Efficient generation of midbrain and hindbrain neurons from mouse embryonic stem cells

Abstract

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.

Electrophysiological evaluation of engrafted stem cell-derived neurons

“…These neurons can be tailored, in cell culture, to display striking morphological and immunophenotypic similarities to cells found in the adult brain, and even to cells lost during the course of brain disease [1][2][3][4][5][6][7][8][9][10] . Because stem cell-derived neurons are born in a culture dish, without the environmental cues of the developing brain, it has become a major quest to determine whether these cells are actually functional, that is, capable of interacting with established neural circuitry.…”

Cell Therapeutics in Parkinson's Disease

“…So far, the most promising results have been obtained using embryonic stem (ES) cells [52][53][54][55][56][57][58][59][60][61][62] or therapeutically cloned ES cells [63]. The examples shown (Fig.…”

Efficient and stable Sendai virus-mediated gene transfer into primate embryonic stem cells with pluripotency preserved

“…31 Day-4 embryoid bodies were plated onto tissue culture dishes and nestin-positive cells were selected in DMEM/F12 medium supplemented with 5 mg/ml of insulin (Sigma), 50 mg/ml of transferrin (Sigma), 30 nM selenium chloride (Sigma), and 5 mg/ml of fibronectin (Sigma) for 5 days. Cells were then trypsinized and plated in polyornithine-coated dishes (15 mg/ml) and expanded in N2 medium 32 supplemented with 1 mg/ml of laminin (Sigma) and 10 mg/ml of basic fibroblast growth factor (bFGF; Roche, Basel, Switzerland) for 6 days.…”