Man scripClick here to ie linked References SummaryClassically considered short-lived, purely defensive leukocytes, neutrophils are unique in their fast and moldable response to stimulation. This plastic behavior may underlie variable and even antagonistic functions during inflammation or cancer, yet the full spectrum of neutrophil properties as they enter healthy tissues remains unexplored. Using a new model to track neutrophil fates, we found short but variable lifetimes across multiple tissues. Through analysis of the receptor, transcriptional and chromatin accessibility landscapes, we identify varying neutrophil states and assign non-canonical functions, including vascular repair and hematopoietic homeostasis. Accordingly, depletion of neutrophils compromised angiogenesis during early age, genotoxic injury and viral infection, and impaired hematopoietic recovery after irradiation. Neutrophils acquired these properties in target tissues, a process that in the lungs occurred in CXCL12-rich areas and relied on CXCR4. Our results reveal that tissues co-opt neutrophils en route for elimination to induce programs that support their physiological demands. circulation (Hidalgo et al., 2019) and reduced transcriptional activity preclude genetic adaptation to tissue environments (Silvestre-Roig et al., 2016). Existing evidence has shown, however, that cancer can instruct different transcriptional profiles, resulting in functions that can either promote, or counteract, tumoral growth and metastasis (Coffelt et al., 2016). Similar heterogeneous behavior has been reported in the context of stroke,

Expansion of human stem cells before cell therapy is typically performed at 20% O 2 . Growth in these pro-oxidative conditions can lead to oxidative stress and genetic instability. Here, we demonstrate that culture of human mesenchymal stem cells at lower, physiological O 2 concentrations significantly increases lifespan, limiting oxidative stress, DNA damage, telomere shortening and chromosomal aberrations. Our gene expression and bioenergetic data strongly suggest that growth at reduced oxygen tensions favors a natural metabolic state of increased glycolysis and reduced oxidative phosphorylation. We propose that this balance is disturbed at 20% O 2 , resulting in abnormally increased levels of oxidative stress. These observations indicate that bioenergetic pathways are intertwined with the control of lifespan and decisively influence the genetic stability of human primary stem cells. We conclude that stem cells for human therapy should be grown under low oxygen conditions to increase biosafety. Cell Death and Differentiation (2012) 19, 743-755; doi:10.1038/cdd.2011; published online 2 December 2011Human mesenchymal stem cells (hMSC) are being evaluated for the treatment of a large variety of pathologies, including traumatic lesions and cardiovascular and autoimmune diseases. 1,2 Although hMSC can be obtained from several tissues, they are scarce and their quantity and quality depends on a patient's clinical history, age, gender and genetic background. Most cell therapy protocols use 10-50 million hMSC per treatment, requiring expansion of extracted stem cells ex vivo for about 8 weeks before implantation. This expansion is typically performed under 'standard' non-physiological culture conditions, which among other factors expose cells to 20% O 2 , roughly 10 times the oxygen concentration encountered in their natural niches. 3,4 Previous studies have shown that exposure of mammalian cells to 20% O 2 concentrations induces DNA damage, thereby contributing decisively to cell senescence and loss of viability. [5][6][7] Conversely, culture of human stem cells over a physiological range of oxygen tensions (1-5%) improves cell growth, alters differentiation processes and extends lifespan. 8 Low oxygen tensions have also been shown to reduce the levels of double-strand breaks (DSB) and chromosomal abnormalities in several types of stem cells. 9,10 This evidence suggests that the poorly defined 'cell culture stress' can be a cause of genetic instability and therefore constitute a biological risk for cell therapy protocols. In agreement with this notion, we have found that short-term growth of hMSC at 20% O 2 significantly increases oxidative stress and DNA damage markers, DSB, chromosomal aberrations, aneuploidy and telomere shortening rates compared with cells grown at 3% O 2 . Despite these clear correlations, the mechanisms underlying the generation of genetic instability at high O 2 tension are mostly unknown.Mammalian cells have developed oxygen-sensing mechanisms to maintain cell and tissue homeostasis (reviewed...

In spite of the extensive potential of human mesenchymal stem cells (hMSCs) in cell therapy, little is known about the molecular mechanisms that regulate their therapeutic properties. We aimed to identify microRNAs (miRNAs) involved in controlling the transition between the resting and reparative phenotypes of hMSCs, hypothesizing that these miRNAs must be present in the undifferentiated cells and downregulated to allow initiation of distinct activation/differentiation programs. Differential miRNA expression analyses revealed that miR-335 is significantly downregulated upon hMSC differentiation. In addition, hMSCs derived from a variety of tissues express miR-335 at a higher level than human skin fibroblasts, and overexpression of miR-335 in hMSCs inhibited their proliferation and migration, as well as their osteogenic and adipogenic potential. Expression of miR-335 in hMSCs was upregulated by the canonical Wnt signaling pathway, a positive regulator of MSC self-renewal, and downregulated by interferon-c (IFN-c), a pro-inflammatory cytokine that has an important role in activating the immunomodulatory properties of hMSCs. Differential gene expression analyses, in combination with computational searches, defined a cluster of 62 putative target genes for miR-335 in hMSCs. Western blot and 3 0 UTR reporter assays confirmed RUNX2 as a direct target of miR-335 in hMSCs. These results strongly suggest that miR-335 downregulation is critical for the acquisition of reparative MSC phenotypes. Mesenchymal stem cells (MSCs) are multipotent mesodermderived somatic stem cell (SSC) precursors of non-hematopoietic connective tissues that are present in the stroma of virtually all mammalian organs, especially bone marrow and subcutaneous fat (reviewed in Bernardo et al. 1 ). Upon activation by tissue damage, MSCs contribute to tissue-repair processes through a multitude of activities, including cell proliferation, differentiation and migration, and the regulation of angiogenesis and immune responses. There is growing evidence, in both animal and clinical models, that administration of ex vivo-expanded human MSCs (hMSCs) has potential to ameliorate many degenerative disorders; however, the specific molecular mechanisms underlying this therapeutic potential remain mostly unknown.MicroRNAs (miRNAs) are an extensive family of small (18-24 nucleotide), single-stranded non-coding RNAs, which regulate gene expression in eukaryotic cells by controlling the translation (usually by repression), stability and localization of specific mRNA targets. Computational predictions of miRNA targets indicate that each miRNA regulates hundreds of mRNAs, and that approximately one third of all mammalian protein-coding genes are regulated by miRNAs. 2 Functional studies show that miRNAs participate in virtually each cellular process investigated, and that alterations in their expression levels might underlie human diseases, including cardiovascular disease and cancer. There are also data indicating that mammalian miRNAs can be imported into the nucleu...

Summary Dendrite branching and spine formation determines the function of morphologically distinct and specialized neuronal subclasses. However, little is known about the programs instructing specific branching patterns in vertebrate neurons and whether such programs influence dendritic spines and synapses. Using knockout and knockdown studies combined with morphological, molecular and electrophysiological analysis we show that the homeobox Cux1 and Cux2 are intrinsic and complementary regulators of dendrite branching, spine development and synapse formation in layer II–III neurons of the cerebral cortex. Cux genes control the number and maturation of dendritic spines partly through direct regulation of the expression of Xlr3b and Xlr4b, chromatin remodeling genes previously implicated in cognitive defects. Accordingly, abnormal dendrites and synapses in Cux2−/− mice correlate with reduced synaptic function and defects in working memory. These demonstrate critical roles of Cux in dendritogenesis and highlight novel subclass-specific mechanisms of synapse regulation that contribute to the establishment of cognitive circuits.

In most clinical trials, human mesenchymal stem cells (hMSCs) are expanded in vitro before implantation. The genetic stability of human stem cells is critical for their clinical use. However, the relationship between stem-cell expansion and genetic stability is poorly understood. Here, we demonstrate that within the normal expansion period, hMSC cultures show a high percentage of aneuploid cells that progressively increases until senescence. Despite this accumulation, we show that in a heterogeneous culture the senescence-prone hMSC subpopulation has a lower proliferation potential and a higher incidence of aneuploidy than the non-senescent subpopulation. We further show that senescence is linked to a novel transcriptional signature that includes a set of genes implicated in ploidy control. Overexpression of the telomerase catalytic subunit (human telomerase reverse transcriptase, hTERT) inhibited senescence, markedly reducing the levels of aneuploidy and preventing the dysregulation of ploidy-controlling genes. hMSC-replicative senescence was accompanied by an increase in oxygen consumption rate (OCR) and oxidative stress, but in long-term cultures that overexpress hTERT, these parameters were maintained at basal levels, comparable to unmodified hMSCs at initial passages. We therefore propose that hTERT contributes to genetic stability through its classical telomere maintenance function and also by reducing the levels of oxidative stress, possibly, by controlling mitochondrial physiology. Finally, we propose that aneuploidy is a relevant factor in the induction of senescence and should be assessed in hMSCs before their clinical use.

We applied the directional tag PCR subtractive hybridization method to construct a rat hypothalamic cDNA library from which cerebellar and hippocampal sequences had been depleted, enriching 20-30-fold for sequences expressed selectively in the hypothalamus. We studied a sample of 94 clones selected for enrichment in the subtracted library. These clones corresponded to 43 distinct mRNA species, about half of which were novel. Thirty-eight of these 43 mRNAs (corresponding to 85 of the clones in the sample) exhibited enrichment in the hypothalamus; 23 were highly enriched. In situ hybridization studies revealed that one novel species was restricted to cells in a small bilaterally symmetric area of the paraventricular hypothalamus. Other novel mRNAs showed substantial enrichment in basal diencephalic structures, particularly the hypothalamus, without restriction to single hypothalamic nuclei. The data suggest that the hypothalamus utilizes at least two distinct strategies for employing its selectively expressed proteins. Secretory neuropeptides utilized for intercellular communication are produced by functionally discrete nuclei, while several other proteins are shared by structures that are unrelated in their physiological roles but may share biochemical systems.The hypothalamus is a phylogenetically ancient region of the mammalian brain. In contrast to laminar cortical structures such as the cerebellum and hippocampus whose final functions rely on innervation from the thalamus and brain stem, the hypothalamus is organized as a collection of distinct, autonomously active nuclei with discrete functions. Ablation and electrical stimulation studies and medical malfunctions have implicated several of these nuclei as central regulatory centers for major autonomic and endocrine homeostatic systems mediating processes such as reproduction, lactation, fluid balance, metabolism, and aspects of behaviors, such as circadian rhythmicity, basic emotions, feeding and drinking, mating activities, and responses to stress, as well as normal development of the immune system (1). Distinct hormones and releasing factors have been associated with some of these nuclei, but, at best, the organizations and molecular operations of these structures are only partially understood.A substantial portion of a mammal's genetic endowment is dedicated to the function of its central nervous system, as evidenced by the substantial number of mRNAs selectively expressed in the brain (2). Many of these have been observed to be selectively associated with distinct neural subsets. We hypothesized that ensembles of mRNAs selectively associated with discrete hypothalamic nuclei encode proteins singularly associated with the unique functions of those nuclei. The hypothesis must be at least partially valid given existing knowledge on the expression of specific hypothalamic hormones and releasing factors, but the extent to which it can account for the function of this organ is not known. To address this hypothesis further and to illuminate additional m...

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Interferon-alpha therapy has been shown to be active in the treatment of mycosis fungoides although the individual response to this therapy is unpredictable and dependent on essentially unknown factors. In an effort to better understand the molecular mechanisms of interferon-alpha resistance we have developed an interferon-alpha resistant variant from a sensitive cutaneous T-cell lymphoma cell line. We have performed expression analysis to detect genes differentially expressed between both variants using a cDNA microarray including 6386 cancer-implicated genes. The experiments showed that resistance to interferon-alpha is consistently associated with changes in the expression of a set of 39 genes, involved in signal transduction, apoptosis, transcription regulation, and cell growth. Additional studies performed confirm that STAT1 and STAT3 expression and interferon-alpha induction and activation are not altered between both variants. The gene MAL, highly overexpressed by resistant cells, was also found to be expressed by tumoral cells in a series of cutaneous T-cell lymphoma patients treated with interferon-alpha and/or photochemotherapy. MAL expression was associated with longer time to complete remission. Time-course experiments of the sensitive and resistant cells showed a differential expression of a subset of genes involved in interferon-response (1 to 4 hours), cell growth and apoptosis (24 to 48 hours.), and signal transduction.