“Of Mice and Men” the Cell Cycle in Human Epidermis in Vivo

Gelfant, Seymour

doi:10.1111/1523-1747.ep12507367

Cited by 26 publications

(14 citation statements)

References 10 publications

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“…In our studies we found that intermediate and periderm cells, as well as basal cells, were labeled; and, therefore, we calculated labeling indices for each cell type (see Table 2). Our data, however, agree with previously published results (Stern, 1974) if basal and intermediate cell labeling indices (Potten, 1975;Gelfant, 1982) and that the needed increased rate of epidermal cell production appears to be provided by the intermediate cell layer. It is also interesting to note that the labeling index of adult skin in this organ culture system was approximately 5%, a figure well within the previously reported in uiuo labeling indices of adult skin (Potten, 1975), which suggests that, within the first day, this organ culture system does not measurably alter the proliferative rates of specimens.…”

Section: Discussionsupporting

confidence: 93%

Proliferation of human embryonic and fetal epidermal cells in organ culture

Bickenbach

Holbrook

1986

Am. J. Anat.

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The morphology of human embryonic and fetal skin growth in organ culture at the air-medium interface was examined, and the labeling indices of the epidermal cells in such cultures were determined. The two-layered epidermis of embryonic specimens increased to five or six cell layers after 21 days in culture, and the periderm in such cultures changed from a flat cell type to one with many blebs. The organelles in the epidermal cells remained unchanged. Fetal epidermis, however, differentiated when grown in this organ culture system from three layers (basal, intermediate, and periderm) to an adult-type epidermis with basal, spinous, granular, and cornified cell layers. Keratohyalin granules, lamellar granules, and bundles of keratin filaments, organelles associated with epidermal cell differentiation, were observed in the suprabasal cells of such cultures. The periderm in these fetal cultures formed blebs early but was sloughed with the stratum corneum in older cultures. The rate of differentiation of the fetal epidermis in organ culture was related to the initial age of the specimen cultured, with the older specimens differentiating at a faster rate than the younger specimens. Labeling indices (LIs) of embryonic and fetal epidermis and periderm were determined. The LI for embryonic basal cells was 8.5% and for periderm was 8%. The fetal LIs were 7% for basal cells, 1% for intermediate cells, and 3% for periderm. The ability to maintain viable pieces of skin in organ culture affords a model for studying normal and abnormal human epidermal differentiation from fetal biopsies and for investigating proliferative diseases.

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Section: Discussionsupporting

confidence: 93%

Proliferation of human embryonic and fetal epidermal cells in organ culture

Bickenbach

Holbrook

1986

Am. J. Anat.

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“…This kinetic profile is comparable to the in vivo antigen kinetics observed on DNA tattooing of mice (Bins et al, 2005). This rather transient nature of vaccine-induced antigen expression in skin may possibly be explained by the high turnover of the epidermis (Gelfant, 1982) but could also be due to gene silencing. Importantly, it has previously been shown that fresh skin in culture does not lose its viability in the first 30 hr of culturing, with a viability decrease of 50% after 60 hr in medium at 378C (Castagnoli et al, 2003;Messager et al, 2003).…”

Section: Van Den Berg Et Alsupporting

confidence: 76%

Optimization of Intradermal Vaccination by DNA Tattooing in Human Skin

et al. 2009

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The intradermal administration of DNA vaccines by tattooing is a promising delivery technique for genetic immunization, with proven high immunogenicity in mice and in nonhuman primates. However, the parameters that result in optimal expression of DNA vaccines that are applied by this strategy to human skin are currently unknown. To address this issue we set up an ex vivo human skin model in which DNA vaccine-induced expression of reporter proteins could be monitored longitudinally. Using this model we demonstrate the following: First, the vast majority of cells that express DNA vaccine-encoded antigen in human skin are formed by epidermal keratinocytes, with only a small fraction (about 1%) of antigen-positive epidermal Langerhans cells. Second, using full randomization of DNA tattoo variables we show that an increase in DNA concentration, needle depth, and tattoo time all significantly increase antigen expression ( p < 0.001), with DNA concentration forming the most critical variable influencing the level of antigen expression. Finally, in spite of the marked immunogenicity of this vaccination method in animal models, transfection efficiency of the technique is shown to be extremely low, estimated at approximately 2 to 2000 out of 1Â10 10 copies of plasmid applied. This finding, coupled with the observed dependency of antigen expression on DNA concentration, suggests that the development of strategies that can enhance in vivo transfection efficacy would be highly valuable. Collectively, this study shows that an ex vivo human skin model can be used to determine the factors that control vaccine-induced antigen expression and define the optimal parameters for the evaluation of DNA tattoo or other dermal delivery techniques in phase 1 clinical trials.

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“…Since human skin is a renewable tissue that undergoes loss and replacement of all keratinocytes approximately every 21 days or less in the wound-like environment of a skin graft (17,20,48), successful skin gene therapy requires gene targeting to keratinocyte progenitor cells for persistent expression in a high percentage of keratinocytes. These studies represent the first comprehensive assessment of keratinocyte transduction by lentiviral vectors and also demonstrate the advantages of using grafts of human skin equivalents as an in vivo assay to assess the ex vivo transduction of keratinocyte progenitor cells.…”

Section: Discussionmentioning

confidence: 99%

“…In our ex vivo model, the grafted human keratinocytes would be expected to completely turn over every 2 to 3 weeks, and all genetically engineered keratinocytes would be lost unless progenitor cells were stably transduced (17,19,20,48). Therefore, the ability of lentiviral vectors (or retroviral vectors) to introduce genes into keratinocyte progenitor cells can be determined by the percentage of human keratinocytes expressing GFP after several epidermal turnovers, analogous to biological models assessing hematopoietic stem cell targeting (7,13,34,(52)(53)(54).…”

Section: Lentiviral Vectors Are Superior To Retroviral Vectors In Tramentioning

confidence: 99%

In Vivo Assessment of Gene Delivery to Keratinocytes by Lentiviral Vectors

Kühn

Terunuma

Pfützner

et al. 2002

J Virol

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For skin gene therapy, introduction of a desired gene into keratinocyte progenitor or stem cells could overcome the problem of achieving persistent gene expression in a significant percentage of keratinocytes. Although keratinocyte stem cells have not yet been completely characterized and purified for gene targeting purposes, lentiviral vectors may be superior to retroviral vectors at gene introduction into these stem cells, which are believed to divide and cycle slowly. Our initial in vitro studies demonstrate that lentiviral vectors are able to efficiently transduce nondividing keratinocytes, unlike retroviral vectors, and do not require the lentiviral accessory genes for keratinocyte transduction. When lentiviral vectors expressing green fluorescent protein (GFP) were directly injected into the dermis of human skin grafted onto immunocompromised mice, transduction of dividing basal and nondividing suprabasal keratinocytes could be demonstrated, which was not the case when control retroviral vectors were used. However, flow cytometry analysis demonstrated low transduction efficiency, and histological analysis at later time points provided no evidence for progenitor cell targeting. In an alternative in vivo method, human keratinocytes were transduced in tissue culture (ex vivo) with either lentiviral or retroviral vectors and grafted as skin equivalents onto immunocompromised mice. GFP expression was analyzed in these human skin grafts after several cycles of epidermal turnover, and both the lentiviral and retroviral vector-transduced grafts had similar percentages of GFP-expressing keratinocytes. This ex vivo grafting study provides a good in vivo assessment of gene introduction into progenitor cells and suggests that lentiviral vectors are not necessarily superior to retroviral vectors at introducing genes into keratinocyte progenitor cells during in vitro culture. Achieving persistent and high-level gene expression in a significant percentage of target cells is important for establishing successful clinical applications of gene therapy. For gene therapy of the skin, a renewable tissue undergoing constant turnover, and for achieving long-term expression in a significant percentage of keratinocytes, gene targeting to keratinocyte stem cells (KSC) or long-lasting keratinocyte progenitor cells will be required (36). In ex vivo skin gene therapy, keratinocytes are removed from the donor and transduced with retroviral vectors in vitro without selectively targeting KSC. The genetically modified keratinocytes can then be grafted back onto the donor. Even though progress in skin gene therapy has enabled gene expression from retroviral vectors to be detected for sustained periods in grafted keratinocytes (9,24,32,56), transgene expression in vivo is frequently decreased over time, either in the duration of expression or in the percentage of keratinocytes expressing the gene (12,14,18,35,50). The factors contributing to this decreased expression may be similar to those for other tissues (14,27,41,51) and may include in...

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“Of Mice and Men” the Cell Cycle in Human Epidermis in Vivo

Cited by 26 publications

References 10 publications

Proliferation of human embryonic and fetal epidermal cells in organ culture

Proliferation of human embryonic and fetal epidermal cells in organ culture

Optimization of Intradermal Vaccination by DNA Tattooing in Human Skin

In Vivo Assessment of Gene Delivery to Keratinocytes by Lentiviral Vectors

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