1,720,977 research outputs found
Human conjunctival epithelial precursor cells and their progeny in 3D organotypic culture
No full textWe report on an in vitro organ culture method to investigate human conjunctival epithelial basal precursor cells and their progeny within a more natural three-dimensional microenvironment. Conjunctival fragments were cultured on gelatin sponges in medium with 10% FBS. The conjunctival phenotype of the epithelium was confirmed by the expression and distribution of a panel of markers (p63, CK-13/CK-10, CK-19, Ki-67, PAS for goblet cells, CD45 for infiltrating interlamellar leukocytes and nestin for mesenchymal and ocular epithelial precursor cells). After 7 days, the epithelium had exfoliated its superficial layers (mostly CK-19 positive cells and all goblets), maintaining only 1-2 layers of basal/parabasal cells, p63, CK-13/CK-10 and nestin positive cells, firmly attached to the specimen. After 14 days, a new multilayered epithelium was formed, consisting of p63, CK-13/CK-10, nestin positive cells and in the high-zone CK-19 positive cells with new goblets. Additionally, we found interlamellar leukocytes which had probably migrated from capillaries that continued to be well maintained in the subepithelial stroma. Cells dispersed from conjunctival epithelium and co-cultured with feeder post-mitotic NIH3T3 fibroblasts formed mosaics displaying a basal epithelial phenotype. These cells expressed CD133 as revealed by RT-PCR. These organ cultures provide new opportunities to investigate epithelial reconstitution of the conjunctival surface and changes that may have occurred to their stem/precursor cells during adaptation to varying conditions in vitro
EFFECTS OF REPEATED EXPOSURE TO HIGH-VOLTAGE ELECTRIC-DISCHARGES AND LOW-FREQUENCY ELECTROMAGNETIC-FIELDS ON CULTURED MOUSE P3X63AG8 PLASMACYTOMA CELLS
No full textSelective growth and expansion of human corneal epithelial basal stem cells in a three-dimensional-organ culture
No full textWe report on a three dimensional (3D)-organotypic culture in vitro for selective growth and expansion of human corneal epithelial stem cells. Limbal corneal explants were cultured on porous collagen sponges submerged in Epilife medium containing 10% fetal bovine serum. The fragments were analyzed by immunohistochemistry for the expression and distribution of a spectrum of corneal epithelium markers: p63, CK-19, CK-3, Ki-67, pan-cytokeratins and vimentin. Early in culture the epithelium began to exfoliate losing its differentiated high-zone layers into the medium, maintaining only basal and few parabasal cells (mostly both p63 and CK-19 positive), which had remained attached to the specimen. After 14 days a new epithelium was formed displaying an increasing prominence of basal and suprabasal cells that, sliding onto the whole explant, showed the tendency to underlay stromal tissue and infiltrate into the underlaying sponge. After 21 days, sponge and fragments were incubated with trypsin-EDTA and dispersed epithelial cells were pipetted on a feeder monolayer of mitomycin-c-treated murine NIH.3T3 fibroblasts. Colonies of undifferentiated epithelial cells (p63, CK-19 and Ki-67 positive, CK-3 negative) were obtained: their cells, if seeded onto a collagen matrix containing embedded primary human corneal fibroblasts as feeder, provided the basic building blocks for reconstructing in vitro a 3D-multilayered corneal epithelium
Inner ear rehabilitation in the deafened cochlea of nod-scid mice following transplantation of human pluripotent mesenchymal stem cells
No full textObjectives. Mesenchymal stem cells (MSC) are currently being investigated in numerous pre-clinical and clinical settings of regenerative medicine. We had previously reported (Revoltella et al., Cell Transplant. 2008; 17, 665-678) that human umbilical cord blood CD133+ stem cells transplanted IV into pre-irradiated nod-scid mice made permanently deaf by ototoxic treatment with kanamycin or intense sound, were able to engraft the cochlea and contribute to inner ear restoration, in vivo. We further investigated here whether human adult MSC derived from either bone marrow or fat (lipid suction), if injected IV to deafened nod-scid mice pre-treated with kanamycin , were able to engraft the damaged cochlea regaining hearing.
Materials, Methods & Results. We tested HLA-DQa1 DNA and three human microsatellites (CODIS) as indicators of engrafted cells, finding polymerase chain reaction evidence of chimaerism in various tissues of the host, including the Organ of Corti in the damaged cochlea, at 7, 31 and 60 days following MSC transplant. After 31 days, histology, immunohistochemistry, and lectin staining confirmed the repair process and stimulation ex novo of morphological recovery in the inner ear, contrasting with the lack of morphological repair in control similarly injured but non-transplanted mice. FISH analysis, to detect human genomic sequences from different chromosomes, confirmed persistent engraftment of the regenerating inner ear with a very limited number of chimaeric cells. Dual color FISH analysis provided evidence of a limited positive engraftment in the inner ear and in other mouse tissues, also revealing small numbers of possible heterokaryons, probably resulting from unstable clones derived from fusion of donor with endogenous cells, up to 2 months following transplantation. Stem cells and differentiation pathways focused PCR arrays favoured to select MSC inducing the best response.
Conclusions. These findings support the concept that transplanted MSC migrating to the damaged inner ear area may provide conditions for the resumption of a damaged cochlea , emerging as a potential strategy for hearing rehabilitation
Granulocyte-macrophage colony-stimulating factor as an autocrine survival-growth factor in human gliomas
No full textWe studied the expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) and its receptors (GM-CSF.R) in 20 human brain gliomas with different tumor gradings and demonstrated constitutive high levels of both mRNA gene expression and protein production exclusively in the highest-grade tumors (WHO, III-IV grade). Five astrocytic cell lines were isolated in vitro from glioma cells, which had selectively adhered to plates pre-coated with rhGM-CSF. These cells were tumorigenic when xenografted to athymic mice, and produced GM-CSF constitutively in culture. Two lines, particularly lines AS1 and PG1, each from a patient with glioblastoma multiforme, constitutively over-expressed both GM-CSF and GM-CSF.R genes and secreted into their culture media biologically active GM-CSF. Different clones of the AS1 line, isolated after subsequent passages in vitro and then transplanted to athymic mice, demonstrated higher tumorigenic capacity with increasing passages in vivo. Cell proliferation was stimulated by rhGM-CSF in late-stage malignant clones, whereas apoptosis occurred at high frequency in the presence of blocking anti-GM-CSF antibodies. In contrast, rhGM-CSF did not induce any apparent effect in early-stage clones expressing neither GM-CSF nor GM-CSF.R. The addition of rhGM-CSF or rhIL-1β, to cultures induced the overproduction of both GM-CSF and its receptors and increased gene activation for several functional proteins (e.g. NGF, VEGF, VEGF.R1, G-CSF, MHC-II), indicating that these cells may undergo dynamic changes in response to environmental stimuli. These findings thus revealed: (1) that the co-expression of both autocrine GM-CSF and GM-CSF.R correlates with the advanced tumor stage; (2) that an important contribution of GM-CSF in malignant glioma cells is the prevention of apoptosis. These results imply that GM-CSF has an effective role in the evolution and pathogenesis of gliomas
Interleukin-1 production by a cloned line of human monocyte-like cells (CM-SM). Correlation with state of differentiation.
No full textSelective growth of epithelial basal cells of the human prostate in a three dimensional organ culture
No full textBACKGROUND: A three-dimensional organotypic culture method has been developed for selectively growing epithelial basal cells from human benign prostate.
METHODS: Tissue fragments were cultured on sponges for several weeks and the viability of luminal and basal epithelium and cellular responses to 4,5alpha-dihydrotesterone (DHT) stimulation were studied.
RESULTS: The gland tissue could be successfully maintained showing preservation of ducts and lobules as in vivo. Without DHT, a progressive hyperplasia of basal cells was observed: these cells proliferated with retention of the lumen or forming nests with squamous differentiation, lining the surface of the fragment and migrating to the underlying sponge. In contrast, secretory cells disappeared. Epithelial cells isolated from long-term cultures showed a typical basal cell-immunophenotype. DHT-treated tissues maintained a much higher percentage of luminal cells than untreated tissues.
CONCLUSIONS: These systems allow the study of proliferation and differentiation of basal cells within their natural microenvironment as well as prostate pathobiology
LOW-FREQUENCY ELECTROMAGNETIC-FIELDS DO NOT AFFECT CELL-GROWTH, ERYTHROID-DIFFERENTIATION, AND VIRUS PRODUCTION IN VARIANT LINES OF UNTREATED AND DIMETHYL SULFOXIDE-TREATED FRIEND-ERYTHROLEUKEMIA CELLS
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