Ronald D.G. McKay - US grants

1.1 Generating pluripotent cells from adult mouse and human sources. [unreadable] [unreadable] Our expertise in generating pluripotent cells was demonstrated by the production of a new type of pluripotent cell (EpiS) from the mouse embryo. This cell may represent the epiblast at the time of implantation rather than the pre-implantation inner cell mass that has previously been considered the most likely homologue of ES cells. This new cell is a better model for human ES cells and because it is a later step in development will permit better control of the differentiation of pluripotent cells into distinct somatic cell types. In the last year, our attention has focussed on the production of pluripotent cells from adult tissues. Using different cell types from adult mouse and human tissues, we have generated more than 12 induced pluripotent cells (iPS). The patterns of gene expression in these iPS cells and embryo derived pluripotent cells are remarkably similar. In the next year, we will determine if the same signaling events control the differentiation of iPS, ES and EpiS cells. In this way, we plan to exploit the potential of reprogramming technology to define how genetic change alters signaling and differentiation in human cells. [unreadable] [unreadable] [unreadable] 1.2 Generating pluripotent cell lines from the cat.[unreadable] [unreadable] To date pluripotent embryonic stem cell lines have only been derived in a limited number of species including mouse, chicken, and primate. Our generation of pluripotent cell lines from the mouse epiblast (EpiSCs) may be a step towards a general method to derive ES cells from any vertebrate. We will test this possibility by generating EpiSCs from the cat epiblast and iPS cells from adult cat cells. We initiated work on rat pluripotent cells but with Dr. James Kehler joining the group, an opportunity arose to generate cat pluripotent cells. The cat is an important model in neuroscience and we are making good progress generating iPS cells from cat fibroblasts. [unreadable] [unreadable] [unreadable] 1.3 To define the chromatin state in ES cells and in neural precursors.[unreadable] [unreadable] There is great interest in how the stable epigenetic state of chromatin is maintained in ES cells. The derivation of EpiSCs cells allows a direct comparison of the state of chromatin in two distinct cells that are both pluripoitent. We are using gene array and chromatin immunoprecipitation (ChIP) methods to analyze the epigenetic changes when pluripotent cells differentiate into neural precursors. The differentiation of the cells is controlled by genetic and pharmacological tools. The data show that we have achieved reproducible and robust differentiation of ES cells and this provides a strong basis for a stringent assessment of the differentiation potential of iPS cells.[unreadable] [unreadable] [unreadable] 2.1 Lineages in CNS stem cells.[unreadable] [unreadable] To understand fate specification in neural stem cells we have developed an imaging system to identify every step in the lineage that transforms multipotent cells into astrocytes, oligodendrocytes and neurons. In the standard conditions, tripotent cells produce specified progenitors through bipotent intermediates and, surprisingly, the tripotent state is regenerated from cells with lower potency when cells are passaged. The action of the pro-astrocytic cytokine CNTF demonstrates that the fate specifying events occur rapidly in tri-potent cells. This precise timing of fate specification provides a strong basis for further analysis of the mechanisms that generate specific cell types from stem cells.[unreadable] [unreadable] [unreadable] 2.2 Energy production in neural stem cells[unreadable] [unreadable] It has been proposed that stem cells and their differentiated progeny use different strategies to generate energy. We have shown that neural stem cells and their immediate derivatives use different lactate dehydrogenase genes. In this way, ATP is generated in stem cells by glycolysis and in differentiated cells by oxidative phosphorylation. This finding has important implication in cancer and ischemia.[unreadable] [unreadable] [unreadable] 2.3 An in vitro assay that predicts stem cell activation in vivo.[unreadable] [unreadable] We have shown that Notch ligands stimulate the stem cell survival in vitro and have powerful neurotrophic effects in vivo. Our goal in this project is to determine if in vitro analysis of the survival network predicts the in vivo neurotrophic effect. In the past year, we have shown that insulin and Notch ligands act in a co-operative manner to control stem cell survival in vitro. This result provides an explanbation for a surprising feature of an earlier report from our group. In this report we showed that Notch ligands activate the major growth pathway in a cell. This growth pathway is normally thought to be activated by receptor tyrosine kinases but Notch does not carry this function. By demonstrating that (1) Notch ligands cause tyrosine phosphorylation of the IGFR1 and (2) Notch antagonsists block IGFR1 activation we provide a reciprocal link between Notch and the classic growth pathway in cells. Insulin injection into the adult brain increases stem cell numbers showing that the association between stem cells survival in vitro and in vivo continues to hold. To extend this finding we are developing simple assays that predict the in vivo effects on the stem cell niche. By automatic microscopy, we propose to generate thousands of data points allowing rapid optimization of regenerative therapies.[unreadable] [unreadable] 3.1 To determine neuronal survival mechanisms.[unreadable] [unreadable] To derive and sustain functional neurons it is necessary to understand neuronal survival mechanisms. Using primary hippocampal neurons, we established that young neurons go through a restricted period when they die and neurotrophins are required for their survival. In vitro pharmacological studies suggested that activation of the neurotrophin receptor is only transient and that neuronal survival is achieved by the sustained action of L-type calcium channels and integrins. This work sets up a powerful in vitro assay to study survival signaling in neurons that complements our work in ES cells and CNS stem cells. We have now shown using antibodies and drugs that neurotrophins act through integrins to control the survival of hippocampal pyramidal neurons in vivo. To our knowledge, this is the first definition of the signaling steps controlling the survival of hippocampal neurons in vivo. This work establishes a powerful system to analyze the activity dependant mechanisms controlling neuronal survival in the most intensely studied region of the brain.

1. The role of foxa2 in the survival of dopamine neurons. [unreadable] [unreadable] Animals that have only one copy of the foxa2 gene show spontaneous loss of dopamine neurons with age. This mouse will likely be very widely used because although many mutations have been identified that contribute to Parkinsons disease and related disorders, it has been difficult to reproduce the specific loss of dopamine neurons in an animal model. This animal has the potential to teach us a number of important lessons: why some dopamine neurons are more at risk than others, why the disease is progressive and why dopamine neurons are sensitive to mutations in genes that are widely expressed? [unreadable] [unreadable] Much of what is known about the relationship between the various symptoms in Parkinsons patients and the underlying defects in the nigrostriatal system comes from lesion studies in the rat. The foxa2 heterozygous mouse allows us to study for the first time, in an animal model, motor and cognitive behaviors in the context of a progressive neurodegenerative process. We are currently focused on the cellular role of foxa2 in dopamine neurons. In brain slices, we have shown that the cellular location of the foxa2 gene is controlled by inputs that signal health or stress. This result provides a way measuring the signaling logic that controls the survival of dopamine neurons. [unreadable] [unreadable] 2. Supporting dopamine neuron survival in vivo.[unreadable] [unreadable] We have shown that a single intra-ventricular injection of Notch ligands alone or in combination with other angiogenic factors promotes widespread activation of the stem cell niche in the adult brain and rescues dopaminergic neurons in a model of PD. These data suggest vascular cytokines promote regenerative responses to brain injury. A major goal of our group is to set up in vitro assays that predict the activation of the stem cell compartment in vivo. In this project, we will use our growing understanding of survival signaling in the stem cell niche to rescue injured dopamine neurons in vivo. This approach may allow a more rapid transition to clinical application than cell replacement therapy. In the past year, we have shown that the major regenerative features found in the adult rat brain are also present in adult monkeys. This result encourages our continued belief in the clinical potential of this approach.

NIH Human Stem Cell Facility is a group at the Bethesda campus that is focused on characterizing the conditions for growing human embryonic stem cells. Because hES cells give access to early developmental events and can generate many terminally differentiated cell types, they may form the basis of a powerful new technology. Despite this promise, technical limitations may blunt the wide use of human ES cells. The most fundamental concern would be caused by genetic instability. The NIH-HSCF has routinely grown hES cells from August 2003. In the following year, most of the hES cells on the NIH registry were obtained, expanded in culture and analyzed by karyotype and FACS analysis. In the period to 7/2004, the central achievement of NIH-HSCF was to show that sub-clones of a hES cell line had a stable genome using a high-resolution genome scanning method. This result provides the most convincing evidence to date that at least one hES cell line can be grown for long periods without genetic change. In the reporting period to 7/05, the major focus was to develop general standards to assess the growth state of hES cell lines in an international collaboration organized through the International Stem Cell Consortium. The NIH-HSCF was one of the few sites contributing multiple hES cells from different providers grown under standard conditions. In the most recent reporting period to 7/06, the major achievements are (1) to establish conditions for homolgous recombination in hES cells, (2) to develop molecular tests for stress responses in the undifferentiated state and (3) to assist Dr. Thomas Cimato (NHLBI) in the isolation of human precursors for endothelial and blood cells.