To bioengineer ectodermal organs such as teeth and whisker follicles, we developed a three-dimensional organ-germ culture method. The bioengineered tooth germ generated a structurally correct tooth, after both in vitro organ culture as well as transplantation under a tooth cavity in vivo, showing penetration of blood vessels and nerve fibers. Our method provides a substantial advance in the development of bioengineered organ replacement strategies and regenerative therapies.

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Oligodendrocyte precursor cells (OPCs) differentiate into oligodendrocytes (OLs) in order to form myelin, which is required for the rapid propagation of action potentials in the vertebrate nervous system. In spite of the considerable clinical importance of myelination, little is known about the basic molecular mechanisms underlying OL differentiation and myelination. Here, we show that cyclin-dependent kinase (Cdk) 5 is activated following the induction of differentiation, and that the Cdk5 inhibitor roscovitine inhibits OL differentiation. The complexity of the OL processes is also diminished after knocking down endogenous Cdk5 using RNAi. We also show that the focal adhesion protein paxillin is directly phosphorylated at Ser244 by Cdk5. Transfection of a paxillin construct harboring a Ser244 to Ala mutation dramatically inhibits its morphological effects. Importantly, phosphorylation of paxillin at Ser244 reduces its interaction with focal adhesion kinase (FAK). Taken together, these results suggest that phosphorylation of paxillin by Cdk5 is a key mechanism in OL differentiation and may ultimately regulate myelination.

Although estrogens can stimulate the growth of uterine epithelial cells in vivo, there is no clear effect of estrogens on the in vitro growth of epithelial cells from reproductive tract tissues; thus, we have established a defined culture system for mouse uterine epithelial cells. Pieces of uteri from immature CD-1 mice (21-23 days of age) were treated with trypsin, and the epithelial fragments were separated, enriched by Percoll gradient centrifugation, and seeded on collagen gels prepared from rat tail tendon. Initially, the cells were cultured in a 1:1 mixture of Ham's F-12 and Dulbecco's Modified Eagle's Medium supplemented with epidermal growth factor (EGF; 10 ng/ml), insulin (10 micrograms/ml), transferrin (10 micrograms/ml), hydrocortisone (0.1 micrograms/ml), and vitamin A (10 ng/ml). The cells formed a monolayer on the collagen gel within 1-2 days, but with time, cells began to detach from the gel. Further studies revealed that the attachment and growth of these cells on collagen were markedly influenced by the calcium concentration. It was found that lowering the calcium concentration from 1.05 to 0.05-0.1 mM dramatically suppressed cell detachment; the number of cells doubled after 7 days of culture. Proliferation of uterine epithelial cells was enhanced by EGF, but not by fibroblast growth factor, platelet-derived growth factor, nerve growth factor, multiplication-stimulating activity, or somatomedin-C. The uterine epithelial cells exhibited a single class of high affinity binding sites for [125I]iodo-EGF (Kd, approximately 1.8 nM), with approximately 5 X 10(4) receptors/cell; binding was inhibited by EGF but not by the other polypeptides. This cell culture system will aid in our investigations on hormonal effects on the growth and differentiation of estrogen target cells.