Biomedical subjects

Kohji Nishida

Publications and source records attributed to Kohji Nishida.

At least 19 recordsLinked to original sources

PubMed2006

Expression of Integrin beta3 is correlated to the properties of quiescent hemopoietic stem cells possessing the side population phenotype.

With significant attention paid to the field of tissue-specific stem cells, the identification of stem cell-specific markers is of considerable importance. Previously, the side population (SP) phenotype, with the capacity to efflux the DNA-binding dye Hoechst 33342, has been recognized as a common feature of adult tissue-specific stem cells. In this study, we show that high expression of integrin beta(3) (CD61) is an attribute of SP cells isolated from mouse bone marrow. Additionally, we confirmed that the expression of integrin beta(3) is correlated with properties of quiescent hemopoietic stem cells (HSCs) including the strength of the SP phenotype, cell cycle arrest, expression of HSC markers, and long-term hemopoiesis. Importantly, Lineage(-) (Lin(-))/integrin beta(3)(high) (beta(3)(high)) SP cells have as strong a capacity for long-term hemopoiesis as c-Kit(+)/Sca-1(+)/Lin(-) SP cells, which are regarded as one of the most highly enriched HSC populations. Finally, the integrin beta(3) subunit that is present in SP cells having the properties of HSCs, is associated with integrin alpha(v) (CD51). Therefore, our results demonstrate that high expression of integrin beta(3) is correlated to the properties of quiescent HSCs and suggest that the integrin beta(3) subunit is available as a common surface marker of tissue-specific stem cells.

Animals↗

PubMed2006

Pretreatment of posterior subtenon injection of triamcinolone acetonide has beneficial effects for grid pattern photocoagulation against diffuse diabetic macular oedema.

AIM: To prospectively evaluate the efficacy of subtenon injection of triamcinolone acetonide (TA) before laser grid pattern photocoagulation (G-PC) for the treatment of diffuse diabetic macular oedema (DDME). METHODS: 42 eyes of 37 consecutive patients with DDME were studied. 1 week before G-PC, 21 eyes received TA subtenon injection, and the other eyes served as control. The clinical course of visual acuity (VA) and foveal thickness (FT) was monitored for up to 24 weeks after G-PC. Mean deviation (MD) of perimetry with 30-2 program on Humphrey Perimeter (Zeiss-Humphrey, Dublin, California, USA) was also measured. The average laser intensity was recorded. RESULTS: After TA injection, FT and VA were improved, and subsequent G-PC maintained the improvement for up to 24 weeks without recurrence of diffuse diabetic macular oedema. In contrast, G-PC without TA injection induced transient worsening of FT and VA, then both were gradually improved. At 24 weeks after G-PC, MD in the TA-injected eyes was better than those in control. The required laser intensity in TA-injected eyes was less than that for control. CONCLUSION: Subtenon injection of TA prior to G-PC allows for treatment with a lower intensity of laser spots and also prevents the decrease in central visual field sensitivity, all of which have clinical advantages for G-PC.

Aged↗

PubMed2006

Conversion of mammalian Müller glial cells into a neuronal lineage by in vitro aggregate-culture.

Mammalian Müller glial cells are major glial cells in the retina. Here we report that these glial cells can be redirected towards a neuronal lineage by an aggregate-culture in vitro. Rat and macaque Müller glial cells did not express neuronal markers except after transfer to adhesive conditions. Furthermore, this expression could only take place in the presence of platelet-derived growth factor and valproic acid. We compared a normal monolayer-culture and an aggregate-culture, and rat Müller glial cells could only differentiate into neurons under non-adhesive conditions. However, Müller glial cells did not express the photoreceptor markers in vitro. After transplantation into the subretinal space, a retina-specific niche, rat Müller glial cells expressed the photoreceptor-specific marker, opsin (RET-P1). We demonstrate the potential of mammalian Müller glial cells as a source of photoreceptors, which may possibly contribute to the treatment of degenerative retinal diseases such as retinitis pigmentosa.

Animals↗

PubMed2006

Development of transplantable genetically modified corneal epithelial cell sheets for gene therapy.

The purpose of this study was to establish a method for the fabrication of exogenous gene-transferred, transplantable corneal epithelial cell sheets. Corneo-limbal epithelial cells collected from USA eye bank eyes were transduced with an EGFP-expressing lentiviral vector at differential MOI. Multi-layered corneal epithelial cell sheets were fabricated by co-cultivation of transduced cells and mitomycin C-treated 3T3 feeder layers on temperature-responsive culture dishes. These cultured epithelial cells could be harvested as intact sheets by simply lowering the temperature. The number of EGFP-positive cells was increased as the MOI raised, and at an MOI of 100, nearly 100% of the superficial cells showed strong EGFP expression. Histological analysis revealed that EGFP was expressed in all layers of the cell sheet of which cell source was transduced with the lentiviral vector at an MOI of 100. Immunofluorescence data showed that p63 was also expressed in the basal layer of the same cell sheet. These results suggest that this technique will likely be applicable to ex vivo gene therapies for various corneal disorders.

Cells, Cultured↗

PubMed2006

N-Cadherin is expressed by putative stem/progenitor cells and melanocytes in the human limbal epithelial stem cell niche.

Corneal epithelial stem cells are known to be localized to the basal layer of the limbal epithelium, providing a model system for epithelial stem cell biology; however, the mechanisms regarding the maintenance of these stem cells in their specialized niche remain poorly understood. N-cadherin is a member of the classic cadherin family and has previously been demonstrated to be expressed by hematopoietic stem cells. In the present study, we demonstrate that N-cadherin is expressed by putative stem/progenitor cells, as well as melanocytes, in the human limbal epithelial stem cell niche. In addition, we demonstrate that upon in vitro culture using 3T3 feeder layers, loss of N-cadherin expression occurs with cell proliferation. These results indicate that N-cadherin may be a critical cell-to-cell adhesion molecule between corneal epithelial stem/progenitor cells and their corresponding niche cells in the limbal epithelium.

Adult↗

PubMed2006

Matrix morphogenesis in cornea is mediated by the modification of keratan sulfate by GlcNAc 6-O-sulfotransferase.

Matrix assembly and homeostasis in collagen-rich tissues are mediated by interactions with proteoglycans (PGs) substituted with sulfated glycosaminoglycans (GAGs). The major GAG in cornea is keratan sulfate (KS), which is N-linked to one of three PG core proteins. To ascertain the importance of the carbohydrate chain sulfation step in KS functionality, we generated a strain of mice with a targeted gene deletion in Chst5, which encodes an N-acetylglucosamine-6-O-sulfotransferase that is integral to the sulfation of KS chains. Corneas of homozygous mutants were significantly thinner than those of WT or heterozygous mice. They lacked high-sulfated KS, but contained the core protein of the major corneal KSPG, lumican. Histochemically stained KSPGs coassociated with fibrillar collagen in WT corneas, but were not identified in the Chst5-null tissue. Conversely, abnormally large chondroitin sulfate/dermatan sulfate PG complexes were abundant throughout the Chst5-deficient cornea, indicating an alteration of controlled PG production in the mutant cornea. The corneal stroma of the Chst5-null mouse exhibited widespread structural alterations in collagen fibrillar architecture, including decreased interfibrillar spacing and a more spatially disorganized collagen array. The enzymatic sulfation of KS GAG chains is thus identified as a key requirement for PG biosynthesis and collagen matrix organization.

Animals↗

PubMed2006

The effect of micropores in the surface of temperature-responsive culture inserts on the fabrication of transplantable canine oral mucosal epithelial cell sheets.

Primary canine oral mucosal epithelial cells were cultured on temperature-responsive dishes and cell culture inserts to fabricate transplantable epithelial cell sheets. When 3T3 feeder layers and fetal bovine serum were eliminated from dish culture, the harvested cell sheets became significantly more fragile. In contrast, when epithelial cells were cultured on inserts having submicron-scale pores, cell sheet fragility was eliminated. Keratin expression profiles showed no differences among the harvested cell sheets, but the expression of p63, a putative stem/progenitor marker, was strongly dependent on the presence of 3T3 feeder layers and serum. These results suggest that the maintenance of stem/progenitor cells is influenced by the apical/basal supply of nutrients as well as culture supplements.

Animals↗

PubMed2006

Cell delivery in regenerative medicine: the cell sheet engineering approach.

Recently, cell-based therapies have developed as a foundation for regenerative medicine. General approaches for cell delivery have thus far involved the use of direct injection of single cell suspensions into the target tissues. Additionally, tissue engineering with the general paradigm of seeding cells into biodegradable scaffolds has also evolved as a method for the reconstruction of various tissues and organs. With success in clinical trials, regenerative therapies using these approaches have therefore garnered significant interest and attention. As a novel alternative, we have developed cell sheet engineering using temperature-responsive culture dishes, which allows for the non-invasive harvest of cultured cells as intact sheets along with their deposited extracellular matrix. Using this approach, cell sheets can be directly transplanted to host tissues without the use of scaffolding or carrier materials, or used to create in vitro tissue constructs via the layering of individual cell sheets. In addition to simple transplantation, cell sheet engineered constructs have also been applied for alternative therapies such as endoscopic transplantation, combinatorial tissue reconstruction, and polysurgery to overcome limitations of regenerative therapies and cell delivery using conventional approaches.

Acrylic Resins↗

PubMed2006

Screening of the MERTK gene for mutations in Japanese patients with autosomal recessive retinitis pigmentosa.

PURPOSE: To determine whether mutations in the MERTK gene are present in Japanese patients with autosomal recessive retinitis pigmentosa (arRP). METHODS: The coding sequence of all 19 exons and the adjacent flanking intron sequences of the MERTK gene were directly sequenced in 96 unrelated Japanese patients with arRP. RESULTS: Seventeen sequence variants were found; six missense changes, three isocoding changes, and eight intron changes were also observed. One arRP patient had a novel homozygous Leu12Pro missense mutation in the MERTK gene. CONCLUSIONS: Mutations in the MERTK gene are relatively rare in Japanese patients with arRP.

Asian People↗

PubMed2006

Transplantable retinal pigment epithelial cell sheets for tissue engineering.

The native retinal pigment epithelium (RPE) exists as a monolayer structure and is critically involved in the maintenance of photoreceptors. Damage or destruction of the RPE due to a variety of diseases therefore often results in loss of vision. With regenerative purposes in mind, we have examined various culture conditions such as the initial cell density and the addition of various supplements in an effort to produce transplantable RPE cell sheets that can be harvested without defects. We demonstrate that the cell density in cultured RPE sheets increased linearly with the number of seeded cells and that RPE sheets were harvested without defects and limited contraction due to cytoskeletal reorganization, when TGF-beta2 was added to the growth medium. Results from histological analysis and the measurement of trans-epithelial resistance also demonstrates that these RPE cell sheets exist as monolayer structure, similar to the native RPE, with intact cell-to-cell junctions. Therefore, these methods provide significant insight into the fabrication of transplantable RPE cell sheets that can be applied to RPE regenerative therapies to restore lost vision.

Cell Count↗

PubMed2006

Fabrication of transplantable human oral mucosal epithelial cell sheets using temperature-responsive culture inserts without feeder layer cells.

To exclude bacteria- or animal-derived factors from cultured fabrication of transplantable epithelial cell sheets, primary human oral mucosal epithelial cells were seeded on temperature-responsive culture inserts having submicron-scale pores. Supplying culture medium containing human autologous serum to both apical and basal sides of human epithelial cells allows these cells to grow to confluence. These proliferating cells created stratified epithelial layers even when 3T3 feeder layers and fetal bovine serum were eliminated from culture. Normal keratin expression profiles were obtained with these cells, and basal and midlayer cells expressed p63, a putative stem/progenitor marker. These results suggest that temperature-responsive culture inserts can be useful in clinical settings that require the exclusion of xenogeneic factors.

Animals↗

PubMed2006

Ultrasound biomicroscopic examination of acute hydrops in patients with keratoconus.

PURPOSE: To investigate the possible factors involved in the development of acute hydrops in patients with keratoconus. DESIGN: Prospective interventional case series. METHODS: Thirteen consecutive keratoconic eyes of 13 patients with acute hydrops were examined by ultrasound biomicroscopy (UBM). RESULTS: Ultrasound biomicroscopy (UBM) examinations revealed a rupture of Descemet membrane and intrastromal clefts in all eyes. In 11 of 13 eyes, the intrastromal clefts were connected to the anterior chamber. CONCLUSIONS: Formation of intrastromal clefts may be an important factor in the development of acute hydrops in keratoconic eyes. The clefts may cause severe corneal edema and delay the closure of Descemet membrane during the resolution of corneal edema.

Acute Disease↗

PubMed2006

Corneal epithelial stem cell delivery using cell sheet engineering: not lost in transplantation.

Cell-based therapies have now generated significant interest as novel drug delivery systems, with various adult cell types used in treating a wide range of diseases. To overcome the limits that restrict treatments for corneal surface dysfunction, corneal epithelial stem cells expanded ex vivo have been applied as an alternative approach. While previous studies used various carrier substrates, we present a novel method using cell sheet engineering with temperature-responsive culture dishes to create carrier-free corneal epithelial stem cell sheets that can be transplanted without sutures. Results from clinical trials reveal successful transplantation with the recovery of lost visual acuity in all cases. Cell sheet engineering, therefore, presents a novel method for the delivery of corneal epithelial stem cells, and can also be applied for other approaches of cellular therapeutics.

Corneal Transplantation↗

PubMed2006

Transplantation of tissue-engineered epithelial cell sheets after excimer laser photoablation reduces postoperative corneal haze.

PURPOSE: To apply tissue-engineered cell sheet transplantation after excimer laser keratectomy as a novel approach for the reduction of postoperative corneal haze. METHODS: Limbal biopsy specimens were obtained, and epithelial cells were cultured on temperature-responsive culture inserts without the use of feeder cells. Laser keratectomy (7.0-mm ablation zone and 160-microm depth) was performed in the contralateral eye, and autologous epithelial cell sheets were transplanted to the ablated corneal stroma. Transplant and control group eyes were assessed by slit lamp biomicroscopy, and corneal haze was scored in a masked fashion, according to the Fantes grading scale. For further examination histologic and immunohistochemical analyses were performed. RESULTS: Tissue-engineered cell sheets produced stable attachment to the laser-ablated sites, resulting in epithelialization, 5 minutes after transplantation. Conversely, control corneas required 3 to 5 days for complete re-epithelialization. At both 1 and 2 months after surgery, corneal haze was significantly inhibited in the transplant group. Histologic analyses showed that the number of keratocytes undergoing apoptosis was decreased in the transplant group at 3 days after surgery. Similarly, the expression of both collagen III and alpha-smooth muscle actin, which may enhance corneal haze, were diminished in the transplant group at 2 months. CONCLUSIONS: The transplantation of tissue-engineered epithelial cell sheets can successfully prevent the development of corneal haze after excimer laser keratectomy.

Actins↗

PubMed2005

Functional human corneal endothelial cell sheets harvested from temperature-responsive culture surfaces.

This study reports a new method for fabricating bioengineered human corneal endothelial cell sheets suitable for ocular surgery and repair. We have initially cultured human corneal endothelial cells on type IV collagen-coated dishes and, after several passages, expanded cells were then seeded onto novel temperature-responsive culture dishes. Four weeks after reaching confluence, these cultured endothelial cells were harvested as intact monolayer cell sheets by simple temperature reduction without enzymatic treatment. Scanning electron microscopy indicated that these cells were primarily hexagonal with numerous microvilli and cilia, similar to the native corneal endothelium. The Na+, K+-ATPase pump sites were located at the cell borders as in vivo. Moreover, cell densities and numbers of pump sites were identical to those of in vivo human corneal endothelium under optimized conditions. A 3H-ouabain binding analysis demonstrated a linear proportionality for cell pump density between confluent cell densities of 575 cells/mm2 and 3070 cells/mm2. We also confirmed Na+, K+-ATPase activity in the sheets in vitro. Xenograft transplantation results showed that the fabricated sheets retain their function of maintaining proper stromal hydration in vivo. We have established a regimen to culture and proliferate human corneal endothelial cells and fabricate endothelial sheets ex vivo morphologically and functionally similar to the native corneal endothelium. Our results support the value of harvested cell sheets for clinical applications in ocular reconstructive surgery in patients with ocular endothelial decompensation.

Cell Culture Techniques↗

PubMed2005

p57Kip2 is expressed in quiescent mouse bone marrow side population cells.

Hematopoietic stem cells can be accurately identified by the side population (SP) phenotype. It has been previously shown that hematopoietic stem cells are cell cycle arrested, but the mechanisms involved are currently poorly understood. In the present study, results from quantitative real-time RT-PCR show that while SP cells have increased expression of various cyclins and cyclin-dependent kinases, the increased expression of cyclin-dependent kinase inhibitors, in particular p57(Kip2), is responsible for the observed cell cycle arrest. In addition, gene expression analysis of c-kit(+/)/Sca-1(+)/Lineage- SP (KSL-SP) cells demonstrates that only p57(Kip2) shows both higher expression compared to both SP and non-SP cells. Furthermore, immunostaining also demonstrates significantly higher protein expression in KSL-SP cells. These results demonstrate that the maintenance of bone marrow SP cells in G0/G1 may be carefully controlled by p57(Kip2).

Animals↗

PubMed2005

Rat limbal epithelial side population cells exhibit a distinct expression of stem cell markers that are lacking in side population cells from the central cornea.

The side population (SP) phenotype is shared by stem cells in various tissues and species. Here we demonstrate SP cells with Hoechst dye efflux were surprisingly collected from the epithelia of both the rat limbus and central cornea, unlike in human and rabbit eyes. Our results show that rat limbal SP cells have a significantly higher expression of the stem cell markers ABCG2, nestin, and notch 1, compared to central corneal SP cells. Immunohistochemistry also revealed that ABCG2 and the epithelial stem/progenitor cell marker p63 were expressed only in basal limbal epithelial cells. These results demonstrate that ABCG2 expression is closely linked to the stem cell phenotype of SP cells.

ATP Binding Cassette Transporter, Subfamily G, Mem↗

PubMed2005

Limbal epithelial side-population cells have stem cell-like properties, including quiescent state.

Corneal epithelial (CE) stem cells are believed to reside in the basal layer of the limbal epithelium but remain poorly understood due to the lack of an accepted in vivo reconstitution assay as well as definitive markers for epithelial stem cells. It has been reported that side-population (SP) cells with the ability to efflux the DNA-binding dye Hoechst 33342 have stem cell-like properties and that the SP phenotype accurately represents a quiescent and immature stem cell population in the adult bone marrow. In the present study, we investigated whether SP cells isolated from the limbal epithelium have stem cell-like properties. SP cells, separated by fluorescence-activated cell sorting, comprise approximately 0.4% of all limbal epithelial cells and have markedly higher expression of the stem cell markers ABCG2, Bmi-1, and nestin but no expression of markers for differentiated CE cells compared with non-SP cells. Cell-cycle and telomerase activity analyses revealed that SP cells are growth arrested and reside in the quiescent state. Moreover, limbal epithelial SP cells did not demonstrate proliferative capabilities under typical in vitro epithelial cell culture conditions using 3T3 feeder layers. These findings present the possibility that quiescent limbal epithelial SP cells may represent an extremely immature stem cell population compared with currently defined epithelial stem or progenitor cells.

Animals↗