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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.

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.

We exploited the ability of lentiviral vectors to govern the stable transduction of cells irrespective of their cycling status to induce the reversible immortalization of human primary cells. First, bicistronic HIV-derived lentiviral vectors expressing GFP- and the HSV1 thymidine kinase and containing the LoxP sequence in their LTR (HLox) were used to transduce HeLa cells. Cre expression led to efficient proviral deletion, and unexcised cells could be eliminated by ganciclovir treatment. A human liver biopsy was then exposed to a combination of HLox vectors that harbored either the SV40 large T (TAg) or the human telomerase (hTERT) DNAs in place of GFP. This led to the isolation of liver sinusoidal endothelial cell (LSEC) clones that exhibited an immortalized phenotype while retaining most of the features of primary hLSEC. Complete growth arrest of these cells was observed in 2 days of Cre expression, and the resulting stationary culture could be kept for at least 2 weeks. Transduction of human adult pancreatic islets with HLox vectors coding for Tag and Bmi-1 also induced the proliferation of insulin-positive cells. These results indicate that lentivectors can be used to mediate the reversible immortalization of primary nondividing cells and should allow for the production of large supplies of a wide variety of human cells for both therapeutic and research purposes.

1. Two early signs of human myoblast commitment to fusion are membrane potential hyperpolarization and concomitant expression of a non-inactivating delayed rectifier K+ current, IK(NI). This current closely resembles the outward K+ current elicited by rat ether-à-go-go (r-eag) channels in its range of potential for activation and unitary conductance. 2. It is shown that activation kinetics of IK(NI), like those of r-eag, depend on holding potential and on [Mg2+]o, and that IK(NI), like r-eag, is reversibly inhibited by a rise in [Ca2+]i. 3. Forced expression of an isolated human ether-à-go-go K+ channel (h-eag) cDNA in undifferentiated myoblasts generates single-channel and whole-cell currents with remarkable similarity to IK(NI). 4. h-eag current (Ih-eag) is reversibly inhibited by a rise in [Ca2+]i, and the activation kinetics depend on holding potential and [Mg2+]o. 5. Forced expression of h-eag hyperpolarizes undifferentiated myoblasts from -9 to -50 mV, the threshold for the activation of both Ih-eag and IK(NI). Similarly, the higher the density of IK(NI), the more hyperpolarized the resting potential of fusion-competent myoblasts. 6. It is concluded that h-eag constitutes the channel underlying IK(NI) and that it contributes to the hyperpolarization of fusion-competent myoblasts. To our knowledge, this is the first demonstration of a physiological role for a mammalian eag K+ channel.

An early sign of human myoblast commitment to fusion is the expression of a non-inactivating delayed rectifier K+ current, I(K(NI)), and an associated membrane potential hyperpolarization. We have isolated the full-length coding region of a human ether-a-go-go K+ channel (h-eag) from myoblasts undergoing differentiation. The h-eag gene was localized to chromosome 1q32-41, and is expressed as a approximately 9 kb transcript in myogenic cells and in adult brain tissue. Forced expression of h-eag in undifferentiated myoblasts generates a current with remarkable similarity to I(K(NI)) indicating that h-eag constitutes the channel responsible for this current in vivo.

1. The role of K+ channels and membrane potential in myoblast fusion was evaluated by examining resting membrane potential and timing of expression of K+ currents at three stages of differentiation of human myogenic cells: undifferentiated myoblasts, fusion-competent myoblasts (FCMBs), and freshly formed myotubes. 2. Two K+ currents contribute to a hyperpolarization of myoblasts prior to fusion: IK(NI), a non-inactivating delayed rectifier, and IK(IR), an inward rectifier. 3. IK(NI) density is low in undifferentiated myoblasts, increases in FCMBs and declines in myotubes. On the other hand, IK(IR) is expressed in 28% of the FCMBs and in all myotubes. 4. IK(IR) is reversibly blocked by Ba2+ or Cs+. 5. Cells expressing IK(IR) have resting membrane potentials of -65 mV. A block by Ba2+ or Cs+ induces a depolarization to a voltage determined by IK(NI) (-32 mV). 6. Cs+ and Ba2+ ions reduce myoblast fusion. 7. It is hypothesized that the IK(IR)-mediated hyperpolarization allows FCMBs to recruit Na+, K+ and T-type Ca2+ channels which are present in these cells and would otherwise be inactivated. FCMBs, rendered thereby capable of firing action potentials, could amplify depolarizing signals and may accelerate fusion.

Both specific and housekeeping transcriptional control elements have been successfully used to generate retroviral vectors that express in a stable tissue specific manner. However, no such vectors have been developed specifically for expression in haematopoietic cells. This paper describes the construction of retroviral vectors incorporating sequences from the promoter region of the murine CD45 (leucocyte common antigen) gene and using the human alpha-L-iduronidase cDNA as a reporter gene. These vectors were tested for expression of the alpha-L-iduronidase gene in skin fibroblasts and a lymphoblastoid cell line which is permissive for CD45 expression, derived from patients with mucopolysaccharidosis type I (MPS I, alpha-L-iduronidase deficiency). The results obtained demonstrate that, in the context of a retroviral construct, the activity of the CD45 gene promoter in lymphocytes and fibroblasts parallels the activity of the retroviral long terminal repeat (LTR). These results suggest that additional transcriptional control elements from the CD45 gene, or other haematopoietic specific genes, will be necessary for the construction of a haematopoietic specific vector.

A full-length cDNA clone encoding the lysosomal hydrolase alpha-L-fucosidase was cloned into two retroviral vectors, one using the human cytomegalovirus immediate-early promoter for expression, and the other, the retroviral long terminal repeat (LTR). High-titer amphotropic virus was produced for both constructs by infection of PA317 cells, and used to efficiently transduce the alpha-L-fucosidase gene into both human and canine fucosidosis fibroblasts. This resulted in correction of the alpha-L-fucosidase enzyme deficiency characteristic of these fibroblasts. The high levels of recombinant enzyme produced corrected the degradative defect normally seen in these cells, enabling them to catabolize efficiently the accumulated storage product present in lysosomes. Therefore, these retroviral constructs should allow us to start evaluating the value of gene therapy in treating the central nervous system pathology associated with fucosidosis and other lysosomal storage disorders in humans, using a canine model of fucosidosis.

The human lysosomal storage disorder fucosidosis results from the deficiency of alpha-L-fucosidase, a lysosomal enzyme essential for the catabolism of oligosaccharides containing alpha-L-fucosides. cDNA clones coding for human alpha-L-fucosidase have been isolated from lambda gt10 and lambda gt11 cDNA libraries derived from human liver, placenta and colon. Compilation of cDNA sequences results in a nucleotide sequence of 2053 base pairs encoding alpha-L-fucosidase. The sequence contains an open reading frame of 461 amino acids beginning with the first in-frame methionine and includes 439 amino acids which comprise the mature protein in addition to a hydrophobic signal peptide sequence of 22 amino acids.