A circuit-based gatekeeper for adult neural stem cell proliferation: Parvalbumin-expressing interneurons of the dentate gyrus control the activation and proliferation of quiescent adult neural stem cells.

Newborn neurons are generated in the adult hippocampus from a pool of self-renewing stem cells located in the subgranular zone (SGZ) of the dentate gyrus. Their activation, proliferation, and maturation depend on a host of environmental and cellular factors but, until recently, the contribution of local neuronal circuitry to this process was relatively unknown. In their recent publication, Song and colleagues have uncovered a novel circuit-based mechanism by which release of the neurotransmitter, γ-aminobutyric acid (GABA), from parvalbumin-expressing (PV) interneurons, can hold radial glia-like (RGL) stem cells of the adult SGZ in a quiescent state. This tonic GABAergic signal, dependent upon the activation of γ(2) subunit-containing GABA(A) receptors of RGL stem cells, can thus prevent their proliferation and subsequent maturation or return them to quiescence if previously activated. PV interneurons are thus capable of suppressing neurogenesis during periods of high network activity and facilitating neurogenesis when network activity is low.

Genetic selection system allowing monitoring of myofibrillogenesis in living cardiomyocytes derived from mouse embryonic stem cells

Embryonic stem (ES) cell-derived cardiomyocytes recapitulate cardiomyogenesis in vitro and are a potential source of cells for cardiac repair. However, this requires enrichment of mixed populations of differentiating ES cells into cardiomyocytes. Toward this goal, we have generated bicistronic vectors that express both the blasticidin S deaminase (bsd) gene and a fusion protein consisting of either myosin light chain (MLC)-3f or human alpha-actinin 2A and enhanced green fluorescent protein (EGFP) under the transcriptional control of the alpha-cardiac myosin heavy chain (alpha-MHC) promoter. Insertion of the DNase I-hypersensitive site (HS)-2 element from the beta-globin locus control region, which has been shown to reduce transgene silencing in other cell systems, upstream of the transgene promoter enhanced MLC3f-EGFP gene expression levels in mouse ES cell lines. The alpha-MHC-alpha-actinin-EGFP, but not the alpha-MHC-MLC3f-EGFP, construct resulted in the correct incorporation of the newly synthesized fusion protein at the Z-band of the sarcomeres in ES cell-derived cardiomyocytes. Exposure of embryoid bodies to blasticidin S selected for a relatively pure population of cardiomyocytes within 3 days. Myofibrillogenesis could be monitored by fluorescence microscopy in living cells due to sarcomeric epitope tagging. Therefore, this genetic system permits the rapid selection of a relatively pure population of developing cardiomyocytes from a heterogeneous population of differentiating ES cells, simultaneously allowing monitoring of early myofibrillogenesis in the selected myocytes

Tissue-specific FLAGELLIN-SENSING 2 (FLS2) expression in roots restores immune responses in Arabidopsis fls2 mutants.

The flagellin receptor of Arabidopsis, At-FLAGELLIN SENSING 2 (FLS2), has become a model for mechanistic and functional studies on plant immune receptors. Responses to flagellin or its active epitope flagellin 22 (flg22) have been extensively studied in Arabidopsis leaves. However, the perception of microbe-associated molecular patterns (MAMPs) and the immune responses in roots are poorly understood. Here, we show that isolated root tissue is able to induce pattern-triggered immunity (PTI) responses upon flg22 perception, in contrast to elf18 (the active epitope of elongation factor thermo unstable (EF-Tu)). Making use of fls2 mutant plants and tissue-specific promoters, we generated transgenic Arabidopsis lines expressing FLS2 only in certain root tissues. This allowed us to study the spatial requirements for flg22 responses in the root. Remarkably, the intensity of the immune responses did not always correlate with the expression level of the FLS2 receptor, but depended on the expressing tissue, supporting the idea that MAMP perception and sensitivity in different tissues contribute to a proper balance of defense responses according to the expected exposure to elicitors. In summary, we conclude that each investigated root tissue is able to perceive flg22 if FLS2 is present and that tissue identity is a major element of MAMP perception in roots.

Hematopoietic stem cells reversibly switch from dormancy to self-renewal during homeostasis and repair.

Bone marrow hematopoietic stem cells (HSCs) are crucial to maintain lifelong production of all blood cells. Although HSCs divide infrequently, it is thought that the entire HSC pool turns over every few weeks, suggesting that HSCs regularly enter and exit cell cycle. Here, we combine flow cytometry with label-retaining assays (BrdU and histone H2B-GFP) to identify a population of dormant mouse HSCs (d-HSCs) within the lin(-)Sca1+cKit+CD150+CD48(-)CD34(-) population. Computational modeling suggests that d-HSCs divide about every 145 days, or five times per lifetime. d-HSCs harbor the vast majority of multilineage long-term self-renewal activity. While they form a silent reservoir of the most potent HSCs during homeostasis, they are efficiently activated to self-renew in response to bone marrow injury or G-CSF stimulation. After re-establishment of homeostasis, activated HSCs return to dormancy, suggesting that HSCs are not stochastically entering the cell cycle but reversibly switch from dormancy to self-renewal under conditions of hematopoietic stress.

Detection of functional antigen-specific T cells from urine of non-muscle invasive bladder cancer patients.

To gain further insights into the role of T lymphocytes in immune responses against bladder tumors, we developed a method that monitors the presence of functional antigen-specific T cells in the urine of non-muscle invasive bladder cancer patients. As relatively few immune cells can usually be recovered from urine, we examined different isolation/amplification protocols and took advantage of patients treated with weekly intravesical instillations of Bacillus Calmette-Guérin, resulting in large amounts of immune cells into urine. Our findings demonstrate that, upon in vitro amplification, antigen-specific T cells can be detected by an interferon γ (IFNγ)-specific ELISPOT assay.

Positional bias of general and tissue-specific regulatory motifs in mouse gene promoters

Background: The arrangement of regulatory motifs in gene promoters, or promoterarchitecture, is the result of mutation and selection processes that have operated over manymillions of years. In mammals, tissue-specific transcriptional regulation is related to the presence ofspecific protein-interacting DNA motifs in gene promoters. However, little is known about therelative location and spacing of these motifs. To fill this gap, we have performed a systematic searchfor motifs that show significant bias at specific promoter locations in a large collection ofhousekeeping and tissue-specific genes.Results: We observe that promoters driving housekeeping gene expression are enriched inparticular motifs with strong positional bias, such as YY1, which are of little relevance in promotersdriving tissue-specific expression. We also identify a large number of motifs that show positionalbias in genes expressed in a highly tissue-specific manner. They include well-known tissue-specificmotifs, such as HNF1 and HNF4 motifs in liver, kidney and small intestine, or RFX motifs in testis,as well as many potentially novel regulatory motifs. Based on this analysis, we provide predictionsfor 559 tissue-specific motifs in mouse gene promoters.Conclusion: The study shows that motif positional bias is an important feature of mammalianproximal promoters and that it affects both general and tissue-specific motifs. Motif positionalconstraints define very distinct promoter architectures depending on breadth of expression andtype of tissue.

Aberrant repair and fibrosis development in skeletal muscle

The repair process of damaged tissue involves the coordinated activities of several cell types in response to local and systemic signals. Following acute tissue injury, infiltrating inflammatory cells and resident stem cells orchestrate their activities to restore tissue homeostasis. However, during chronic tissue damage, such as in muscular dystrophies, the inflammatory-cell infiltration and fibroblast activation persists, while the reparative capacity of stem cells (satellite cells) is attenuated. Abnormal dystrophic muscle repair and its end stage, fibrosis, represent the final common pathway of virtually all chronic neurodegenerative muscular diseases. As our understanding of the pathogenesis of muscle fibrosis has progressed, it has become evident that the muscle provides a useful model for the regulation of tissue repair by the local microenvironment, showing interplay among muscle-specific stem cells, inflammatory cells, fibroblasts and extracellular matrix components of the mammalian wound-healing response. This article reviews the emerging findings of the mechanisms that underlie normal versus aberrant muscle-tissue repair.

Cardiac repair with allogeneic mesenchymal stem cells after myocardial infarction.

Over the past decade, use of autologous bone marrow-derived mononuclear cells (BMCs) has proven to be safe in phase-I/II studies in patients with myocardial infarction (MI). Taken as a whole, results support a modest yet significant improvement in cardiac function in cell-treated patients. Skeletal myoblasts, adipose-derived stem cells, and bone marrow-derived mesenchymal stem cells (MSCs) have also been tested in clinical studies. MSCs expand rapidly in vitro and have a potential for multilineage differentiation. However, their regenerative capacity decreases with aging, limiting efficacy in old patients. Allogeneic MSCs offer several advantages over autologous BMCs; however, immune rejection of allogeneic cells remains a key issue. As human MSCs do not express the human leukocyte antigen (HLA) class II under normal conditions, and because they modulate T-cell-mediated responses, it has been proposed that allogeneic MSCs may escape immunosurveillance. However, recent data suggest that allogeneic MSCs may switch immune states in vivo to express HLA class II, present alloantigen and induce immune rejection. Allogeneic MSCs, unlike syngeneic ones, were eliminated from rat hearts by 5 weeks, with a loss of functional benefit. Allogeneic MSCs have also been tested in initial clinical studies in cardiology patients. Intravenous allogeneic MSC infusion has proven to be safe in a phase-I trial in patients with acute MI. Endoventricular allogeneic MSC injection has been associated with reduced adverse cardiac events in a phase-II trial in patients with chronic heart failure. The long-term safety and efficacy of allogeneic MSCs for cardiac repair remain to be established. Ongoing phase-II trials are addressing these issues.

Qualitative modeling identifies IL-11 as a novel regulator in maintaining self-renewal in human pluripotent stem cells.

Pluripotency in human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) is regulated by three transcription factors-OCT3/4, SOX2, and NANOG. To fully exploit the therapeutic potential of these cells it is essential to have a good mechanistic understanding of the maintenance of self-renewal and pluripotency. In this study, we demonstrate a powerful systems biology approach in which we first expand literature-based network encompassing the core regulators of pluripotency by assessing the behavior of genes targeted by perturbation experiments. We focused our attention on highly regulated genes encoding cell surface and secreted proteins as these can be more easily manipulated by the use of inhibitors or recombinant proteins. Qualitative modeling based on combining boolean networks and in silico perturbation experiments were employed to identify novel pluripotency-regulating genes. We validated Interleukin-11 (IL-11) and demonstrate that this cytokine is a novel pluripotency-associated factor capable of supporting self-renewal in the absence of exogenously added bFGF in culture. To date, the various protocols for hESCs maintenance require supplementation with bFGF to activate the Activin/Nodal branch of the TGFβ signaling pathway. Additional evidence supporting our findings is that IL-11 belongs to the same protein family as LIF, which is known to be necessary for maintaining pluripotency in mouse but not in human ESCs. These cytokines operate through the same gp130 receptor which interacts with Janus kinases. Our finding might explain why mESCs are in a more naïve cell state compared to hESCs and how to convert primed hESCs back to the naïve state. Taken together, our integrative modeling approach has identified novel genes as putative candidates to be incorporated into the expansion of the current gene regulatory network responsible for inducing and maintaining pluripotency.

Telomere fluorescence measurements in granulocytes and T lymphocyte subsets point to a high turnover of hematopoietic stem cells and memory T cells in early childhood.

To study telomere length dynamics in hematopoietic cells with age, we analyzed the average length of telomere repeat sequences in diverse populations of nucleated blood cells. More than 500 individuals ranging in age from 0 to 90 yr, including 36 pairs of monozygous and dizygotic twins, were analyzed using quantitative fluorescence in situ hybridization and flow cytometry. Granulocytes and naive T cells showed a parallel biphasic decline in telomere length with age that most likely reflected accumulated cell divisions in the common precursors of both cell types: hematopoietic stem cells. Telomere loss was very rapid in the first year, and continued for more than eight decades at a 30-fold lower rate. Memory T cells also showed an initial rapid decline in telomere length with age. However, in contrast to naive T cells, this decline continued for several years, and in older individuals lymphocytes typically had shorter telomeres than did granulocytes. Our findings point to a dramatic decline in stem cell turnover in early childhood and support the notion that cell divisions in hematopoietic stem cells and T cells result in loss of telomeric DNA.

Melan-A/MART-1-specific CD4 T cells in melanoma patients: identification of new epitopes and ex vivo visualization of specific T cells by MHC class II tetramers.

Over the past decade, many efforts have been made to identify MHC class II-restricted epitopes from different tumor-associated Ags. Melan-A/MART-1(26-35) parental or Melan-A/MART-1(26-35(A27L)) analog epitopes have been widely used in melanoma immunotherapy to induce and boost CTL responses, but only one Th epitope is currently known (Melan-A51-73, DRB1*0401 restricted). In this study, we describe two novel Melan-A/MART-1-derived sequences recognized by CD4 T cells from melanoma patients. These epitopes can be mimicked by peptides Melan-A27-40 presented by HLA-DRB1*0101 and HLA-DRB1*0102 and Melan-A25-36 presented by HLA-DQB1*0602 and HLA-DRB1*0301. CD4 T cell clones specific for these epitopes recognize Melan-A/MART-1+ tumor cells and Melan-A/MART-1-transduced EBV-B cells and recognition is reduced by inhibitors of the MHC class II presentation pathway. This suggests that the epitopes are naturally processed and presented by EBV-B cells and melanoma cells. Moreover, Melan-A-specific Abs could be detected in the serum of patients with measurable CD4 T cell responses specific for Melan-A/MART-1. Interestingly, even the short Melan-A/MART-1(26-35(A27L)) peptide was recognized by CD4 T cells from HLA-DQ6+ and HLA-DR3+ melanoma patients. Using Melan-A/MART-1(25-36)/DQ6 tetramers, we could detect Ag-specific CD4 T cells directly ex vivo in circulating lymphocytes of a melanoma patient. Together, these results provide the basis for monitoring of naturally occurring and vaccine-induced Melan-A/MART-1-specific CD4 T cell responses, allowing precise and ex vivo characterization of responding T cells.

Is the macromolecule signal tissue-specific in healthy human brain? A (1)H MRS study at 7 Tesla in the occipital lobe.

PURPOSE: The macromolecule signal plays a key role in the precision and the accuracy of the metabolite quantification in short-TE (1) H MR spectroscopy. Macromolecules have been reported at 1.5 Tesla (T) to depend on the cerebral studied region and to be age specific. As metabolite concentrations vary locally, information about the profile of the macromolecule signal in different tissues may be of crucial importance. METHODS: The aim of this study was to investigate, at 7T for healthy subjects, the neurochemical profile differences provided by macromolecule signal measured in two different tissues in the occipital lobe, predominantly composed of white matter tissue or of grey matter tissue. RESULTS: White matter-rich macromolecule signal was relatively lower than the gray matter-rich macromolecule signal from 1.5 to 1.8 ppm and from 2.3 to 2.5 ppm with mean difference over these regions of 7% and 12% (relative to the reference peak at 0.9 ppm), respectively. The neurochemical profiles, when using either of the two macromolecule signals, were similar for 11 reliably quantified metabolites (CRLB < 20%) with relatively small concentration differences (< 0.3 μmol/g), except Glu (± 0.8 μmol/g). CONCLUSION: Given the small quantification differences, we conclude that a general macromolecule baseline provides a sufficiently accurate neurochemical profile in occipital lobe at 7T in healthy human brain.

Phenotypic and functional analysis of human fetal liver hematopoietic stem cells in culture.

Steady-state hematopoiesis and hematopoietic transplantation rely on the unique potential of stem cells to undergo both self-renewal and multilineage differentiation. Fetal liver (FL) represents a promising alternative source of hematopoietic stem cells (HSCs), but limited by the total cell number obtained in a typical harvest. We reported that human FL nonobese diabetic/severe combined immunodeficient (NOD/SCID) repopulating cells (SRCs) could be expanded under simple stroma-free culture conditions. Here, we sought to further characterize FL HSC/SRCs phenotypically and functionally before and following culture. Unexpanded or cultured FL cell suspensions were separated into various subpopulations. These were tested for long-term culture potential and for in vivo repopulating function following transplantation into NOD/SCID mice. We found that upon culture of human FL cells, a tight association between classical stem cell phenotypes, such as CD34(+) /CD38(-) and/or side population, and NOD/SCID repopulating function was lost, as observed with other sources. Although SRC activity before and following culture consistently correlated with the presence of a CD34(+) cell population, we provide evidence that, contrary to umbilical cord blood and adult sources, stem cells present in both CD34(+) and CD34(-) FL populations can sustain long-term hematopoietic cultures. Furthermore, upon additional culture, CD34-depleted cell suspensions, devoid of SRCs, regenerated a population of CD34(+) cells possessing SRC function. Our studies suggest that compared to neonatal and adult sources, the phenotypical characteristics of putative human FL HSCs may be less strictly defined, and reinforce the accumulated evidence that human FL represents a unique, valuable alternative and highly proliferative source of HSCs for clinical applications.