Cultures of differentiated melanocytes can readily be grown from the dissociated epidermis of neonatal mice, and immortal cell lines often develop from these. However, the first cells that grow and transiently dominate the cultures, while similar to melanocytes, are unpigmented. These have been shown to be precursors of melanocytes and may be termed melanoblasts. Under our previous standard culture conditions, involving the use of keratinocyte feeder cells, foetal calf serum, the phorbol ester 12-O-tetradecanoyl phorbol acetate (TPA) and cholera toxin, all the melanoblasts spontaneously differentiated to pigmented melanocytes within about 3 weeks. We now describe some factors affecting the proliferation and differentiation of diploid murine melanoblasts in the presence of serum. Murine stem cell factor/steel factor (SCF), basic fibroblast growth factor (bFGF) and murine leukaemia inhibitory factor/differentiation-inhibiting activity (LIF/DIA) all increased melanoblast numbers. SCF and LIF also slightly inhibited melanoblast differentiation, while cholera toxin and TPA promoted differentiation. Using some of these findings, and by regular replacement of keratinocyte or fibroblastoid feeder cells, we have established a clonal line of immortal murine melanoblasts, ‘melb-a’. These cells express tyrosinase-related protein-2 but not, in general, tyrosinase. They can be induced to differentiate irreversibly to functional melanocytes (also proliferative and immortal) by plating in the absence of feeder cells. Thus a new immortal melanocyte line, ‘melan-a2′, has also been produced.

Reference

Abdel-Malek
Z.
,
Swope
V.
,
Collins
C.
,
Boissy
R.
,
Zhao
H.
,
Nordlund
J.
(
1993
)
Contribution of melanogenic proteins to the heterogeneous pigmentation of human melanocytes.
J. Cell Sci
106
,
1323
1331
Baroffio
A.
,
Dupin
E.
,
Le Douarin
N. M.
(
1988
)
Clone-forming ability and differentiation potential of migratory neural crest cells.
Proc. Natl. Acad. Sci. USA
85
,
5325
5329
Bennett
D. C.
(
1993
)
Genetics, development and malignancy of melanocytes.
Int. Rev. Cytol
146
,
191
260
Bennett
D. C.
,
Bridges
K.
,
McKay
I. A.
(
1985
)
Clonal separation of mature melanocytes from premelanocytes in a diploid human cell strain: spontaneous and induced pigmentation of premelanocytes.
J. Cell Sci
77
,
167
183
Bennett
D. C.
,
Cooper
P. J.
,
Hart
I. R.
(
1987
)
A line of non-tumorigenic mouse melanocytes, syngeneic with the B16 melanoma and requiring a tumour promoter for growth.
Int. J. Cancer
39
,
414
418
Bennett
D. C.
,
Cooper
P. J.
,
Dexter
T. J.
,
Devlin
L. M.
,
Heasman
J.
,
Nester
B.
(
1989
)
Cloned mouse melanocyte lines carrying the germline mutations albino and brown: complementation in culture.
Development
105
,
379
385
Bennett
D. C.
,
Huszar
D.
,
Laipis
P. J.
,
Jaenisch
R.
,
Jackson
I. J.
(
1990
)
Phenotypic rescue of mutant brown melanocytes by a retrovirus carrying a wild-type tyrosinase-related protein gene.
Development
110
,
471
475
Eisinger
M.
,
Marko
O.
(
1982
)
Selective proliferation of normal human melanocytes in the presence of phorbol ester and cholera toxin.
Proc. Natl. Acad. Sci. USA
79
,
2018
2022
Flanagan
J. G.
,
Leder
P.
(
1990
)
The kit ligand: a cell surface molecule altered in steel mutant fibroblasts.
Cell
63
,
185
194
Flanagan
J. G.
,
Chan
D. C.
,
Leder
P.
(
1991
)
Transmembrane form of the kit ligand growth factor is determined by alternative splicing and is missing in the Sld mutant.
Cell
64
,
1025
1035
Funasaka
Y.
,
Boulton
T.
,
Cobb
M.
,
Yarden
Y.
,
Fan
B.
,
Lyman
S. D.
,
Williams
D. E.
,
Anderson
D. M.
,
Zakut
R.
,
Mishima
Y.
,
Halaban
R.
(
1992
)
c-Kit-kinase induces a cascade of protein tyrosine phosphorylation in normal human melanocytes in response to mast cell growth factor and stimulates mitogen-activated protein kinase but is down-regulated in melanomas.
Mol. Biol. Cell
3
,
197
209
Gabbianelli
M.
,
Sargiacomo
M.
,
Pelosi
E.
,
Testa
U.
,
Isacchi
G.
,
Peschle
C.
(
1990
)
‘Pure’ human hematopoietic progenitors: permissive action of basic fibroblast growth factor.
Science
249
,
1561
1564
Halaban
R.
,
Ghosh
S.
,
Baird
A.
(
1987
)
bFGF is the putative natural growth factor for human melanocytes.
In Vitro Cell Dev. Biol
23
,
47
52
Halaban
R.
,
Moellmann
G.
,
Tamura
A.
,
Kwon
B. S.
,
Kuklinska
E.
,
Pomerantz
S.
,
Lerner
A. B.
(
1988
)
Tyrosinases of murine melanocytes with mutations at the albino locus.
Proc. Natl. Acad. Sci. USA
85
,
7241
7245
Herlyn
M.
,
Mancianti
M. R.
,
Jambrosic
J.
,
Bolen
J. B.
,
Koprowski
H.
(
1988
)
Regulatory factors that determine growth and phenotype of normal human melanocytes.
Exp. Cell Res
179
,
322
331
Hilton
D. J.
(
1992
)
LIF: lots of interesting functions.
Trends Biochem. Sci
17
,
72
76
Hirobe
T.
(
1991
)
Selective growth and serial passage of mouse melanocytes from neonatal epidermis in a medium supplemented with bovine pituitary extract.
J. Exp. Zool
257
,
184
194
Hirobe
T.
(
1992
)
Basic fibroblast growth factor stimulates the sustained proliferation of mouse epidermal melanoblasts in a serum-free medium in the presence of dibutyryl cyclic AMP and keratinocytes.
Development
114
,
435
445
Hirobe
T.
(
1994
)
Keratinocytes are involved in regulating the developmental changes in the proliferative activity of mouse epidermal melanoblasts in serum-free culture.
Dev. Biol
161
,
59
69
Hunt
G.
,
Todd
C.
,
Kyne
S.
,
Thody
A. J.
(
1994
)
ACTH stimulates melanogenesis in cultured human melanocytes.
J. Endocrinol
140
,
1
–.
Huszar
D.
,
Sharpe
A.
,
Jaenisch
R.
(
1991
)
Migration and proliferation of cultured neural crest cells in W mutant neural crest chimeras.
Development
112
,
131
141
Ito
K.
,
Morita
T.
,
Sieber-Blum
M.
(
1993
)
In vitro clonal analysis of mouse neural crest development.
Dev. Biol
157
,
517
525
Jackson
I. J.
,
Bennett
D. C.
(
1990
)
Identification of the albino mutation of mouse tyrosinase by analysis of an in vitro revertant.
Proc. Natl. Acad. Sci. USA
87
,
7010
7014
Matsui
Y.
,
Zsebo
K.
,
Hogan
B. L. M.
(
1992
)
Derivation of pluripotent embryonic stem cells from murine primordial germ cells in culture.
Cell
70
,
841
847
Mayer
T. C.
(
1982
)
The control of embryonic pigment cell proliferation in culture by cyclic AMP.
Dev. Biol
94
,
509
514
Morrison-Graham
K.
,
Weston
J. A.
(
1993
)
Transient steel factor dependence by neural crest-derived melanocyte precursors.
Dev. Biol
159
,
346
352
Murphy
M.
,
Reid
K.
,
Hilton
D. J.
,
Bartlett
P. F.
(
1991
)
Generation of sensory neurons is stimulated by leukemia inhibitory factor.
Proc. Natl. Acad. Sci. USA
88
,
3498
3501
Neer
E. J.
,
Clapham
D. E.
(
1988
)
Roles of G protein subunits in transmembrane signalling.
Nature
333
,
129
134
Parchment
R. E.
,
Lewellyn
D.
,
Swartzendruber
D.
,
Pierce
G. B.
(
1990
)
Serum amine oxidase activity contributes to crisis in mouse embryo cell lines.
Proc. Natl. Acad. Sci. USA
87
,
4340
4344
Pavan
W. J.
,
Tilghman
S. M.
(
1994
)
Piebald lethal (sl acts early to disrupt the development of neural crest-derived melanocytes.
Proc. Natl. Acad. Sci. USA
91
,
7159
7163
Resnick
J. L.
,
Bixler
L. S.
,
Cheng
L.
,
Donovan
P. J.
(
1992
)
Longterm proliferation of mouse primordial germ cells in culture.
Nature
359
,
550
551
Rheinwald
J. G.
,
Green
H.
(
1975
)
Formation of a keratinizing epithelium in culture by a cloned cell line derived from a teratoma.
Cell
6
,
317
330
Sieber-Blum
M.
,
Cohen
A. M.
(
1980
)
Clonal analysis of quail neural crest cells: they are pluripotent and differentiate in the absence of noncrest cells.
Dev. Biol
80
,
96
106
Spanakis
E.
,
Lamina
P.
,
Bennett
D. C.
(
1992
)
Effects of the developmental colour mutations silver and recessive spotting on proliferation of diploid and immortal mouse melanocytes in culture.
Development
114
,
675
680
Steel
K. P.
,
Davidson
D. R.
,
Jackson
I. J.
(
1992
)
TRP-2/DT, a new early melanoblast marker, shows that steel growth factor (c-kit ligand) is a survival factor.
Development
115
,
1111
1119
Stemple
D. L.
,
Anderson
D. J.
(
1993
)
Lineage diversification of the neural crest: In vitro investigations.
Dev. Biol
159
,
12
23
Tang
A.
,
Eller
M. S.
,
Hara
M.
,
Yaar
M.
,
Hirohashi
S.
,
Gilchrest
B. A.
(
1994
)
E-cadherin is the major mediator of human melanocyte adhesion to keratinocytes in vitro.
J. Cell Sci
107
,
983
992
Tsukamoto
K.
,
Jackson
I.
,
Urabe
K.
,
Montague
P.
,
Hearing
V.
(
1992
)
A second tyrosinase-related protein, TRP-2, is a melanogenic enzyme termed dopachrome tautomerase.
EMBO J
11
,
519
526
Wakeling
W. F.
,
Greetham
J.
,
Devlin
L. M.
,
Bennett
D. C.
(
1992
)
Suppression of properties associated with malignancy in murine melanoma-melanocyte hybrid cells.
Br. J. Cancer
65
,
529
537
Warburton
M. J.
,
Mitchell
D.
,
Ormerod
E. J.
,
Rudland
P. S.
(
1982
)
Distribution of myoepithelial cells and basement membrane proteins in the resting, pregnant, lactating, and involuting rat mammary gland.
J. Histochem. Cytochem
30
,
667
676
White
R.
,
Hu
F.
,
Roman
N. A.
(
1983
)
False dopa reaction in studies of mammalian tyrosinase: some characteristics and precautions.
Stain Technol
58
,
13
19
Yamamoto
H.
,
Takeuchi
S.
,
Kudo
T.
,
Sato
C.
,
Takeuchi
T.
(
1989
)
Melanin production in cultured albino melanocytes transfected with mouse tyrosinase cDNA.
Jpn. J. Genet
64
,
121
135
Zhou
B.-K.
,
Kobayashi
T.
,
Donatien
P. D.
,
Bennett
D. C.
,
Hearing
V. J.
,
Orlow
S. J.
(
1994
)
Identification of a melanosomal matrix protein encoded by the murine si (silver) locus using ‘organelle scanning’.
Proc. Natl. Acad. Sci. USA
91
,
7076
7080
This content is only available via PDF.