Differences between niche cells and limbal stromal cells in maintenance of corneal limbal stem cells.

PURPOSE: To investigate the differing characteristics of limbal niche cells (LNCs) and limbal stromal cells (LSCs) in the maintenance of limbal epithelial stem/progenitor cells in the cornea. METHODS: Limbal niche cells were obtained from direct dissection of the human corneal limbus, and LSCs were obtained from explant cultures of limbal stromal tissues under the same culture conditions. The resulting cultures were examined for their ability to support the growth of limbal stem/progenitor cells in colony-forming capacity, stratified epithelial cell sheet formation, maintenance of limbal epithelial stem/progenitor cell characteristics, and gene expression levels of factors that supported the limbal epithelial stem/progenitor cells. RESULTS: The colony-forming efficiency of limbal epithelial stem/progenitor cells in the LNC group (6.57 ± 1.54%) was significantly higher than that in the LSC group (1.43 ± 0.47%). The epithelial cell sheets in the LNC group stratified into four or five layers compared with two or three stratified layers in the LSC group. Staining of both the colonies and the epithelial cell sheets in the LNC group showed a higher intensity of the limbal stem cell marker ΔNp63 than in the LSC group. Moreover, reverse transcription polymerase chain reaction analysis revealed that compared with the common expression of EGF and so on, the LNCs showed a higher expression level of E-cadherin and a lower expression level of neurotrophin-3 (NT3) than the LSCs. CONCLUSIONS: LNCs have a different role compared to LSCs in their ability to support epithelial stem/progenitor cells and epithelial cellular sheet formation.

Development of genetically modified eliminable human dermal fibroblast feeder cells for ocular surface regeneration medicine.

PURPOSE: Cultured human corneal limbal stem/progenitor cells are usually established and maintained on feeder layers. However, animal feeder cells are associated with viral infection, pathogen transmission, and xenogenic contamination. All feeder cells also can be mixed easily into cell-sheet production, causing self-contamination. We developed a line of labeled, immortalized, eliminable human dermal fibroblast cells to eliminate these problems. METHODS: The enhanced green fluorescent protein gene, human-derived telomerase reverse transcriptase gene, and herpes simplex virus thymidine kinase gene were transfected into human dermal fibroblast cells to establish labeled, immortalized, eliminable feeder cells. Established eliminable dermal fibroblasts (TERT+TK-D) were treated with mitomycin, cocultured with human limbal stem/progenitor cells to regenerate epithelium sheets, and compared with 3T3 feeder cells. RESULTS: Established TERT+TK-D feeder cells maintained immortalization, visualization, and eliminable characteristics during 6 months of continuous passages. The colony-forming efficiency of limbal stem/progenitor cells was similar in the TERT+TK-D group (11.77 ± 0.21%) and the 3T3 group (12.8 ± 1.61%) (P = 0.332). All cell sheets were well stratified into 4 to 5 layers. The TERT+TK-D group colonies and epithelial cell sheets showed weaker staining of corneal epithelium differentiation marker K3 than the 3T3 group and quantitative analysis of mRNA transcripts. Moreover, PCR analysis against the long terminal repeat sequence of the lentiviral vector integrated into the genetically modified feeder cells showed no contamination of ganciclovir-treated regeneration epithelial sheets. CONCLUSIONS: Genetically modified, labeled, immortalized, eliminable human dermal feeder cells are promising substitutes for 3T3 feeder cells for xenogeny-free ocular surface regeneration.

New culture technique of human eliminable feeder-assisted target cell sheet production.

Cultured cell sheets for transplantation generally have been co-cultured with animal feeder cells, which carry risks because of different species and results in non-contact culture between the feeder and target cells. We developed a new technique to produce human eliminable feeder-assisted target cell sheets by novel human-derived genetically modified feeder cells. Three genes (human-derived telomerase reverse transcriptase gene, enhanced green fluorescent protein gene, and herpes simplex virus thymidine kinase gene) were transducted into human stromal cells, which enabled genetically modified feeder cells to be immortalized, labeled, and eliminated as needed. A target cell sheet was produced as one sheet by assisting the genetically modified feeder cells and successfully transplanted in vivo without their contamination. Genetically modified human eliminable feeder cells could be a promising tool for cultivated cell sheet transplantation.

Two cases of acanthamoeba keratitis diagnosed only by real-time polymerase chain reaction.

PURPOSE: To report 2 cases of Acanthamoeba keratitis whose causative pathogen was detected only by real-time polymerase chain reaction (PCR). METHODS: Histological examinations of corneal scrapings were stained with Fungiflora Y. Corneal scrapings were also cultured on nonnutrient agar. Real-time PCR analyses of corneal scrapings were also performed. RESULTS: Both cases had clinical signs and risk factors of Acanthamoeba infection. Histological examinations of corneal scrapings with Fungiflora Y staining were negative, and the cultures did not grow any pathogens. Real-time PCR analysis was positive for Acanthamoeba DNA from 2 corneal scrapings. Antiamoebic treatments led to excellent clinical improvements. CONCLUSIONS: These results indicate that PCR analyses can detect the DNA of Acanthamoeba in corneal scrapings and may be a valuable supplemental examination. This method is especially helpful when clinical signs and risk factors of Acanthamoeba infection are present but histological examinations with Fungiflora Y staining and cultures are negative.

Prevalence and features of keratitis with quantitative polymerase chain reaction positive for cytomegalovirus.

PURPOSE: To assess corneal scrapings and aqueous humor samples analyzed by polymerase chain reaction (PCR) that were positive for cytomegalovirus (CMV) in patients with keratitis of unknown origin and to investigate their clinical manifestations. DESIGN: Retrospective, interventional case series. PARTICIPANTS: Seventy-eight patients with epithelial (n=37), stromal (n=12), or endothelial keratitis (n=29) of unknown origin examined at the Osaka University Medical Hospital. METHODS: Clinical examination and tears, corneal scrapings, and aqueous humor specimens were evaluated by real-time PCR for CMV. MAIN OUTCOME MEASURES: Quantification of CMV DNA at the diagnosis of each type of keratitis with unknown origin and monitoring during the therapeutic course for CMV-positive cases. RESULTS: No cases of epithelial or stromal keratitis had CMV DNA. Seven of 29 corneal endotheliitis cases (24.1%) were positive for CMV. Cytomegalovirus-positive cases of corneal endotheliitis characterized by localized corneal edema and keratic precipitates included 4 patients who had undergone penetrating keratoplasty and were refractory to the treatment for graft rejection and 3 patients with idiopathic endotheliitis. Cytomegalovirus DNA copy numbers were estimated and ranged from 6.3x10(4) to 3.6x10(6)/ml. In all positive cases, the numbers of CMV DNA copies decreased within weeks during treatment with systemic and topical ganciclovir (GCV) combined with a topical steroid. Five eyes (62.5%) had clinical improvement. In cases of endothelial keratitis, diabetes mellitus was significantly higher in patients positive for CMV (71.4%) than in patients negative for CMV (18.2%, P=0.016, chi-square test). CONCLUSIONS: A total of 24.1% of cases with corneal edema of unknown origin were CMV positive and should be included in the differential diagnosis of idiopathic corneal endotheliitis or graft edema after penetrating keratoplasty, especially for bullous keratopathy. Real-time PCR for CMV, based on the diagnosis and monitoring of the clinical course, may be useful. Cytomegalovirus corneal endotheliitis requires early appropriate treatment using GCV. Because clinical remission after GCV may depend on the area of normal endothelium, early diagnosis and therapy are important for CMV corneal endotheliitis.

Induction of fibroblast growth factor-2 by electrical stimulation in cultured retinal Mueller cells.

It has been demonstrated that retinal Mueller cells, the predominant glial cells, produce neurotrophic factors including basic fibroblast growth factor (FGF-2), and that electrical stimulation enhances the transcription of FGF-2 in the central nervous system. In this study, the effect of electrical stimulation on the induction of FGF-2 in cultured rat Mueller cells was investigated by quantitative real-time polymerase chain reaction and western blotting. Both the messenger RNA and protein of FGF-2 were significantly upregulated after electrical stimulation compared with that of controls. These results suggest that electrical stimulation may directly induce the production of FGF-2 in the retina.

Prolonged protective effect of basic fibroblast growth factor-impregnated nanoparticles in royal college of surgeons rats.

PURPOSE: To investigate the protective effect of intravitreal injection of basic fibroblast growth factor-impregnated nanoparticles (bFGF-NPs) against photoreceptor degeneration in Royal College of Surgeons (RCS) rats. METHODS: Three-week-old RCS rats received intravitreal injection of PBS, blank NPs, bFGF (2.5 microg), or bFGF-NPs (2.5 microg). Eyes were assessed by morphologic, immunohistochemical, and physiological analyses for the following 8 weeks. Cell death was examined using the TUNEL assay, and bFGF protein levels in the retina were measured by Western blot analysis. Rhodamine (Rh)-labeled bFGF-NPs were injected intravitreally and visualized by confocal microscopy to determine the localization of the nanoparticles in the retina. RESULTS: Intravitreally injected Rh-labeled bFGF-NPs were found in the outer nuclear layer 6 and 8 weeks after injection. ERG a- and b-wave amplitudes in bFGF-NP-treated retinas were greater than amplitudes in retinas receiving other treatment. Immunocytochemical analysis showed consistently greater opsin preservation in bFGF-NP-treated retinas, and a significantly higher number of photoreceptors and significantly fewer TUNEL-positive cells were present after bFGF-NP treatment than after bFGF treatment. Western blot analysis showed a significant increase in the bFGF level in bFGF-NP-treated retinas. CONCLUSIONS: The results suggest that intravitreally injected bFGF-NPs prevent photoreceptor degeneration by inhibiting apoptosis in the RCS rat retina because of targeting and sustained release of bFGF. This novel drug delivery system for bFGF may serve as a potential short-term treatment for photoreceptor degeneration in humans.

Further studies on the hyperphosphorylated form (p40) of the rabies virus nominal phosphoprotein (P).

We investigated possible mechanisms involved in production of a hyperphosphorylated form (p40) of rabies virus P protein, to which two dimensional (2-D) gel electrophoresis was applied. The P gene products produced in Escherichia coli cells could be detected as a single spot of unphosphorylated 37-kDa form (termed as p37-0) in a 2-D gel. The 37-kDa proteins in the virus-infected cells are composed of some phosphorylated forms, including a major p37-1 and more phosphorylated minor forms (e.g., p37-2, p37-3, etc.), but little p37-0 is detected (Eriguchi et al., 2002). When the E. coli -produced P protein analogues were incubated with BHK-21 cell lysates, heparin-sensitive phosphorylation occurred as described previously (Takamatsu et al., 1998), giving an additional 40-kDa spot. However, such a p40-like derivative displayed a little more basic pI value than that of the authentic p40 produced in the infected cells; hence, the former was termed p40-0 (pI=4.78), while the latter, p40-1 (pI=4.73). In contrast, p40 produced in the P cDNAtransfected animal cell was detected at the p40-1 position. In addition, staurosporine did not affect the p40-1 production in virus-infected nor the P cDNA-transfected animal cells, while the agent reduced production of hyperphosphorylated forms of p37, resulting in accumulation of p37-1, but not of p37-0. These results suggest that, although p37-0 may become a substrate for the heparin-sensitive protein kinase (PK) in vitro, only p37-1 is a substrate for p40 production catalyzed by heparin-sensitive PK in animal cells, and staurosporine-sensitive PK is involved in the production of more phosphorylated forms of p37, but not in p37-1 production from p37-0.