Using high-throughput immunoblotting to identify proteins involved in the differentiation of ES cells along the hair follicle lineage in vitro.

The hair follicles develop from a stem cell population in the surface ectoderm that feeds a complexe terminal differentiation pathway. We have developed a two-step high density culture scheme in which pluripotent mouse ES cells are induced first to ectoderm phenotype and then give rise to morphologically three dimensional nodule-like structures that express hair keratin in the center of them suggesting that they are progressing along the terminal differentiation program of the hair follicle in vitro. Using this model system we have now analyzed the protein expression profile using a high throughput western blotting method (BP Power Blot). This protocol provides an invaluable system in which to study both the mechanisms that direct stem cells along the hair follicle pathway as well as those that influence their subsequent epidermal differentiation in vitro.