Spontaneous immortalization of human epidermal cells with naturally elevated telomerase.

This work explores spontaneous immortalization in keratinocytes, derived from two skin samples, that display naturally elevated telomerase activity. Serially passaged with 3T3 feeder layer support, the keratinocytes were examined for colony-forming ability, telomerase activity, telomere length, and finally gene expression using Affymetrix DNA microarrays. The cells initially exhibited normal karyotypes and low colony-forming efficiencies typical of normal epidermal cells, but after 40 passages (approximately 400 generations) colony-forming ability increased markedly, yielding immortalized lines exhibiting a small number of chromosomal aberrations and functionally normal p53. An improved protocol for analysis of microarray data permitted detection of 707 transcriptional changes accompanying immortalization including reduced p16(INK4A) mRNA. Telomerase activity was clearly elevated in cells even at low passage from both samples, and telomerase catalytic subunit mRNA was greatly elevated in those with elevated colony-forming ability. The data raise the possibility of an unusual natural phenotype in which aberrant telomerase regulation extends keratinocyte lifespan until rare variants evade senescence. In addition to revealing a potential tumor-prone syndrome, the findings emphasize the desirability of carefully minimizing the degree or timing of elevated expression of telomerase used to immortalize cells for therapeutic purposes.

An immortalized drug-resistant cell line established from 12-13-day mouse embryos for the propagation of human embryonic stem cells.

Human embryonic stem (ES) cells are usually co-cultivated with supporting cells consisting of short-term cultures of fibroblasts (not an immortalized line) in a medium lacking serum. This method has promoted important progress in the field, but suffers from certain disadvantages. By serial cultivation for 27 consecutive transfers and about 63 cell generations, we have evolved an immortalized line from fibroblastic cells of 12-13-day mouse embryos. This line (MMM) supports the multiplication of H9 cells better than the 3T3 line. It supports the growth of H9 cells as well as do available short-term fibroblast cultures, but maintains more effectively the stem cell character of the H9 cells, judging by their better retention of Oct4. We have made MMM cells resistant to blasticidin and zeocin, the most efficient antibiotics for selection of stable transformants. In the presence of zeocin, the resistant MMM were able to support multiplication and selection of ES cells transfected with an exogenous gene encoding zeocin resistance.

Immortalized keratinocyte lines derived from human embryonic stem cells.

Cells of the human embryonic stem (hES) cell line H9, when cultured in the form of embryoid bodies, give rise to cells with markers of the keratinocyte of stratified squamous epithelia. Keratinocytes also form in nodules produced in scid mice by injected H9 cells; the hES-derived keratinocytes could be recovered in culture, where their colonies underwent a peculiar form of fragmentation. Whether formed from embryoid bodies or in nodules, hES-derived keratinocytes differed from postnatal keratinocytes in their much lower proliferative potential in culture; isolated single keratinocytes could not be expanded into mass cultures. Although their growth was not improved by transduction with the hTERT gene, these keratinocytes were immortalized by transduction with the E6E7 genes of HPV16. Clonally derived lines isolated from E6E7-transduced keratinocytes continued to express markers of the keratinocyte lineage, but the frequency with which they terminally differentiated was reduced compared with keratinocytes cultured from postnatal human epidermis. If other hES-derived somatic cell types also prove to be restricted in growth potential, not identical to the corresponding postnatal cell types, and to require immortalization for clonal isolation and expansion, these properties will have to be considered in planning their therapeutic use.

Marker succession during the development of keratinocytes from cultured human embryonic stem cells.

Human embryonic stem cells injected into scid mice produce nodules containing differentiated somatic tissues. From the trypsinized cells of such a nodule, we have recovered keratinocytes that can be grown in cell culture. The method of recovery is sensitive enough to detect small numbers of keratinocytes formed in the nodule, but for purposes of analysis, it is preferable to study the development of the entire keratinocyte lineage in culture. The principle of our analysis is the successive appearance of markers, including transcription factors with considerable specificity for the keratinocyte (p63 and basonuclin) and differentiation markers characteristic of its final state (keratin 14 and involucrin). We have determined the order of marker succession during the time- and migration-dependent development of keratinocytes from single embryoid bodies in cell culture. Of the markers we have examined, p63 was the earliest to appear in the keratinocyte lineage. The successive accumulation of later markers provides increasing certainty of emergence of the definitive keratinocyte.