Primary cultured epithelial cells that are used for basic research are often cultivated on plastic whereas those used for clinical purposes are usually cultured in the presence of a feeder layer. Here, we examined the influence of a feeder layer on the expression, affinity and DNA binding ability of the transcription factors, Sp1 and Sp3 in primary cultures of human skin keratinocytes. Co-culturing both newborn and adult skin keratinocytes with lethally irradiated 3T3 cells as a feeder layer contributed to maintain the cell's morphological and growth characteristics and delayed terminal differentiation in vitro. 3T3 also stabilized the DNA binding properties of Sp1 without altering its transcription. Stimulation of Sp1/Sp3 expression appears to be mediated through cell-cell interactions and by factors secreted by 3T3. Thus, we propose that the feeder layer delay terminal differentiation of primary cultured skin keratinocytes by preventing extinction of transcription factors, like Sp1 and Sp3, which play pivotal functions in the cell cycle.

The Merkel cell is a highly specialized cell that primarily acts as a slowly adapting mechanoreceptor. Merkel cells are scarce in normal skin but can be identified by the expression of distinct keratin filaments. Merkel cells constitute a very unique population and many questions still remain as to their origin, number, proliferative capacity, and functions in cutaneous biology. The dissociation of epidermal cells from skin is a widely used technique to extract and culture keratinocytes. We took advantage of a two-step extraction method to quantify keratin-20-expressing Merkel cells among total cutaneous cells obtained from either hairy or glabrous skin biopsies. Flow cytometry analysis revealed that keratin-20-labeled Merkel cells represent between 3.6% and 5.7% of freshly dissociated basal epidermal cells. No significant differences were seen between samples derived from glabrous palmar and hairy anatomic sites, from children and adult, respectively. We also report on the presence of Merkel cells in primary and first subcultures of epidermal cells indicating their capacity to remain viable after extraction from skin of various anatomic sites. To our knowledge, this is the first demonstration of nontumorigenic human Merkel cells in culture in vitro. The persistence of a small number of Merkel cells in culture suggests that, with the development of appropriate culture conditions, these cells could be amplified and further studied to unravel long-standing questions relative to their paracrine function or epithelial origin.

To adequately and permanently restore organ function after grafting, human tissue-engineered skin substitutes (TESs) must ultimately contain and preserve functional epithelial stem cells (SCs). It is therefore essential that a maximum of SCs be preserved during each in vitro step leading to the production of TESs such as the culture process and the elaboration of a skin cell bank by cryopreservation. To investigate the presence and functionality of epithelial SCs within the human TESs made by the self-assembly approach, slow-cycling cells were identified using 5'-bromo-2'-deoxyuridine (BrdU) in the three-dimensional construct. A subset of basal epithelial cells retained the BrdU label and was positive for the SC-associated marker keratin 19 within TESs after a chase of 21 days in culture post-BrdU labeling. Moreover, keratinocytes harvested from TESs gave rise to SC-like colonies in secondary monolayer subcultures, indicating that SCs were preserved within TESs. To evaluate the effect of cryopreservation with dimethyl sulfoxide and storage in liquid nitrogen on SCs, human epithelial cells were extracted from skin samples, amplified in culture, and used to produce TESs, before cryopreservation as well as after thawing. We found that the proportion and the growth potential of epithelial SCs in monolayer culture and in TESs remained constant before and after cryopreservation. Further, the functionality of these substitutes was demonstrated by successfully grafting human TESs on athymic mice for 6 months. We conclude that human epithelial skin SCs are adequately preserved upon human tissue reconstruction. Thus, these TESs produced by the self-assembly approach are suitable for clinical applications.

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