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)
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;
