Abstract:

BACKGROUND: The proliferation and differentiation of hair-follicle-generating stem cells are influenced by the joint action of their own genes and external signals. Wnt/β-catenin signaling pathway plays an important role in the development of hair follicles, but the detailed mechanisms are not yet clear.
OBJECTIVE: To investigate, with interruption of keratinocyte growth factor and lithium chloride, the function and the interrelationship of Wnt/β-catenin signaling pathway with other signal factors when human hair-follicle-generating stem cells differentiate into dermal papilla cells or epidermal cells.
METHODS: Hair-follicle-generating stem cells were isolated from the bulge and cultivated. Then the growth curve of hair-follicle-generating stem cells was tested and formed in order to observe the cell proliferation ability cultivated at different densities (1×10⁶/L, 1×10⁷/L, 1×10⁸/L, 1×10⁹/L) at each time. Immunoflurorescene staining was performed to identify hair-follicle-generating stem cells and their differentiated cells. Lithium chloride (0, 0.5, 1.5, 10, 25 mmol/L individually) and keratinocyte growth factor (0, 10, 25, 50, 100 μg/L individually) were used to induce the differentiation of hair-follicle-generating stem cells. Then, we contrasted and analyzed the proliferation ability in each case, thereby investigating the most appropriate concentration of keratinocyte growth factor and lithium chloride to spur the differentiation of hair-follicle-generating stem cells. At days 3, 5, 7 and 9, we tested and compared the mRNA expressions of β-catenin, APC, GSK-3β, Axin and Lef1 from cells in control group, 10 mmol/L lithium chloride group and 10 μg/L keratinocyte growth factor group.
RESULTS AND CONCLUSION: Isolating cultured hair-follicle-generating stem cells still had a great reproductive activity and multi-lineage potential even after various times subculture in vitro. With higher lithium chloride concentration, the proliferation ability of hair-follicle-generating stem cells declined; while it increased when keratinocyte growth factor concentration increased. In K-SFM medium which contained lithium chloride, the transformation of hair-follicle-generating stem cells was obvious, showing distinct differences among groups. Especially, the level of β-catenin reached the peak when lithium chloride > 10 mmol/L. However, in K-SFM medium which contained keratinocyte growth factor, hair-follicle-generating stem cells differentiated into epidermal cells and the level of β-catenin changed slightly. We found that, while spurring the differentiation of hair-follicle-generating stem cells, lithium chloride could activate Wnt/β-catenin signal pathway and inhibit GSK-3β, a vital component of degradation compound. This facilitated β-catenin expressing in the cytoplasm to translocate into the nucleus. As a result, the transcription of target gene increased. It is the most appropriate concentration to spur hair-follicle-generating stem cells differentiation when lithium chloride level is > 10 mmol/L, but the proliferation ability declines correspondingly. Keratinocyte growth factor, which can facilitate hair-follicle-generating stem cells differentiated into epidermal cells, is a key factor to accelerate proliferation ability and migration of hair-follicle-generating stem cells, re-epithelialization and healing of wound. The mechanisms of hair-follicle-generating stem cells oriented differentiation induced by lithium chloride and keratinocyte growth factor are activating Wnt/β-catenin signal pathway, inducing change of β-catenin expression, and activating the transcription of target gene related to Wnt/β-catenin signaling pathway.

Key words: stem cells, hair follicle, lithium chloride, Wnt proteins