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Human embryonic stem (hES) cells can generate cells expressing p63, K14, and involucrin, which have been proposed to be keratinocytes. Although these hES-derived, keratinocyte-like (hESderK) cells form epithelioid colonies when cultured in a fibroblast feeder system optimal for normal tissue-derived keratinocytes, they have a very short replicative lifespan unless engineered to express HPV16 E6E7. We report here that hESderK cells undergo senescence associated with p16(INK4A) expression, unrelated to telomere status. Transduction to express bmi1, a repressor of the p16(INK4A)/p14(ARF) locus, conferred upon hESderK cells and keratinocytes a substantially extended lifespan. When exposed to transforming growth factor beta or to an incompletely processed form of Laminin-332, three lifespan-extended or immortalized hESderK lines that we studied became directionally hypermotile, a wound healing and invasion response previously characterized in keratinocytes. In organotypic culture, hESderK cells stratified and expressed involucrin and K10, as do epidermal keratinocytes in vivo. However, their growth requirements were less stringent than keratinocytes. We then extended the comparison to endoderm-derived, p63(+)/K14(+) urothelial and tracheobronchial epithelial cells. Primary and immortalized lines of these cell types had growth requirements and hypermotility responses similar to keratinocytes and bmi1 expression facilitated their immortalization by engineering to express the catalytic subunit of telomerase (TERT). In organotypic culture, they stratified and exhibited squamous metaplasia, expressing involucrin and K10. Thus, hESderK cells proved to be distinct from all three normal p63(+) cell types tested. These results indicate that hESderK cells cannot be identified conclusively as keratinocytes or even as ectodermal cells, but may represent an incomplete form of, or deviation from, normal p63(+) lineage development.
Pubmed ID: 19489101
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American chemical, life science and biotechnology company owned by Merck KGaA. Merger of Sigma Chemical Company and Aldrich Chemical Company. Provides organic and inorganic chemicals, building blocks, reagents, advanced materials and stable isotopes for chemical synthesis, medicinal chemistry and materials science, antibiotics, buffers, carbohydrates, enzymes, forensic tools, hematology and histology, nucleotides, proteins, peptides, amino acids and their derivatives.
View all literature mentionsA commercial organization in regenerative medicine, focused in the areas of bio-active wound healing and soft tissue regeneration.
View all literature mentionsCommercial instrument and chemical vendor. Developer and manufacturer of specialized technological products for life science research and clinical diagnostics markets.
View all literature mentionsA second-generation retrovirus producer lines for the generation of helper free ecotropic and amphotropic retroviruses. The lines are based on the 293T cell line (a human embryonic kidney line transformed with adenovirus E1a and carrying a temperature sensitive T antigen co-selected with neomycin). The unique feature of this cell line is that it is highly transfectable with either calcium phosphate mediated transfection or lipid-based transfection protocols-- up to 50% or higher of cells can be transiently transfected. The lines were created by placing into 293T cells constructs capable of producing gag-pol, and envelope protein for ecotropic and amphotropic viruses. The lines offered advantages over previous stable systems in that virus can be produced in just a few days. Academic and non-profit laboratories may obtain the Phoenix cells from either Allele Biotechnology or the National Gene Vector Bank. The vectors may be obtained from Addgene. They are no longer distributing these reagents from the lab.
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