Regulation of the stem cell leukemia (SCL) gene: A tale of two fishes

The stem cell leukemia (SCL) gene encodes a tissue-specific basic helix–loop–helix (bHLH) protein with a pivotal role in hemopoiesis and vasculogenesis. Several enhancers have been identified within the murine SCL locus that direct reporter gene expression to subdomains of the normal SCL expression pattern, and long-range sequence comparisons of the human and murine SCL loci have identified additional candidate enhancers. To facilitate the characterization of regulatory elements, we have sequenced and analyzed 33 kb of the SCL genomic locus from the pufferfish Fugu rubripes, a species with a highly compact genome. Although the pattern of SCL expression is highly conserved from mammals to teleost fish, the genes flanking pufferfish SCL were unrelated to those known to flank both avian and mammalian SCL genes. These data suggest that SCL regulatory elements are confined to the region between the upstream and downstream flanking genes, a region of 65 kb in human and 8.5 kb in pufferfish. Consistent with this hypothesis, the entire 33-kb pufferfish SCL locus directed appropriate expression to hemopoietic and neural tissue in transgenic zebrafish embryos, as did a 10.4-kb fragment containing the SCL gene and extending to the 5′ and 3′ flanking genes. These results demonstrate the power of combining the compact genome of the pufferfish with the advantages that zebrafish provide for studies of gene regulation during development. Furthermore, the pufferfish SCL locus provides a powerful tool for the manipulation of hemopoiesis and vasculogenesis in vivo.

The level of mRNA encoding the amphotropic retrovirus receptor in mouse and human hematopoietic stem cells is low and correlates with the efficiency of retrovirus transduction.

The low level of amphotropic retrovirus-mediated gene transfer into human hematopoietic stem cells (HSC) has been a major impediment to gene therapy for hematopoietic diseases. In the present study, we have examined amphotropic retrovirus receptor (amphoR) and ecotropic retrovirus receptor mRNA expression in highly purified populations of mouse and human HSC. Murine HSC with low to undetectable levels of amphoR mRNA and relatively high levels of ecotropic retrovirus receptor mRNA were studied. When these HSC were analyzed simultaneously for ecotropic and amphotropic retrovirus transduction, ecotropic provirus sequences were detected in 10 of 13 long-term repopulated animals, while amphotropic proviral sequences were detected in only one recipient. A second distinct population of murine HSC were isolated that express 3-fold higher levels of amphoR mRNA. When these HSC were analyzed simultaneously for ecotropic and amphotropic retrovirus transduction, 11 of 11 repopulated mice contained ecotropic provirus and 6 of 11 contained amphotropic provirus sequences, a significant increase in the amphotropic retrovirus transduction (P = 0.018). These results indicate that, among the heterogeneous populations of HSC present in adult mouse bone marrow, the subpopulation with the highest level of amphoR mRNA is more efficiently transduced by amphotropic retrovirus. In a related study, we found low levels of human amphoR mRNA in purified populations of human HSC (CD34+ CD38-) and higher levels in committed progenitor cells (CD34+ CD38+). We conclude that the amphoR mRNA level in HSC correlates with amphotropic retrovirus transduction efficiency.

The stem cell antigen CD34 functions as a regulator of hemopoietic cell adhesion.

Although the CD34 antigen is widely used in the identification and purification of hemopoietic stem and progenitor cells, its function within hemopoiesis is unknown. We have investigated this issue by ectopically expressing human (hu) CD34 on the surface of murine hemopoietic cells. Forced expression of hu-CD34 in the thymocytes of transgenic mice did not appear to affect the development, maturation, or distribution of murine T cells but did significantly increase their ability to adhere to bone marrow stromal layers of human but not mouse origin. Ectopic expression of hu-CD34 on murine 416B cells, a multipotential progenitor that expresses murine CD34, yielded similar results. In both cases hu-CD34-dependent adhesion was enhanced by molecular engagement of the hu-CD34 protein using anti-CD34 antibodies. These results provide evidence that CD34 promotes the adhesive interactions of hemopoietic cells with the stromal microenvironment of the bone marrow thereby implicating CD34 in regulation and compartmentalization of stem cells. We propose that CD34 regulates these processes in part via an indirect mechanism, signaling changes in cellular adhesion in response to molecular recognition of an as yet unidentified stromal CD34 counterreceptor or ligand.

Identification of a unique membrane-bound molecule on a hemopoietic stem cell line and on multipotent progenitor cells.

Hemopoietic stem cells are a distinct population of cells that can differentiate into multilineages of hemopoietic cells and have long-term repopulation capability. A few membrane-bound molecules have been found to be preferentially, but not uniquely, present on the surface of these primitive cells. We report here the identification of a unique 105-kDa glycoprotein on the surface of hemopoietic stem cell line BL3. This molecule, recognized by the absorbed antiserum, is not present on the surface of myeloid progenitors 32D and FDC-P1 cells, EL4 T cells, and NIH 3T3 fibroblasts. This antiserum can also be used to block the proliferation of BL3 cells even in the presence of mitogen-stimulated spleen cell conditioned medium, which is known to have a stimulating activity on BL3 cells. It can also inhibit development of in vitro, fetal liver cell-derived multilineage colonies, but not other types of colonies, and of in vivo bone marrow cell-derived colony-forming unit spleen foci. These data suggest that gp105 plays an important role in hemopoietic stem cell differentiation.

Isolation of a primate embryonic stem cell line.

Embryonic stem cells have the ability to remain undifferentiated and proliferate indefinitely in vitro while maintaining the potential to differentiate into derivatives of all three embryonic germ layers. Here we report the derivation of a cloned cell line (R278.5) from a rhesus monkey blastocyst that remains undifferentiated in continuous passage for > 1 year, maintains a normal XY karyotype, and expresses the cell surface markers (alkaline phosphatase, stage-specific embryonic antigen 3, stage-specific embryonic antigen 4, TRA-1-60, and TRA-1-81) that are characteristic of human embryonal carcinoma cells. R278.5 cells remain undifferentiated when grown on mouse embryonic fibroblast feeder layers but differentiate or die in the absence of fibroblasts, despite the presence of recombinant human leukemia inhibitory factor. R278.5 cells allowed to differentiate in vitro secrete bioactive chorionic gonadotropin into the medium, express chorionic gonadotropin alpha- and beta-subunit mRNAs, and express alpha-fetoprotein mRNA, indicating trophoblast and endoderm differentiation. When injected into severe combined immunodeficient mice, R278.5 cells consistently differentiate into derivatives of all three embryonic germ layers. These results define R278.5 cells as an embryonic stem cell line, to our knowledge, the first to be derived from any primate species.

WW6: an embryonic stem cell line with an inert genetic marker that can be traced in chimeras.

Mutant mice produced by gene targeting in embryonic stem (ES) cells often have a complex or embryonic lethal phenotype. In these cases, it would be helpful to identify tissues and cell types first affected in mutant embryos by following the contribution to chimeras of ES cells homozygous for the mutant allele. Although a number of strategies for following ES cell development in vivo have been reported, each has limitations that preclude its general application. In this paper, we describe ES cell lines that can be tracked to every nucleated cell type in chimeras at all developmental stages. These lines were derived from blastocysts of mice that carry an 11-Mb beta-globin transgene on chromosome 3. The transgene is readily detected by DNA in situ hybridization, providing an inert, nuclear-localized marker whose presence is not affected by transcriptional or translational controls. The "WW" series of ES lines possess the essential features of previously described ES lines, including giving rise to a preponderance of male chimeras, all of which have to date exhibited germ-line transmission. In addition, clones selected for single or double targeting events form strong chimeras, demonstrating the feasibility of using WW6 cells to identify phenotypes associated with the creation of a null mutant.

Banho de clorexidina para prevenção de colonização e infecção por micro-organismos multirresistentes na unidade de transplante de células tronco e hematopoiéticas

Introdução: Infecções relacionadas à assistência de saúde (IRAS) representam hoje um dos principais desafios da qualidade do cuidado do paciente, principalmente em pacientes submetido a transplante de células tronco e hematopoiéticas (TCTH) O banho diário com a clorexidina (CHG) degermante a 2% tem sido proposto principalmente em unidades de terapia intensivas (UTIs) para diminuir a colonização bacteriana do paciente e assim diminuir IRAS. O objetivo deste estudo foi avaliar o impacto do banho com CHG degermante a 2% em unidade de internação de TCTH na incidência de infecção e colonização por patógenos multirresistentes e ainda avaliar seu impacto na sensibilidade das bactérias ao antisséptico. Métodos: Foi realizado um estudo quasi-experimental, com duração de 9 anos, com início em janeiro/2005 até dezembro/2013. A intervenção foi iniciada em agosto de 2009, sendo que os períodos pré e pós-intervenção tiveram duração de 4,5 anos. As taxas de IRAS, infecção por gram-negativos multirresistentes e infecção e colonização por enterococo resistente a vancomicina (VRE) foram avaliadas através de série temporal, para estudar o impacto da intervenção. As concentrações inibitórias mínimas (CIM) das bactérias para a CHG com e sem o inibidor de bomba de efluxo (CCCP) foram avaliadas nos dois períodos. Os genes de resistência a CHG foram estudados por meio da PCR e a clonalidade dos isolados por eletroforese em campo pulsátil. Resultados: Foi observada redução significativa na incidência de infecção e colonização de VRE na unidade no período pós-intervenção (p: 0,001). Essa taxa permaneceu estável em outras UTIs clínicas do hospital. Contudo as taxas de infecção por Gram negativos multirresistentes aumentou nos últimos anos na unidade. Não ocorreu diminuição na taxa de IRAS na unidade. As CIMs testadas de CHG aumentaram nas amostras de VRE e K. pneumoniae após o período de exposição ao antisséptico, com queda importante da CIM após o uso do CCCP, revelando ser a bomba de efluxo, um importante mecanismo de resistência à CHG. As amostras de A. baumannii e P. aeruginosa não apresentaram aumento da CIM após período de exposição à clorexidina. As bombas de efluxo Ade A, B e C estiveram presentes na maioria dos A. baumannii do grupo controle (66%). A bomba cepA foi encontrada em 67% de todas as K. pneumoniae testadas e em 44,5% das P. aeruginosas do grupo pré intervenção. Observamos uma relação positiva entre a presença da CepA nas amostras de K. pneumoniae e a resposta ao CCCP: de todas as 49 amostras CepA positivas 67,3% obtiveram redução do seu MIC em 4 diluições após adição do CCCP. A avaliação de clonalidade demonstrou padrão policlonal das amostras de VRE, K. pneumoniae e A. baumannii avaliadas. Em relação às amostras de P. aeruginosa foi observado que no período pós-intervenção ocorreu predominância de um clone com > 80% semelhança em 10 das 22 amostras avaliadas pelo dendrograma. Conclusões: O banho de clorexidina teve impacto na redução da incidência de infecção e colonização por VRE na unidade de TCTH, e não teve o mesmo impacto nas bactérias gram-negativas. Os mecanismos moleculares de resistência à clorexidina estão intimamente ligados à presença de bomba de efluxo, sendo provavelmente o principal mecanismo de resistência e tolerância das bactérias ao antisséptico

Patterns of Stem Cell Divisions Contribute to Plant Longevity

The lifespan of plants ranges from a few weeks in annuals to thousands of years in trees. It is hard to explain such extreme longevity considering that DNA replication errors inevitably cause mutations. Without purging through meiotic recombination, the accumulation of somatic mutations will eventually result in mutational meltdown, a phenomenon known as Muller’s ratchet. Nevertheless, the lifespan of trees is limited more often by incidental disease or structural damage than by genetic aging. The key determinants of tree architecture are the axillary meristems, which form in the axils of leaves and grow out to form branches. The number of branches is low in annual plants, but in perennial plants iterative branching can result in thousands of terminal branches. Here, we use stem cell ablation and quantitative cell-lineage analysis to show that axillary meristems are set aside early, analogous to the metazoan germline. While neighboring cells divide vigorously, axillary meristem precursors maintain a quiescent state, with only 7–9 cell divisions occurring between the apical and axillary meristem. During iterative branching, the number of branches increases exponentially, while the number of cell divisions increases linearly. Moreover, computational modeling shows that stem cell arrangement and positioning of axillary meristems distribute somatic mutations around the main shoot, preventing their fixation and maximizing genetic heterogeneity. These features slow down Muller’s ratchet and thereby extend lifespan.

Donor treatment with pegylated G-CSF augments the generation of IL-10-producing regulatory T cells and promotes transplantation tolerance

We investigated whether the protection from graft-versus-host disease (GVHD) afforded by donor treatment with granulocyte colony-stimulating factor (G-CSF) could be enhanced by dose escalation. Donor treatment with human G-CSIF prevented GVHD in the B6 --> B6D2F1 murine model in a dose-dependent fashion, and murine G-CSF provided equivalent protection from GVHD at 10-fold lower doses. Donor pretreatment with a single dose of pegylated G-CSF (peg-G-CSF) prevented GVHD to a significantly greater extent than standard G-CSIF (survival, 75% versus 11%, P < .001). Donor T cells from peg-G-CSF-treated donors failed to proliferate to alloantigen and inhibited the responses of control T cells in an interleukin 10 (IL-10)-dependent-fashion in vitro. T cells from peg-GCSF-treated IL-10(-/-) donors induced lethal GVHD; T cells from peg-G-CSF-treated wild-type (wt) donors promoted long-term survival. Whereas T cells from peg-G-CSF wt donors were able to regulate GVHD induced by T cells from control-treated donors, T cells from G-CSF-treated wt donors and peg-G-CSF-treated IL-10(-/-) donors did not prevent mortality. Thus, peg-G-CSF is markedly superior to standard G-CSF for the prevention of GVHD following allogeneic stem cell transplantation (SCT), due to the generation of IL-10-producing regulatory T cells. These data support prospective clinical trials of peg-G-CSF-mobilized allogeneic blood SCT. (C) 2004 by The American Society of Hematology.

An in vivo comparative study of sonic, desert and Indian hedgehog reveals that hedgehog pathway activity regulates epidermal stem cell homeostasis

Despite the well-characterised role of sonic hedgehog (Shh) in promoting interfollicular basal cell proliferation and hair follicle downgrowth, the role of hedgehog signalling during epidermal stem cell fate remains largely uncharacterised. In order to determine whether the three vertebrate hedgehog molecules play a role in regulating epidermal renewal we overexpressed sonic (Shh), desert (Dhh) and Indian (Ihh) hedgehog in the basal cells of mouse skin under the control of the human keratin 14 promoter. We observed no overt epidermal morphogenesis phenotype in response to Ihh overexpression, however Dhh overexpression resulted in a range of embryonic and adult skin manifestations indistinguishable from Shh overexpression. Two distinct novel phenotypes were observed amongst Shh and Dhh transgenics, one exhibiting epidermal progenitor cell hyperplasia with the other displaying a complete loss of epidermal tissue renewal indicating deregulation of stem cell activity. These data suggest that correct temporal regulation of hedgehog activity is a key factor in ensuring epidermal stem cell maintenance. In addition, we observed Shh and Dhh transgenic skin from both phenotypes developed lesions reminiscent of human basal cell carcinoma (BCC), indicating that BCCs can be generated despite the loss of much of the proliferative (basal) compartment. These data suggest the intriguing possibility that BCC can arise outside the stem cell population. Thus the elucidation of Shh (and Dhh) target gene activation in the skin will likely identify those genes responsible for increasing the proliferative potential of epidermal basal cells and the mechanisms involved in regulating epidermal stem cell fate.

The adult mouse hippocampal progenitor is neurogenic but not a stem cell

The aim of this investigation was to characterize the proliferative precursor cells in the adult mouse hippocampal region. Given that a very large number of new hippocampal cells are generated over the lifetime of an animal, it is predicted that a neural stem cell is ultimately responsible for maintaining this genesis. Although it is generally accepted that a proliferative precursor resides within the hippocampus, contradictory reports exist regarding the classification of this cell. Is it a true stem cell or a more limited progenitor? Using a strict functional definition of a neural stem cell and a number of in vitro assays, we report that the resident hippocampal precursor is a progenitor capable of proliferation and multipotential differentiation but is unable to self-renew and thus proliferate indefinitely. Furthermore, the mitogen FGF-2 stimulates proliferation of these cells to a greater extent than epidermal growth factor ( EGF). In addition, we found that BDNF was essential for the production of neurons from the hippocampal progenitor cells, being required during proliferation to trigger neuronal fate. In contrast, a bona fide neural stem cell was identified in the lateral wall of the lateral ventricle surrounding the hippocampus. Interestingly, EGF proved to be the stronger mitogenic factor for this cell, which was clearly a different precursor from the resident hippocampal progenitor. These results suggest that the stem cell ultimately responsible for adult hippocampal neurogenesis resides outside the hippocampus, producing progenitor cells that migrate into the neurogenic zones and proliferate to produce new neurons and glia.

Optimization of a transplant model to assess skin reconstitution from stem cell-enriched primary human keratinocyte populations

Given that an important functional attribute of stem cells in vivo is their ability to sustain tissue regeneration, we set out to establish a simple and easy technique to assess this property from candidate populations of human keratinocyte stem cells in an in vivo setting. Keratinocytes were inoculated into devitalized rat tracheas and transplanted subcutaneously into SCID mice, and the epithelial lining regenerated characterized to establish the validity of this heterotypic model. Furthermore, the rate and quality of epidermal tissue reconstitution obtained from freshly isolated unfractionated vs. keratinocyte stem cell-enriched populations was tested as a function of (a) cell numbers inoculated; and (b) the inclusion of irradiated support keratinocytes and dermal cells. Rapid and sustained epidermal tissue regeneration from small numbers of freshly isolated human keratinocyte stem cells validates the utilization of this simple and reliable model system to assay for enrichment of epidermal tissue-reconstituting cells.