Previous culture systems for melanocytes have employed serum-supplemented medium and uncoated plastic dishes, prohibiting examination of possible substrate influences on cellular morphology and function. We now report, using a sensitive serum-free system and a quantitative procedure for evaluating cellular morphology, that modification of the plating surface affects human epidermal melanocyte attachment rate and subsequent morphology in vitro. Melanocytes attach and spread more rapidly on surfaces coated with fibronectin or Type I/III collagen or on surfaces previously conditioned by human keratinocytes, dermal fibroblasts, melanocytes, or melanoma cells than do melanocytes on untreated control surfaces. Type IV collagen and laminin, although minimally beneficial for cell attachment, do support a characteristic melanocyte morphology that differs from that seen either on the other coated surfaces or on uncoated plastic controls. Addition of fetal bovine serum at the time of inoculation has no appreciable effect on attachment but markedly improves cell spreading on untreated surfaces, while addition of nerve growth factor with or without serum to this system fails to affect cell attachment or spreading. Our data establish that human epidermal melanocytes are indeed capable of responding morphologically to substrate signals. The ability of several biochemically unrelated surfaces to enhance melanocyte attachment rate and spreading suggests that melanocytes have surface receptors with a variety of specificities. This work is relevant to the development of improved culture systems for melanocytes in vitro and to understanding melanocyte behavior in vivo.
Interferons (IF) are a family of glycoproteins known for their antiviral activity and the ability to inhibit growth and alter behavior of various normal and transformed cell types, both in vivo and in vitro. Because cultured human keratinocytes (HK) produce IF in response to viral infection, we undertook studies of alpha-IF and beta-IF effects on HK. Cloned human IF were added at time of seeding and at each feeding to paired dishes of keratinocytes maintained in serum-free hormone-supplemented medium. At 7 days significant inhibition of growth was observed for both alpha-IF and beta-IF, as determined by cell counts, total protein, and appearance of stained colonies, and was sustained for at least two weeks during continuous IF exposure. The inhibition was substantially blocked by prior addition of cholera toxin to the medium, consistent with competition for a common cell surface receptor. Growth of a single human epidermal carcinoma cell line was much less affected by IF than was growth of the normal keratinocytes. IF also promoted terminal differentiation of keratinocytes as assessed by desquamation rate of cells from the colony surface and by proportion of total cells having cornified envelopes. IF effect on both growth and differentiation was completely reversible within days of its removal from medium. These findings suggest that IF may function as a physiologic regulator of epidermal growth in vivo with properties of a negative growth factor or chalone.
A sensitive serum-free culture system was used to demonstrate that cells derived from normal human skin release soluble mediators which can modulate keratinocyte, melanocyte, and fibroblast growth in vitro. In M199 supplemented with epidermal growth factor, hydrocortisone, insulin, transferrin, triidothyronine, bovine serum albumin, and an extract of bovine hypothalamus, keratinocytes underwent approximately three to five cumulative population doublings (CPD) over a 7-day period. Addition of 20% keratinocyte-conditioned medium more than doubled keratinocyte growth and increased fibroblast growth 25-40% above controls. Dermal fibroblasts maintained in the same serum-free hormone-supplemented medium (SFHSM) alone underwent approximately 4.5-5.5 CPD over a 7-day period; 20% fibroblast-conditioned medium increased fibroblast growth 50-80% and increased keratinocyte growth 30-50%. Melanocytes maintained in the same SFHSM underwent approximately 1 CPD over a 14-day period and 2 CPD if the medium was supplemented with 2% fetal bovine serum (FBS). Addition of 20% melanocyte-conditioned medium more than doubled melanocyte growth in either SFHSM or in medium containing 2% FBS, but decreased both keratinocyte and fibroblast growth up to 30-60%. None of the conditioned media altered cellular morphology. These data provide the first demonstration of mutual growth modulation by cell types normally contiguous in vivo and expand existing evidence for autocrine and paracrine growth regulation by normal human cells.
An extract of bovine hypothalamus is known to be mitogenic for human keratinocytes in vitro. In order to identify the responsible substance(s), biochemical characterization and subsequent bioassay of the extract in a serum-free culture system were performed. The keratinocyte growth-promoting activity of the hypothalamic extract was unaffected by heating (100 degrees C, 10 min); acidification to pH 3.3; or by exposure to lipase, RNAase, or proteolytic enzymes; but was abolished by alkalinization to pH 11. An approximate molecular weight of 1,700 daltons was determined by elution on a calibrated Sephadex G-25 column, and an approximate pl of 3.5 was determined by isoelectric focusing. Optimal concentrations of the crude extract (150-300 micrograms/ml) increased keratinocyte growth approximately 50-fold compared to control cultures lacking the extract. Partial purification resulted in a preparation biologically active at 30 ng/ml protein equivalent and was consistent with the presence of a single mitogen which we have termed keratinocyte growth factor (KGF). Mitogenic activity for human melanocytes, dermal fibroblasts, and endothelial cells, present in the crude hypothalamic extract, was lacking in heat-treated preparations that contained KGF. Optimal concentrations of purified epidermal growth factor and ethanolamine, the only remotely similar substances previously reported to augment keratinocyte growth in vitro, could not substitute for KGF in the serum-free culture system. Keratinocyte growth-promoting activity comparable to that observed in bovine hypothalamic extracts was present in human hypothalamic extracts prepared in the same manner.
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