Compositions and methods for the generation of melanocytes through direct reprogramming
Compositions and methods for generating melanocytes through direct reprogramming are disclosed. Also disclosed are methods of use of such compositions for the treatment of vitiligo and other hypopigmentation disorders. In accordance with the present invention, a method for producing melanocytes suitable for use in human patients is provided. An exemplary method comprises providing cells capable of transdifferentiation into melanocytes, culturing said cells in a chemically defined culture medium, introducing at least two of microphthalmia-associated transcription factor (MITF), SRY-related HMG-box (SOX10) transcription factor and paired box-3 (PAX-3) transcription factor and paired box-3 (PAX-3) transcription factor, or nucleic acids encoding said transcription factors into said cells, wherein expression of said factors induces the cells to transdifferentiae into melanocytes expressing melanocyte markers TYR, DCT, S-100 and Melan-A.
1. A method for producing melanocytes comprising:
a) providing fibroblast cells capable of transdifferentiation into melanocytes;
b) culturing said fibroblast cells in melanocyte culture medium for transdifferentiation;
c) introducing nucleic acids encoding each of at least microphthalmia-associated transcription factor (MITF), and SRY-related HMG-box (SOX10) transcription factors and, optionally a nucleic acid encoding paired box-3 (PAX-3) transcription factor, into said cells, wherein said transcription factors are expressed to cause transdifferentiation into melanocytes expressing melanocyte markers TYR, DCT, S-100 and Melan-A; and, optionally
d) isolating said melanocytes.
2. The method of claim 1 , wherein nucleic acid encoding all three transcription factors are introduced.
3. The method of claim 1 , wherein said cells are mammalian in origin.
4. The method of claim 3 , wherein said cells are human in origin.
5. The method of claim 2 , wherein said fibroblast cells are fetal or adult fibroblasts.
6. The method of claim 1 , wherein said transcription factors are encoded by one or more recombinant expression vectors.
7. The method of claim 1 , wherein said nucleic acid is synthetic mRNA.
8. The method of claim 1 , where is said culture media comprises Dulbecco's modified Eagle medium, fetal bovine serum, non-essential amino acids, sodium pyruvate and penicillin/streptomycin.
9. The method of claim 1 , wherein said fibroblasts cells are obtained from a patient in need of treatment with melanocytes.