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In vitro dedifferentiation of melanocytes from adult epidermis.

Kormos B, Belso N, Bebes A, Szabad G, Bacsa S, Széll M, Kemény L, Bata-Csörgo Z - PLoS ONE (2011)

Bottom Line: In Mel-mix medium the cultured melanocytes are bipolar, unpigmented and highly proliferative.TRP-1 mRNA expression was induced in dedifferentiated melanocytes by UV-B irradiated keratinocyte supernatants, however direct UV-B irradiation of the cells resulted in further decrease of TRP-1 mRNA expression.These dedifferentiated, easily accessible cultured melanocytes provide a good model for studying melanocyte differentiation and possibly transdifferentiation.

View Article: PubMed Central - PubMed

Affiliation: Dermatological Research Group of the Hungarian Academy of Sciences, Szeged, Hungary. kormosbetti@mail.derma.szote.u-szeged.hu

ABSTRACT
In previous work we described a novel culture technique using a cholera toxin and PMA-free medium (Mel-mix) for obtaining pure melanocyte cultures from human adult epidermis. In Mel-mix medium the cultured melanocytes are bipolar, unpigmented and highly proliferative. Further characterization of the cultured melanocytes revealed the disappearance of c-Kit and TRP-1 and induction of nestin expression, indicating that melanocytes dedifferentiated in this in vitro culture. Cholera toxin and PMA were able to induce c-Kit and TRP-1 protein expressions in the cells, reversing dedifferentiation. TRP-1 mRNA expression was induced in dedifferentiated melanocytes by UV-B irradiated keratinocyte supernatants, however direct UV-B irradiation of the cells resulted in further decrease of TRP-1 mRNA expression. These dedifferentiated, easily accessible cultured melanocytes provide a good model for studying melanocyte differentiation and possibly transdifferentiation. Because melanocytes in Mel-mix medium can be cultured with human serum as the only supplement, this culture system is also suitable for autologous cell transplantation.

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Switching the PMA-containing M254 medium to PMA-free Mel-mix medium results in cell morphology changes and loss of c-Kit and TRP-1 expressions.When melanocyte cultures were initiated in PMA-containing M254 medium, cells were dendritic, and expressed TRP-1 as well as c-Kit proteins (100% of the cells expressed TRP-1 and c-Kit). At week 4, when cells were in 4th passage, half of the cultures were switched to PMA-free Mel-mix medium. Cells in Mel-mix medium became bipolar (B), lost TRP-1 (D) and c-Kit (F), and increased EGFR (H) expressions in two weeks, while melanocytes continuously cultured in M254 medium remained dendritic (A) expressed both TRP-1 (C) and c-Kit (E) proteins, and showed only a week EGFR staining (G). Bar: 100 µm.
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pone-0017197-g002: Switching the PMA-containing M254 medium to PMA-free Mel-mix medium results in cell morphology changes and loss of c-Kit and TRP-1 expressions.When melanocyte cultures were initiated in PMA-containing M254 medium, cells were dendritic, and expressed TRP-1 as well as c-Kit proteins (100% of the cells expressed TRP-1 and c-Kit). At week 4, when cells were in 4th passage, half of the cultures were switched to PMA-free Mel-mix medium. Cells in Mel-mix medium became bipolar (B), lost TRP-1 (D) and c-Kit (F), and increased EGFR (H) expressions in two weeks, while melanocytes continuously cultured in M254 medium remained dendritic (A) expressed both TRP-1 (C) and c-Kit (E) proteins, and showed only a week EGFR staining (G). Bar: 100 µm.

Mentions: In order to study the possible dedifferentiation of melanocytes, normal human adult epidermal melanocytes were cultured in commercially available PMA containing M254 medium for four weeks. These cells showed dendrite-rich morphology, expressed TRP-1 and c-Kit proteins uniformly and EGFR weakly. After four weeks, cultures were split into two; one part was continuously cultured in M254 medium; the other part was switched to Mel-mix medium for the following two weeks. Melanocytes cultured in the PMA containing M254 medium remained dendritic (Figure 2A) and expressed TRP-1 (100% of the cells were positive, Figure 2C) and c-Kit (100% of the cells were positive, Figure 2E) proteins and showed a generally week EGFR staining (only 32% of the cells showed weak positivity, Figure 2G), while cells cultured in Mel-mix for two weeks became bipolar (Figure 2B), their TRP-1 (Figure 2D) and c-Kit (Figure 2F) protein expressions drastically decreased (TRP-1+cells: 16%, c-Kit+cells: 25%), and their EGFR expression strongly increased (EGFR+ cell: 80%) (Figure 2H).


In vitro dedifferentiation of melanocytes from adult epidermis.

Kormos B, Belso N, Bebes A, Szabad G, Bacsa S, Széll M, Kemény L, Bata-Csörgo Z - PLoS ONE (2011)

Switching the PMA-containing M254 medium to PMA-free Mel-mix medium results in cell morphology changes and loss of c-Kit and TRP-1 expressions.When melanocyte cultures were initiated in PMA-containing M254 medium, cells were dendritic, and expressed TRP-1 as well as c-Kit proteins (100% of the cells expressed TRP-1 and c-Kit). At week 4, when cells were in 4th passage, half of the cultures were switched to PMA-free Mel-mix medium. Cells in Mel-mix medium became bipolar (B), lost TRP-1 (D) and c-Kit (F), and increased EGFR (H) expressions in two weeks, while melanocytes continuously cultured in M254 medium remained dendritic (A) expressed both TRP-1 (C) and c-Kit (E) proteins, and showed only a week EGFR staining (G). Bar: 100 µm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3044174&req=5

pone-0017197-g002: Switching the PMA-containing M254 medium to PMA-free Mel-mix medium results in cell morphology changes and loss of c-Kit and TRP-1 expressions.When melanocyte cultures were initiated in PMA-containing M254 medium, cells were dendritic, and expressed TRP-1 as well as c-Kit proteins (100% of the cells expressed TRP-1 and c-Kit). At week 4, when cells were in 4th passage, half of the cultures were switched to PMA-free Mel-mix medium. Cells in Mel-mix medium became bipolar (B), lost TRP-1 (D) and c-Kit (F), and increased EGFR (H) expressions in two weeks, while melanocytes continuously cultured in M254 medium remained dendritic (A) expressed both TRP-1 (C) and c-Kit (E) proteins, and showed only a week EGFR staining (G). Bar: 100 µm.
Mentions: In order to study the possible dedifferentiation of melanocytes, normal human adult epidermal melanocytes were cultured in commercially available PMA containing M254 medium for four weeks. These cells showed dendrite-rich morphology, expressed TRP-1 and c-Kit proteins uniformly and EGFR weakly. After four weeks, cultures were split into two; one part was continuously cultured in M254 medium; the other part was switched to Mel-mix medium for the following two weeks. Melanocytes cultured in the PMA containing M254 medium remained dendritic (Figure 2A) and expressed TRP-1 (100% of the cells were positive, Figure 2C) and c-Kit (100% of the cells were positive, Figure 2E) proteins and showed a generally week EGFR staining (only 32% of the cells showed weak positivity, Figure 2G), while cells cultured in Mel-mix for two weeks became bipolar (Figure 2B), their TRP-1 (Figure 2D) and c-Kit (Figure 2F) protein expressions drastically decreased (TRP-1+cells: 16%, c-Kit+cells: 25%), and their EGFR expression strongly increased (EGFR+ cell: 80%) (Figure 2H).

Bottom Line: In Mel-mix medium the cultured melanocytes are bipolar, unpigmented and highly proliferative.TRP-1 mRNA expression was induced in dedifferentiated melanocytes by UV-B irradiated keratinocyte supernatants, however direct UV-B irradiation of the cells resulted in further decrease of TRP-1 mRNA expression.These dedifferentiated, easily accessible cultured melanocytes provide a good model for studying melanocyte differentiation and possibly transdifferentiation.

View Article: PubMed Central - PubMed

Affiliation: Dermatological Research Group of the Hungarian Academy of Sciences, Szeged, Hungary. kormosbetti@mail.derma.szote.u-szeged.hu

ABSTRACT
In previous work we described a novel culture technique using a cholera toxin and PMA-free medium (Mel-mix) for obtaining pure melanocyte cultures from human adult epidermis. In Mel-mix medium the cultured melanocytes are bipolar, unpigmented and highly proliferative. Further characterization of the cultured melanocytes revealed the disappearance of c-Kit and TRP-1 and induction of nestin expression, indicating that melanocytes dedifferentiated in this in vitro culture. Cholera toxin and PMA were able to induce c-Kit and TRP-1 protein expressions in the cells, reversing dedifferentiation. TRP-1 mRNA expression was induced in dedifferentiated melanocytes by UV-B irradiated keratinocyte supernatants, however direct UV-B irradiation of the cells resulted in further decrease of TRP-1 mRNA expression. These dedifferentiated, easily accessible cultured melanocytes provide a good model for studying melanocyte differentiation and possibly transdifferentiation. Because melanocytes in Mel-mix medium can be cultured with human serum as the only supplement, this culture system is also suitable for autologous cell transplantation.

Show MeSH
Related in: MedlinePlus