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Endogenous neural stem/precursor cells (NPCs) are considered a functional reservoir for promoting tissue homeostasis and repair after injury, therefore regenerative strategies that mobilize these cells have recently been proposed. Despite evidence of increased neurogenesis upon acute inflammatory insults (e.g. ischaemic stroke), the plasticity of the endogenous brain stem cell compartment in chronic CNS inflammatory disorders remains poorly characterized. Here we show that persistent brain inflammation, induced by immune cells targeting myelin, extensively alters the proliferative and migratory properties of subventricular zone (SVZ)-resident NPCs in vivo leading to significant accumulation of non-migratory neuroblasts within the SVZ germinal niche. In parallel, we demonstrate a quantitative reduction of the putative brain stem cells proliferation in the SVZ during persistent brain inflammation, which is completely reversed after in vitro culture of the isolated NPCs. Together, these data indicate that the inflamed brain microenvironment sustains a non cell-autonomous dysfunction of the endogenous CNS stem cell compartment and challenge the potential efficacy of proposed therapies aimed at mobilizing endogenous precursors in chronic inflammatory brain disorders.

MSCs are known to have an extensive proliferative potential and ability to differentiate in various cell types. Osteoblastic differentiation from mesenchymal progenitor cells is an important step of bone formation, though the pattern of gene expression during differentiation is not yet well understood. Here, to investigate the possibility to obtain a model for in vitro bone differentiation using mesenchymal stem cells (hMSCs) from human subjects non-invasively, we developed a method to obtain hMSCs-like cells from peripheral blood by a two step method that included an enrichment of mononuclear cells followed by depletion of unwanted cells. Using these cells, we analyzed the expression of transcription factor genes (runt-related transcription factor 2 (RUNX2) and osterix (SP7)) and bone related genes (osteopontin (SPP1), osteonectin (SPARC) and collagen, type I, alpha 1 (COLIA1)) during osteoblastic differentiation. Our results demonstrated that hMSCs can be obtained from peripheral blood and that they are able to generate CFU-F and to differentiate in osteoblast and adipocyte; in this study, we also identified a possible gene expression timing during osteoblastic differentiation that provided a powerful tool to study bone physiology.

PURPOSE: The Aurora kinase family plays pivotal roles in mitotic integrity and cell cycle. We sought to determine the effects of inhibiting Aurora kinase on ovarian cancer growth in an orthotopic mouse model using a small molecule pan-Aurora kinase inhibitor, MK-0457. EXPERIMENTAL DESIGN: We examined cell cycle regulatory effects and ascertained the therapeutic efficacy of Aurora kinase inhibition both alone and combined with docetaxel using both in vitro and in vivo ovarian cancer models. RESULTS: In vitro cytotoxicity assays with HeyA8 and SKOV3ip1 cells revealed textgreater10-fold greater docetaxel cytotoxicity in combination with MK-0457. After in vivo dose kinetics were determined using phospho-histone H3 status, therapy experiments with the chemosensitive HeyA8 and SKOV3ip1 as well as the chemoresistant HeyA8-MDR and A2780-CP20 models showed that Aurora kinase inhibition alone significantly reduced tumor burden compared with controls (P valuestextless0.01). Combination treatment with docetaxel resulted in significantly improved reduction in tumor growth beyond that afforded by docetaxel alone (P textlessor= 0.03). Proliferating cell nuclear antigen immunohistochemistry revealed that MK-0457 alone and in combination with docetaxel significantly reduced cellular proliferation (P valuestextless0.001). Compared with controls, treatment with MK-0457 alone and in combination with docetaxel also significantly increased tumor cell apoptosis by approximately 3-fold (Ptextless0.01). Remarkably, compared with docetaxel monotherapy, MK-0457 combined with docetaxel resulted in significantly increased tumor cell apoptosis. CONCLUSIONS: Aurora kinase inhibition significantly reduces tumor burden and cell proliferation and increases tumor cell apoptosis in this preclinical orthotopic model of ovarian cancer. The role of Aurora kinase inhibition in ovarian cancer merits further investigation in clinical trials.

TGF-beta can be a potent suppressor of lymphocyte effector cell functions and can mediate these effects via distinct molecular pathways. The role of TGF-beta in regulating CD16-mediated NK cell IFN-gamma production and antibody-dependent cellular cytotoxicity (ADCC) is unclear, as are the signaling pathways that may be utilized. Treatment of primary human NK cells with TGF-beta inhibited IFN-gamma production induced by CD16 activation with or without IL-12 or IL-2, and it did so without affecting the phosphorylation/activation of MAP kinases ERK and p38, as well as STAT4. TGF-beta treatment induced SMAD3 phosphorylation, and ectopic overexpression of SMAD3 resulted in a significant decrease in IFN-gamma gene expression following CD16 activation with or without IL-12 or IL-2. Likewise, NK cells obtained from smad3(-/-) mice produced more IFN-gamma in response to CD16 activation plus IL-12 when compared with NK cells obtained from wild-type mice. Coactivation of human NK cells via CD16 and IL-12 induced expression of T-BET, the positive regulator of IFN-gamma, and T-BET was suppressed by TGF-beta and by SMAD3 overexpression. An extended treatment of primary NK cells with TGF-beta was required to inhibit ADCC, and it did so by inhibiting granzyme A and granzyme B expression. This effect was accentuated in cells overexpressing SMAD3. Collectively, our results indicate that TGF-beta inhibits CD16-mediated human NK cell IFN-gamma production and ADCC, and these effects are mediated via SMAD3.

Spinal muscular atrophy (SMA), a motor neuron disease (MND) and one of the most common genetic causes of infant mortality, currently has no cure. Patients with SMA exhibit muscle weakness and hypotonia. Stem cell transplantation is a potential therapeutic strategy for SMA and other MNDs. In this study, we isolated spinal cord neural stem cells (NSCs) from mice expressing green fluorescent protein only in motor neurons and assessed their therapeutic effects on the phenotype of SMA mice. Intrathecally grafted NSCs migrated into the parenchyma and generated a small proportion of motor neurons. Treated SMA mice exhibited improved neuromuscular function, increased life span, and improved motor unit pathology. Global gene expression analysis of laser-capture-microdissected motor neurons from treated mice showed that the major effect of NSC transplantation was modification of the SMA phenotype toward the wild-type pattern, including changes in RNA metabolism proteins, cell cycle proteins, and actin-binding proteins. NSC transplantation positively affected the SMA disease phenotype, indicating that transplantation of NSCs may be a possible treatment for SMA.

Cancer in humans is the result of a multi-step process. This process often involves the activation of oncogenes and/or the inactivation of tumor suppressor genes. These two steps arise not only due to mutations, but can also be the result of a translocation or an altered transcription rate. One important mechanism is the occurrence of epigenetic alterations like promotor methylation (which may lead to tumor suppressor silencing) or decreased histone acetylation (which can result in the downregulation of proteins involved in apoptosis). Today, histone acetylation and DNA methylation are epigenetic modifications which have been linked closely to the pathology of human cancers and inhibitors of both enzyme classes for clinical use are at hand. In contrast, other fields of epigenetics still lack of similarly thorough knowledge. This is especially true for the group of histone methyltransferases and their inhibitors. Since connections between histone methylation patterns and cancer progression have been recognized, histone methyltransferases represent promising targets for future cancer treatment.

Human B cells can be cultured ex vivo for a few weeks, following stimulation of the CD40 cell surface molecule in the presence of recombinant cytokines such as interleukin-4 (IL-4). However, attempts to produce polyclonal antigen-specific human antibodies by in vitro culture of human B cells obtained from immunized donors have not been successful. It has been shown in mice that lipopolysaccharide (LPS) is a potent mitogen for B cells and plays an important role in the generation of antigen-specific antibody responses. Although it has long been believed that LPS has no direct effect on human B cells, recent data indicating that IL-4-activated human B cells are induced to express Toll-like receptor-4, the main LPS receptor, prompted us to study the effects of LPS on the proliferation and antibody secretion of human B cells. Our results showed that LPS caused a reduction in the expansion of CD40-activated human B cells, accompanied by an increase in antigen-specific antibody secretion. This result suggested that some, but not all, B cells were able to differentiate into antibody-secreting cells in response to LPS. This increased differentiation could be explained by the observation that LPS-stimulated human B cells were induced to secrete higher amounts of IL-6, a pleiotropic cytokine well-known for its B-cell differentiation activity. In vivo, the effect of LPS on cytokine secretion by B cells may not only enhance B-cell differentiation but also help to sustain a local ongoing immune response to invading Gram-negative bacteria, until all pathogens have been cleared from the organism.

We report a system for Cre-regulated expression of RNA interference in vivo. Expression cassettes comprise selectable and FACS-sortable markers in tandem with additional marker genes and shRNAs in the antisense orientation. The cassettes are flanked by tandem LoxP sites arranged so that Cre expression inverts the marker-shRNA construct, allowing its regulated expression (and, at the same time, deletes the original selection/marker genes). The cassettes can be incorporated into retroviral or lentiviral vectors and delivered to cells in culture or used to generate transgenic mice. We describe cassettes incorporating various combinations of reporter genes, miRNA-based RNAi (including two shRNA constructs at once), and oncogenes and demonstrate the delivery of effective RNA interference in cells in culture, efficient transduction into hematopoietic stem cells with cell-type-specific knockdown in their progeny, and rapid generation of regulated shRNA knockdown in transgenic mice. These vector systems allow regulated combinatorial manipulation (both overexpression and loss of function) of gene expression in multiple systems in vitro and in vivo.

Tissue fibrosis is a major cause of death in SSc, but therapies that target selectively fibrosis are not yet available for routine clinical use. Recent pre-clinical studies suggest that selective tyrosine kinase inhibitors that target c-Abl, PDGF receptor or Src kinases might be promising targets for anti-fibrotic approaches. Dual inhibition of c-Abl and PDGF receptor by imatinib and nilotinib, and inhibition of Src kinases either selectively by SU6656 or in combination with c-Abl and PDGF by dasatinib exerted potent anti-fibrotic effects. Imatinib, nilotinib, dasatinib and SU6656 reduced dose-dependently the synthesis of extracellular matrix protein in human dermal fibroblasts in vitro and prevented fibrosis in the mouse model of bleomycin-induced skin fibrosis. Clinical data from patients with chronic myelogenous leukaemia suggest that imatinib, nilotinib and dasatinib are well tolerated. Based on the promising pre-clinical data, imatinib is currently evaluated in clinical trials for the treatment of fibrosis in SSc and trials with other tyrosine kinase inhibitors are in preparation.

The developmental potential of human ES cells makes them an important tool in developmental, pharmacological, and clinical research. For human ES cell technology to be fully exploited, however, culture efficiency must be improved, large-scale culture enabled, and safety ensured. Traditional human ES cell culture systems have relied on serum products and mouse feeder layers, which limit the scale, present biological variability, and expose the cells to potential contaminants. Defined, feeder-independent culture systems improve the safety and efficiency of ES cell technology, enabling translational research. The protocols herein are designed with the standard research laboratory in mind. They contain recipes for the formulation of mTeSR (a defined medium for human ES cell culture) and detailed protocols for the culture, transfer, and passage of cells grown in these feeder-independent conditions. They provide a basis for routine feeder-independent culture, and a starting point for additional optimization of culture conditions.

Direct reprogramming of human fibroblasts to a pluripotent state has been achieved through ectopic expression of the transcription factors OCT4, SOX2, and either cMYC and KLF4 or NANOG and LIN28. Little is known, however, about the mechanisms by which reprogramming occurs, which is in part limited by the low efficiency of conversion. To this end, we sought to create a doxycycline-inducible lentiviral system to convert primary human fibroblasts and keratinocytes into human induced pluripotent stem cells (hiPSCs). hiPSCs generated with this system were molecularly and functionally similar to human embryonic stem cells (hESCs), demonstrated by gene expression profiles, DNA methylation status, and differentiation potential. While expression of the viral transgenes was required for several weeks in fibroblasts, we found that 10 days was sufficient for the reprogramming of keratinocytes. Using our inducible system, we developed a strategy to induce hiPSC formation at high frequency. Upon addition of doxycycline to hiPSC-derived differentiated cells, we obtained secondary" hiPSCs at a frequency at least 100-fold greater than the initial conversion. The ability to reprogram cells at high efficiency provides a unique platform to dissect the underlying molecular and biochemical processes that accompany nuclear reprogramming."

Current approaches to reprogram human somatic cells to pluripotent iPSCs utilize viral transduction of different combinations of transcription factors. These protocols are highly inefficient because only a small fraction of cells carry the appropriate number and stoichiometry of proviral insertions to initiate the reprogramming process. Here we have generated genetically homogeneous secondary" somatic cells�