Subversion of host genome integrity by bacterial pathogens.

Mammalian cells possess sophisticated genome surveillance and repair mechanisms, executed by the so-called DNA damage response (DDR), failure of which leads to accumulation of DNA damage and genomic instability. Mounting evidence suggests that bacterial infections can elicit DNA damage in host cells, and certain pathogens induce such damage as part of their multi-faceted infection programme. Bacteria-mediated DNA damage can occur either directly through the formation of toxins with genotoxic activities or indirectly as a result of the activation of cell-autonomous or immune defence mechanisms against the pathogen. Moreover, host-cell signalling routes involved in the DDR can be altered in response to an infection, and this, in the context of DNA damage elicited by the pathogen, has the potential to trigger mutations and cancer.

Gastric cancer pathogenesis.

Gastric cancer (GC) results from a multistep process that is influenced by Helicobacter pylori infection, genetic susceptibility of the host, as well as of other environmental factors. GC results from the accumulation of numerous genetic and epigenetic alterations in oncogenes and tumor suppressor genes, leading to dysregulation of multiple signaling pathways, which disrupt the cell cycle and the balance between cell proliferation and cell death. For this special issue, we have selected to review last year's advances related to three main topics: the cell of origin that initiates malignant growth in GC, the mechanisms of direct genotoxicity induced by H. pylori infection, and the role of aberrantly expressed long noncoding RNAs in GC transformation. The understanding of the molecular basis of GC development is of utmost importance for the identification of novel targets for GC prevention and treatment.

Modulation of Host Cell Metabolism by Chlamydia trachomatis.

Propagation of the intracellular bacterial pathogen Chlamydia trachomatis is strictly bound to its host cells. The bacterium has evolved by minimizing its genome size at the cost of being completely dependent on its host. Many of the vital nutrients are synthesized only by the host, and this has complex implications. Recent advances in loss-of-function analyses and the metabolomics of human infected versus noninfected cells have provided comprehensive insight into the molecular changes that host cells undergo during the stage of infection. Strikingly, infected cells acquire a stage of high metabolic activity, featuring distinct aspects of the Warburg effect, a condition originally assigned to cancer cells. This condition is characterized by aerobic glycolysis and an accumulation of certain metabolites, altogether promoting the synthesis of crucial cellular building blocks, such as nucleotides required for DNA and RNA synthesis. The altered metabolic program enables tumor cells to rapidly proliferate as well as C. trachomatis-infected cells to feed their occupants and still survive. This program is largely orchestrated by a central control board, the tumor suppressor protein p53. Its downregulation in C. trachomatis-infected cells or mutation in cancer cells not only alters the metabolic state of cells but also conveys the prevention of programmed cell death involving mitochondrial pathways. While this points toward common features in the metabolic reprogramming of infected and rapidly proliferating cells, it also forwards novel treatment options against chronic intracellular infections involving well-characterized host cell targets and established drugs.

Human stem cells for CNS repair.

Although most peripheral tissues have at least a limited ability for self-repair, the central nervous system (CNS) has long been known to be relatively resistant to regeneration. Small numbers of stem cells have been found in the adult brain but do not appear to be able to affect any significant recovery following disease or insult. In the last few decades, the idea of being able to repair the brain by introducing new cells to repair damaged areas has become an accepted potential treatment for neurodegenerative diseases. This review focuses on the suitability of various human stem cell sources for such treatments of both slowly progressing conditions, such as Parkinson's disease, Huntington's disease and multiple sclerosis, and acute insult, such as stroke and spinal cord injury. Despite stem cell transplantation having now moved a step closer to the clinic with the first trials of autologous mesenchymal stem cells, the effects shown are moderate and are not yet at the stage of development that can fulfil the hopes that have been placed on stem cells as a means to replace degenerating cells in the CNS. Success will depend on careful investigation in experimental models to enable us to understand not just the practicalities of stem cell use, but also the underlying biological principles.

Is there a place for human fetal-derived stem cells for cell replacement therapy in Huntington's disease?

Huntington's disease (HD) is a neurodegenerative disease that offers an excellent paradigm for cell replacement therapy because of the associated relatively focal cell loss in the striatum. The predominant cells lost in this condition are striatal medium spiny neurons (MSNs). Transplantation of developing MSNs taken from the fetal brain has provided proof of concept that donor MSNs can survive, integrate and bring about a degree of functional recovery in both pre-clinical studies and in a limited number of clinical trials. The scarcity of human fetal tissue, and the logistics of coordinating collection and dissection of tissue with neurosurgical procedures makes the use of fetal tissue for this purpose both complex and limiting. Alternative donor cell sources which are expandable in culture prior to transplantation are currently being sought. Two potential donor cell sources which have received most attention recently are embryonic stem (ES) cells and adult induced pluripotent stem (iPS) cells, both of which can be directed to MSN-like fates, although achieving a genuine MSN fate has proven to be difficult. All potential donor sources have challenges in terms of their clinical application for regenerative medicine, and thus it is important to continue exploring a wide variety of expandable cells. In this review we discuss two less well-reported potential donor cell sources; embryonic germ (EG) cells and fetal neural precursors (FNPs), both are which are fetal-derived and have some properties that could make them useful for regenerative medicine applications.

Insights in spatio-temporal characterization of human fetal neural stem cells.

Primary human fetal cells have been used in clinical trials of cell replacement therapy for the treatment of neurodegenerative disorders such as Huntington's disease (HD). However, human fetal primary cells are scarce and difficult to work with and so a renewable source of cells is sought. Human fetal neural stem cells (hfNSCs) can be generated from human fetal tissue, but little is known about the differences between hfNSCs obtained from different developmental stages and brain areas. In the present work we characterized hfNSCs, grown as neurospheres, obtained from three developmental stages: 4-5, 6-7 and 8-9weeks post conception (wpc) and four brain areas: forebrain, cortex, whole ganglionic eminence (WGE) and cerebellum. We observed that, as fetal brain development proceeds, the number of neural precursors is diminished and post-mitotic cells are increased. In turn, primary cells obtained from older embryos are more sensitive to the dissociation process, their viability is diminished and they present lower proliferation ratios compared to younger embryos. However, independently of the developmental stage of derivation proliferation ratios were very low in all cases. Improvements in the expansion rates were achieved by mechanical, instead of enzymatic, dissociation of neurospheres but not by changes in the seeding densities. Regardless of the developmental stage, neurosphere cultures presented large variability in the viability and proliferation rates during the initial 3-4 passages, but stabilized achieving significant expansion rates at passage 5 to 6. This was true also for all brain regions except cerebellar derived cultures that did not expand. Interestingly, the brain region of hfNSC derivation influences the expansion potential, being forebrain, cortex and WGE derived cells the most expandable compared to cerebellar. Short term expansion partially compromised the regional identity of cortical but not WGE cultures. Nevertheless, both expanded cultures were multipotent and kept the ability to differentiate to region specific mature neuronal phenotypes.

The effects of various concentrations of FGF-2 on the proliferation and neuronal yield of murine embryonic neural precursor cells in vitro.

Embryonic neural precursors (ENPs), also termed neural stem cells or "neurospheres," are an attractive potential source of tissue for neural transplantation, because of their capacity to expand in number in vitro while retaining the ability to develop into the major phenotypes of the CNS. ENPs are isolated from the developing brain and proliferate in the presence of mitogens such as FGF-2 and EGF. Subsequent withdrawal of these mitogens and exposure to a suitable substrate results in differentiation into the major cell types of the CNS. As well as its role in precursor cell expansion, FGF-2 also plays a key role in the division of astrocytes, and in neuronal differentiation. Thus, it is important to establish the optimal concentrations of this factor for expansion and differentiation of neuronal phenotypes. Here we explore the effect of FGF-2 concentrations ranging from 1 to 20 ng/ml on the expansion and differentiation capacity of ENPs isolated from the cortex and striatum of E14 mice. ENP expansion was seen under all conditions, but was greatest at 10 and 20 ng/ml and least at 1 ng/ml. The numbers of neurons (as a proportion of total cell number) differentiating from these ENP populations appeared to be greatest at 1 ng/ml. However, once adjustments were made for the amount of expansion at each dose, final neuronal yield was maximum at the highest concentration of FGF-2 used (20 ng/ml).

The release of excitatory amino acids, dopamine, and potassium following transplantation of embryonic mesencephalic dopaminergic grafts to the rat striatum, and their effects on dopaminergic neuronal survival in vitro.

A major limitation to the effectiveness of grafts of fetal ventral mesencephalic tissue for parkinsonism is that about 90-95% of grafted dopaminergic neurones die. In rats, many of the cells are dead within 1 day and most cell death is complete within 1 week. Our previous results suggest that a major cause of this cell death is the release of toxins from the injured CNS tissue surrounding the graft, and that many of these toxins have dissipated within 1 h of inserting the grafting cannula. In the present experiments we measured the change over time in the concentration of several potential toxins around an acutely implanted grafting cannula. We also measured the additional effect of injecting suspensions of embryonic mesencephalon, latex microspheres, or vehicle on these concentrations. Measurements of glutamate, aspartate, and dopamine by microdialysis showed elevated levels during the first 20-60 min, which then declined to baseline. In the first 20 min glutamate levels were 10.7 times, aspartate levels 5 times, and dopamine levels 24.3 times baseline. Potassium levels increased to a peak of 33 +/- 10.6 mM 4-5 min after cannula insertion, returning to baseline of <5 mM by 30 min. Injection of cell suspension, latex microspheres, or vehicle had no significant effect on these levels. We then assayed the effect of high concentrations of glutamate, aspartate, dopamine, and potassium on dopaminergic neuronal survival in E14 ventral mesencephalic cultures. In monolayer cultures only dopamine at 200 microM showed toxicity. In three-dimensional cultures only the combination of raised potassium, dopamine, glutamate, and aspartate together decreased dopaminergic neuronal survival. We conclude that toxins other than the ones measured are the main cause of dopaminergic cell death after transplantation, or the effects of the toxins measured are enhanced by anoxia and metabolic challenges affecting newly inserted grafts.

Long-term expansion of human foetal neural progenitors leads to reduced graft viability in the neonatal rat brain.

We previously reported that early passage human foetal neural progenitors (hFNPs) survive long-term in the rodent host brain whereas late passage cells disappear at later post-graft survival times. The extent to which this finding is related to changes in the expanded FNPs or in the adult host brain environment was not determined. Here we report the effect of expanding hFNPs for different periods of time in vitro on their ability to survive transplantation into the neonatal rat hippocampus, a generally more permissive environment than the adult rat brain. After 2 and 8 weeks in vitro, transplanted hFNPs formed large grafts, most of which survived well until at least 12 weeks. However, following continued expansion, hFNPs formed smaller grafts, and cells transplanted after 20 weeks expansion produced no surviving grafts, even at early survival times. To determine whether this could be due to a dilution of "true" neural stem cells through more differentiated progeny over time in culture, we derived homogeneous neural stem (NS) cells grown as a monolayer from the 8 week expanded hFNPs. These cells homogeneously expressed the neural stem cell markers sox-2, 3CB2 and nestin and were expanded for 5 months before transplantation into the neonatal rat brain. However, these cells exhibited a similar survival profile to the long-term expanded FNPs. These results indicate that, while the cellular phenotype of neural stem cells may appear to be stable in vitro using standard markers, expansion profoundly influences the ability of such cells to form viable grafts.

Stem cell transplantation for neurodegenerative diseases.

PURPOSE OF REVIEW: To review recent developments in the application of stem cells for transplantation therapies in neurodegenerative diseases. RECENT FINDINGS: Stem cell transplantation has the potential to improve function by replacing cells lost to the disease and reconstructing elements of neural circuitry or by providing support for host cells (e.g. by secretion of trophic factors). Other mechanisms, such as modulation of the immune system by bone marrow stem cell transplantation, pertinent to conditions such as multiple sclerosis, are emerging as therapies but will not be discussed here. There have been substantial advances in our understanding of stem cell biology and some recent important advances in controlling their differentiated phenotype. Using stem cells to provide trophic support places less stringent requirements on the cells and this is the area in which many of the first clinical studies are taking place. SUMMARY: There are real prospects of stem cell technology having a place in clinical management of neurodegenerative conditions, but directing the differentiation of stem cells towards the appropriate neural phenotype remains a challenge. This is a relatively new and rapidly evolving area, and caution should be applied when advising patients.

The survival of neural precursor cell grafts is influenced by in vitro expansion.

Embryonic neural precursor cells (ENPs) provide a potential alternative for transplantation in neurodegenerative diseases, as they can be expanded in culture, avoiding many of the practical obstacles that limit the application of transplanting primary neurones. However, grafts of ENPs into animal models show variable survival and limited differentiation into neurones. The effect of expansion time on their ability to survive and differentiate may be an important factor in this and has not been examined directly. In these experiments, murine and human ENPs were expanded for short (4 weeks) and long (20 weeks) periods before transplantation into the adult rat striatum. Whereas grafts of both short- and long-term expanded human ENPs survived for 4 weeks following transplantation, by 20 weeks all long-term expanded grafts had disappeared. Murine ENPs behaved similarly: only grafts of short-term expanded ENPs survived at 12 weeks following transplantation. RT-PCR analysis of ENP cultures after 4 and 20 weeks of expansion demonstrated changes in expression of a number of different groups of genes. We conclude that long-term expansion of ENPs profoundly impairs their ability to survive long-term after transplantation into the adult brain. This has implications for the potential use of these cells for neural transplantation strategies.