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F E Kruse

Publications and source records attributed to F E Kruse.

86 records · Page 5Linked to original sources
PubMed1993

Growth factors modulate clonal growth and differentiation of cultured rabbit limbal and corneal epithelium.

PURPOSE: To compare the extent of modulation by various growth factors on clonal growth and differentiation between corneal and stem cell-containing limbal epithelium. METHODS: A reported serum-free clonal growth assay was used. The mitogenic response was measured by colony forming efficiency, colony size, and bromodeoxyuridine labelling index; the differentiation was assessed by AE-5 monoclonal antibody staining. RESULTS: As compared to controls in an insulin-containing basic medium, the addition of epidermal growth factor, acidic fibroblast growth factor, basic fibroblast growth factor, and a high dose of nerve growth factor was mitogenic for both epithelia. Cholera toxin was also mitogenic, but platelet-derived growth factor might not have, and insulin-like growth factor type I lacked an additive mitogenic effect with insulin. The mitogenic effect of epidermal growth factor differed from the others in its dose-dependent down-regulation and in having more migratory cells in epidermal growth factor-stimulated colonies. In contrast to these mitogens, transforming growth factor-beta 1 exhibited a clear inhibitory effect in the controls as well as in epidermal growth factor- or fibroblast growth factor-stimulated cultures. Transforming growth factor-beta 1's inhibitory effect was correlated with strong AE-5 staining, whereas the mitogenic effect of epidermal growth factors, fibroblast growth factors or nerve growth factors was correlated with the presence of negative AE-5 colonies. CONCLUSION: There exist potential dynamic interactions among various growth-modifying cytokines in controlling epithelial growth and differentiation. The lack of specific activation of limbal stem cells in this culture assay also suggests that other unidentified factor(s) might be regulating stem cell functions.

Animals↗
PubMed1992

Proliferative and differentiative response of corneal and limbal epithelium to extracellular calcium in serum-free clonal cultures.

An increasing concentration of extracellular Ca2+ ([Ca2+]e) consistently induces epithelial differentiation, but its effect on proliferation remains variable. We investigated the effect of [Ca2+]e on two different cell populations: the peripheral corneal (PC) and limbal (L) epithelia, the latter containing corneal stem cells. Primary clonal (18 cells/cm2) cultures from rabbit limbal and peripheral corneal epithelia were established in serum-free MCDB 151 medium containing growth-promoting agents and 0.03, 0.3, or 1.8 mM Ca2+. During early culture life, colony size and the BrdU labelling-index of L and PC, assayed on day 6, increased in response to increasing [Ca2+]e; cell attachment and colony-forming efficiency remained unchanged for both L and PC epithelia. These results indicate that increasing [Ca2+]e, under these defined conditions, stimulates the proliferation of transient amplifying cells, but does not stimulate the differentiation of stem cells into clonal proliferation. A 10-fold increase of the seeding density or prolongation of the culture up to day 14 or 21 changed the response to [Ca2+]e allowing better proliferation in lower [Ca2+]e. Only cells grown as a monolayer in 0.03 mM Ca2+ could still be passaged on day 14, whereas cells in higher [Ca2+]e showed increasing stratification and cell detachment and could not be passaged. Normal cellular differentiation accessed by the expression of a cornea-type K3 keratin, recognized by the monoclonal antibody AE-5, was enhanced by increasing [Ca2+]e. Abnormal differentiation featured by the formation of cornified envelopes was only observed in higher [Ca2+]e. These results indicate that [Ca2+]e promotes the proliferation of relatively undifferentiated transient amplifying cells under clonal, serum-free culture conditions. Factors that enhance differentiation, such as seeding density or prolonged culture life, can modify this response and allow better proliferation in low [Ca2+]e.

Animals↗
PubMed1991

A serum-free clonal growth assay for limbal, peripheral, and central corneal epithelium.

The stem cells and transient amplifying cells of the corneal epithelium are thought to be localized in the limbal and corneal basal epithelium, respectively. To study the differential regulation of proliferation of these progenitor cells, a defined, serum-free, clonal growth assay was developed for central (CC) and peripheral (PC) corneal and limbal (L) epithelial cells. After incubation in Dispase II (1.2 U/ml; 1 hr for PC and CC and 3 hr for L) and subsequent brief trypsin-ethylenediaminetetraacetic acid digestion, 18 or 180 single cells/cm2 were seeded in MCDB 151 medium supplemented with insulin, transferrin, selenium, hydrocortisone, epidermal growth factor (EGF), phosphoethanolamine, ethanolamine, and calcium. During the first week of culture, the cells gradually developed an increasing number of colonies, and the mean colony-forming efficiency on day 6 for L was 4.2 +/- 2.4%, significantly lower than 11.4 +/- 5.9% for PC and 12.8 +/- 7.6% for CC (P less than 0.003). Colony morphology was identical in L, PC, and CC with small, elongated cells more cohesive in the center but more migratory in the periphery. There were no differences in immunofluorescent staining with monoclonal antibody AE-5, indicating the corneal derivation of all colonies. Cultures could be passaged on day 14 and grown for more than 3 weeks with increasing desquamation. Addition of a mixture of trace metals to yield MCDB 153 did not enhance growth; increased selenium concentrations were inhibitory. Elimination of EGF from the supplement abolished most of the clonal growth. The lower rate of L proliferation might be explained by the absence of serum and stromal mitogens. This culture system seems preferentially to support transient amplifying cells and allows investigation of the differentiation of isolated corneal stem cells to transient amplifying cells or the proliferation and differentiation of transient amplifying cells by various factors without the interaction of undefined serum components or paracrine influences from other cells.

Animals↗
PubMed1991

[The limbus epithelium in vitro].

The limbal basal epithelium contains the stem cells of the corneal epithelium. In order to study the regulation of these cells we developed models allowing their investigation. While an explant model based on n-heptanol debridement did not allow outgrowth of corneal epithelium onto culture dishes, a serum-free single-cell clonal growth supported colony growth in limbal cells. MCDB 151 medium was supplemented with insulin, transferrin, selenium, hydrocortisone, phospho-/ethanolamine and calcium. Single cells (5,000) were seeded onto 60 mm plastic dishes following combined digestion with dispase and trypsin/EDTA. Increasing CFE was noted until day 6, allowing increasing recruitment into proliferation. This model will serve as a tool for the further investigation of factors that might govern the regulation of the differentiation and proliferation of limbal epithelial cells.

Animals↗
PubMed1990

Conjunctival transdifferentiation is due to the incomplete removal of limbal basal epithelium.

Previous studies have shown that using n-heptanol to create a total corneal epithelial defect beyond the limbus results in two different healing patterns with an unpredictable incidence. Between 14-68% of these wounded rabbit corneas (n = 287, combining various reports) showed extensive vascularization and conjunctivalization, whereas the remaining were not vascularized and had conjunctival transdifferentiation with a cornea-like epithelium. To investigate the role of the limbal epithelium in these two healing patterns, the authors treated rabbit eyes for various durations with n-heptanol and additional scraping. Histology showed that treatment for up to 120 seconds removed both the corneal and conjunctival epithelia but left the limbal basal cells intact. To prove viability, they cultured the treated limbal explants on collagen gel. After 14 days of culture, increased stratification of the limbal epithelium and an epithelial outgrowth onto the corneal stroma was observed. The latter was proven to be of corneal origin (positive to AE-5 but negative to AM-3 monoclonal antibody staining). The authors then surgically removed the entire limbal zone including 2 mm of peripheral cornea and 3 mm of adjacent conjunctiva in addition to n-heptanol debridement of the entire corneal epithelium in 54 rabbit eyes and observed a high incidence (96%) of corneal vascularization and conjunctivalization of the resultant epithelial phenotype (positive to AM-3, but negative to AE-5 monoclonal antibody staining). These results support the hypothesis that corneal epithelial stem cells are located in the limbus and indicate that an incomplete removal of the basal limbal epithelium by n-heptanol leads to unvascularized corneas with conjunctival transdifferentiation. Conversely, complete removal of such cells results in corneal vascularization and conjunctivalization.

Alcohols↗
PubMed1989

Reproducibility of topographic measurements of the optic nerve head with laser tomographic scanning.

Topographic analysis and measurement of the optic nerve head is important for the diagnosis and follow-up of glaucoma. To quantify structures of the optic nerve head the new technique of laser tomographic scanning was used. A laser beam was focused onto the surface of the optic nerve head and the reflected light was detected in a confocal detection unit. The consequent change of focus produced a tomographic scanning series and allowed measurement of three-dimensional structures. To analyze the reproducibility of optic cup measurements the authors did ten recordings of one eye of eight normal volunteers. The mean standard deviation of the measurements was +/- 0.015 mm3 and the mean coefficient of variation was 9.5%. Confocal laser tomographic scanning is a safe, effective, convenient method to measure and document the topography of the optic nerve head and should be a valuable technique for follow-up of glaucoma patients.

Humans↗
PubMed1989

[3-dimensional biomorphometry of the papilla using a laser tomography scanning procedure--initial experiences with pathologic papillar findings].

Three-dimensional topographical analysis of the optic nerve head and the parapapillary region was performed with a confocal laser tomographic scanner. Four patients with various disorders of the optic nerve head (glaucoma, optic disk pit, morning glory syndrome and a parapapillary neoplasm) were investigated as examples of application of the method in a clinical setting.

Adult↗
PubMed1986

[Impression cytology as a noninvasive method of conjunctival biopsy and its results].

Impression cytology is a non-invasive, repeatable, and uncomplicated method of examining the conjunctiva. Cellulose-acetate filters are used to remove the superficial layers of the conjunctiva. This technique causes no subjective discomfort to the patient. The method combines the advantages of a biopsy with those of a "Häutchenpräparat." The authors used 3 different stains for the conjunctival imprints: PAS-hematoxylin, Alcian blue and a combination of Alcian blue and PAS. From the different staining characteristics of the goblet cells it was possible to draw conclusions about different functional cell conditions. The metachromatic properties of a few cells among the large number of epithelial cells led to speculation on the existence of different cell types and their possible correlation with the various cell types described by Rohen for the conjunctiva of monkeys. In patients with keratoconjunctivitis sicca or Sjögren's syndrome pathologic nuclear changes occur that lead to chromatin taking on a snake-like appearance, the so-called "snakes." To the authors' surprise, similar chromatin changes were found in a great number of cells of contact lens wearers, with frequency depending on the type of contact lens. Impression cytology is a special method of non-invasive biopsy providing new possibilities for diagnosis, differential diagnosis, and therapeutic control of conjunctival disorders.

Biopsy↗
PubMed1986

[Scanning electron microscopy image of the cells of the conjunctival epithelium in specimens from impression cytology].

Impression cytology specimens of the conjunctiva can be examined by scanning electron microscopy. If a cellulose-acetate filter is pressed onto the conjunctiva, the microvilli-bearing epithelium becomes so firmly attached to the fine meshwork of the filter that the most superficial conjunctival cell layers adhere to the filter. When the filter is removed intercellular connections are destroyed without any damage to the cell membranes being detectable in the SEM image. Since the surface of the corneal epithelium is spread out on the filter and is firmly attached to it the lateral bond between the cells is preserved. Shrinkage is minimal. The posterior surface of the epithelial cells, facing the observer, consists of smooth, porous cell membranes. The nuclei of many cells are clearly distinguishable. Since it can be directly compared with light-microscopic specimens, the SEM image can help provide answers to morphological and pathological questions, and may represent a useful diagnostic aid.

Adult↗
PubMedDate not supplied

Irradiation for inhibition of endothelial cell growth in vitro.

The clinical effect of ionising radiation on ocular neovascularisations is controversial, not only because of the variety of treatment modalities. The aim of our study was to investigate an experimental model which allows to evaluate radiation parameters and to study the mechanism of the inhibitory effect on neoangiogenesis. In vitro experiments were performed using human umbilical vein endothelial cells and human corneal keratocytes. The cells were irradiated with different doses of photon beam radiation and counted daily after treatment. Cell cultures confirmed the efficiency of irradiation in inhibiting proliferation of vascular endothelial cells. Radiosensitivity was shown to be higher in vascular endothelial cells than in corneal stromal keratocytes. This might be helpful in investigating therapy parameters for treating ocular neovascularisations, as well as underlying mechanisms.

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