Abstract
BackgroundThe generation of functional human epidermal melanocytes (HEM) from stem cells provides 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 the safety and scalability characteristics essential for their translational application.MethodsHere, we report a rapid and efficient protocol based on defined culture conditions capable of differentiating adult adipose-derived stem cells (ADSC) to scalable amounts of proliferative melanocyte precursors (PreMel) within 30 days. PreMel were characterized in vitro through qPCR, Western blot, flow cytometry, biochemical assays, and in vivo assays in immunocompromised mice (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ, or NSG).ResultsAfter 30 days of differentiation, the stem cell-derived PreMel were defined as CD105neg CD73low according to immunophenotypic changes in comparison with parental stem cell markers. In addition, expression of microphthalmia-associated transcription factor (MITF), active tyrosinase (TYR), and the terminal differentiation-involved premelanosome protein (PMEL) were detected. Furthermore, PreMel had the potential to synthesize melanin and package it into melanosomes both in vitro and in vivo in NSG mice skin.ConclusionsThis study proposes a rapid and scalable protocol for the generation of proliferative melanocyte precursors (PreMel) from ADSC. These PreMel display the essential functional characteristics of bona fide HEM, opening a new path for an autologous cellular therapy for vitiligo patients.
Highlights
The generation of functional human epidermal melanocytes (HEM) from stem cells provides an unprecedented source for cell-based therapy in vitiligo
adipose-derived stem cells (ADSC) were positive for CD105, CD90, CD73, and CD13 in more than 95% of the cell population; CD44 was positive in a minority of the population (Additional file 1: Figure S1A-E)
As ADSC present a differentiation potential in vitro, we developed a defined medium to induce the differentiation of ADSC into HEM and evaluated the changes in the expression of the abovementioned markers by flow cytometry
Summary
The generation of functional human epidermal melanocytes (HEM) from stem cells provides an unprecedented source for cell-based therapy in vitiligo. Human epidermal melanocytes (HEM) produce melanin, a pigment responsible for both skin color and protection from ultraviolet radiation (UVR) [1]. Melanin is synthetized in melanosomes and delivered to the surrounding keratinocytes, cells that reside in the epidermis and constitute the defense barrier against UVR [2]. HEM development and differentiation begin with the migration of multipotent progenitors from the neural crest. Expression of transcription factors including paired box gene 3 (PAX3), sex-determining region Y-box 10 (SOX10), lymphoid enhancer-binding factor 1 (LEF1), and microphthalmia-associated transcription factor (MITF) induce the commitment of progenitor cells into melanoblasts and subsequent differentiation into melanocytes when the cells reach the epidermis [3]. There is abundant literature regarding HEM human biology, which is fundamental for the understanding of processes relevant to their development and pathologies such as vitiligo.
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