Prepatterning in the Stem Cell Compartment

The mechanism by which an apparently uniform population of cells can generate a heterogeneous population of differentiated derivatives is a fundamental aspect of pluripotent and multipotent stem cell behaviour. One possibility is that the environment and the differentiation cues to which the cells are exposed are not uniform. An alternative, but not mutually exclusive possibility is that the observed heterogeneity arises from the stem cells themselves through the existence of different interconvertible substates that pre-exist before the cells commit to differentiate. We have tested this hypothesis in the case of apparently homogeneous pluripotent human embryonal carcinoma (EC) stem cells, which do not follow a uniform pattern of differentiation when exposed to retinoic acid. Instead, they produce differentiated progeny that include both neuronal and non-neural phenotypes. Our results suggest that pluripotent NTERA2 stem cells oscillate between functionally distinct substates that are primed to select distinct lineages when differentiation is induced

Functional and molecular characterisation of mammary side population cells

BACKGROUND: Breast cancer is thought to arise in mammary epithelial stem cells. However, the identity of these stem cells is unknown. METHODS: Studies in the haematopoetic and muscle systems show that stem cells have the ability to efflux the dye Hoechst 33342. Cells with this phenotype are referred to as the side population (SP). We have adapted the techniques from the haematopoetic and muscle systems to look for a mammary epithelial SP. RESULTS: Of mammary epithelial cells isolated from both the human and mouse mammary epithelia, 0.2–0.45% formed a distinct SP. The SP was relatively undifferentiated but grew as typical differentiated epithelial clones when cultured. Transplantation of murine SP cells at limiting dilution into cleared mammary fat pads generated epithelial ductal and lobuloalveolar structures. CONCLUSION: These data demonstrate the existence of an undifferentiated SP in human and murine mammary epithelium. Purified SP cells are a live single-cell population that retain the ability to differentiate in vitro and in vivo. Studies of haematopoetic cells have suggested that the SP phenotype constitutes a universal stem cell marker. This work therefore has implications for mammary stem cell biology

DH and JH usage in murine fetal liver mirrors that of human fetal liver

In mouse and human, the regulated development of antibody repertoire diversity during ontogeny proceeds in parallel with the development of the ability to generate antibodies to an array of specific antigens. Compared to adult, the human fetal antibody repertoire limits N addition and uses specifically positioned VDJ gene segments more frequently, including V6-1 the most DH-proximal VH, DQ52, the most JH-proximal DH, and JH2, which is DH-proximal. The murine fetal antibody repertoire also limits the incorporation of N nucleotides and uses its most DH proximal VH, VH81X, more frequently. To test whether DH and JH also follow the pattern observed in human, we used the scheme of Hardy to sort B lineage cells from BALB/c fetal and neonatal liver, RT-PCR cloned and sequenced VH7183-containing VDJCμ transcripts, and then assessed VH7183-DH-JH and complementary determining region 3 of the immunoglobulin heavy chain (CDR-H3) content in comparison to the previously studied adult BALB/c mouse repertoire. Due to the deficiency in N nucleotide addition, perinatal CDR-H3s manifested a distinct pattern of amino acid usage and predicted loop structures. As in the case of adult bone marrow, we observed a focusing of CDR-H3 length and CDR-H3 loop hydrophobicity, especially in the transition from the early to late pre-B cell stage, a developmental checkpoint associated with expression of the pre-B cell receptor. However, fetal liver usage of JH-proximal DHQ52 and DH-proximal JH2 was markedly greater than that of adult bone marrow. Thus, the early pattern of DH and JH usage in mouse feta liver mirrors that of human

Breach of the nuclear lamina during assembly of herpes simplex viruses

Beneath the inner nuclear membrane lies the dense meshwork of the nuclear lamina, which provides structural support for the nuclear envelope and serves as an important organizing center for a number of nuclear and cytoplasmic constituents and processes. Herpesviruses have a significant and wide-ranging impact on human health, and their capacity to replicate and cause disease includes events that occur in the host cell nucleus. Herpes viruses begin assembly of progeny virus in the nuclei of infected cells and their capsids must escape the confines of the nucleus by traversing the inner nuclear membrane (INM) to proceed with later stages of virion assembly and egress. Access of viral capsids to the INM thus necessitates disruption of the dense nuclear lamina. We review herpesvirus effects on the nuclear lamina and in particular the roles of the herpes simplex virus-encoded nuclear egress complex and viral kinases on phosphorylation and dissociation of lamina components, and nucleocapsid envelopment at the INM

Evolution of human immunodeficiency virus type 1 nef and long terminal repeat sequences over 4 years in vivo and in vitro

The evolution of an 851-bp segment of the human immunodeficiency virus type 1 (HIV-1) genome encoding the nef open reading frame and U3/R elements of the long terminal repeat has been followed over a 4-year period in vivo and in vitro. The population of viral sequences at any given time was established by sequencing cloned polymerase chain reaction products. The samples studied were derived from the same man for whom a detailed analysis of the tat gene was previously described (A. Meyerhans, R. Cheynier, J. Albert, M. Seth, S. Kwok, J. Sninsky, L. Morfeldt-Manson, B. Asjö, and S. Wain-Hobson, Cell 58:901-910, 1989). Once again in vitro culture resulted in the selection of minor forms. Over a 4-year period in vivo, there was no obvious selection for, or outgrowth of, any particular nef or U3/R sequence. Few defective nef protein sequences were observed, which argues against nef acting as a negative regulatory factor. Although no functionally defective promoter/trans-activation-responsive elements were identified, the transactivation efficiencies varied between 0.2 and 2 times that of the control. The sequence encoding the most efficient trans-activation-responsive region did not outgrow others. The extreme genetic heterogeneity of the different samples of the locus, either in vivo or in vitro, indicates that there is no such thing as a single, distinct HIV sequence. It is suggested that different HIV-1 loci evolve independently, recombination being responsible for their uncoupling.</jats:p

Nonhomogeneous distribution of human immunodeficiency virus type 1 proviruses in the spleen

A nonhomogeneous spatial distribution of human immunodeficiency virus type 1 proviruses in an infected spleen was observed. Antigenic stimulation of infected cells might explain this partition.</jats:p