Generation of Functional Stem Cell-Derived CD105neg CD73low Melanocyte Precursors Guided by Defined Culture Conditions

Abstract

Background: The generation of functional human epidermal melanocytes (HEM) from stem cells would provide an unprecedented source for cell-based therapy in vitiligo. Despite the important efforts exerted to obtain melanin-producing cells from stem cells, pre-clinical results still lack safety or scalability characteristics, essential for their translational application. Methods: Here, we report a rapid and efficient protocol based on defined culture conditions capable of differentiating adipose-derived stem cells (ADSC) to scalable amounts of proliferative melanocyte precursors (PreMel), within 30 days. PreMel were characterized in vitro through PCR, Western blot, flow cytometry and biochemical assays and in in vivo assays in immunocompromised mice (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ or NSG). Findings: After 30 days of differentiation, the stem-cell-derived PreMel were defined as CD105negCD73low according to changes measured in the expression of parental surface markers. In addition, the expression of MITF and active tyrosinase (TYR) and PMEL, a melanosome related protein for terminal differentiation, were also detected. Furthermore, cells were capable of synthetizing melanin and pack it into melanosomes both in vitro and in the skin of a transplanted mouse model. Interpretation: These results evidence a short and scalable protocol for the obtaining of proliferative melanocyte precursors or PreMel, displaying essential functional characteristics of bona fide HEM. Funding Statement: This work was funded by Consorcio REGENERO. Declaration of Interests: GZ y DM receives stipends from Consorcio REGENERO, Chilean Biotechnology Company for cell therapy development. MK receives stipends from Cells for Cells, Chilean Biotechnology Company for cell therapy development. CS, RC and WG declare no conflict of interest. Ethics Approval Statement: All the procedures presented in this work were approved for the Ethics Committee of Universidad de los Andes. All the personal involved in animal studies was trained internally using courses available at www.aalas.org and assays were performed according to ARRIVE guidelines of the NC3Rs (National Centre for the Replacement, Refinement & Reduction of Animals in Research).