The Experts below are selected from a list of 31620 Experts worldwide ranked by ideXlab platform
Shigeru Kinoshita - One of the best experts on this subject based on the ideXlab platform.
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PAX6 regulates human corneal epithelium cell identity.
Experimental eye research, 2016Co-Authors: Koji Kitazawa, Takahiro Nakamura, Shigeru Kinoshita, Chie Sotozono, Takafusa Hikichi, Shinji MasuiAbstract:PAX6, a paired box transcription factor, is necessary for eye development. However, how it regulates the cell identity of human corneal epithelial cells (CECs) is not well understood. We aimed to clarify the function of PAX6 in human CECs using gene knockout via the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR associated protein 9 (Cas9) system. We designed guide RNAs for different targets in PAX6. PAX6-depleted CECs maintained the epithelial morphology, but became larger. Global analyses using microarray revealed that down-regulated genes were primarily CEC-specific and included Keratin 12, Keratin 3, clusterin (CLU), aldehyde dehydrogenase 3 family member A1 (ALDH3A1), angiopoietin-like 7 (ANGPTL7) and transketolase (TKT), while up-regulated genes were primarily epidermis-related and included Keratin 10, Keratin 1, involucrin (IVL), filaggrin (FLG). These findings suggest that PAX6 maintains CEC identity by regulating differentiation.
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long term phenotypic study after allogeneic cultivated corneal limbal epithelial transplantation for severe ocular surface diseases
Ophthalmology, 2010Co-Authors: Takahiro Nakamura, Chie Sotozono, Noriko Koizumi, Tsutomu Inatomi, Nigel J Fullwood, Adam J Bentley, Sachiko Mano, Shigeru KinoshitaAbstract:Purpose To determine the long-term epithelial lineage of origin of surgically removed grafts after allogeneic cultivated corneal limbal epithelial transplantation (CLET). Design Interventional case reports. Participants We studied 2 eyes from 2 patients with total corneal stem cell destruction; 1 eye was from a patient with Stevens-Johnson syndrome and 1 eye had sustained chemical injury. Methods Allogeneic cultivated corneal limbal epithelial sheets on human amniotic membrane (AM) were transplanted onto the ocular surface. Regrafting (1 eye, 42 months later) or penetrating keratoplasty (1 eye, 75 months later) were performed after the initial transplantation procedure for further visual rehabilitation. Main Outcome Measures The excised grafts were subjected to clinical evaluation and to light- and transmission electron microscopy (TEM) examination and to immunohistochemical analysis. Results In clinically conjunctival grafts, TEM and immunohistochemical analysis disclosed only small areas where the original cultivated corneal epithelial cells persisted. Neighboring conjunctival epithelial cells had apparently invaded a large portion of the corneal surface (Keratin 3/12(−), Muc5ac(+)). In clinically corneal grafts, transplanted allogeneic cultivated corneal epithelial cells clearly survived for a long period of time (Keratin 3/12(+), Muc5ac(−)); there was no infiltration by inflammatory cells, nor was there dissolution of the AM substrate. Conclusions We theorize that the process of graft opacification after allogeneic CLET is responsible for the loss of transplanted cultivated corneal epithelial cells and that this is followed by conjunctival cell invasion onto the corneal surface. The results of this study confirmed that in the clinically evaluated corneal graft, transplanted cultivated corneal epithelial cells indeed survived for a long period of time on the corneal surface and maintained ocular surface integrity, even though the transplanted cells were allogeneic. Financial Disclosure(s) The authors have no proprietary or commercial interest in any of the materials discussed in this article.
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phenotypic investigation of human eyes with transplanted autologous cultivated oral mucosal epithelial sheets for severe ocular surface diseases
Ophthalmology, 2007Co-Authors: Takahiro Nakamura, Tsutomu Inatomi, Leanne J Cooper, Helen Rigby, Nigel J Fullwood, Shigeru KinoshitaAbstract:Purpose To determine the epithelial lineage of origin of surgically removed grafts after autologous cultivated oral mucosal epithelial transplantation (COMET). Design Retrospective comparative case series. Participants We studied 6 eyes from 5 patients with total corneal stem cell destruction; 3 eyes were from patients with Stevens–Johnson syndrome and 3 eyes had sustained chemical injury. Methods Autologous cultivated oral mucosal epithelial sheets on human amniotic membrane (AM) were transplanted onto the ocular surface. Regrafting (2 eyes) or penetrating keratoplasty (4 eyes) was performed after the initial transplantation procedure for further visual rehabilitation. Main Outcome Measures The excised grafts were subjected to clinical evaluation and to light, scanning, and transmission electron microscopic (EM) study and to immunohistochemical analysis. Results In clinically failed grafts, EM and immunohistochemical analysis disclosed only small areas where the original cultivated oral epithelial cells persisted. Neighboring conjunctival epithelial cells had apparently invaded a large portion of the corneal surface (Keratin 3[−], Muc5ac[+]); there were many blood vessels and inflammatory cells. In clinically successful grafts, transplanted cultivated oral epithelial cells survived and had adapted well to the host corneal tissues (Keratin 3[+], Muc5ac[−]); there was no infiltration by inflammatory cells, nor was there dissolution of the AM substrate. Conclusions We posit that the process of graft opacification after COMET is responsible for the loss of transplanted cultivated oral epithelial cells and that this is followed by conjunctival cell invasion onto the corneal surface. We confirmed that in clinically successfully grafted eyes, autologous cultivated oral epithelial cells survived on the corneal surface and maintained ocular surface integrity.
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transplantable cultivated mucosal epithelial sheet for ocular surface reconstruction
Experimental Eye Research, 2004Co-Authors: Shigeru Kinoshita, Noriko Koizumi, Takahiro NakamuraAbstract:Ocular surface reconstruction by tissue engineering using somatic stem cells is a second-generation therapeutic modality. In view of future treatment of bilaterally affected, severe ocular surface disorders, two types of transplantable cultivated mucosal epithelial sheets can be used for reconstruction. One is an allogeneic corneal epithelial stem cell sheet, and the other is an autologous oral mucosal epithelial cell sheet. We first investigated the feasibility of amniotic membrane as an epithelial carrier, and found that denuded amniotic membrane was the most appropriate substrate for this purpose. Thus, cultivated corneal epithelial stem cell sheets were created by co-culturing with 3T3 fibroblast and air-lifting on amniotic membrane. These epithelial sheets demonstrated positive Keratin 3 and 12 specific to in vivo corneal epithelium, tight junction related proteins and proliferative activity. The transplanted allogeneic human corneal epithelial sheets existed successfully on the corneal surface, and were quite effective in achieving ocular surface stability in severe ocular surface disorders. A few cases, however, developed immunological reactions or opportunistic infections, etc. Secondly, we established transplantable autologous cultivated oral mucosal epithelial sheets in rabbits. The in vitro oral mucosal epithelial sheets showed histological characteristics similar to those of in vivo corneal epithelial sheets; for example, positive Keratin 3 expression. Based on the fact that, the transplanted autologous oral mucosal epithelial sheets resembled corneal epithelium and that we achieved the recovery of corneal transparency in rabbits, we propose that cultivated oral mucosal epithelium may become the substitute for corneal epithelium in ocular surface reconstruction.
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Cultivated oral mucosal epithelial transplantation for ocular surface reconstruction
Ensho Saisei, 2004Co-Authors: Takahiro Nakamura, Shigeru KinoshitaAbstract:Autologous cultivated mucosal epithelial cells from a non-ocular surface origin were investigated to determine their possible use in ocular surface reconstruction. An ocular-surface injury was created in one eye of each of the adult albino rabbits used, via lamellar keratectomy. Next, oral mucosal biopsies were taken from these rabbits and then cultivated for 2-3 weeks on denuded amniotic membrane. The cultivated oral epithelial sheets were examined by electron microscopy (EM) and immunohistochemically labeled for several Keratins. EM revealed that the epithelial cells were very similar in appearance to those of in vivo normal corneal epithelium. It also showed that they had numerous desmosomal junctions and were strongly attached to a basement membrane with hemi-desmosomes. Immunohistochemistry confirmed the presence of non-Keratinized, stratified Keratins 4/13 and cornea-specific Keratin 3. All of the corneas that were transplanted with the autologous cultivated oral epithelial sheets were clear and epithelialized 10 days after transplantation. From these results, we conclude that the autologous transplantation of cultivated oral epithelium is a feasible procedure for ocular surface reconstruction.
Takahiro Nakamura - One of the best experts on this subject based on the ideXlab platform.
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PAX6 regulates human corneal epithelium cell identity.
Experimental eye research, 2016Co-Authors: Koji Kitazawa, Takahiro Nakamura, Shigeru Kinoshita, Chie Sotozono, Takafusa Hikichi, Shinji MasuiAbstract:PAX6, a paired box transcription factor, is necessary for eye development. However, how it regulates the cell identity of human corneal epithelial cells (CECs) is not well understood. We aimed to clarify the function of PAX6 in human CECs using gene knockout via the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR associated protein 9 (Cas9) system. We designed guide RNAs for different targets in PAX6. PAX6-depleted CECs maintained the epithelial morphology, but became larger. Global analyses using microarray revealed that down-regulated genes were primarily CEC-specific and included Keratin 12, Keratin 3, clusterin (CLU), aldehyde dehydrogenase 3 family member A1 (ALDH3A1), angiopoietin-like 7 (ANGPTL7) and transketolase (TKT), while up-regulated genes were primarily epidermis-related and included Keratin 10, Keratin 1, involucrin (IVL), filaggrin (FLG). These findings suggest that PAX6 maintains CEC identity by regulating differentiation.
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long term phenotypic study after allogeneic cultivated corneal limbal epithelial transplantation for severe ocular surface diseases
Ophthalmology, 2010Co-Authors: Takahiro Nakamura, Chie Sotozono, Noriko Koizumi, Tsutomu Inatomi, Nigel J Fullwood, Adam J Bentley, Sachiko Mano, Shigeru KinoshitaAbstract:Purpose To determine the long-term epithelial lineage of origin of surgically removed grafts after allogeneic cultivated corneal limbal epithelial transplantation (CLET). Design Interventional case reports. Participants We studied 2 eyes from 2 patients with total corneal stem cell destruction; 1 eye was from a patient with Stevens-Johnson syndrome and 1 eye had sustained chemical injury. Methods Allogeneic cultivated corneal limbal epithelial sheets on human amniotic membrane (AM) were transplanted onto the ocular surface. Regrafting (1 eye, 42 months later) or penetrating keratoplasty (1 eye, 75 months later) were performed after the initial transplantation procedure for further visual rehabilitation. Main Outcome Measures The excised grafts were subjected to clinical evaluation and to light- and transmission electron microscopy (TEM) examination and to immunohistochemical analysis. Results In clinically conjunctival grafts, TEM and immunohistochemical analysis disclosed only small areas where the original cultivated corneal epithelial cells persisted. Neighboring conjunctival epithelial cells had apparently invaded a large portion of the corneal surface (Keratin 3/12(−), Muc5ac(+)). In clinically corneal grafts, transplanted allogeneic cultivated corneal epithelial cells clearly survived for a long period of time (Keratin 3/12(+), Muc5ac(−)); there was no infiltration by inflammatory cells, nor was there dissolution of the AM substrate. Conclusions We theorize that the process of graft opacification after allogeneic CLET is responsible for the loss of transplanted cultivated corneal epithelial cells and that this is followed by conjunctival cell invasion onto the corneal surface. The results of this study confirmed that in the clinically evaluated corneal graft, transplanted cultivated corneal epithelial cells indeed survived for a long period of time on the corneal surface and maintained ocular surface integrity, even though the transplanted cells were allogeneic. Financial Disclosure(s) The authors have no proprietary or commercial interest in any of the materials discussed in this article.
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phenotypic investigation of human eyes with transplanted autologous cultivated oral mucosal epithelial sheets for severe ocular surface diseases
Ophthalmology, 2007Co-Authors: Takahiro Nakamura, Tsutomu Inatomi, Leanne J Cooper, Helen Rigby, Nigel J Fullwood, Shigeru KinoshitaAbstract:Purpose To determine the epithelial lineage of origin of surgically removed grafts after autologous cultivated oral mucosal epithelial transplantation (COMET). Design Retrospective comparative case series. Participants We studied 6 eyes from 5 patients with total corneal stem cell destruction; 3 eyes were from patients with Stevens–Johnson syndrome and 3 eyes had sustained chemical injury. Methods Autologous cultivated oral mucosal epithelial sheets on human amniotic membrane (AM) were transplanted onto the ocular surface. Regrafting (2 eyes) or penetrating keratoplasty (4 eyes) was performed after the initial transplantation procedure for further visual rehabilitation. Main Outcome Measures The excised grafts were subjected to clinical evaluation and to light, scanning, and transmission electron microscopic (EM) study and to immunohistochemical analysis. Results In clinically failed grafts, EM and immunohistochemical analysis disclosed only small areas where the original cultivated oral epithelial cells persisted. Neighboring conjunctival epithelial cells had apparently invaded a large portion of the corneal surface (Keratin 3[−], Muc5ac[+]); there were many blood vessels and inflammatory cells. In clinically successful grafts, transplanted cultivated oral epithelial cells survived and had adapted well to the host corneal tissues (Keratin 3[+], Muc5ac[−]); there was no infiltration by inflammatory cells, nor was there dissolution of the AM substrate. Conclusions We posit that the process of graft opacification after COMET is responsible for the loss of transplanted cultivated oral epithelial cells and that this is followed by conjunctival cell invasion onto the corneal surface. We confirmed that in clinically successfully grafted eyes, autologous cultivated oral epithelial cells survived on the corneal surface and maintained ocular surface integrity.
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transplantable cultivated mucosal epithelial sheet for ocular surface reconstruction
Experimental Eye Research, 2004Co-Authors: Shigeru Kinoshita, Noriko Koizumi, Takahiro NakamuraAbstract:Ocular surface reconstruction by tissue engineering using somatic stem cells is a second-generation therapeutic modality. In view of future treatment of bilaterally affected, severe ocular surface disorders, two types of transplantable cultivated mucosal epithelial sheets can be used for reconstruction. One is an allogeneic corneal epithelial stem cell sheet, and the other is an autologous oral mucosal epithelial cell sheet. We first investigated the feasibility of amniotic membrane as an epithelial carrier, and found that denuded amniotic membrane was the most appropriate substrate for this purpose. Thus, cultivated corneal epithelial stem cell sheets were created by co-culturing with 3T3 fibroblast and air-lifting on amniotic membrane. These epithelial sheets demonstrated positive Keratin 3 and 12 specific to in vivo corneal epithelium, tight junction related proteins and proliferative activity. The transplanted allogeneic human corneal epithelial sheets existed successfully on the corneal surface, and were quite effective in achieving ocular surface stability in severe ocular surface disorders. A few cases, however, developed immunological reactions or opportunistic infections, etc. Secondly, we established transplantable autologous cultivated oral mucosal epithelial sheets in rabbits. The in vitro oral mucosal epithelial sheets showed histological characteristics similar to those of in vivo corneal epithelial sheets; for example, positive Keratin 3 expression. Based on the fact that, the transplanted autologous oral mucosal epithelial sheets resembled corneal epithelium and that we achieved the recovery of corneal transparency in rabbits, we propose that cultivated oral mucosal epithelium may become the substitute for corneal epithelium in ocular surface reconstruction.
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Cultivated oral mucosal epithelial transplantation for ocular surface reconstruction
Ensho Saisei, 2004Co-Authors: Takahiro Nakamura, Shigeru KinoshitaAbstract:Autologous cultivated mucosal epithelial cells from a non-ocular surface origin were investigated to determine their possible use in ocular surface reconstruction. An ocular-surface injury was created in one eye of each of the adult albino rabbits used, via lamellar keratectomy. Next, oral mucosal biopsies were taken from these rabbits and then cultivated for 2-3 weeks on denuded amniotic membrane. The cultivated oral epithelial sheets were examined by electron microscopy (EM) and immunohistochemically labeled for several Keratins. EM revealed that the epithelial cells were very similar in appearance to those of in vivo normal corneal epithelium. It also showed that they had numerous desmosomal junctions and were strongly attached to a basement membrane with hemi-desmosomes. Immunohistochemistry confirmed the presence of non-Keratinized, stratified Keratins 4/13 and cornea-specific Keratin 3. All of the corneas that were transplanted with the autologous cultivated oral epithelial sheets were clear and epithelialized 10 days after transplantation. From these results, we conclude that the autologous transplantation of cultivated oral epithelium is a feasible procedure for ocular surface reconstruction.
Junzo Tanaka - One of the best experts on this subject based on the ideXlab platform.
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amniotic membrane immobilized poly vinyl alcohol hybrid polymer as an artificial cornea scaffold that supports a stratified and differentiated corneal epithelium
Journal of Biomedical Materials Research Part B, 2007Co-Authors: Yuichi Uchino, Hideyuki Miyashita, Shigeto Shimmura, Hisatoshi Kobayashi, Tetsushi Taguchi, Jun Shimazaki, Junzo TanakaAbstract:Poly(vinyl alcohol) (PVA) is a biocompatible, transparent hydrogel with physical strength that makes it promising as a material for an artificial cornea. In our previous study, type I collagen was immobilized onto PVA (PVA-COL) as a possible artificial cornea scaffold that can sustain a functional corneal epithelium. The cellular adhesiveness of PVA in vitro was improved by collagen immobilization; however, stable epithelialization was not achieved in vivo. To improve epithelialization in vivo, we created an amniotic membrane (AM)-immobilized polyvinyl alcohol hydrogel (PVA-AM) for use as an artificial cornea material. AM was attached to PVA-COL using a tissue adhesive consisting of collagen and citric acid derivative (CAD) as a crosslinker. Rabbit corneal epithelial cells were air-lift cultured with 3T3 feeder fibroblasts to form a stratified epithelial layer on PVA-AM. The rabbit corneal epithelial cells formed 3–5 layers of Keratin-3-positive epithelium on PVA-AM. Occludin-positive cells were observed lining the superficial epithelium, the gap-junctional protein connexin43-positive cells was localized to the cell membrane of the basal epithelium, while both collagen IV were observed in the basement membrane. Epithelialization over implanted PVA-AM was complete within 2 weeks, with little inflammation or opacification of the hydrogel. Corneal epithelialization on PVA-AM in rabbit corneas improved over PVA-COL, suggesting the possibility of using PVA-AM as a biocompatible hybrid material for keratoprosthesis. © 2006 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2006
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amniotic membrane immobilized poly vinyl alcohol hybrid polymer as an artificial cornea scaffold that supports a stratified and differentiated corneal epithelium
Journal of Biomedical Materials Research Part B, 2007Co-Authors: Yuichi Uchino, Hideyuki Miyashita, Shigeto Shimmura, Hisatoshi Kobayashi, Tetsushi Taguchi, Jun Shimazaki, Junzo TanakaAbstract:Poly(vinyl alcohol) (PVA) is a biocompatible, transparent hydrogel with physical strength that makes it promising as a material for an artificial cornea. In our previous study, type I collagen was immobilized onto PVA (PVA-COL) as a possible artificial cornea scaffold that can sustain a functional corneal epithelium. The cellular adhesiveness of PVA in vitro was improved by collagen immobilization; however, stable epithelialization was not achieved in vivo. To improve epithelialization in vivo, we created an amniotic membrane (AM)-immobilized polyvinyl alcohol hydrogel (PVA-AM) for use as an artificial cornea material. AM was attached to PVA-COL using a tissue adhesive consisting of collagen and citric acid derivative (CAD) as a crosslinker. Rabbit corneal epithelial cells were air-lift cultured with 3T3 feeder fibroblasts to form a stratified epithelial layer on PVA-AM. The rabbit corneal epithelial cells formed 3-5 layers of Keratin-3-positive epithelium on PVA-AM. Occludin-positive cells were observed lining the superficial epithelium, the gap-junctional protein connexin43-positive cells was localized to the cell membrane of the basal epithelium, while both collagen IV were observed in the basement membrane. Epithelialization over implanted PVA-AM was complete within 2 weeks, with little inflammation or opacification of the hydrogel. Corneal epithelialization on PVA-AM in rabbit corneas improved over PVA-COL, suggesting the possibility of using PVA-AM as a biocompatible hybrid material for keratoprosthesis.
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Collagen-immobilized poly(vinyl alcohol) as an artificial cornea scaffold that supports a stratified corneal epithelium.
Journal of biomedical materials research. Part B Applied biomaterials, 2006Co-Authors: Hideyuki Miyashita, Shigeto Shimmura, Hisatoshi Kobayashi, Tetsushi Taguchi, Naoko Asano-kato, Yuichi Uchino, Masabumi Kato, Jun Shimazaki, Junzo Tanaka, Kazuo TsubotaAbstract:The cornea is a transparent tissue of the eye, which is responsible for the refraction of incoming light. Both biological corneal equivalents and synthetic keratoprostheses have been developed to replace donor tissue as a means to restore vision. However, both designs have drawbacks in terms of stability and biocompatibility. Clinically available synthetic devices do not support an intact epithelium, which poses a risk of microbial infection or protrusion of the prosthesis. In the present study, type I collagen was immobilized onto poly(vinyl alcohol) (PVA-COL) as a possible artificial cornea scaffold that can sustain a functional corneal epithelium. Human and rabbit corneal epithelial cells were air-lift cultured with 3T3 feeder fibroblasts to form a stratified epithelial layer on PVA-COL. The epithelial sheet expressed Keratin 3/12 differentiation markers, the tight junction protein occludin, and had characteristic microvilli structures on transmission electron microscopy. Functionally, the stratified epithelium contained normal glycogen levels, and an apical tight-junction network was observed to exclude the diffusion of horseradish peroxidase. Furthermore, the epithelium-PVA-COL composite was suturable in the rabbit cornea, suggesting the possibility of using PVA-COL as a biocompatible material for keratoprosthesis.
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collagen immobilized poly vinyl alcohol as an artificial cornea scaffold that supports a stratified corneal epithelium
Journal of Biomedical Materials Research Part B, 2006Co-Authors: Hideyuki Miyashita, Shigeto Shimmura, Hisatoshi Kobayashi, Tetsushi Taguchi, Yuichi Uchino, Masabumi Kato, Jun Shimazaki, Junzo Tanaka, Naoko Asanokato, Kazuo TsubotaAbstract:The cornea is a transparent tissue of the eye, which is responsible for the refraction of incoming light. Both biological corneal equivalents and synthetic keratoprostheses have been developed to replace donor tissue as a means to restore vision. However, both designs have drawbacks in terms of stability and biocompatibility. Clinically available synthetic devices do not support an intact epithelium, which poses a risk of microbial infection or protrusion of the prosthesis. In the present study, type I collagen was immobilized onto poly(vinyl alcohol) (PVA-COL) as a possible artificial cornea scaffold that can sustain a functional corneal epithelium. Human and rabbit corneal epithelial cells were air-lift cultured with 3T3 feeder fibroblasts to form a stratified epithelial layer on PVA-COL. The epithelial sheet expressed Keratin 3/12 differentiation markers, the tight junction protein occludin, and had characteristic microvilli structures on transmission electron microscopy. Functionally, the stratified epithelium contained normal glycogen levels, and an apical tight-junction network was observed to exclude the diffusion of horseradish peroxidase. Furthermore, the epithelium-PVA-COL composite was suturable in the rabbit cornea, suggesting the possibility of using PVA-COL as a biocompatible material for keratoprosthesis. © 2005 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2006
Kohji Nishida - One of the best experts on this subject based on the ideXlab platform.
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PAX6 Isoforms, along with Reprogramming Factors, Differentially Regulate the Induction of Cornea-specific Genes
Scientific Reports, 2016Co-Authors: Yuzuru Sasamoto, Satoshi Kawasaki, Andrew J. Quantock, Ryuhei Hayashi, Sung-joon Park, Mihoko Saito-adachi, Yutaka Suzuki, Kenta Nakai, Motokazu Tsujikawa, Kohji NishidaAbstract:PAX6 is the key transcription factor involved in eye development in humans, but the differential functions of the two PAX6 isoforms, isoform-a and isoform-b, are largely unknown. To reveal their function in the corneal epithelium, PAX6 isoforms, along with reprogramming factors, were transduced into human non-ocular epithelial cells. Herein, we show that the two PAX6 isoforms differentially and cooperatively regulate the expression of genes specific to the structure and functions of the corneal epithelium, particularly Keratin 3 (KRT3) and Keratin 12 (KRT12). PAX6 isoform-a induced KRT3 expression by targeting its upstream region. KLF4 enhanced this induction. A combination of PAX6 isoform-b, KLF4 and OCT4 induced KRT12 expression. These new findings will contribute to furthering the understanding of the molecular basis of the corneal epithelium specific phenotype.
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Characteristics of the human ocular surface epithelium.
Progress in retinal and eye research, 2001Co-Authors: Shigeru Kinoshita, Wakako Adachi, Kohji Nishida, Andrew J. Quantock, Chie Sotozono, Norihiko Yokoi, Kousaku OkuboAbstract:An appreciation of the biological characteristics of the human ocular surface epithelium affords us a great insight into the physiology of the human ocular surface in health and disease. Here, we review five important aspects of the human ocular surface epithelium. First, we recognize the discovery of corneal epithelial stem cells, and note how the palisades of Vogt have been suggested as a clinical marker of their presence. Second, we introduce the concept of the gene expression profile of the ocular surface epithelium as arrived at using a new strategy for the systematic analysis of active genes. We also provide a summary of several genes abundantly or uniquely expressed in the human corneal epithelium, namely clusterin, Keratin 3, Keratin 12, aldehyde dehydrogenase 3 (ALDH3), troponin-I fast-twitch isoform, sig-h3, cathepsin L2 (cathepsin V), uroplakin Ib, and Ca2+-activated chloride channel. Genes related to limbal and conjunctival epithelia are also described. Third, we touch upon the genetic abnormalities thought to be involved with epithelial dysfunction in Meesmann's dystrophy, gelatinous drop-like corneal dystrophy, and the sig-h3-mutated corneal dystrophies. Fourth, we provide an update regarding the current state of knowledge of the role of cytokines, growth factors and apoptosis in relation to ocular surface homeostasis and tissue reconstruction; the main factors being epidermal growth factor (EGF), Keratinocyte growth factor (KGF), hepatocyte growth factor (HGF), transforming growth factor-s (TGF-s), and some inflammatory cytokines. Fifth, corneal epithelial barrier function and dysfunction as measured by fluorophotometry is remarked upon, with an explanation of the FL-500 fluorophotometer and its ability to detect corneal epithelial dysfunction at a subclinical level. The research described in this review has undoubtedly generated a complete understanding of corneal epithelial pathophysiology—an understanding that, directly or indirectly, has helped advance the development of new therapeutic modalities for ocular surface reconstruction.
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Isolation and chromosomal localization of a cornea-specific human Keratin 12 gene and detection of four mutations in Meesmann corneal epithelial dystrophy.
American Journal of Human Genetics, 1997Co-Authors: Kohji Nishida, Yoichi Honma, Wakako Adachi, Hisashi Hosotani, Shuji Yamamoto, Takahiro Nakamura, Atsuyoshi Dota, Satoshi Kawasaki, Andrew J. Quantock, Masaki OkadaAbstract:Summary Keratin 12 (K12) is an intermediate-filament protein expressed specifically in corneal epithelium. Recently, we isolated K12 cDNA from a human corneal epithelial cDNA library and determined its full sequence. Herein, we present the exon-intron boundary structure and chromosomal localization of human K12. In addition, we report four K12 mutations in Meesmann corneal epithelial dystrophy (MCD), an autosomal dominant disorder characterized by intraepithelial microcysts and corneal epithelial fragility in which mutations in Keratin 3 (K3) and K12 have recently been implicated. In the human K12 gene, we identified seven introns, defining eight individual exons that cover the coding sequence. Together the exons and introns span ∼6 kb of genomic DNA. Using FISH, we found that the K12 gene mapped to 17q12, where a type I Keratin cluster exists. In this study, four new K12 mutations (Arg135Gly, Arg135Ile, Tyr429Asp, and Leu140Arg) were identified in three unrelated MCD pedigrees and in one individual with MCD. All mutations were either in the highly conserved α-helix–initiation motif of rod domain 1A or in the α-helix–termination motif of rod domain 2B. These sites are essential for Keratin filament assembly, suggesting that the mutations described above may be causative for MCD. Of particular interest, one of these mutations (Tyr429Asp), detected in both affected individuals in one of our pedigrees, is the first mutation to be identified within the α-helix–termination motif in type I Keratin.
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Isolation and chromosomal localization of a cornea-specific human Keratin 12 gene and detection of four mutations in Meesmann corneal epithelial dystrophy.
American journal of human genetics, 1997Co-Authors: Kohji Nishida, Yoichi Honma, Wakako Adachi, Hisashi Hosotani, Shuji Yamamoto, Takahiro Nakamura, Atsuyoshi Dota, Satoshi Kawasaki, Andrew J. Quantock, Masaki OkadaAbstract:Keratin 12 (K12) is an intermediate-filament protein expressed specifically in corneal epithelium. Recently, we isolated K12 cDNA from a human corneal epithelial cDNA library and determined its full sequence. Herein, we present the exon-intron boundary structure and chromosomal localization of human K12. In addition, we report four K12 mutations in Meesmann corneal epithelial dystrophy (MCD), an autosomal dominant disorder characterized by intraepithelial microcysts and corneal epithelial fragility in which mutations in Keratin 3 (K3) and K12 have recently been implicated. In the human K12 gene, we identified seven introns, defining eight individual exons that cover the coding sequence. Together the exons and introns span approximately 6 kb of genomic DNA. Using FISH, we found that the K12 gene mapped to 17q12, where a type I Keratin cluster exists. In this study, four new K12 mutations (Arg135Gly, Arg135Ile, Tyr429Asp, and Leu140Arg) were identified in three unrelated MCD pedigrees and in one individual with MCD. All mutations were either in the highly conserved alpha-helix-initiation motif of rod domain 1A or in the alpha-helix-termination motif of rod domain 2B. These sites are essential for Keratin filament assembly, suggesting that the mutations described above may be causative for MCD. Of particular interest, one of these mutations (Tyr429Asp), detected in both affected individuals in one of our pedigrees, is the first mutation to be identified within the alpha-helix-termination motif in type I Keratin.
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a gene expression profile of human corneal epithelium and the isolation of human Keratin 12 cdna
Investigative Ophthalmology & Visual Science, 1996Co-Authors: Kohji Nishida, Wakako Adachi, Shigeru Kinoshita, K Mizuno, K Matsubara, A Shimizumatsumoto, Kousaku OkuboAbstract:PURPOSE: To describe the quantitative and qualitative aspects of gene expression in human corneal epithelium and to discover novel cornea-specific genes. METHODS: A 3'-directed cDNA library was constructed with messenger RNA prepared from normal human corneal epithelial cells, and inserts in 1069 randomly chosen clones were sequenced. These sequences were compared with each other to determine the frequency of appearance and were searched against GenBank for identification. The resultant expression profile, a list of gene species and their recurrences, reflected the composition of mRNA in the cornea. Recurrently appearing sequences, representing abundant transcripts, were compared with sequences in expression profiles obtained from seven other tissues and from those in dbEST to discover cornea-specific genes. RESULTS: The expression profile of human corneal epithelium showed that the most abundant transcript in this tissue was that for apolipoprotein J. Altogether 62 genes were suggested to be very active, including calcyclin, alpha-enolase, Keratin 3, connexin 43, and 12 novel genes. The expression of four of these 12 novel genes seemed to be limited to cornea because they were not found in seven other expression profiles nor in dbEST. Full-length cDNA corresponding to one of these (GS8025), isolated from a separately made cDNA library, contained open reading frame highly homologous to mouse Keratin 12, which is known to be cornea specific. CONCLUSIONS: An expression profile of corneal epithelium provides probes to monitor physiological and pathologic conditions of this tissue in terms of gene expression. Furthermore, by comparing this profile with those of other tissues, probes to isolate genes uniquely transcribed in corneal epithelium are determined. These genes are assumed to carry unique functions for this tissue and are candidate genes for inherited diseases that manifest only in cornea. As an example, human cornea-specific Keratin was isolated, and partial cDNA sequences for three more cornea-specific genes were presented.
Hisatoshi Kobayashi - One of the best experts on this subject based on the ideXlab platform.
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amniotic membrane immobilized poly vinyl alcohol hybrid polymer as an artificial cornea scaffold that supports a stratified and differentiated corneal epithelium
Journal of Biomedical Materials Research Part B, 2007Co-Authors: Yuichi Uchino, Hideyuki Miyashita, Shigeto Shimmura, Hisatoshi Kobayashi, Tetsushi Taguchi, Jun Shimazaki, Junzo TanakaAbstract:Poly(vinyl alcohol) (PVA) is a biocompatible, transparent hydrogel with physical strength that makes it promising as a material for an artificial cornea. In our previous study, type I collagen was immobilized onto PVA (PVA-COL) as a possible artificial cornea scaffold that can sustain a functional corneal epithelium. The cellular adhesiveness of PVA in vitro was improved by collagen immobilization; however, stable epithelialization was not achieved in vivo. To improve epithelialization in vivo, we created an amniotic membrane (AM)-immobilized polyvinyl alcohol hydrogel (PVA-AM) for use as an artificial cornea material. AM was attached to PVA-COL using a tissue adhesive consisting of collagen and citric acid derivative (CAD) as a crosslinker. Rabbit corneal epithelial cells were air-lift cultured with 3T3 feeder fibroblasts to form a stratified epithelial layer on PVA-AM. The rabbit corneal epithelial cells formed 3–5 layers of Keratin-3-positive epithelium on PVA-AM. Occludin-positive cells were observed lining the superficial epithelium, the gap-junctional protein connexin43-positive cells was localized to the cell membrane of the basal epithelium, while both collagen IV were observed in the basement membrane. Epithelialization over implanted PVA-AM was complete within 2 weeks, with little inflammation or opacification of the hydrogel. Corneal epithelialization on PVA-AM in rabbit corneas improved over PVA-COL, suggesting the possibility of using PVA-AM as a biocompatible hybrid material for keratoprosthesis. © 2006 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2006
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amniotic membrane immobilized poly vinyl alcohol hybrid polymer as an artificial cornea scaffold that supports a stratified and differentiated corneal epithelium
Journal of Biomedical Materials Research Part B, 2007Co-Authors: Yuichi Uchino, Hideyuki Miyashita, Shigeto Shimmura, Hisatoshi Kobayashi, Tetsushi Taguchi, Jun Shimazaki, Junzo TanakaAbstract:Poly(vinyl alcohol) (PVA) is a biocompatible, transparent hydrogel with physical strength that makes it promising as a material for an artificial cornea. In our previous study, type I collagen was immobilized onto PVA (PVA-COL) as a possible artificial cornea scaffold that can sustain a functional corneal epithelium. The cellular adhesiveness of PVA in vitro was improved by collagen immobilization; however, stable epithelialization was not achieved in vivo. To improve epithelialization in vivo, we created an amniotic membrane (AM)-immobilized polyvinyl alcohol hydrogel (PVA-AM) for use as an artificial cornea material. AM was attached to PVA-COL using a tissue adhesive consisting of collagen and citric acid derivative (CAD) as a crosslinker. Rabbit corneal epithelial cells were air-lift cultured with 3T3 feeder fibroblasts to form a stratified epithelial layer on PVA-AM. The rabbit corneal epithelial cells formed 3-5 layers of Keratin-3-positive epithelium on PVA-AM. Occludin-positive cells were observed lining the superficial epithelium, the gap-junctional protein connexin43-positive cells was localized to the cell membrane of the basal epithelium, while both collagen IV were observed in the basement membrane. Epithelialization over implanted PVA-AM was complete within 2 weeks, with little inflammation or opacification of the hydrogel. Corneal epithelialization on PVA-AM in rabbit corneas improved over PVA-COL, suggesting the possibility of using PVA-AM as a biocompatible hybrid material for keratoprosthesis.
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Collagen-immobilized poly(vinyl alcohol) as an artificial cornea scaffold that supports a stratified corneal epithelium.
Journal of biomedical materials research. Part B Applied biomaterials, 2006Co-Authors: Hideyuki Miyashita, Shigeto Shimmura, Hisatoshi Kobayashi, Tetsushi Taguchi, Naoko Asano-kato, Yuichi Uchino, Masabumi Kato, Jun Shimazaki, Junzo Tanaka, Kazuo TsubotaAbstract:The cornea is a transparent tissue of the eye, which is responsible for the refraction of incoming light. Both biological corneal equivalents and synthetic keratoprostheses have been developed to replace donor tissue as a means to restore vision. However, both designs have drawbacks in terms of stability and biocompatibility. Clinically available synthetic devices do not support an intact epithelium, which poses a risk of microbial infection or protrusion of the prosthesis. In the present study, type I collagen was immobilized onto poly(vinyl alcohol) (PVA-COL) as a possible artificial cornea scaffold that can sustain a functional corneal epithelium. Human and rabbit corneal epithelial cells were air-lift cultured with 3T3 feeder fibroblasts to form a stratified epithelial layer on PVA-COL. The epithelial sheet expressed Keratin 3/12 differentiation markers, the tight junction protein occludin, and had characteristic microvilli structures on transmission electron microscopy. Functionally, the stratified epithelium contained normal glycogen levels, and an apical tight-junction network was observed to exclude the diffusion of horseradish peroxidase. Furthermore, the epithelium-PVA-COL composite was suturable in the rabbit cornea, suggesting the possibility of using PVA-COL as a biocompatible material for keratoprosthesis.
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collagen immobilized poly vinyl alcohol as an artificial cornea scaffold that supports a stratified corneal epithelium
Journal of Biomedical Materials Research Part B, 2006Co-Authors: Hideyuki Miyashita, Shigeto Shimmura, Hisatoshi Kobayashi, Tetsushi Taguchi, Yuichi Uchino, Masabumi Kato, Jun Shimazaki, Junzo Tanaka, Naoko Asanokato, Kazuo TsubotaAbstract:The cornea is a transparent tissue of the eye, which is responsible for the refraction of incoming light. Both biological corneal equivalents and synthetic keratoprostheses have been developed to replace donor tissue as a means to restore vision. However, both designs have drawbacks in terms of stability and biocompatibility. Clinically available synthetic devices do not support an intact epithelium, which poses a risk of microbial infection or protrusion of the prosthesis. In the present study, type I collagen was immobilized onto poly(vinyl alcohol) (PVA-COL) as a possible artificial cornea scaffold that can sustain a functional corneal epithelium. Human and rabbit corneal epithelial cells were air-lift cultured with 3T3 feeder fibroblasts to form a stratified epithelial layer on PVA-COL. The epithelial sheet expressed Keratin 3/12 differentiation markers, the tight junction protein occludin, and had characteristic microvilli structures on transmission electron microscopy. Functionally, the stratified epithelium contained normal glycogen levels, and an apical tight-junction network was observed to exclude the diffusion of horseradish peroxidase. Furthermore, the epithelium-PVA-COL composite was suturable in the rabbit cornea, suggesting the possibility of using PVA-COL as a biocompatible material for keratoprosthesis. © 2005 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2006
