Reconstituted skin in culture:a simple method with optimal differentiation

Basset-Séguin, Nicole; Culard, Jean Francois; Kerai, Cécile.; Bernard, Frédéric; Watrin, Annette; Demaille, Jacques; Guilhou, Jean Jacques

doi:10.1111/j.1432-0436.1990.tb00622.x

Cited by 38 publications

(19 citation statements)

References 30 publications

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“…The final number of cells per field could not be scored accurately because of stratification but was estimated as ∼1000 for K106ER cells in complete medium and ∼500 for K106ER cells in FAD without supplements. The final number of KmycER cells per field was slightly lower than that of K106ER (see ture plastic prevents analysis of the complete terminal differentiation pathway; therefore, we seeded keratinocytes on dead, de-epidermized dermis and cultured them at the air-medium interface to achieve a degree of histological differentiation that is as close as possible to epidermis in vivo (Prunié ras et al 1983;Basset-Sé guin et al 1990;Rikimaru et al 1997). If cells are being driven out of the stem cell compartment, there should be evidence of premature (accelerated) terminal differentiation and depletion of the proliferative population.…”

Section: Premature Terminal Differentiation In Epidermis Reconstructementioning

confidence: 99%

c-Myc promotes differentiation of human epidermal stem cells

Gandarillas¹,

Watt²

1997

Genes Dev.

302

301

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The epidermis contains two types of proliferative keratinocyte: stem cells, with unlimited self-renewal capacity, and transit amplifying cells, those daughters of stem cells that are destined to withdraw from the cell cycle and terminally differentiate after a few rounds of division. In a search for factors that regulate exit from the stem cell compartment, we constitutively expressed c-Myc in primary human keratinocytes by use of wild-type and steroid-activatable constructs. In contrast to its role in other cell types, activation of c-Myc in keratinocytes caused a progressive reduction in growth rate, without inducing apoptosis, and a marked stimulation of terminal differentiation. Keratinocytes can be enriched for stem or transit amplifying cells on the basis of ␤ 1 integrin expression and by use of this method to fractionate cells prior to c-Myc activation, we found that c-Myc acted selectively on stem cells, driving them into the transit amplifying compartment. As a result, activation of c-Myc in epidermis reconstituted on a dermal equivalent led to premature execution of the differentiation program. The transcriptional regulatory domain of c-Myc was required for these effects because a deletion within that domain acted as a dominant-negative mutation. Our results reveal a novel biological role for c-Myc and provide new insights into the mechanism regulating epidermal stem cell fate.

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Section: Premature Terminal Differentiation In Epidermis Reconstructementioning

confidence: 99%

c-Myc promotes differentiation of human epidermal stem cells

Gandarillas¹,

Watt²

1997

Genes Dev.

302

301

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“…NOE monolayers were also found to produce a sub-basal extracellular matrix (Figure 4) which has strong analogy to the basement membrane that epithelial cells reside on in vivo. The in vitro synthesis of a basement membrane has been noted for epidermal keratinocytes in organ and reconstituted skin equivalent systems [16,17], models with strong analogy to in vivo. The composition of and biological significance of this matrix remains to be determined but its presence would be expected to influence the biology of NOE cells and signifies that this in vitro model is allowing in vivo-like processes to occur.…”

Section: Biological Characterization Of Noe Cells (A) Morphology-exprmentioning

confidence: 99%

A simplified method for the routine culture of normal oral epithelial (NOE) cells from upper aerodigestive tract mucosa

Schantz

Edelstein

et al. 1996

Methods Cell Sci

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A method has been established for the routine culture for normal oral epithelial (NOE) cells based on simple methodology and commercially available materials. Normal mucosa was obtained from surgical resections of cancer patients, oropharyngeal mucosa, or pediatric tonsillectomies, anterior tonsillar pillar. NOE cells were allowed to outgrow using explant outgrowth techniques on whole mucosa or the dispased epithelial component. Outgrowth in AmnioMax-C100 medium (Gibco) followed by cell passaging in KGM (Clonetics) using Primaria (Falcon) tissue culture dishes produced reproducible growth, a reflection of a high rate of cell proliferation. Using this technique, cells maintain log phase growth for 2 cell passages, a minimum of 10-20 population doublings, and over 108 cells can be easily obtained from most specimens, if desired. The growth potential of NOE cells from individual clinical specimens can be predicted by cell size on a particle counter. If the mean population diameter is approximately 15 ~tm or less, cells exhibit good log phase growth whereas if the diameter is greater than 16 um, growth is poor. NOE cells cultured by this method show typical epithelial morphology, have desmosomal junctions, rest on an extracellular matrix, are positive for cytokeratin expression and are growth inhibited by TGF-[~, a normal inhibitory growth regulator. In summary, using this technique, epithelial cells from human oral mucosa can be easily generated in sufficient quantity and quality for cell biological, biochemical and molecular studies.

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“…This approach obviates the unnecessary use of large numbers of live animals and, in addition, has a number of advantages over animal models (19). The study of reconstruction of skin is of particular importance in relation to the treatment of extensive lesions such as burns or leg ulcers, and in vitro epithelial preparations also are valuable as a tool for studies of cell proliferation, differentiation, and transformation under controlled conditions (4). This technique has been used to study the keratin expression of oral epithelial cells from different sites (26).…”

Section: Introductionmentioning

confidence: 99%

“…This technique has been used to study the keratin expression of oral epithelial cells from different sites (26). Early attempts at producing a normal, stratified squamous epithelium included culture on air-exposed, de-epidermized human dermis with intact basement membrane (4,24). More recent refinements have used collagen/fibroblast matrices (5), with air/liquid interphase leading to the construction of a multilayered stratified epithelium on the matrix (2,6,7).…”

Section: Introductionmentioning

confidence: 99%

Reconstituted human oral and esophageal mucosa in culture

Oda

Savard

Eng

et al. 1998

In Vitro Cell.Dev.Biol.-Animal

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We have successfully established monolayer and organotypic culture techniques for growing human oral and esophageal epithelial cells. Cells in monolayer culture were grown in serum-free medium, modified from techniques previously reported by our group. The organotypic cultures were grown in a defined medium supplemented with 10% fetal calf serum. Oral and esophageal cells were maintained in keratinocyte basal medium with pituitary extract and other supplements, and 0.05 mM calcium for 7-9 and 9-11 passages, respectively. Both cell types had similar morphology by phase contrast microscopy. When confluent, the cells were predominantly small, basaloid, and uniform and interspersed with larger, differentiated cells. By immunohistochemistry, both cell types in monolayer were positive to AE1, AE3, and 34BE12 antibodies to keratins of stratified epithelia. Oral epithelial cells in monolayer also were positive to 35BH11, representative of simple epithelial keratins, while esophageal cells were not. The esophageal cells were focally positive to K13, while the oral cells were negative. Both were negative for K19. When comparing monolayer to organotypic cultures and to in vivo specimens, there was a significant difference in the expression of keratins. Using organotypic cultures, AE1, AE3, and 34BE12 were strongly positive in both oral and esophageal cells, similar to in vivo tissues. In contrast to monolayers, both were also focally positive for K19. Esophageal cells were strongly positive for K13, while the oral cells were mildly but uniformly positive. Both were negative for keratins of simple epithelia. These two cell culture techniques offer unique opportunities to study the pathobiology, including carcinogenesis, of stable cell systems from the oral and esophageal epithelia.

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Reconstituted skin in culture:a simple method with optimal differentiation

Cited by 38 publications

References 30 publications

c-Myc promotes differentiation of human epidermal stem cells

c-Myc promotes differentiation of human epidermal stem cells

A simplified method for the routine culture of normal oral epithelial (NOE) cells from upper aerodigestive tract mucosa

Reconstituted human oral and esophageal mucosa in culture

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