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| L-Wnt3a cell culture system with increased contents of Wnt3a in supernatant and its efficacy of inducing differentiation of pluripotent stem cells into melanocytes |
| QU Bowei1， LIU Liping1， LU Hao2， LI Yumei1 |
(1. Department of Dermatology, Affiliated Hospital of Jiangsu University, Zhenjiang Jiangsu 212001; 2. Department of Dermatology, Affiliated Hospital of Inner Mongolia Medical University, Hohhot Inner Mongolia 010107, China)
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Abstract Objective： To optimize the collection protocol and calibrate the Wnt3a protein concentration of L-Wnt3a conditioned medium (L-Wnt3a CM) and verify its role as a source of Wnt3a in pluripotent stem cell (PSC) to melanocytes differentiation system. Methods： The growth characteristics of L-Wnt3a cells and the characteristics of their secreted Wnt3a protein amount were observed to set the parameters of the collection method of L-Wnt3a conditioned medium, and the collected conditioned medium was calibrated for Wnt3a protein concentration using ELISA, and melanocyte differentiation medium with a defined Wnt3a concentration was formulated for the embryonic stem cell line WA09 and the induced pluripotent stem cell line WTC-11 cells were subjected to suspension-induced differentiation of melanocytes, and the obtained induced melanocytes were subjected to melanin gene expression assay and Masson-Fontana staining. Results： The new collection system was able to obtain Wnt3a protein stably and efficiently, and the differentiation system with a defined Wnt3a concentration was able to induce melanocytes successfully in WA09 and WTC-11, and the corresponding concentration of recombinant Wnt3a protein was able to achieve similar results. Conclusion： The precise concentration of L-Wnt3a CM could be efficiently obtained by optimizing the collection protocol and the use of a culture system with defined Wnt3a concentration also successfully induced of PSC differentiation into functional melanocytes.
［Key words］melanocytes; pluripotent stem cells; Wnt3a protein; induced pluripotent stem cells; cell differentiation;
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Received: 23 March 2022
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