Mesenchymal stem cells from mouse bone marrow are committed to differentiate into active melanocytes in vitro: an experimental study / 中华皮肤科杂志

ABSTRACT

Objective To explore the possibility of bone marrow mesenchymal stem cells (MSCs) differentiating into active melanocytes in vitro.Methods Bone marrow stromal cells were harvested from femoral marrow of 6-week-old black male C57BL/6 mice,and subjected to a primary culture.After 6-passage subculture,an induction medium containing hydrocortisone,recombinant human insulin,transferrin and fibroblast growth factor was used to induce the differentiation of MSCs into melanocytes.Inverted light microscopy was applied to observe the process of cell differentiation,transmission electron microscopy to observe melanosome formation and maturation,and immunocytochemistry to determine the expression of melanocyte-associated epitopes,and flow cytometry to analyze cell cycles and yield of differentiated melanocytes.Results The total number of MSCs was close to 109 after 6 passages of subculture,and immunofluorescent studies showed an expression rate of 94.3％ for CD44 and 82.3％ for CD105 in these MSCs.After 180-day cultivation in the induction medium,the MSCs showed a morphological similarity to melanocytes with an increase in dendrites,formation of melanosome-like structures,and cell growth cycle was shortened to 3-4 days.Brown/black cell sediments were visualized by naked eyes.Electron microscopy revealed that intracellular melanosomes were mainly in Ⅳ phase.Immunofluorescence studies of the differentiated melanocytes showed a positive staining for tyrosinase related protein-1 (TRP-1),TRP-2,and microphthalmia-associated transcription factor (MITF).Flow cytometric analysis showed that most of the melanocytes differentiated from the MSCs were in G1 and S phases,and TRP-1-positive melanocytes amounted to 80％ of gate cells.Conclusions Bone marrow MSCs can be largely differentiated into melanocytes with a close similarity to normal melanocytes in morphology,ultrastructure and specific epitopes and a certain degree of proliferative activity.