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1.
J Vis Exp ; (150)2019 08 10.
Article in English | MEDLINE | ID: mdl-31449232

ABSTRACT

The protocol described here is a reliable method of harvesting primary keratinocytes from adult female mice (54 ± 2 days old) yielding approximately 30 x 106 viable cells per mouse. Primary adult mouse keratinocytes are harvested from the dorsal skin of female mice. Male mice (~6 weeks old) can be used for keratinocyte harvesting depending on the requirements of the experiment. Euthanized mice are shaved and sterilized with serial washes in povidone iodine and ethanol solutions (70% alcohol). After disinfecting the mice, the dorsal skin is removed and the subcutaneous fat and muscle are removed with a scalpel and discarded. The skins are cut into small pieces and treated with a mild, low temperature trypsinization to detach the lower dermis from the epidermis. The scraped epidermises are stirred at low speed, filtered to remove the hairs, counted, and re-suspended in culture medium. This method provides an excellent single cell suspension of highly culturable cells for many downstream applications.


Subject(s)
Cell Culture Techniques/methods , Cell Separation/methods , Epidermal Cells/cytology , Keratinocytes/cytology , Animals , Cell Count , Female , Mice
2.
Nat Commun ; 9(1): 5293, 2018 12 13.
Article in English | MEDLINE | ID: mdl-30546048

ABSTRACT

We used allogeneic bone marrow transplantation (BMT) and a mouse multistage cutaneous carcinogenesis model to probe recruitment of bone marrow-derived epithelial cells (BMDECs) in skin tumors initiated with the carcinogen, dimethylbenz[a]anthracene (DMBA), and promoted with 12-O-tetradecanolyphorbol-13-acetate (TPA). BMDECs clustered in the lesional epithelium, expressed cytokeratins, proliferated, and stratified. We detected cytokeratin induction in plastic-adherent bone marrow cells (BMCs) cultured in the presence of filter-separated keratinocytes (KCs) and bone morphogenetic protein 5 (BMP5). Lineage-depleted BMCs migrated towards High Mobility Group Box 1 (HMGB1) protein and epidermal KCs in ex vivo invasion assays. Naive female mice receiving BMTs from DMBA-treated donors developed benign and malignant lesions after TPA promotion alone. We conclude that BMDECs contribute to the development of papillomas and dysplasia, demonstrating a systemic contribution to these lesions. Furthermore, carcinogen-exposed BMCs can initiate benign and malignant lesions upon tumor promotion. Ultimately, these findings may suggest targets for treatment of non-melanoma skin cancers.


Subject(s)
Bone Marrow Cells/pathology , Cell Transformation, Neoplastic/chemically induced , Cell Transformation, Neoplastic/pathology , Epithelial Cells/pathology , Skin Neoplasms/pathology , 9,10-Dimethyl-1,2-benzanthracene/toxicity , Animals , Bone Marrow Cells/cytology , Bone Marrow Transplantation , Bone Morphogenetic Protein 5/metabolism , Cell Movement , Cell Plasticity/physiology , Coculture Techniques , Epithelial Cells/cytology , Female , HMGB1 Protein/metabolism , Hair Follicle/cytology , Keratinocytes/pathology , Keratins/metabolism , Male , Mice , Mice, Inbred C57BL , Neoplasm Invasiveness/pathology , Papilloma/pathology , Stem Cells/cytology , Stem Cells/pathology , Tetradecanoylphorbol Acetate/toxicity , Tumor Cells, Cultured
3.
Photochem Photobiol ; 88(5): 1099-110, 2012.
Article in English | MEDLINE | ID: mdl-22211846

ABSTRACT

The mammalian skin is a complex dynamic organ composed of thin multilayered epidermis and a thick underlying connective tissue layer dermis. The epidermis undergoes continuous renewal throughout life. The stems cells uniquely express particular surface markers utilized for their identification, isolation and localization in specific niches in epidermis as well as hair follicles (HFs). The two stage skin carcinogenesis model involves stepwise accumulation of genetic alterations and ultimately leading to malignancy. Whereas early research on skin carcinogenesis focused on the molecular nature of carcinogens and tumor promoters, more recent studies have focused on the identification of the target cells and tumor promoting cells for both chemical and physical carcinogens and promoters. Recent studies support the hypothesis that keratinocyte stem cells are the targets in skin carcinogenesis. In this review, we discuss briefly the localization of stem cells in the epidermis and HFs, and review the possibility that skin papillomas and carcinomas are derived from stem cells, as well as from other cells in the cutaneous epithelium whose stem cell properties are not well known.


Subject(s)
Carcinoma, Basal Cell/pathology , Carcinoma, Squamous Cell/pathology , Keratinocytes/pathology , Neoplastic Stem Cells/pathology , Papilloma/pathology , Skin Neoplasms/pathology , Animals , Biomarkers/metabolism , Carcinogens/pharmacology , Carcinoma, Basal Cell/metabolism , Carcinoma, Squamous Cell/metabolism , Cell Transformation, Neoplastic/drug effects , Dermis/drug effects , Dermis/metabolism , Dermis/pathology , Epidermis/drug effects , Epidermis/metabolism , Epidermis/pathology , Hair Follicle/drug effects , Hair Follicle/metabolism , Hair Follicle/pathology , Humans , Keratinocytes/metabolism , Mice , Neoplastic Stem Cells/metabolism , Papilloma/metabolism , Skin Neoplasms/metabolism
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