Immortalized keratinocyte lines derived from human embryonic stem cells

Abstract

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 hESderived 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.hES-derived keratinocytes ͉ HPV16 E6E7 ͉ immortalization

Retinoic Acid and Bone Morphogenetic Protein Signaling Synergize to Efficiently Direct Epithelial Differentiation of Human Embryonic Stem Cells

“…6C). As has previously been described [3], these epithelial cultures exhibited a lower expansion potential than primary cultures; cryopreserved hESC-derived keratinocytes could be propagated for approximately 10 population doublings (Fig. 6D).…”

“…6D). However, we were able to obtain colonyforming keratinocytes at a frequency of at least 0.003 from differentiated hESCs plated, a sixfold increase over previously reported methods that used implantation into immunocompromised mice [3]. It must also be noted that our growth analysis used feeder-free culture in serum-free medium, and it is unclear how these results might translate to more routinely used protocols.…”

“…Previous studies have demonstrated that spontaneous keratinocyte differentiation in EBs occurs inefficiently [2,4]. These hESC derivatives exhibited a lower proliferative capacity compared with primary keratinocytes, and immortalization was required to extend the lifespan of these cultures [3]. Although RA-induced hESC cultures produced keratinocytes with a replicative potential similar to that of controls, the high efficiency of this process provides an alternative to transformation, which can interfere with biological functions and complicates clinical use.…”

“…Human ESCs can differentiate to the keratinocyte lineage [2], although current methods are inefficient and isolated keratinocytes have a reduced expansion potential [3,4]. The hESC differentiation process toward keratinocytes is marked by expression of p63, a transcription factor necessary for maintenance of regenerative epithelia (i.e., the epidermis) [5][6][7], and keratin 14 (K14), an intermediate filament present in the basal layer of stratified squamous epithelia [8].…”

Progress and prospects: gene transfer into embryonic stem cells

“…52,53 Although hESCs have a stable and significant proliferative capacity, this may not be true of hESC-derived cells. Some evidence has suggested, for instance, that hESC-derived hematopoietic progenitors and keratinocytes 53,54 have much lower proliferative potential than their somatic counterparts. In the latter case, gene …”