1.1 Generating pluripotent cells from adult mouse and human sources. The current interest in human stem cells rests on the belief that they can be maintained in a stable state in cell culture over many cell divisions. The earliest cell fate decisions in the mammalian embryo generate the inner cell mass, trophoblast and primitive endoderm. A remarkable feature of these results is that major embryonic and extra-embryonic cell types of the developing embryo can be expanded as cell lines. The embryo is derived from pluripotent cells and there are now four pluripotent cell types that have been derived from the mouse developing embryonic stem (ES) cells, embryonic germ (EG) cells, epiblast stem (EpiS) cells. Work in LMB identified the EpiS cells and this cell is now studied in many laboratories including our own. The generation of pluripotent cells from adult sources has transformed the ethical and technical status of the stem cell field. These induced pluripotent stem (iPS) cells are attracting great interest as they provide a powerful substrate for the functional analysis of the human genome. In LMB, we have a specific interest in the pathways that control stem cell self-renewal and differentiation. Work from our lab shows that Fox genes specify dopamine neurons at the earliest stages of development and sustain these cells throughout life. In the last year, we have developed a series of reagents to further explore the functions of Fox genes. Our experiments are currently focused on how Fox genes controlling self-renewal and the dopaminergic fate choice in the mouse. 1.2 Generating new pluripotent cell lines. By generating pluripotent cells from adult tissues we propose to generate new research models. In collaboration with the laboratory of Michael Gottesman (NCI) we are generating iPS cells from the human mammary epithelium to generate new models of breast cancer. In addition, we are establishing contacts with physicians with an interest in neurodegenerative disease to establish long-term collaborations where we will analyze disease mechanisms using iPS cells as a central resource. 1.3 Epigenetics of pluripotent and adult stem cells. 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. The transcription factors Oct4 and Sox2 play a central role in pluripotent and neural stem cells. There are many laboratories, our own among them, analyzing gene expression in ES, EpiS and iPS. As they play a central role in the reprogramming that generates iPS cells, the role of Oct4 and Sox2 in adult cells is also of general interest. We have developed simple treatments with growth factors generate increased numbers of Sox2-positive cells and promote recovery in models of stroke and Parkinsons disease. To better understand the function of Oct4 and Sox2 in adult tissue, we have developed strains of mice where these transcription can be induced by small molecules. Our data suggest these mice will be valuable tools to define how these transcription factors promote epigenetic alterations in chromatin that promote reprogramming and regenerative responses. 2.1 Lineages in CNS stem cells 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. By real-time imaging we have defined the lineage patterns that link neural stem cells to their neuronal and glial progeny. These results show cell fate is specified at an earlier time than was previously thought providing a strong rationale for further analysis of these early steps in the lineage of neural stem cells. The precise control of later steps in neuronal and glial differentiation is also evident in these real-time imaging data. To gain a better insight into these later stages we have developed the tools to measure and analyze calcium responses in neural stem cells lineages. This assay takes advantage of our investment in the technology required to monitor the behavior of neural stem cells constantly as they generate functional progeny. 2.2 Energy production in neural stem cells 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. We continue to use neural stem cells to explore the important implications of this finding in cancer and ischemia. 2.3 An in vitro assay that predicts stem cell activation in vivo. We have shown that insulin and Notch ligands act in a co-operative manner to control stem cell survival in vitro and in the past year we have published data suggesting that this pathway can be readily and beneficially activated in vivo. An earlier report from our group showed that Notch ligands activate the major growth pathway normally studied downstream of receptor tyrosine kinases, including the insulin receptor. To extend our understanding of the link stem cell survival in vivo and in vitro we have demonstrated the expression and functional role of the Tie2 receptor in neural stem cells. This angiopoietin receptor regulates hematopoiesis and angiogenesis. We have demonstrated that angiopoietin2 (Ang2) has powerful effects on adult neural stem cells in vivo. We have now demonstrated that Ang2 and ligands for the Notch receptor co-operate to promote the in vitro growth of adult neural stem cells. The definition of an in vitro assay for this receptor is an important step in our continuing search for a comprehensive understanding of the signals in the stem cell niche. 3.1 Mechanisms regulating neuronal survival. During the neonatal period, as many as a half of the neurons in the brain are eliminated. This naturally occurring neuronal death is thought to be a component of a negative selection process that plays an important role in refining neuro-circuitry. The goal of this project is to understand the selection mechanism that shapes the neuro-circuitry in the brain. Using an in vitro system, we found that survival signaling initiated by neurotrophins requires calcium influx through L-type channels and integrin receptors. This test demonstrates that neurotrophins increase the number of spontaneously active neurons and increases the frequency of neuronal activity. In the past year, we have demonstrated that this mechanism also regulates neuronal survival in vivo.
