Effects of serum and serum-derived factors on growth and differentiation of mouse keratinocytes

Abstract

Mouse epidermal keratinocytes (MK cells) were grown as replicating subcultures at clonal density, in a serum-free, low calcium basal medium supplemented with seven different growth factors (Bertolero et al., Exp. Cell. Res. 155:64-80, 1984). This serum-free system was used to investigate the activity of fetal bovine serum (FBS) and of serum-derived factors on the growth and differentiation of MK cells. Unfractionated, whole FBS inhibited growth and induced terminal differentiation of normal MK cells. The growth inhibitory activity was considerably reduced by passing whole FBS over a resin (Chelex) to remove Ca2+ and other di- and trivalent cations. It is not known whether this treatment removed other factors. Addition of individual serum components either stimulated or inhibited cell-growth and differentiation. Fetuin, a major alpha-globulin of FBS, and high density lipoprotein strongly inhibited the colony forming efficiency (CFE) of MK cells, whereas bovine serum albumin increased the CFE 4.5-fold and stimulated the growth rate as well. The addition of impure commercial preparations of platelet-derived growth factor inhibited the CFE and induced the morphological features of squamous terminally-differentiating keratinocytes. As reported in other systems, transforming growth factor beta (TGF-beta) inhibited the growth of secondary keratinocytes in a dose-dependent manner. Thus, at least three factors present in FBS inhibited growth whereas others were stimulatory. These observations explain the difficulties in obtaining replicating subcultures of mouse keratinocytes in serum-supplemented media and emphasize the importance of a serum-free system for studies on growth control and carcinogenesis in keratinocytes.

Control of growth and squamous differentiation in normal human bronchial epithelial cells by chemical and biological modifiers and transferred genes.

“…The data implied that TGF-p1 is an important differentiationinducing serum factor in NHBE cells. Similar findings have been reported for rat hepatocytes (44), mouse or human epiderminal keratinocytes (24,45), and rabbit tracheal epithelial cells (46,47).…”

“…Serum-Free Culture System for NHBE Cells Serum-free culture systems have been developed for human and mouse keratinocytes (22)(23)(24) and for normal (25)(26)(27) and neoplastic epithelial cells (28)(29)(30). Serum-free media permit studies of individual elements controlling growth and differentiation in a well-characterized environment.…”

Growth and differentiation of normal and transformed human bronchial epithelial cells

“…In these systems less than 1% serum shows growth inhibitory effect. In some of these cell culture systems, TGF-6 is growth inhibitory as is serum, i.e., NHBE cells (Masui et al, 19861, human epidermal keratinocytes (Moses et al, 1985), rat hepatocytes (Hayashi and Carr, 19851, and mouse epidermal keratinocytes (Bertolero et al, 1986). Thus, it is reasonable to consider TGF-fi as the factor in serum that inhibits growth of these primary epithelial cells listed in culture.…”

Binding and biological effects of tumor necrosis factor alpha on cultured human neonatal foreskin keratinocytes.

“…A similar process is observed in vitro with cultured epidermal cells or keratinocytes (1)(2)(3), making these cells a useful model for the study of growth and differentiation. Cytokines such as epidermal growth factor (4), transforming growth factors (5), and IFN (6), as well as retinoids (7), vitamin D metabolites (8,9), calcium (10, 1 1), and cAMP (12) are all known to alter the growth and differentiation of keratinocytes. Tumor necrosis factor alpha (TNF alpha),' a cytokine secreted by stimulated macrophages, is known to bind to and regulate the growth and function of a number of cells in vitro (13)(14)(15)(16)(17)(18).…”