The tongue represents a very accessible source of tissue-specific epithelial stem cells of endodermal origin. However, little is known about the properties of these cells and the mechanisms ...regulating their proliferation and differentiation. Foxa2, an endodermal marker, is expressed throughout the tongue epithelium during embryonic development but becomes confined to a minority of basal cells and some taste bud sensory cells in the adult tongue. Using a previously described line of transgenic mice in which enhanced green fluorescent protein (eGFP) is expressed under the control of a human keratin 5 promoter region (Krt5-eGFP), we have isolated a subpopulation of cells in the basal epithelial layer of the mouse tongue with a high efficiency of generating holoclones of undifferentiated cells in culture with a feeder layer. Krt5-GFPhi cells can both self renew and give rise to differentiated stratified keratinized epithelial cells when cultured on an air-liquid interface.
The cornea at the front of the eye is covered by an epithelium. This epithelium is maintained by stem cells located at the periphery of the cornea, in a region known as the limbus. Because this ...region harbors the stem cells for the corneal epithelium, the so-called limbal stem cells, its culture provides considerable interest. Limbal epithelial culture is used for two main reasons. The first is to further our understanding of limbal stem-cell biology. The second is for the culture expansion of limbal stem cells for transplantation purposes in patients with limbal stem-cell deficiency. However, considerable variations in the culture methods for limbal epithelium exist. These include culture media, sera used in the culture, use of 3T3 fibroblasts or amniotic membrane or both, the culture of whole pieces of limbal tissue or enzymatically digested tissue, and the use of airlifting.
Objective: Although oral mucosal epithelial stem cells are thought to reside in the basal layer, such cells have not yet been isolated. We isolated a population of rabbit oral epithelial progenitor ...cells containing putative stem cells.
Materials and methods: Epithelial cells harvested from rabbit buccal mucosa were allowed to adhere to dishes coated with collagen IV for periods ranging from 10 min to 16 h. The properties of individual cell populations were evaluated using BrdU, Ki‐67, integrin β1, integrin α6 and keratin 13 using colony forming efficiency (CFE).
Results: Cells that adhered to collagen IV‐coated dishes within 10 min were enriched about sixfold in terms of BrdU incorporation, Ki‐67, integrin α6 and integrin β1 were strongly expressed. Interestingly, keratin 13 was faintly expressed. The CFE of rapidly adherent cells among oral epithelial cells was significant compared with other cell populations.
Conclusions: These results suggested that rabbit oral epithelial cells could be isolated by depending on adhesiveness to collagen IV, especially when segregated according to progenitor cell properties. Putative progenitor cells with stem cell properties were most effectively harvested within 10 min. Our separation procedure should be a useful tool with which to isolate epithelial stem cells for regenerative medicine.
Aim: To evaluate the use of human serum (HS) in supporting the in vitro and in vivo proliferation of human conjunctival epithelial cells, and compare it with fetal bovine serum (FBS) and bovine ...pituitary extract (BPE). Methods: Conjunctival epithelial cells were cultivated in media supplemented with HS (5%, 10%), FBS (5%, 10%), and BPE (70 μg/ml, 140 μg/ml). The colony forming efficiency (CFE), bromodeoxyuridine (BrdU) ELISA proliferation assay, and cell generations were analysed. Cells were evaluated for keratin (K4, K19, and K3) and MUC5AC expression by immunostaining and RT-PCR. Conjunctival equivalents constructed on amniotic membranes were transplanted onto severe combined immune deficient (SCID) mice for 10 days and analysed histologically. Results: The proliferation assays of HS supplemented cultures (CFE, 6.7% (SD 1.8%); BrdU absorbance, 0.86 (0.16)) were comparable to FBS supplemented (CFE, 9.3% (1.8%); BrdU absorbance, 1.11 (0.18)) and BPE supplemented cultures (CFE, 5.9 (1.5); BrdU absorbance, 0.65 (0.12)). Goblet cell densities for HS, FBS, and BPE supplemented media were 52 cells/cm2, 60 cells/cm2, and 50 cells/cm2, respectively. HS supplemented cultures formed stratified epithelial sheets in vivo following transplantation. Conclusions: The proliferative capacity of conjunctival epithelial cells cultivated in HS supplemented cultures was comparable to FBS and BPE supplemented cultures. The elimination of animal material from the culture system is advantageous when cultivating cells for clinical transplantation.
Genomic instability has been associated with cancer development. Oxidative DNA damage seems to contribute to genetic instability observed in cancer. We have used human lung cancer cell lines carrying ...a plasmid vector containing a (CA)13 microsatellite sequence to study frameshift mutations mediated by ROS-generating chemicals paraquat and hydrogen peroxide. Exposure of the cells to both paraquat and hydrogen peroxide resulted in significantly higher mutation frequencies compared with untreated control cells. Mutation frequencies up to 27-fold higher than the spontaneous mutation frequencies were obtained. The majority of the reversion mutants contained frameshift mutations within the target sequence. However, the pattern of deletions and additions was significantly different in the two cell lines. These results indicate that oxidative damage may play a role in instability of microsatellite sequences in vivo.
A number of clinical observations have indicated that the regenerative potential and overall function of the epidermis is modified with age. The epidermis becomes thinner, repairs itself less ...efficiently after wounding, and presents modified barrier function recovery. In addition, the dermal papillae fatten out with increasing age, suggesting a modification in the interaction between epidermal and dermal compartments. As the epidermal regenerative capacity is dependent upon stem and progenitor cell function, it is naturally of interest to identify and understand age-related changes in these particular keratinocyte populations. Previous studies have indicated that the number of stem cells does not decrease with age in mouse models but little solid evidence is currently available concerning human skin. The objective of this study was to evaluate the clonogenic potential of keratinocyte populations isolated from the epidermis of over 50 human donors ranging from 18 to 71 years old. The data indicate that the number of epidermal cells presenting high regenerative potential does not dramatically decline with age in human skin. The authors believe that changes in the microenvironment controlling epidermal basal cell activity are more likely to explain the differences in epidermal function observed with increasing age.
Micro-arc oxidation (MAO) is a novel technique to deposit ceramic coatings on the surface of valve metals such as Al, Mg, Ti and their alloys. The oxide films prepared by this technique have a dense ...structure, high adhesion and excellent integrated mechanical properties. This technique is especially suitable for the surface treatment of machine parts that work at high speed and have a higher demand for resistance to wear and corrosion. There have been many studies in this field in the last few years. The development of micro-arc oxidation and its applications in industrial field was reviewed. Based on conventional micro-arc oxidation, an innovative scanning micro-arc oxidation technology with visible advantages was developed in this paper. Moreover, verification experiment on scanning micro-arc oxidation was carried out, and the experimental results suggested that scanning micro-arc oxidation could be conduced at a high efficiency but low cost way. At last, the effects of scanning times on film thickness were discussed.
The purpose of this study was to evaluate the influence of fibrin glue and aprotinin on the growth of adult human skin keratinocytes in defined serum-free conditions. The keratinocytes were cultured ...on cell culture plastics and on a fibrin matrix prepared from fibrin glue. The cell growth was measured by MTT assay, while the growth of clonogenic keratinocytes was evaluated by colony assay and expressed as colony-forming efficiency (CFE). The clonogenic potential of keratinocytes released from subconfluent and confluent cultures grown on fibrin glue was also studied by the colony assay. In comparison to a plastic culture surface the fibrin glue had significantly (
P
<
0.05) increased the clonogenic potential of keratinocytes, as well as enhanced their growth. Keratinocytes released from subconfluent cultures grown on fibrin glue attained a significantly (
P
<
0.05) higher percentage of clonogenic cells than their confluent parallels. At 75, 150, 300 and 450
KIU/ml aprotinin did not influence the growth of keratinocytes (
P
>
0.2). A fibrin-based skin substitute produced in the defined keratinocyte medium could be safely used to treat a number of skin defects.
While convincing data clearly suggest the presence of stem cells in the basal limbal epithelium in vivo, testing the proliferation, self-renewal, and differentiation capacity of stem cells relies on ...the development of methodologies that allow for their isolation and extensive propagation in vitro. Clonal analysis involving differentiation between short-lived transient cell clones and long-lived stem cell clones is an invaluable technique to identify stem cells in vitro, and allows cells to be expanded over multiple passages. This chapter describes a protocol for the isolation, expansion, and clonal analysis of limbal epithelial stem cells. The cultivation method described may be essential for long-term restoration of the damaged ocular surface in patients with limbal stem cell deficiency.
When treating extensively burned patients using cultured epidermal sheets, the main problem is the time required for its production. Conventional keratinocyte isolation is usually done using Trypsin. ...We used a modification of the conventional isolation method in order to improve this process and increase the number of colonies from the isolated epidermal cell population.
To compare the action of trypsin and thermolysin in the keratinocyte isolation using newborn foreskin.
This method used thermolysin as it selectively digests the dermo-epidermal junction. After dermis separation, the epidermis was digested by trypsin in order to obtain a cell suspension.
Compared to the conventional procedure, these experiments demonstrated that in the thermolysin group, the epidermis was easily detached from the dermis, there was no fibroblast contamination and there were a larger number of keratinocyte colonies which had a significant statistical difference.
The number of colonies in the thermolysin group was significantly greater than in the trypsin group.
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