Integrated control of growth and differentiation of normal human prokeratinocytes cultured in serum‐free medium: Clonal analyses, growth kinetics, and cell cycle studies

Abstract

The effects of growth factors, hormones, and calcium on the growth and differentiation of secondary cultures of normal human prokeratinocytes, i.e., proliferative keratinocytes, derived from adult or neonatal skin were determined by culture in serum-free basal medium, MCDB 153. Clonal growth was achieved when MCDB 153 was supplemented with either epidermal growth factor (EGF) or bovine pituitary extract (BPE), provided insulin was present. In the absence of insulin, however, both EGF and BPE were required for clonal growth. Using this assay, it was established that colony-forming efficiency is independent of calcium concentrations above 0.03 mM and averages 56%; colony size, however, was influenced by calcium and EGF concentrations. Optimal clonal growth occurred in medium containing 10 ng/ml EGF and 0.3 mM calcium. By contrast, differentiation was enhanced by the combination of low EGF (0.1 ng/ml) and high calcium (2 mM). This suggests that an inverse relationship exists between the growth response (extent of clonal growth) and the differentiation response (extent of differentiation). These results suggest that proliferation and differentiation are regulated in an integrated manner. Detailed kinetic studies and cytofluorimetric and autoradiographic analyses also showed that exponentially growing secondary cultures of adult and neonatal prokeratinocytes have a 24-hour cell generation time with G1, S, G2, and M phases of 12, 8, 3, and 1 hours, respectively. In addition, the data show that such cells can be growth arrested in medium that does not induce differentiation and that such a procedure significantly limits the cell's subsequent proliferative potential. Furthermore, prolonged culture of adult (greater than 30 population doublings) and neonatal prokeratinocytes (greater than 50 population doublings) is associated with senescence and the G1 arrest of noncycling cells.

Switch from p53 to MDM2 as differentiating human keratinocytes lose their proliferative potential and increase in cellular size

“…An induction of MDM2 preceded that of p53, and occurred when an increase of cellular size started to become evident (see 24 h, Figure 3b). p53 and MDM2 expression during spontaneous keratinocyte differentiation Primary keratinocytes do not stratify when cultured in a de®ned serum-free low calcium medium (DKSFM) in the absence of a feeder layer but instead, they form a monolayer of basal-like proliferative cells (Hennings et al, 1980;Wille et al, 1984). However, terminal di erentiation is not suppressed and a proportion of cells within this monolayer express involucrin and eventually shed into the culture medium (Drozdo and Pledger, 1993;Watt and Green, 1982; Figure 4; unpublished results).…”

Multistep Process of Squamous Differentiation in Tracheobronchial Epithelial Cells In Vitro: Analogy with Epidermal Differentiation

“…In contrast, tracheobronchial epithelial and human epidermal cells exhibit optimal growth at calcium concentrations > 100 ,4M (33,43,84,85) and maintain a high colony-forming efficiency. However, high calcium does induce a reversible morphological change in these cells (33,43,84,85) Transforming Growth Factor ( Transforming growth factor p (TGF-p) is a polypeptide hormone that is expressed and released by many normal and neoplastic cells (123,124). It consists of two 12 kD chains linked by disulfide bonds and is synthesized as part of a larger secretory precursor (123).…”

Isolation and cultivation of primary keratinocytes from piglet skin for compartmentalized co-culture with dorsal root ganglion neurons

“…Different culture media have been used to improve cell culture yield and the viability of keratinocytes from humans, mice and adult pigs [2,6,[20][21][22], but no medium has been described yet for keratinocytes isolated from neonatal pigs.…”

“…In vitro, keratinocyte differentiation has been successfully induced by several factors, such as osmotic stress, growth factors, high calcium concentrations, or serum supplementation [16,22,23,27,46]. To investigate whether the keratinocytes isolated from piglets retain the ability to differentiate in vitro after the isolation procedure, normal keratinocyte growth medium (EKGM) was replaced with a differentiation medium.…”

“…Bovine pituitary extract and insulin are other nutrients found in most keratinocyte media and can increase keratinocyte proliferation. BPE can stimulate keratinocyte proliferation in medium deficient in EGF (0.1 ng/ml) [22]. However, in combination with other nutrients they can also prevent growth.…”