Lentivector-mediated transfer of Bmi-1 and telomerase in muscle satellite cells yields a duchenne myoblast cell line with long-term genotypic and phenotypic stability.

Conditionally immortalized human cells are valuable substrates for basic biologic studies, as well as for the production of specific proteins and for the creation of bioartificial organs. We previously demonstrated that the lentivector-mediated transduction of immortalizing genes into human primary cells is an efficient method for obtaining such cell lines. Here, we used human muscle satellite cells as model targets to examine the impact of the transduced genes on the genotypic and phenotypic characteristics of the immortalized cells. The most commonly used immortalizing gene, the SV40 large T antigen (T-Ag), was extremely efficient at inducing the continuous growth of primary myoblasts, but the resulting cells rapidly accumulated major chromosomal aberrations and exhibited profound phenotypic changes. In contrast, the constitutive expression of telomerase and Bmi-1 in satellite cells from a control individual and from a patient suffering from Duchenne's muscular dystrophy yielded cell lines that remained diploid and conserved their growth factor dependence for proliferation. However, despite the absence of detectable cytogenetic abnormalities, clones derived from satellite cells of a control individual exhibited a differentiation block in vitro. In contrast, a Duchenne-derived cell line exhibited all the phenotypic characteristics of its primary parent, including an ability to differentiate fully into myotubes when placed in proper culture conditions. This cell line should constitute a useful reagent for a wide range of studies aimed at this disease.

Hyaluronan is synthesized by primitive hemopoietic cells, participates in their lodgment at the endosteum following transplantation, and is involved in the regulation of their proliferation and differentiation in vitro.

The localization of adult hemopoiesis to the marrow involves developmentally regulated interactions between hemopoietic stem cells and the stromal cell-mediated hemopoietic microenvironment. Although primitive hemopoietic cells exhibit a broad repertoire of adhesion molecules, little is known about the molecules influencing the site of cell lodgment within the marrow following transplantation. However, our recent studies indicate that hierarchically dependent patterns of migration of transplanted hemopoietic cells result in the retention of primitive cells within the endosteal and lineage-committed cells in the central marrow regions. Herein, we now demonstrate that these 2 subpopulations exhibit a striking difference in the expression of a cell surface adhesion molecule, with populations enriched for murine and human hemopoietic stem cells expressing the carbohydrate hyaluronic acid (HA). Furthermore, the presence of this glycosaminoglycan appears critical for the spatial distribution of transplanted stem cells in vivo. In addition, we also demonstrate that the binding of HA by a surrogate ligand results in marked inhibition of primitive hemopoietic cell proliferation and granulocyte differentiation. Collectively, these data describe an important yet previously unrecognized role for HA in the biology of primitive hemopoietic progenitor cells.