Bone morphogenetic proteins 4 and 2/7 induce osteogenic differentiation of mouse skin derived fibroblast and dermal papilla cells.

Heterotopic ossification is a pathological condition in which bone forms outside the skeletal system. It can also occur in skin, which is the case in some genetic disorders. In addition to precursor cells and the appropriate tissue environment, heterotopic ossification requires inductive signals such as bone morphogenetic proteins (BMP). BMPs are growth and differentiation factors that have the ability to induce cartilage and bone formation in ectopic sites. The objective of this study is to explore the effect of the BMP-4 homodimer and BMP-2/7 heterodimer on the osteogenic differentiation of primary mouse skin fibroblasts and hair follicle dermal papilla (DP) cells. Osteogenic differentiation was induced by osteogenic induction medium (OS) containing 10 nM dexamethasone. The effect of BMP-4 and BMP-2/7 was studied using alkaline phosphatase (ALP) and calcium assays after 1.5, 3 and 5 weeks of differentiation. Fibroblasts and DP cells were able to differentiate into osteoblast-like matrix mineralizing cells. The first visible sign of differentiation was the change of morphology from rounded to more spindle-shaped cells. BMP-4 and BMP-2/7 exposure elevated ALP activity and calcium production significantly more than OS alone. The osteogenic response to BMP-4 and BMP-2/7 was similar in fibroblasts, whereas, in DP cells, BMP-2/7 was more potent than BMP-4. OS alone could not induce osteogenic differentiation in DP cells. Clear and consistent results show that dermal fibroblasts and stem cells from the dermal papilla were capable of osteogenic differentiation. The BMP-2/7 heterodimer was significantly more effective on hair follicular dermal stem cell differentiation.

Isolation, purification and expansion of myelination-competent, neonatal mouse Schwann cells.

Most studies of peripheral nerve myelination using culture models are performed with dorsal root ganglion neurons and Schwann cells pre-purified from the rat. The potential of this model is severely compromised by the lack of rat myelin mutants and the published protocols work poorly with mouse cells, for which numerous myelin mutants are available. This is partly due to difficulties in obtaining sufficient quantities of myelination-competent mouse Schwann cells. Here, we describe the isolation, purification and expansion of wild-type, myelination-competent Schwann cells from the sciatic nerves of 4-day-old mouse pups. The method consistently yields 1.9-3.3 x 10(6) of approximately 95% pure Schwann cells from the sciatic nerves of 12-15 4-day-old mouse pups, within 14-20 days. The Schwann cell proliferation rate ranges from 2.7- to 4.30-fold growth/week. Proliferation ceases within 4 weeks, when the cells become quiescent. Growth is reinduced by the presence of neurons; neuregulin is not sufficient for this effect. The Schwann cells isolated by this protocol are able to form compact myelin in culture, as judged by the segregated expression patterns of early (myelin-associated glycoprotein) and late (myelin basic protein) myelination markers in a three-dimensional neuron/Schwann cell coculture model. The Schwann cell batch yields are sufficient to perform 100-150 individual myelinating coculture assays. Employing mixed phenotype/genotype mouse neuron/Schwann cell cocultures, it will be possible to analyse the cell specificity of a mutation, and the cumulative effects of different mutations, without having to cross-breed the animals.