnam
| | - Kasem Theerakittayakorn
- Embryo Technology and Stem Cell Research Center, School of Biotechnology, Institute of Agricultural Technology, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand; (H.T.N.); (K.T.); (S.S.)
| | - Sirilak Somredngan
- Embryo Technology and Stem Cell Research Center, School of Biotechnology, Institute of Agricultural Technology, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand; (H.T.N.); (K.T.); (S.S.)
| | - Apichart Ngernsoungnern
- School of Preclinical Sciences, Institute of Science, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand; (A.N.); (P.N.); (P.S.)
| | - Piyada Ngernsoungnern
- School of Preclinical Sciences, Institute of Science, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand; (A.N.); (P.N.); (P.S.)
| | - Pishyaporn Sritangos
- School of Preclinical Sciences, Institute of Science, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand; (A.N.); (P.N.); (P.S.)
| | - Mariena Ketudat-Cairns
- School of Biotechnology, Institute of Agricultural Technology, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand; (M.K.-C.); (S.I.)
| | - Sumeth Imsoonthornruksa
- School of Biotechnology, Institute of Agricultural Technology, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand; (M.K.-C.); (S.I.)
| | - Juthaporn Assawachananont
- School of Ophthalmology, Institute of Medicine, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand;
| | - Nattawut Keeratibharat
- School of Surgery, Institute of Medicine, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand;
| | - Rangsirat Wongsan
- The Center for Scientific and Technological Equipment, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand;
| | - Ruttachuk Rungsiwiwut
- Department of Anatomy, Faculty of Medicine, Srinakharinwirot University, Bangkok 10000, Thailand;
| | - Chuti Laowtammathron
- Siriraj Center of Excellence for Stem Cell Research, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10000, Thailand;
| | | | - Rangsun Parnpai
- Embryo Technology and Stem Cell Research Center, School of Biotechnology, Institute of Agricultural Technology, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand; (H.T.N.); (K.T.); (S.S.)
- Correspondence: ; Tel.: +66-442-242-34
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21
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Jiang Z, Lin FY, Jiang K, Nguyen H, Chang CY, Lin CC. Dissolvable microgel-templated macroporous hydrogels for controlled cell assembly. BIOMATERIALS ADVANCES 2022; 134:112712. [PMID: 35581097 PMCID: PMC9358784 DOI: 10.1016/j.msec.2022.112712] [Citation(s) in RCA: 15] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/19/2021] [Revised: 12/05/2021] [Accepted: 02/08/2022] [Indexed: 12/22/2022]
Abstract
Mesenchymal stem cells (MSCs)-based therapies have been widely used to promote tissue regeneration and to modulate immune/inflammatory response. The therapeutic potential of MSCs can be further improved by forming multi-cellular spheroids. Meanwhile, hydrogels with macroporous structures are advantageous for improving mass transport properties for the cell-laden matrices. Herein, we report the fabrication of MSC-laden macroporous hydrogel scaffolds through incorporating rapidly dissolvable spherical cell-laden microgels. Dissolvable microgels were fabricated by tandem droplet-microfluidics and thiol-norbornene photopolymerization using a novel fast-degrading macromer poly(ethylene glycol)-norbornene-dopamine (PEGNB-Dopa). The cell-laden PEGNB-Dopa microgels were subsequently encapsulated within another bulk hydrogel matrix, whose porous structure was generated efficiently by the rapid degradation of the PEGNB-Dopa microgels. The cytocompatibility of this in situ pore-forming approach was demonstrated with multiple cell types. Furthermore, adjusting the stiffness and cell adhesiveness of the bulk hydrogels afforded the formation of solid cell spheroids or hollow spheres. The assembly of solid or hollow MSC spheroids led to differential activation of AKT pathway. Finally, MSCs solid spheroids formed in situ within the macroporous hydrogels exhibited robust secretion of HGF, VEGF-A, IL-6, IL-8, and TIMP-2. In summary, this platform provides an innovative method for forming cell-laden macroporous hydrogels for a variety of future biomedical applications.
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Affiliation(s)
- Zhongliang Jiang
- Department of Biomedical Engineering, Indiana University-Purdue University Indianapolis, Indianapolis, IN 46202 USA
| | - Fang-Yi Lin
- Department of Biomedical Engineering, Indiana University-Purdue University Indianapolis, Indianapolis, IN 46202 USA
| | - Kun Jiang
- Department of Biomedical Engineering, Indiana University-Purdue University Indianapolis, Indianapolis, IN 46202 USA
| | - Han Nguyen
- Weldon School of Biomedical Engineering, Purdue University, West Lafayette, IN 47907 USA
| | - Chun-Yi Chang
- Weldon School of Biomedical Engineering, Purdue University, West Lafayette, IN 47907 USA
| | - Chien-Chi Lin
- Department of Biomedical Engineering, Indiana University-Purdue University Indianapolis, Indianapolis, IN 46202, USA; Weldon School of Biomedical Engineering, Purdue University, West Lafayette, IN 47907, USA; Indiana University Simon Comprehensive Cancer Center, Indianapolis, IN 46202, USA.
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22
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Derivation and Characterization of EGFP-Labeled Rabbit Limbal Mesenchymal Stem Cells and Their Potential for Research in Regenerative Ophthalmology. Biomedicines 2021; 9:biomedicines9091134. [PMID: 34572321 PMCID: PMC8465718 DOI: 10.3390/biomedicines9091134] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/04/2021] [Revised: 08/24/2021] [Accepted: 08/28/2021] [Indexed: 12/13/2022] Open
Abstract
The development of cell-based approaches to the treatment of various cornea pathologies, including limbal stem cell deficiency (LSCD), is an area of current interest in regenerative biomedicine. In this context, the shortage of donor material is urgent, and limbal mesenchymal stem cells (L-MSCs) may become a promising cell source for the development of these novel approaches, being established mainly within the rabbit model. In this study, we obtained and characterized rabbit L-MSCs and modified them with lentiviral transduction to express the green fluorescent protein EGFP (L-MSCs-EGFP). L-MSCs and L-MSCs-EGFP express not only stem cell markers specific for mesenchymal stem cells but also ABCG2, ABCB5, ALDH3A1, PAX6, and p63a specific for limbal epithelial stem cells (LESCs), as well as various cytokeratins (3/12, 15, 19). L-MSCs-EGFP have been proven to differentiate into adipogenic, osteogenic, and chondrogenic directions, as well as to transdifferentiate into epithelial cells. The possibility of using L-MSCs-EGFP to study the biocompatibility of various scaffolds developed to treat corneal pathologies was demonstrated. L-MSCs-EGFP may become a useful tool for studying regenerative processes occurring during the treatment of various corneal pathologies, including LSCD, with the use of cell-based technologies.
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23
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Liu XN, Mi SL, Chen Y, Wang Y. Corneal stromal mesenchymal stem cells: reconstructing a bioactive cornea and repairing the corneal limbus and stromal microenvironment. Int J Ophthalmol 2021; 14:448-455. [PMID: 33747824 DOI: 10.18240/ijo.2021.03.19] [Citation(s) in RCA: 9] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/22/2020] [Accepted: 11/17/2020] [Indexed: 02/07/2023] Open
Abstract
Corneal stroma-derived mesenchymal stem cells (CS-MSCs) are mainly distributed in the anterior part of the corneal stroma near the corneal limbal stem cells (LSCs). CS-MSCs are stem cells with self-renewal and multidirectional differentiation potential. A large amount of data confirmed that CS-MSCs can be induced to differentiate into functional keratocytes in vitro, which is the motive force for maintaining corneal transparency and producing a normal corneal stroma. CS-MSCs are also an important component of the limbal microenvironment. Furthermore, they are of great significance in the reconstruction of ocular surface tissue and tissue engineering for active biocornea construction. In this paper, the localization and biological characteristics of CS-MSCs, the use of CS-MSCs to reconstruct a tissue-engineered active biocornea, and the repair of the limbal and matrix microenvironment by CS-MSCs are reviewed, and their application prospects are discussed.
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Affiliation(s)
- Xian-Ning Liu
- Department of Ophthalmology, First Hospital of Xi'an; Shaanxi Institute of Ophthalmology, Shaanxi Provincial Key Lab of Ophthalmology, Clinical Research Center for Ophthalmology Diseases of Shaanxi Province, the First Affiliated Hospital of Northwest University, Xi'an 710002, Shaanxi Province, China
| | - Sheng-Li Mi
- Open FIESTA Center, Graduate School at Shenzhen, Tsinghua University, Shenzhen 518055, Guangdong Province, China.,Biomanufacturing Engineering Laboratory, Graduate School at Shenzhen, Tsinghua University, Shenzhen 518055, Guangdong Province, China
| | - Yun Chen
- Open FIESTA Center, Graduate School at Shenzhen, Tsinghua University, Shenzhen 518055, Guangdong Province, China
| | - Yao Wang
- Department of Ophthalmology, First Hospital of Xi'an; Shaanxi Institute of Ophthalmology, Shaanxi Provincial Key Lab of Ophthalmology, Clinical Research Center for Ophthalmology Diseases of Shaanxi Province, the First Affiliated Hospital of Northwest University, Xi'an 710002, Shaanxi Province, China
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24
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Cen YJ, You DB, Wang W, Feng Y. Preliminary studies of constructing a tissue-engineered lamellar corneal graft by culturing mesenchymal stem cells onto decellularized corneal matrix. Int J Ophthalmol 2021; 14:10-18. [PMID: 33469478 DOI: 10.18240/ijo.2021.01.02] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/06/2020] [Accepted: 07/28/2020] [Indexed: 12/28/2022] Open
Abstract
AIM To construct a competent corneal lamellar substitute in order to alleviate the shortage of human corneal donor. METHODS Rabbit mesenchymal stem cells (MSCs) were isolated from bone marrow and identified by flow cytometric, osteogenic and adipogenic induction. Xenogenic decellularized corneal matrix (XDCM) was generated from dog corneas. MSCs were seeded and cultured on XDCM to construct the tissue-engineered cornea. Post-transplantation biocompatibility of engineered corneal graft were tested by animal experiment. Rabbits were divided into two groups then underwent lamellar keratoplasty (LK) with different corneal grafts: 1) XDCM group (n=5): XDCM; 2) XDCM-MSCs groups (n=4): tissue-engineered cornea made up with XDCM and MSCs. The ocular surface recovery procedure was observed while corneal transparency, neovascularization and epithelium defection were measured and compared. In vivo on focal exam was performed 3mo postoperatively. RESULTS Rabbit MSCs were isolated and identified. Flow cytometry demonstrated isolated cells were CD90 positive and CD34, CD45 negative. Osteogenic and adipogenic induction verified their multipotent abilities. MSC-XDCM grafts were constructed and observed. In vivo transplantation showed the neovascularization in XDCM-MSC group was much less than that in XDCM group postoperatively. Post-transplant 3-month confocal test showed less nerve regeneration and bigger cell-absent area in XDCM-MSC group. CONCLUSION This study present a novel corneal tissue-engineered graft that could reduce post-operatively neovascularization and remain transparency, meanwhile shows that co-transplantation of MSCs may help increase corneal transplantation successful rate and enlarge the source range of corneal substitute to overcome cornea donor shortage.
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Affiliation(s)
- Yu-Jie Cen
- Department of Ophthalmology, Peking University Third Hospital, Beijing 100191, China.,Beijing Key Laboratory of Restoration of Damaged Ocular Nerve, Peking University Third Hospital, Beijing 100191, China
| | - De-Bo You
- Department of Ophthalmology, Peking University Third Hospital, Beijing 100191, China.,Beijing Key Laboratory of Restoration of Damaged Ocular Nerve, Peking University Third Hospital, Beijing 100191, China
| | - Wei Wang
- Department of Ophthalmology, Peking University Third Hospital, Beijing 100191, China.,Beijing Key Laboratory of Restoration of Damaged Ocular Nerve, Peking University Third Hospital, Beijing 100191, China
| | - Yun Feng
- Department of Ophthalmology, Peking University Third Hospital, Beijing 100191, China.,Beijing Key Laboratory of Restoration of Damaged Ocular Nerve, Peking University Third Hospital, Beijing 100191, China
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25
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Park IS, Kim BK, Truong MD, Yang HS, Park SH, Park HS, Choi BH, Won BH, Min BH. Corneal Repair with Adhesive Cell Sheets of Fetal Cartilage-Derived Stem Cells. Tissue Eng Regen Med 2021; 18:187-198. [PMID: 33415672 DOI: 10.1007/s13770-020-00317-w] [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: 09/27/2020] [Revised: 10/22/2020] [Accepted: 10/29/2020] [Indexed: 11/28/2022] Open
Abstract
BACKGROUND Corneal scarring or disease may lead to severe corneal opacification and consequently, severe loss of vision due to the complete loss of corneal epithelial cells. We studied the use of epithelial cell sheets differentiated from fetal cartilage-derived stem cells (FCSC) to resurface damaged cornea. METHODS The FCSC were isolated from the femoral head of immature cartilage tissue. The ability of the FCSCs to differentiate into corneal epithelial cells was evaluated using differentiation media at 2 days and 7 days post-seeding. A sheet fabricated of FCSCs was also used for the differentiation assay. The results of the in vitro studies were evaluated by immunocytochemistry and Western blots for corneal epithelial cell markers (CK3/12 and Pax6) and limbal epithelial stem cell markers (ABCG2 and p63). To test the material in vivo, an FCSC-sheet was applied as a treatment in a chemically burned rabbit model. The healing ability was observed histologically one week after treatment. RESULTS The in vitro experiments showed morphological changes in the FCSCs at two and seven days of culture. The differentiated cells from the FCSCs or the FCSC-sheet expressed corneal epithelial cells markers. FCSC were create cell sheet that successfully differentiated into corneal epithelial cells and had sufficient adhesion so that it could be fused to host tissue after suture to the ocular surface with silk suture. The implanted cell sheet maintained its transparency and the cells were alive a week after implantation. CONCLUSION These results suggest that carrier-free sheets fabricated of FCSCs have the potential to repair damaged corneal surfaces.
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Affiliation(s)
- In-Su Park
- Cell Therapy Center, Ajou University School of Medicine, 164 World cup-ro, Yeongtong-gu, Suwon, Gyeonggi-do, 16499, Republic of Korea
| | - Byeong Kook Kim
- Cell Therapy Center, Ajou University School of Medicine, 164 World cup-ro, Yeongtong-gu, Suwon, Gyeonggi-do, 16499, Republic of Korea
| | - Minh-Dung Truong
- Cell Therapy Center, Ajou University School of Medicine, 164 World cup-ro, Yeongtong-gu, Suwon, Gyeonggi-do, 16499, Republic of Korea
| | - Hong Seok Yang
- Ophthalmologic Department, Ajou University School of Medicine, 164 World cup-ro, Yeongtong-gu, Suwon, Gyeonggi-do, 16499, Republic of Korea
| | - Sang-Hyug Park
- Department of Biomedical Engineering, Pukyong National University, Busan, 48513, Republic of Korea
| | - Hyo Soon Park
- Nunevit Eye Clinic, 772 Gayadea-ro, Busanjin-gu, Busan, 47285, Republic of Korea
| | - Byung Hyune Choi
- Department of Physiology, Inha University College of Medicine, 100 Inha-ro, Michuhol-gu, Incheon, 22212, Republic of Korea
| | - Bae Hie Won
- Department of Orthopedic Surgery, Ajou University School of Medicine, 164 World cup-ro, Yeongtong-gu, Suwon, Gyeonggi-do, 16499, Republic of Korea
| | - Byoung-Hyun Min
- Cell Therapy Center, Ajou University School of Medicine, 164 World cup-ro, Yeongtong-gu, Suwon, Gyeonggi-do, 16499, Republic of Korea. .,Department of Orthopedic Surgery, Ajou University School of Medicine, 164 World cup-ro, Yeongtong-gu, Suwon, Gyeonggi-do, 16499, Republic of Korea. .,Department of Molecular Science and Technology, Ajou University, 164 World cup-ro, Yeongtong-gu, Suwon, Gyeonggi-do, 16499, Republic of Korea.
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26
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Comparison of similar cells: Mesenchymal stromal cells and fibroblasts. Acta Histochem 2020; 122:151634. [PMID: 33059115 PMCID: PMC7550172 DOI: 10.1016/j.acthis.2020.151634] [Citation(s) in RCA: 41] [Impact Index Per Article: 8.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/28/2020] [Revised: 09/03/2020] [Accepted: 09/23/2020] [Indexed: 12/23/2022]
Abstract
Almost from all organs, both mesenchymal stromal cells and fibroblasts can be isolated. Mesenchymal stromal cells (MSCs) are the most preferred cellular therapeutic agents with the regenerative potential, and fibroblasts are one of the most abundant cell types with the ability to maintain homeostasis. Because of the promising properties of MSCs, they have been well studied and their differentiation potentials, immunomodulatory potentials, gene expression profiles are identified. It has been observed that fibroblasts and mesenchymal stromal cells have similar morphology, gene expression patterns, surface markers, proliferation, differentiation, and immunomodulatory capacities. Thus, it is hard to distinguish these two cell types. Epigenetic signatures, i.e., methylation patterns of cells, are the only usable promising difference between them. Such significant similarities show that these two cells may be related to each other.
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27
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Xeno-free approach for the expansion of human adipose derived mesenchymal stem cells for ocular therapies. Exp Eye Res 2020; 202:108358. [PMID: 33207223 DOI: 10.1016/j.exer.2020.108358] [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: 08/21/2020] [Revised: 11/05/2020] [Accepted: 11/09/2020] [Indexed: 11/21/2022]
Abstract
To restore corneal transparency and vision loss after an injury on the ocular surface, the use of human stem cells from different origins has been recently proposed. Mesenchymal stem cells (MSCs) seem to be an appropriate adult source of autologous stem cells due to their accessibility, high proliferation rate, and multipotent capacity. In this work, we developed a simple culture system to prepare a graft based on a fibrin membrane seeded with human MSCs. A commercial kit, PRGF Endoret®, was used to prepare both, the growth factors used as culture media supplement and the fibrin membrane grafts. Adipose-derived MSCs (Ad-MSCs) were expanded, characterised by flow cytometry and their multilineage differentiation potential confirmed by inducing adipogenesis, osteogenesis and chondrogenesis. Ad-MSCs seeded on the fibrin membranes were grafted onto athymic mice showing good biocompatibility with no adverse reactions observed during the follow up period. These findings support the assumption that a system in which all the biological components (cells, grow factors and carrier) are autologous, could potentially be used for future ex vivo expansion of Ad-MSCs to treat ocular conditions such as an inflammatory milieu, traumatic scars and loss of the regenerative capacity of the corneal epithelium that compromise the quality of vision.
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28
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Theerakittayakorn K, Thi Nguyen H, Musika J, Kunkanjanawan H, Imsoonthornruksa S, Somredngan S, Ketudat-Cairns M, Parnpai R. Differentiation Induction of Human Stem Cells for Corneal Epithelial Regeneration. Int J Mol Sci 2020; 21:E7834. [PMID: 33105778 PMCID: PMC7660084 DOI: 10.3390/ijms21217834] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/25/2020] [Revised: 10/16/2020] [Accepted: 10/17/2020] [Indexed: 12/13/2022] Open
Abstract
Deficiency of corneal epithelium causes vision impairment or blindness in severe cases. Transplantation of corneal epithelial cells is an effective treatment but the availability of the tissue source for those cells is inadequate. Stem cells can be induced to differentiate to corneal epithelial cells and used in the treatment. Multipotent stem cells (mesenchymal stem cells) and pluripotent stem cells (embryonic stem cells and induced pluripotent stem cells) are promising cells to address the problem. Various protocols have been developed to induce differentiation of the stem cells into corneal epithelial cells. The feasibility and efficacy of both human stem cells and animal stem cells have been investigated for corneal epithelium regeneration. However, some physiological aspects of animal stem cells are different from those of human stem cells, the protocols suited for animal stem cells might not be suitable for human stem cells. Therefore, in this review, only the investigations of corneal epithelial differentiation of human stem cells are taken into account. The available protocols for inducing the differentiation of human stem cells into corneal epithelial cells are gathered and compared. Also, the pathways involving in the differentiation are provided to elucidate the relevant mechanisms.
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Affiliation(s)
- Kasem Theerakittayakorn
- Embryo Technology and Stem Cell Research Center, School of Biotechnology, Institute of Agricultural Technology, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand; (K.T.); (H.T.N.); (J.M.); (S.I.); (S.S.); (M.K.-C.)
| | - Hong Thi Nguyen
- Embryo Technology and Stem Cell Research Center, School of Biotechnology, Institute of Agricultural Technology, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand; (K.T.); (H.T.N.); (J.M.); (S.I.); (S.S.); (M.K.-C.)
| | - Jidapa Musika
- Embryo Technology and Stem Cell Research Center, School of Biotechnology, Institute of Agricultural Technology, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand; (K.T.); (H.T.N.); (J.M.); (S.I.); (S.S.); (M.K.-C.)
| | - Hataiwan Kunkanjanawan
- Medeze Research and Development Co., Ltd. 28/9 Moo 8, Phutthamonthon Sai 4 Rd., Krathum Lom, Sam Phran, Nakhon Pathom 73220, Thailand;
| | - Sumeth Imsoonthornruksa
- Embryo Technology and Stem Cell Research Center, School of Biotechnology, Institute of Agricultural Technology, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand; (K.T.); (H.T.N.); (J.M.); (S.I.); (S.S.); (M.K.-C.)
| | - Sirilak Somredngan
- Embryo Technology and Stem Cell Research Center, School of Biotechnology, Institute of Agricultural Technology, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand; (K.T.); (H.T.N.); (J.M.); (S.I.); (S.S.); (M.K.-C.)
| | - Mariena Ketudat-Cairns
- Embryo Technology and Stem Cell Research Center, School of Biotechnology, Institute of Agricultural Technology, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand; (K.T.); (H.T.N.); (J.M.); (S.I.); (S.S.); (M.K.-C.)
| | - Rangsun Parnpai
- Embryo Technology and Stem Cell Research Center, School of Biotechnology, Institute of Agricultural Technology, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand; (K.T.); (H.T.N.); (J.M.); (S.I.); (S.S.); (M.K.-C.)
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29
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Mesenchymal Stem Cells in Synovial Fluid Increase in Knees with Degenerative Meniscus Injury after Arthroscopic Procedures through the Endogenous Effects of CGRP and HGF. Stem Cell Rev Rep 2020; 16:1305-1315. [DOI: 10.1007/s12015-020-10047-0] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 09/25/2020] [Indexed: 12/22/2022]
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30
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Tseng SCG, Chen SY, Mead OG, Tighe S. Niche regulation of limbal epithelial stem cells: HC-HA/PTX3 as surrogate matrix niche. Exp Eye Res 2020; 199:108181. [PMID: 32795525 DOI: 10.1016/j.exer.2020.108181] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/27/2020] [Revised: 06/15/2020] [Accepted: 07/31/2020] [Indexed: 12/13/2022]
Abstract
Homeostasis of the corneal epithelium is ultimately maintained by stem cells that reside in a specialized microenvironment within the corneal limbus termed palisades of Vogt. This limbal niche nourishes, protects, and regulates quiescence, self-renewal, and fate decision of limbal epithelial stem/progenitor cells (LEPCs) toward corneal epithelial differentiation. This review focuses on our current understanding of the mechanism by which limbal (stromal) niche cells (LNCs) regulate the aforementioned functions of LEPCs. Based on our discovery and characterization of a unique extracellular matrix termed HC-HA/PTX3 (Heavy chain (HC1)-hyaluronan (HA)/pentraxin 3 (PTX3) complex, "-" denotes covalent linkage; "/" denotes non-covalent binding) in the birth tissue, i.e., amniotic membrane and umbilical cord, we put forth a new paradigm that HC-HA/PTX3 serves as a surrogate matrix niche by maintaining the in vivo nuclear Pax6+ neural crest progenitor phenotype to support quiescence and self-renewal but prevent corneal fate decision of LEPCs. This new paradigm helps explain how limbal stem cell deficiency (LSCD) develops in aniridia due to Pax6-haplotype deficiency and further explains why transplantation of HC-HA/PTX3-containing amniotic membrane prevents LSCD in acute chemical burns and Stevens Johnson syndrome, augments the success of autologous LEPCs transplantation in patients suffering from partial or total LSCD, and assists ex vivo expansion (engineering) of a graft containing LEPCs. We thus envisage that this new paradigm based on regenerative matrix HC-HA/PTX3 as a surrogate niche can set a new standard for regenerative medicine in and beyond ophthalmology.
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Affiliation(s)
- Scheffer C G Tseng
- Research & Development Department, TissueTech, Inc., Miami, FL, 33126, USA; Ocular Surface Center and Ocular Surface Research & Education Foundation, Miami, FL, 33126, USA.
| | - Szu-Yu Chen
- Research & Development Department, TissueTech, Inc., Miami, FL, 33126, USA
| | - Olivia G Mead
- Research & Development Department, TissueTech, Inc., Miami, FL, 33126, USA
| | - Sean Tighe
- Research & Development Department, TissueTech, Inc., Miami, FL, 33126, USA; Department of Biochemistry and Molecular Biology, University of Miami Miller School of Medicine, Miami, FL, 33136, USA; Department of Ophthalmology, Herbert Wertheim College of Medicine, Florida International University, Miami, FL, 33199, USA
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31
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Nieto-Nicolau N, Martín-Antonio B, Müller-Sánchez C, Casaroli-Marano RP. In vitro potential of human mesenchymal stem cells for corneal epithelial regeneration. Regen Med 2020; 15:1409-1426. [PMID: 32352350 DOI: 10.2217/rme-2019-0067] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023] Open
Abstract
Aim: To determine the potential of mesenchymal stem cells (MSC) for corneal epithelial regeneration in vitro. Materials & methods: Bone marrow MSC (BM-MSC) and adipose tissue MSC were analyzed for corneal epithelial and mesenchymal markers, using limbal stem cells and corneal cells as controls. MSC with better potential were cultured with specific mediums for epithelial induction. Transepithelial electric resistance and wound healing assay with human corneal epithelial cells were performed. Results: BM-MSC showed better potential, increased corneal markers, and higher transepithelial electric resistance values when induced with limbal epithelial culture medium. Induced BM-MSC promoted better wound healing of human corneal epithelial cells by paracrine secretion. Conclusion: BM-MSC has potential for corneal epithelial induction in a protocol compatible with human application.
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Affiliation(s)
| | | | | | - Ricardo P Casaroli-Marano
- Barcelona Tissue Bank, Banc de Sang I Teixits (BST), Barcelona, Spain.,Department of Surgery, School of Medicine & Hospital Clinic de Barcelona, University of Barcelona, Barcelona, Spain.,Institute of Biomedical Research Sant Pau (IIB-Sant Pau), Barcelona, Spain
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32
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Wang W, Li S, Xu L, Jiang M, Li X, Zhang Y, Tighe S, Zhu Y, Li G. Differential Gene Expression between Limbal Niche Progenitors and Bone Marrow Derived Mesenchymal Stem Cells. Int J Med Sci 2020; 17:549-557. [PMID: 32174786 PMCID: PMC7053302 DOI: 10.7150/ijms.40881] [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] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/03/2019] [Accepted: 01/20/2020] [Indexed: 12/21/2022] Open
Abstract
Purpose: To compare the difference in gene expression between human limbal niche cells (LNC) and bone marrow derived mesenchymal stem cells (BMMSC). Methods: LNC were isolated by collagenase and expanded in modified embryonic stem cell medium (MESCM) on a Matrigel coated plastic plate. Cell diameters were measured with Image J software. Relative gene expression levels between LNC and BMMSC were compared using Affymetrix Human Primer View Gene Expression Array. A subset of differentially expressed genes was verified by RT-qPCR. The protein level of LAMA1 and COL4A1 was confirmed by Western blot and immunostaining. Results: The average diameter of LNC was 10.2±2.4 μm, which was significantly smaller than that of BMMSC (14 ±3.4 μm) (p<0.0001). Expression of 20,432 genes was examined by Gene Expression Array, among which expression of 349 genes in LNC was 10-fold or higher than that of BMMSC and expression of 8 genes in LNC was 100-fold or higher than that of BMMSC, while expression of 3 genes in BMMSC was 100-fold higher than that of LNC. GO analysis and pathway analysis showed that the differentially expressed genes were mainly enriched in the extracellular matrix receptor interaction pathway and Wnt signaling pathway. In addition, RT-qPCR results demonstrated that the expression of CD73, CD90, CD105, PDGFRβ, Vimentin, SCF, KIT (CD117), COL14A1, LAMA2, THBS2, FZD1, BMP2 and CXCL12 genes in LNC were at least 2 folds higher than BMMSC. The protein level of LAMA1 was higher but the protein level of COL4A1 was lower in LNC than that in BMMSC. Conclusion: LNC exhibit differential gene expression from BMMSC in the extracellular matrix (ECM) receptor interaction pathway and Wnt signaling pathway, suggesting that LNC have their unique signaling pathways to support limbal stem cell niches.
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Affiliation(s)
- Wei Wang
- Department of Ophthalmology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei Province 430030, China
| | - Shen Li
- Department of Ophthalmology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei Province 430030, China
| | - Lingjuan Xu
- Department of Ophthalmology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei Province 430030, China
| | - Menglin Jiang
- Department of Ophthalmology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei Province 430030, China
| | - Xinyu Li
- Department of Ophthalmology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei Province 430030, China
| | - Yuan Zhang
- Tissue Tech Inc, Miami, Florida, 33126 USA
| | - Sean Tighe
- Tissue Tech Inc, Miami, Florida, 33126 USA
| | | | - Guigang Li
- Department of Ophthalmology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei Province 430030, China
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Han Y, Li X, Zhang Y, Han Y, Chang F, Ding J. Mesenchymal Stem Cells for Regenerative Medicine. Cells 2019; 8:E886. [PMID: 31412678 PMCID: PMC6721852 DOI: 10.3390/cells8080886] [Citation(s) in RCA: 715] [Impact Index Per Article: 119.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/12/2019] [Revised: 08/05/2019] [Accepted: 08/06/2019] [Indexed: 02/06/2023] Open
Abstract
In recent decades, the biomedical applications of mesenchymal stem cells (MSCs) have attracted increasing attention. MSCs are easily extracted from the bone marrow, fat, and synovium, and differentiate into various cell lineages according to the requirements of specific biomedical applications. As MSCs do not express significant histocompatibility complexes and immune stimulating molecules, they are not detected by immune surveillance and do not lead to graft rejection after transplantation. These properties make them competent biomedical candidates, especially in tissue engineering. We present a brief overview of MSC extraction methods and subsequent potential for differentiation, and a comprehensive overview of their preclinical and clinical applications in regenerative medicine, and discuss future challenges.
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Affiliation(s)
- Yu Han
- Department of Orthopedics, The Second Hospital of Jilin University, 218 Ziqiang Street, Changchun 130041, China
- Key Laboratory of Polymer Ecomaterials, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, 5625 Renmin Street, Changchun 130022, China
| | - Xuezhou Li
- Department of Orthopedics, The Second Hospital of Jilin University, 218 Ziqiang Street, Changchun 130041, China
- Key Laboratory of Polymer Ecomaterials, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, 5625 Renmin Street, Changchun 130022, China
| | - Yanbo Zhang
- Department of Orthopedics, China-Japan Union Hospital of Jilin University, 126 Xiantai Street, Changchun 130033, China.
| | - Yuping Han
- Department of Urology, China-Japan Union Hospital of Jilin University, 126 Xiantai Street, Changchun 130033, China.
| | - Fei Chang
- Department of Orthopedics, The Second Hospital of Jilin University, 218 Ziqiang Street, Changchun 130041, China.
| | - Jianxun Ding
- Key Laboratory of Polymer Ecomaterials, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, 5625 Renmin Street, Changchun 130022, China
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Venugopal B, Shenoy SJ, Mohan S, Anil Kumar PR, Kumary TV. Bioengineered corneal epithelial cell sheet from mesenchymal stem cells-A functional alternative to limbal stem cells for ocular surface reconstruction. J Biomed Mater Res B Appl Biomater 2019; 108:1033-1045. [PMID: 31400069 DOI: 10.1002/jbm.b.34455] [Citation(s) in RCA: 25] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/23/2018] [Revised: 05/25/2019] [Accepted: 07/17/2019] [Indexed: 12/13/2022]
Abstract
Limbal stem cell deficiency (LSCD) is the loss of limbal stem cells that reside in the corneoscleral junction resulting in vision loss or blindness. Bilateral LSCD is usually treated by allogeneic corneal transplantation, with instances of tissue rejection or failure in long-term follow-up. This study aims to use adipose mesenchymal stem cells (ASC) as an alternative autologous cell source for treating bilateral limbal deficiency conditions. ASCs derived from rabbit fat tissue were differentiated into corneal epithelial lineage using limbal explant condition media. Apart from transdifferentiation, ASC sheets were developed to facilitate effective delivery of these cells to the damage site. A thermoresponsive polymer N-isopropylacrylamide-co-glycidylmethacrylate (NGMA) was synthesized and characterized to demonstrate ASC sheet formation. Transdifferentiated ASCs showed positive expression of corneal epithelial marker CK3/12 on immunostaining, supported by gene expression studies. in vivo studies by transplanting cell sheet in rabbit models of corneal injury showed clear and smooth cornea in comparison to the sham models. Histology revealed a sheet of cells aligned and integrated on to the injured corneal surface, 1 month posttransplantation. Identifying ASCs as an alternative cell source along with cell sheet technology will be a novel step in the field of corneal surface therapies.
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Affiliation(s)
- Balu Venugopal
- Division of Tissue Culture, Biomedical Technology Wing, Sree Chitra Tirunal Institute for Medical Sciences and Technology, Trivandrum, Kerala, India
| | - Sachin J Shenoy
- Division of in vivo Modes and Testing, Biomedical Technology Wing, Sree Chitra Tirunal Institute for Medical Sciences and Technology, Trivandrum, Kerala, India
| | - Sumitha Mohan
- Division of Tissue Culture, Biomedical Technology Wing, Sree Chitra Tirunal Institute for Medical Sciences and Technology, Trivandrum, Kerala, India
| | - P R Anil Kumar
- Division of Tissue Culture, Biomedical Technology Wing, Sree Chitra Tirunal Institute for Medical Sciences and Technology, Trivandrum, Kerala, India
| | - T V Kumary
- Division of Tissue Culture, Biomedical Technology Wing, Sree Chitra Tirunal Institute for Medical Sciences and Technology, Trivandrum, Kerala, India
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Chen Y, Yin YW, Zhao Y, Wu XY, Young K, Song WT, Xia XB, Wen D. Differentiation of human embryonic stem cells derived mesenchymal stem cells into corneal epithelial cells after being seeded on decellularized SMILE-derived lenticules. Int J Ophthalmol 2019; 12:717-724. [PMID: 31131228 DOI: 10.18240/ijo.2019.05.04] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/07/2018] [Accepted: 03/27/2019] [Indexed: 12/14/2022] Open
Abstract
AIM To evaluate the feasibility of mesenchymal stem cells (MSCs) to differentiate into corneal epithelial cells after being seeded on the decellularized small incision lenticule extraction (SMILE)-derived lenticules. METHODS The fresh lenticules procured from patients undergoing SMILE for the correction of myopia were decellularized. The MSCs were subsequently cultivated on those denuded lenticules. The MSCs without lenticules were used as a control. The proliferation activity of the MSCs after seeding 24h was quantitatively determined with the Cell Counting Kit-8 (CCK-8) assay. Immunofluorescence staining and quantitative reverse transcription polymerase chain reaction (qRT-PCR) were used to assess the marker expression in differentiated MSCs. RESULTS The data showed that both fresh and decellularized lenticules could significantly promote the proliferation of MSCs, compared to that in control (P=0.02 for fresh lenticules, P=0.001 for decellularize ones, respectively). The MSCs seeded on both lenticules were positive for cytokeratin 3 (CK3) staining. The expression of CK3 increased 5-fold in MSCs seeded on fresh lenticules and 18-fold on decellularized ones, compared to that in control. There was a significant difference in the expression of CK3 in MSCs seeded on fresh and decellularized lenticules (P<0.001). The expression of CK8 and CK18 was similar in pure MSCs and MSCs seeded on fresh lenticules (P>0.05), while the expression of these markers was decreased in MSCs seeded on decellularized ones. CONCLUSION These results suggest that the decellularized lenticules might be more suitable for MSCs to differentiate into corneal epithelial cells, which offers the prospect of a novel therapeutic modality of SMILE-derived lenticules in regenerative corneal engineering.
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Affiliation(s)
- Yao Chen
- Department of Ophthalmology, Xiangya Hospital, Central South University, Changsha 410008, Hunan Province, China
| | - Ye-Wei Yin
- Department of Ophthalmology, Xiangya Hospital, Central South University, Changsha 410008, Hunan Province, China
| | - Yang Zhao
- Department of Ophthalmology, Xiangya Hospital, Central South University, Changsha 410008, Hunan Province, China
| | - Xiao-Ying Wu
- Department of Ophthalmology, Xiangya Hospital, Central South University, Changsha 410008, Hunan Province, China
| | - Kelly Young
- Department of Veterans Affairs, Miami VA Healthcare System, Miami, Florida 33125, USA
| | - Wei-Tao Song
- Department of Ophthalmology, Xiangya Hospital, Central South University, Changsha 410008, Hunan Province, China
| | - Xiao-Bo Xia
- Department of Ophthalmology, Xiangya Hospital, Central South University, Changsha 410008, Hunan Province, China
| | - Dan Wen
- Department of Ophthalmology, Xiangya Hospital, Central South University, Changsha 410008, Hunan Province, China
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Comparative gene expression profiling reveals key pathways and genes different in skin epidermal stem cells and corneal epithelial cells. Genes Genomics 2019; 41:679-688. [DOI: 10.1007/s13258-019-00814-w] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/30/2017] [Accepted: 03/26/2019] [Indexed: 01/20/2023]
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Alió del Barrio JL, Alió JL. Cellular therapy of the corneal stroma: a new type of corneal surgery for keratoconus and corneal dystrophies. EYE AND VISION (LONDON, ENGLAND) 2018; 5:28. [PMID: 30410944 PMCID: PMC6211455 DOI: 10.1186/s40662-018-0122-1] [Citation(s) in RCA: 30] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 06/01/2018] [Accepted: 10/17/2018] [Indexed: 12/13/2022]
Abstract
Cellular therapy of the corneal stroma, with either ocular or extraocular stem cells, has been gaining a lot of interest over the last decade. Multiple publications from different research groups are showing its potential benefits in relation to its capacity to improve or alleviate corneal scars, improve corneal transparency in metabolic diseases by enhancing the catabolism of the accumulated molecules, generate new organized collagen within the host stroma, and its immunosuppressive and immunomodulatory properties. Autologous extraocular stem cells do not require a healthy contralateral eye and they do not involve any ophthalmic procedures for their isolation. Mesenchymal stem cells have been the most widely assayed and have the best potential to differentiate into functional adult keratocytes in vivo and in vitro. While embryonic stem cells have been partially abandoned due to ethical implications, the discovery of the induced pluripotent stem cells (iPSC) has opened a new and very promising field for future research as they are pluripotent cells with the capacity to theoretically differentiate into any cell type, with the special advantage that they are obtained from adult differentiated cells. Cellular delivery into the corneal stroma has been experimentally assayed in vivo in multiple ways: systemic versus local injections with or without a carrier. Encouraging preliminary human clinical data is already available although still very limited, and further research is necessary in order to consolidate the clinical applications of this novel therapeutic line.
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Affiliation(s)
- Jorge L. Alió del Barrio
- Cornea, Cataract and Refractive Surgery Unit, Vissum Corporación, Alicante, Spain
- Division of Ophthalmology, Universidad Miguel Hernández, Alicante, Spain
- Vissum, Instituto Oftalmologico de Alicante, Avda de Denia s/n, 03016 Alicante, Spain
| | - Jorge L. Alió
- Cornea, Cataract and Refractive Surgery Unit, Vissum Corporación, Alicante, Spain
- Division of Ophthalmology, Universidad Miguel Hernández, Alicante, Spain
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Abstract
Mesenchymal Stem Cells (MSCs) are a heterogeneous population of fibroblast-like cells which maintain self-renewability and pluripotency to differentiate into mesodermal cell lineages. The use of MSCs in clinical settings began with high enthusiasm and the number of MSC-based clinical trials has been rising ever since. However; the very unique characteristics of MSCs that made them suitable to for therapeutic use, might give rise to unwanted outcomes, including tumor formation and progression. In this paper, we present a model of carcinogenesis initiated by MSCs, which chains together the tissue organization field theory, the stem cell theory, and the inflammation-cancer chain. We believe that some tissue resident stem cells could be leaked cells from bone marrow MSC pool to various injured tissue, which consequently transform and integrate in the host tissue. If the injury persists or chronic inflammation develops, as a consequence of recurring exposure to growth factors, cytokines, etc. the newly formed tissue from MSCs, which still has conserved their mesenchymal and stemness features, go through rapid population expansion, and nullify their tumor suppressor genes, and hence give rise to neoplastic cell (carcinomas, sarcomas, and carcino-sarcomas). Considering the probability of this hypothesis being true, the clinical and therapeutic use of MSCs should be with caution, and the recipients' long term follow-up seems to be insightful.
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Affiliation(s)
- Abbas Ghaderi
- Shiraz Institute for Cancer Research, School of Medicine, Shiraz University of Medical Sciences, PO.Box: 71345-1798, Shiraz, Iran.
| | - Shabnam Abtahi
- Shiraz Institute for Cancer Research, School of Medicine, Shiraz University of Medical Sciences, PO.Box: 71345-1798, Shiraz, Iran
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39
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Coculture of conjunctiva derived mesenchymal stem cells (CJMSCs) and corneal epithelial cells to reconstruct the corneal epithelium. Biologicals 2018; 54:39-43. [DOI: 10.1016/j.biologicals.2018.04.005] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/18/2018] [Revised: 03/26/2018] [Accepted: 04/25/2018] [Indexed: 11/21/2022] Open
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40
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Trans-differentiation Induction of Human-mesenchymal Stem Cells Derived from Different Tissue Origin and Evaluation of their Potential for Differentiation into Corneal Epithelial-like Cells. JOURNAL OF ANIMAL REPRODUCTION AND BIOTECHNOLOGY 2018. [DOI: 10.12750/jet.2018.33.2.85] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022] Open
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41
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Soundararajan M, Kannan S. Fibroblasts and mesenchymal stem cells: Two sides of the same coin? J Cell Physiol 2018; 233:9099-9109. [DOI: 10.1002/jcp.26860] [Citation(s) in RCA: 84] [Impact Index Per Article: 12.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/01/2018] [Accepted: 05/23/2018] [Indexed: 12/13/2022]
Affiliation(s)
| | - Suresh Kannan
- Department of Biomedical Sciences Sri Ramachandra University Chennai Tamil Nadu India
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42
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Dohlman CH, Cade F, Regatieri CV, Zhou C, Lei F, Crnej A, Harissi-Dagher M, Robert MC, Papaliodis GN, Chen D, Aquavella JV, Akpek EK, Aldave AJ, Sippel KC, DʼAmico DJ, Dohlman JG, Fagerholm P, Wang L, Shen LQ, González-Andrades M, Chodosh J, Kenyon KR, Foster CS, Pineda R, Melki S, Colby KA, Ciolino JB, Vavvas DG, Kinoshita S, Dana R, Paschalis EI. Chemical Burns of the Eye: The Role of Retinal Injury and New Therapeutic Possibilities. Cornea 2018; 37:248-251. [PMID: 29135604 PMCID: PMC8728745 DOI: 10.1097/ico.0000000000001438] [Citation(s) in RCA: 30] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/26/2022]
Abstract
PURPOSE To propose a new treatment paradigm for chemical burns to the eye - in the acute and chronic phases. METHODS Recent laboratory and clinical data on the biology and treatment of chemical burns are analyzed. RESULTS Corneal blindness from chemical burns can now be successfully treated with a keratoprosthesis, on immediate and intermediate bases. Long term outcomes, however, are hampered by early retinal damage causing glaucoma. New data suggest that rapid diffusion of inflammatory cytokines posteriorly (TNF-α, etc) can severely damage the ganglion cells. Prompt anti-TNF-α treatment is markedly neuroprotective. Long term profound reduction of the intraocular pressure is also vital. CONCLUSION A new regimen, in addition to standard treatment, for severe chemical burns is proposed. This involves tumor necrosis factor alpha (TNF-α) inhibition promptly after the accident (primarily for retinal neuroprotection), prophylactic maximal lowering of the intraocular pressure (starting immediately), and keratoprosthesis implantation in a later quiet state.
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Affiliation(s)
- Claes H Dohlman
- Cornea Service and Boston Keratoprosthesis Laboratory, Massachusetts Eye and Ear Infirmary and Schepens Eye Research Institute, Harvard Medical School, Boston, MA
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Guo Y, Ma X, Wu W, Shi M, Ma J, Zhang Y, Zhao E, Yang X. Coordinated microRNA/mRNA expression profiles reveal a putative mechanism of corneal epithelial cell transdifferentiation from skin epidermal stem cells. Int J Mol Med 2017; 41:877-887. [PMID: 29207049 PMCID: PMC5752239 DOI: 10.3892/ijmm.2017.3304] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/16/2016] [Accepted: 11/15/2017] [Indexed: 01/09/2023] Open
Abstract
Skin epidermal stem cells (SESCs), which share a single origin with corneal epithelial cells (CECs), are considered to be one of the most ideal seed cells for the construction of tissue engineered corneas. However, the mechanism underlying the transdifferentiation of SESCs to CECs has not been fully elucidated. In the present study, to identify critical microRNAs (miRNAs/miRs) and genes that regulate the transdifferentiation of SESCs to CECs, SESCs and CECs were collected from sheep and used for small RNA sequencing and mRNA microarray analyses. Among the differentially expressed miRNAs and genes, 36 miRNAs were downregulated and 123 genes were upregulated in the CECs compared with those in the SESCs. miR-10b exhibited the largest change in expression between the cell types. Target genes of the 36 downregulated miRNAs were predicted and a computational approach demonstrated that these target genes may be involved in several signaling pathways, including the 'PI3K signaling pathway', the 'Wnt signaling pathway' and the 'MAPK signaling pathway', as well as in 'focal adhesion'. Comparison of these target genes to the 123 upregulated genes identified 43 intersection genes. A regulatory network of these 43 intersection genes and its correlative miRNAs were constructed, and three genes (dedicator of cytokinesis 9, neuronal differentiation 1 and activated leukocyte cell adhesion molecule) were found to have high interaction frequencies. The expression levels of 7 randomly selected miRNAs and the 3 intersection genes were further validated by reverse transcription-quantitative polymerase chain reaction. It was found that miR-10b, the Wnt signaling pathway and the 3 intersection genes may act together and serve a critical role in the transdifferentiation process. This study identified miRNAs and genes that were expressed in SESCs and CECs that may assist in uncovering its underlying molecular mechanism, as well as promote corneal tissue engineering using epidermal stem cells for clinical applications.
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Affiliation(s)
- Yanjie Guo
- Life Science College, Luoyang Normal University, Luoyang, Henan 471934, P.R. China
| | - Xiya Ma
- Life Science College, Luoyang Normal University, Luoyang, Henan 471934, P.R. China
| | - Weini Wu
- Life Science College, Luoyang Normal University, Luoyang, Henan 471934, P.R. China
| | - Mingyan Shi
- Life Science College, Luoyang Normal University, Luoyang, Henan 471934, P.R. China
| | - Junlong Ma
- Life Science College, Luoyang Normal University, Luoyang, Henan 471934, P.R. China
| | - Yaping Zhang
- Life Science College, Luoyang Normal University, Luoyang, Henan 471934, P.R. China
| | - Erkang Zhao
- Life Science College, Luoyang Normal University, Luoyang, Henan 471934, P.R. China
| | - Xueyi Yang
- Life Science College, Luoyang Normal University, Luoyang, Henan 471934, P.R. China
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Gonzalez G, Sasamoto Y, Ksander BR, Frank MH, Frank NY. Limbal stem cells: identity, developmental origin, and therapeutic potential. WILEY INTERDISCIPLINARY REVIEWS-DEVELOPMENTAL BIOLOGY 2017; 7. [PMID: 29105366 DOI: 10.1002/wdev.303] [Citation(s) in RCA: 82] [Impact Index Per Article: 10.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 05/20/2017] [Revised: 08/22/2017] [Accepted: 09/03/2017] [Indexed: 12/15/2022]
Abstract
The cornea is our window to the world and our vision is critically dependent on corneal clarity and integrity. Its epithelium represents one of the most rapidly regenerating mammalian tissues, undergoing full-turnover over the course of approximately 1-2 weeks. This robust and efficient regenerative capacity is dependent on the function of stem cells residing in the limbus, a structure marking the border between the cornea and the conjunctiva. Limbal stem cells (LSC) represent a quiescent cell population with proliferative capacity residing in the basal epithelial layer of the limbus within a cellular niche. In addition to LSC, this niche consists of various cell populations such as limbal stromal fibroblasts, melanocytes and immune cells as well as a basement membrane, all of which are essential for LSC maintenance and LSC-driven regeneration. The LSC niche's components are of diverse developmental origin, a fact that had, until recently, prevented precise identification of molecularly defined LSC. The recent success in prospective LSC isolation based on ABCB5 expression and the capacity of this LSC population for long-term corneal restoration following transplantation in preclinical in vivo models of LSC deficiency underline the considerable potential of pure LSC formulations for clinical therapy. Additional studies, including genetic lineage tracing of the developmental origin of LSC will further improve our understanding of this critical cell population and its niche, with important implications for regenerative medicine. WIREs Dev Biol 2018, 7:e303. doi: 10.1002/wdev.303 This article is categorized under: Adult Stem Cells, Tissue Renewal, and Regeneration > Stem Cells and Disease Adult Stem Cells, Tissue Renewal, and Regeneration > Tissue Stem Cells and Niches Adult Stem Cells, Tissue Renewal, and Regeneration > Regeneration.
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Affiliation(s)
- Gabriel Gonzalez
- Department of Medicine, VA Boston Healthcare System, Boston, MA, USA.,Division of Genetics, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA
| | - Yuzuru Sasamoto
- Department of Medicine, VA Boston Healthcare System, Boston, MA, USA
| | - Bruce R Ksander
- Massachusetts Eye and Ear, Schepens Eye Research Institute, Harvard Medical School, Boston, MA, USA
| | - Markus H Frank
- Transplant Research Program, Boston Children's Hospital, Harvard Medical School, Boston, MA, USA.,Harvard Stem Cell Institute, Harvard University, Cambridge, MA, USA.,School of Medical and Health Sciences, Edith Cowan University, Perth, Western Australia, Australia
| | - Natasha Y Frank
- Department of Medicine, VA Boston Healthcare System, Boston, MA, USA.,Division of Genetics, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA
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Soleimanifar F, Mortazavi Y, Nadri S, Soleimani M. Conjunctiva derived mesenchymal stem cell (CJMSCs) as a potential platform for differentiation into corneal epithelial cells on bioengineered electrospun scaffolds. J Biomed Mater Res A 2017; 105:2703-2711. [DOI: 10.1002/jbm.a.36123] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/27/2017] [Revised: 05/16/2017] [Accepted: 05/23/2017] [Indexed: 11/05/2022]
Affiliation(s)
- Fatemeh Soleimanifar
- Medical Biotechnology and Nanotechnology Department, Faculty of Medicine; Zanjan University of Medical Sciences; Zanjan Iran
| | - Yousef Mortazavi
- Medical Biotechnology and Nanotechnology Department, Faculty of Medicine; Zanjan University of Medical Sciences; Zanjan Iran
| | - Samad Nadri
- Medical Biotechnology and Nanotechnology Department, Faculty of Medicine; Zanjan University of Medical Sciences; Zanjan Iran
| | - Masoud Soleimani
- Department of Hematology and Blood Banking, Faculty of Medicine; Tarbiat Modaress University; Tehran Iran
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Wound-Healing Studies in Cornea and Skin: Parallels, Differences and Opportunities. Int J Mol Sci 2017; 18:ijms18061257. [PMID: 28604651 PMCID: PMC5486079 DOI: 10.3390/ijms18061257] [Citation(s) in RCA: 139] [Impact Index Per Article: 17.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/13/2017] [Revised: 05/24/2017] [Accepted: 05/31/2017] [Indexed: 02/06/2023] Open
Abstract
The cornea and the skin are both organs that provide the outer barrier of the body. Both tissues have developed intrinsic mechanisms that protect the organism from a wide range of external threats, but at the same time also enable rapid restoration of tissue integrity and organ-specific function. The easy accessibility makes the skin an attractive model system to study tissue damage and repair. Findings from skin research have contributed to unravelling novel fundamental principles in regenerative biology and the repair of other epithelial-mesenchymal tissues, such as the cornea. Following barrier disruption, the influx of inflammatory cells, myofibroblast differentiation, extracellular matrix synthesis and scar formation present parallel repair mechanisms in cornea and skin wound healing. Yet, capillary sprouting, while pivotal in proper skin wound healing, is a process that is rather associated with pathological repair of the cornea. Understanding the parallels and differences of the cellular and molecular networks that coordinate the wound healing response in skin and cornea are likely of mutual importance for both organs with regard to the development of regenerative therapies and understanding of the disease pathologies that affect epithelial-mesenchymal interactions. Here, we review the principal events in corneal wound healing and the mechanisms to restore corneal transparency and barrier function. We also refer to skin repair mechanisms and their potential implications for regenerative processes in the cornea.
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47
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Aharony I, Michowiz S, Goldenberg-Cohen N. The promise of stem cell-based therapeutics in ophthalmology. Neural Regen Res 2017; 12:173-180. [PMID: 28400789 PMCID: PMC5361491 DOI: 10.4103/1673-5374.200793] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
Abstract
The promising role of cellular therapies in the preservation and restoration of visual function has prompted intensive efforts to characterize embryonic, adult, and induced pluripotent stem cells for regenerative purposes. Three main approaches to the use of stem cells have been described: sustained drug delivery, immunomodulation, and differentiation into various ocular structures. Studies of the differentiation capacity of all three types of stem cells into epithelial, neural, glial and vascular phenotypes have reached proof-of-concept in culture, but the correction of vision is still in the early developmental stages, and the requirements for effective in vivo implementation are still unclear. We present an overview of some of the preclinical findings on stem-cell rescue and regeneration of the cornea and retina in acute injury and degenerative disorders.
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Affiliation(s)
- Israel Aharony
- The Krieger Eye Research Laboratory, Felsenstein Medical Research Center, Petach Tikva, Israel; Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel
| | - Shalom Michowiz
- The Krieger Eye Research Laboratory, Felsenstein Medical Research Center, Petach Tikva, Israel; Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel; Department of Neurosurgery, Rabin Medical Center - Beilinson Hospital, Petach Tikva, Israel
| | - Nitza Goldenberg-Cohen
- The Krieger Eye Research Laboratory, Felsenstein Medical Research Center, Petach Tikva, Israel; Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel; Department of Ophthalmology, Bnai Zion Medical Center, Haifa, Israel
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Katikireddy KR, Schmedt T, Price MO, Price FW, Jurkunas UV. Existence of Neural Crest-Derived Progenitor Cells in Normal and Fuchs Endothelial Dystrophy Corneal Endothelium. THE AMERICAN JOURNAL OF PATHOLOGY 2016; 186:2736-50. [PMID: 27639969 DOI: 10.1016/j.ajpath.2016.06.011] [Citation(s) in RCA: 34] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 04/13/2016] [Revised: 06/16/2016] [Accepted: 06/21/2016] [Indexed: 12/13/2022]
Abstract
Human corneal endothelial cells are derived from neural crest and because of postmitotic arrest lack competence to repair cell loss from trauma, aging, and degenerative disorders such as Fuchs endothelial corneal dystrophy (FECD). Herein, we identified a rapidly proliferating subpopulation of cells from the corneal endothelium of adult normal and FECD donors that exhibited features of neural crest-derived progenitor (NCDP) cells by showing absence of senescence with passaging, propensity to form spheres, and increased colony forming efficacy compared with the primary cells. The collective expression of stem cell-related genes SOX2, OCT4, LGR5, TP63 (p63), as well as neural crest marker genes PSIP1 (p75(NTR)), PAX3, SOX9, AP2B1 (AP-2β), and NES, generated a phenotypic footprint of endothelial NCDPs. NCDPs displayed multipotency by differentiating into microtubule-associated protein 2, β-III tubulin, and glial fibrillary acidic protein positive neurons and into p75(NTR)-positive human corneal endothelial cells that exhibited transendothelial resistance of functional endothelium. In conclusion, we found that mitotically incompetent ocular tissue cells contain adult NCDPs that exhibit a profile of transcription factors regulating multipotency and neural crest progenitor characteristics. Identification of normal NCDPs in FECD-affected endothelium holds promise for potential autologous cell therapies.
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Affiliation(s)
| | - Thore Schmedt
- Schepens Eye Research Institute, Massachusetts Eye and Ear, Boston, Massachusetts; AbbVie Deutschland GmbH & Co KG, Ludwigshafen, Germany
| | | | | | - Ula V Jurkunas
- Schepens Eye Research Institute, Massachusetts Eye and Ear, Boston, Massachusetts.
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Tomasello L, Musso R, Cillino G, Pitrone M, Pizzolanti G, Coppola A, Arancio W, Di Cara G, Pucci-Minafra I, Cillino S, Giordano C. Donor age and long-term culture do not negatively influence the stem potential of limbal fibroblast-like stem cells. Stem Cell Res Ther 2016; 7:83. [PMID: 27296060 PMCID: PMC4906894 DOI: 10.1186/s13287-016-0342-z] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/16/2015] [Revised: 01/14/2016] [Accepted: 05/16/2016] [Indexed: 12/13/2022] Open
Abstract
BACKGROUND In regenerative medicine the maintenance of stem cell properties is of crucial importance. Ageing is considered a cause of reduced stemness capability. The limbus is a stem niche of easy access and harbors two stem cell populations: epithelial stem cells and fibroblast-like stem cells. Our aim was to investigate whether donor age and/or long-term culture have any influence on stem cell marker expression and the profiles in the fibroblast-like stem cell population. METHODS Fibroblast-like stem cells were isolated and digested from 25 limbus samples of normal human corneo-scleral rings and long-term cultures were obtained. SSEA4 expression and sphere-forming capability were evaluated; cytofluorimetric assay was performed to detect the immunophenotypes HLA-DR, CD45, and CD34 and the principle stem cell markers ABCG2, OCT3/4, and NANOG. Molecular expression of the principal mesenchymal stem cell genes was investigated by real-time PCR. Two-dimensional gel electrophoresis and mass spectrometric sequencing were performed and a stable proteomic profile was identified. The proteins detected were explored by gene ontology and STRING analysis. The data were reported as means ± SD, compared by Student's unpaired t test and considering p < 0.05 as statistically significant. RESULTS The isolated cells did not display any hematopoietic surface marker (CD34 and CD45) and HLA-DR and they maintained these features in long-term culture. The expression of the stemness genes and the multilineage differentiation under in-vitro culture conditions proved to be well maintained. Proteomic analysis revealed a fibroblast-like stem cell profile of 164 proteins with higher expression levels. Eighty of these showed stable expression levels and were involved in maintenance of "the stem gene profile"; 84 were differentially expressed and were involved in structural activity. CONCLUSIONS The fibroblast-like limbal stem cells confirmed that they are a robust source of adult stem cells and that they have good plasticity, good proliferative capability, and long-term maintenance of stem cell properties, independently of donor age and long-term culture conditions. Our findings confirm that limbal fibroblast-like stem cells are highly promising for application in regenerative medicine and that in-vitro culture steps do not influence their stem cell properties. Moreover, the proteomic data enrich our knowledge of fibroblast-like stem cells.
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Affiliation(s)
- Laura Tomasello
- Laboratory of Regenerative Medicine, Section of Endocrinology, Diabetology and Metabolism, Di.Bi.M.I.S., University of Palermo, Piazza delle Cliniche 2, 90127, Palermo, Italy
| | - Rosa Musso
- Centro di Oncobiologia Sperimentale (COBS), Palermo, Italy
| | - Giovanni Cillino
- Department of Ophthalmology, University of Palermo, Palermo, Italy
| | - Maria Pitrone
- Laboratory of Regenerative Medicine, Section of Endocrinology, Diabetology and Metabolism, Di.Bi.M.I.S., University of Palermo, Piazza delle Cliniche 2, 90127, Palermo, Italy
| | - Giuseppe Pizzolanti
- Laboratory of Regenerative Medicine, Section of Endocrinology, Diabetology and Metabolism, Di.Bi.M.I.S., University of Palermo, Piazza delle Cliniche 2, 90127, Palermo, Italy
- ATeN (Advanced Technologies Network Center), University of Palermo, Palermo, Italy
| | - Antonina Coppola
- Laboratory of Regenerative Medicine, Section of Endocrinology, Diabetology and Metabolism, Di.Bi.M.I.S., University of Palermo, Piazza delle Cliniche 2, 90127, Palermo, Italy
| | - Walter Arancio
- Laboratory of Regenerative Medicine, Section of Endocrinology, Diabetology and Metabolism, Di.Bi.M.I.S., University of Palermo, Piazza delle Cliniche 2, 90127, Palermo, Italy
| | | | | | | | - Carla Giordano
- Laboratory of Regenerative Medicine, Section of Endocrinology, Diabetology and Metabolism, Di.Bi.M.I.S., University of Palermo, Piazza delle Cliniche 2, 90127, Palermo, Italy.
- ATeN (Advanced Technologies Network Center), University of Palermo, Palermo, Italy.
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Mathan JJ, Ismail S, McGhee JJ, McGhee CNJ, Sherwin T. Sphere-forming cells from peripheral cornea demonstrate the ability to repopulate the ocular surface. Stem Cell Res Ther 2016; 7:81. [PMID: 27250558 PMCID: PMC4888426 DOI: 10.1186/s13287-016-0339-7] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/22/2016] [Revised: 04/25/2016] [Accepted: 05/06/2016] [Indexed: 02/07/2023] Open
Abstract
BACKGROUND The limbus forms the outer rim of the cornea at the corneoscleral junction and harbours a population of stem cells for corneal maintenance. Injuries to the limbus, through disease or accidents such as chemical injuries or burns, may lead to significant visual impairment due to depletion of the native stem cells of the tissue. METHODS Sphere-forming cells were isolated from peripheral cornea for potential use as transplantable elements for limbal stem cell repopulation and limbal reconstruction. Immunocytochemistry, live cell imaging and quantitative PCR were used to characterize spheres and elucidate activity post implantation into human cadaveric corneal tissue. RESULTS Spheres stained positively for stem cell markers ∆NP63α, ABCG2 and ABCB5 as well as the basal limbal marker and putative niche marker, notch 1. In addition, spheres also stained positively for markers of corneal cells, vimentin, keratin 3, keratocan and laminin, indicating a heterogeneous mix of stromal and epithelial-origin cells. Upon implantation into decellularized corneoscleral tissue, 3D, polarized and radially orientated cell migration with cell proliferation was observed. Cells migrated out from the spheres and repopulated the entire corneal surface over 14 days. Post-implantation analysis revealed qualitative evidence of stem, stromal and epithelial cell markers while quantitative PCR showed a quantitative reduction in keratocan and laminin expression indicative of an enhanced progenitor cell response. Proliferation, quantified by PCNA expression, significantly increased at 4 days subsequently followed by a decrease at day 7 post implantation. CONCLUSION These observations suggest great promise for the potential of peripheral corneal spheres as transplantable units for corneal repair, targeting ocular surface regeneration and stem cell repopulation.
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Affiliation(s)
- Jeremy John Mathan
- Department of Ophthalmology, New Zealand National Eye Centre, Faculty of Medical and Health Sciences, The University of Auckland, Private Bag 92019, Auckland, 1010, New Zealand
| | - Salim Ismail
- Department of Ophthalmology, New Zealand National Eye Centre, Faculty of Medical and Health Sciences, The University of Auckland, Private Bag 92019, Auckland, 1010, New Zealand
| | - Jennifer Jane McGhee
- Department of Ophthalmology, New Zealand National Eye Centre, Faculty of Medical and Health Sciences, The University of Auckland, Private Bag 92019, Auckland, 1010, New Zealand
| | - Charles Ninian John McGhee
- Department of Ophthalmology, New Zealand National Eye Centre, Faculty of Medical and Health Sciences, The University of Auckland, Private Bag 92019, Auckland, 1010, New Zealand
| | - Trevor Sherwin
- Department of Ophthalmology, New Zealand National Eye Centre, Faculty of Medical and Health Sciences, The University of Auckland, Private Bag 92019, Auckland, 1010, New Zealand.
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