Although the source of embryonic stem (ES) cells presents ethical concerns, their use may lead to many clinical benefits if differentiated cell types can be derived from them and used to assemble functional organs. In pancreas, insulin is produced and secreted by specialized structures, islets of Langerhans. Diabetes, which affects 16 million people in the United States, results from abnormal function of pancreatic islets. We have generated cells expressing insulin and other pancreatic endocrine hormones from mouse ES cells. The cells self-assemble to form three-dimensional clusters similar in topology to normal pancreatic islets where pancreatic cell types are in close association with neurons. Glucose triggers insulin release from these cell clusters by mechanisms similar to those employed in vivo. When injected into diabetic mice, the insulin-producing cells undergo rapid vascularization and maintain a clustered, islet-like organization.

Parkinson's disease is a widespread condition caused by the loss of midbrain neurons that synthesize the neurotransmitter dopamine. Cells derived from the fetal midbrain can modify the course of the disease, but they are an inadequate source of dopamine-synthesizing neurons because their ability to generate these neurons is unstable. In contrast, embryonic stem (ES) cells proliferate extensively and can generate dopamine neurons. If ES cells are to become the basis for cell therapies, we must develop methods of enriching for the cell of interest and demonstrate that these cells show functions that will assist in treating the disease. Here we show that a highly enriched population of midbrain neural stem cells can be derived from mouse ES cells. The dopamine neurons generated by these stem cells show electrophysiological and behavioural properties expected of neurons from the midbrain. Our results encourage the use of ES cells in cell-replacement therapy for Parkinson's disease.

Embryonic stem (ES) cells are clonal cell lines derived from the inner cell mass of the developing blastocyst that can proliferate extensively in vitro and are capable of adopting all the cell fates in a developing embryo. Clinical interest in the use of ES cells has been stimulated by studies showing that isolated human cells with ES properties from the inner cell mass or developing germ cells can provide a source of somatic precursors. Previous studies have defined in vitro conditions for promoting the development of specific somatic fates, specifically, hematopoietic, mesodermal, and neurectodermal. In this study, we present a method for obtaining dopaminergic (DA) and serotonergic neurons in high yield from mouse ES cells in vitro. Furthermore, we demonstrate that the ES cells can be obtained in unlimited numbers and that these neuron types are generated efficiently. We generated CNS progenitor populations from ES cells, expanded these cells and promoted their differentiation into dopaminergic and serotonergic neurons in the presence of mitogen and specific signaling molecules. The differentiation and maturation of neuronal cells was completed after mitogen withdrawal from the growth medium. This experimental system provides a powerful tool for analyzing the molecular mechanisms controlling the functions of these neurons in vitro and in vivo, and potentially for understanding and treating neurodegenerative and psychiatric diseases.

Identifying the signals that regulate stem cell differentiation is fundamental to understanding cellular diversity in the brain. In this paper we identify factors that act in an instructive fashion to direct the differentiation of multipotential stem cells derived from the embryonic central nervous system (CNS). CNS stem cell clones differentiate to multiple fates: neurons, astrocytes, and oligodendrocytes. The differentiation of cells in a clone is influenced by extracellular signals: Platelet-derived growth factor (PDGF-AA, -AB, and -BB) supports neuronal differentiation. In contrast, ciliary neurotrophic factor and thyroid hormone T3 act instructively on stem cells to generate clones of astrocytes and oligodendrocytes, respectively. Adult stem cells had remarkably similar responses to these growth factors. These results support a simple model in which transient exposure to extrinsic factors acting through known pathways initiates fate decisions by muhipotential CNS stem cells.