Abstract
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 p16INK4A expression, unrelated to telomere status. Transduction to express bmi1, a repressor of the p16INK4A/p14ARF 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.
Highlights
Since their initial cultivation and characterization [1, 2], human embryonic stem cell lines have attracted interest for their potential to produce functional somatic cells for cell replacement therapy and to elucidate mechanisms of lineage development [3]
The p63þ/K14þ cell population recovered from H9 Human embryonic stem (hES) teratomas by cultivation in semidefined keratinocyte media or with 3T3 feeder support could not be propagated beyond 10–15 PD [35, 37]
Our extensive analysis and comparison with three structurally and developmentally different p63þ epithelial cell types has disclosed that this identification was incorrect. hESderK cells share with keratinocytes, urothelial cells, and tracheobronchial epithelial cells a set of hypermotility, marker expression, and histogenic characteristics in culture
Summary
Since their initial cultivation and characterization [1, 2], human embryonic stem (hES) cell lines have attracted interest for their potential to produce functional somatic cells for cell replacement therapy and to elucidate mechanisms of lineage development [3]. A major obstacle to evaluating normality, quality, and suitability of such cells for human use is the absence of optimized culture media for many normal cell types, necessary to evaluate proliferative and differentiation potential of hES-derived cells against proper standards. Some cell types, such as epidermal keratinocytes, can be expanded greatly in culture from small biopsies from the intended recipient, obviating the necessity to derive them from hES cells for therapy. This substantial proliferative potential of postnatal epidermal keratinocytes in culture and their ability to undergo histogenesis in experimental transplant [8,9,10] and organotypic culture [11,12,13] models and to re-establish normal, functioning, and permanent tissue in autologous transplants [14,15,16] provides a strong rationale for generating keratinocytes from hES cells to obtain proof-of-principal for hES-derived cell therapy.
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