When transplanted into the central nervous system, Schwann cells (Scs) promote regeneration of axons and remyelination of demyelinated axons. Autologous transplantation of human SCs generated by growth in vitro from small nerve biopsies might thus be of significant clinical value. A procedure for growing adult human SCs and testing their function by transplantation into immune-deficient rodents was developed. SCs are stimulated to divide rapidly by the addition of a combination of the growth factor, heregulin, and an activator of adenylyl cyclase, forskolin, but human SCs appear to growth arrest after 12-14 population doublings and their ability to myelinate axons decreases after growth with mitogens. These observations suggest that the molecular and functional properties of human SCs are changed by growth with soluble mitogens in vitro. Experiments are proposed to test this hypothesis and to characterize changes in the properties of human SCs. First, tissue culture effects on expression of heregulin receptors and receptor-mediated signal transduction will be evaluated. Second, the mechanism by which forskolin potentiates the mitogenic effect of heregulin and whether prolonged exposure to combined mitogens alters the SCs response to forskolin will be examined. Third, experiments will test whether arrest of human SC growth reflects their entry into a state of replicative senescence. These studies will focus on the ability of heregulin/forskolin to activate senescence-related proteins, p53 and p21. Effect on phosphorylation of the growth-regulating protein, pRb, will be determined. The role of telomere shortening will be assessed and prevention of growth arrest by introduction of the catalytic subunit of telomerase into early passage human SCs will be tested. Fourth, changes in the human SC's ability to interact with axons by growth in vitro will be evaluated Effects on myelination capability in vivo will be examined and an in vitro model developed for study of the earliest SC-axon interactions: the initial contact, recognition, and association of the SC with the axonal surface. Experiments include an assessment of adhesion molecules, N-cadherin and L1, and on the ability of the SC to assemble an extracellular matrix. These studies explore the potential for use of human SCs in clinical applications, while expanding our knowledge of the biological and molecular properties of Schwann cells.
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