The Proliferation and Differentiation of Neonatal Epidermal Melanocytes in F₁ Hairless Mice of HR‐1 × HR/De in Serum‐Free Culture

Abstract

To investigate the characteristics of the proliferation and differentiation of epidermal melanocytes in F1 hairless mice of HR-1 x HR/De parents in vitro, cell suspensions of the neonatal epidermis were cultured in a serum-free medium supplemented with dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP) and/or basic fibroblast growth factor (bFGF). The differentiation of melanocytes was induced by treatment with DBcAMP. In contrast, the sustained proliferation of melanoblasts was induced by combined treatment with DBcAMP and bFGF. The melanoblasts could be subcultured in serum-free medium supplemented with the two factors in the presence of keratinocytes, but not in their absence. This is the first report of successful culture of melanoblasts and melanocytes from hairless mice; it is expected to be useful in understanding the mechanism of the development of pigmented spots in the epidermis of (HR-1 x HR/De)F1 mice, which are reported to be induced by repeated exposure to ultraviolet light B.

Immunohistochemical survey of the distribution of epidermal melanoblasts and melanocytes during the development of UVB-induced pigmented spots

“…Our previous study indicated that melanoblasts exist in primary-cultured epidermal cell suspensions derived from the same hairless mouse strain [27]. This hairless mouse characteristically possesses many melanoblasts in the epidermis of neonatal [24] and adult [27] skin and the melanoblasts proliferate well in culture. The study of melanocyte stem cells in this hairless mouse may contribute to our understanding of the mechanisms of the existence and maintenance of human stem cells.…”

“…Naganuma et al [23] reported that repeated UVBexposures induce pigmented spots in Hos:(HR-1 Â HR/De)F 1 mice long after the cessation of irradiation. Hos:(HR-1 Â HR/De)F 1 mice have many melanocytes in the epidermis [24] and are suitable for the study of melanocyte function. Pigmented spots induced by UV irradiation in mice may be a good animal model for human lentigines and freckles.…”

Granulocyte‐Macrophage Colony‐Stimulating Factor (GM‐CSF) Controls the Proliferation and Differentiation of Mouse Epidermal Melanocytes from Pigmented Spots Induced by Ultraviolet Radiation B

“…Epidermal cell suspensions from the control and from the UV‐irradiated skins were cultured with 2 ml MDMD2 for 2 d. At this time, cultures consisted of numerous keratinocyte colonies. They were then provided with 1 ml of fresh MDMD2, incubated for another 2 d, and the resulting culture media from multiple paired dishes (6–11) were collected with a Pasteur pipette and stored in plastic centrifuge tubes (Becton Dickinson) at −80°C. Similarly, culture media from 4 to 6 and 6 to 8 d (increase in the size and number of keratinocyte colonies as well as the initiation of melanocyte proliferation) were collected.…”

“…Disaggregated epidermal cell suspensions derived from the control and from the UV-irradiated mice were cultured in MDMD2. Culture media from multiple paired dishes (6)(7)(8)(9)(10)(11) were collected and assayed for GM-CSF using an ELISA Kit. As shown in Fig.…”

Purification and growth of melanocortin 1 receptor (Mc1r)-defective primary murine melanocytes is dependent on stem cell factor (SFC) from keratinocyte-conditioned media

“…Their poor growth has been attributed to faulty MSH responsiveness and decreased survival. Accordingly, current protocols for culturing primary murine melanocytes rely on pharmacologic manipulation of intracellular cAMP with α-MSH (either in recombinant form or as bovine pituitary extract), dibutyryl cAMP, cholera toxin and/or IBMX (Boissy and Halaban 1985; Halaban et al 1986; Abdel-Malek et al 1992; Furuya et al 1998) as well as stimulation of the protein kinase C pathway with phorbol esters (Kitano 1976; Pittelkow and Shipley 1989). Even with the use of these agents, however, the growth rate of Mc1r -defective melanocytes is delayed when compared to melanocytes with intact MC1R signaling suggesting that MC1R is involved in cell growth in ways other than by simply raising cytoplasmic cAMP levels (Abdel-Malek et al 1995; Suzuki et al 1996).…”