ABSTRACT
During neural development caudalization and dorsoventral patterning of the neural tube is directed by several inductive factors including retinoic acid, sonic hedgehog (Shh), bone morphogenetic proteins (BMPs), and Wnt signaling. The purpose of the current study was to investigate whether dorsal interneurons specific for the spinal cord can be generated from mouse embryonic stem (ES) cells using known inductive signals. Here we show that specific combination of developmental signaling molecules including all trans-retinoic acid, Shh, bone morphogenetic protein 2 (BMP2), and Wnt3A can direct differentiation of ES cells into dorsal interneurons possessing appropriate neuronal markers, synaptic proteins and functional neurotransmitter machineries. We introduce a concept that Wnt3A morphogenic action relies on crosstalk with both Shh and BMP2 signaling pathways.
Subject(s)
Cell Differentiation/physiology , Embryo, Mammalian/cytology , Ganglia, Spinal/cytology , Interneurons/cytology , Stem Cells/cytology , Animals , Cell Line , Eye Proteins/metabolism , Gene Expression Regulation, Developmental/physiology , Immunoglobulins/biosynthesis , Interneurons/physiology , Membrane Glycoproteins/biosynthesis , Membrane Glycoproteins/metabolism , Mice , Mitogen-Activated Protein Kinase 1/biosynthesis , Neurites/metabolism , Neurons/chemistry , Neurons/metabolism , Neurotransmitter Agents/biosynthesis , Proteins/physiology , Stem Cells/metabolism , Stem Cells/physiology , Synapsins/biosynthesis , Wnt Proteins , Wnt3 Protein , Wnt3A ProteinABSTRACT
Existing protocols show a variety in the percentage of neurons that can be generated from mouse embryonic stem (ES) cells. In the current study, we compared effects of various differentiating conditions, including gelatin and poly-l-ornithine/fibronectin coatings, and NGF and 17beta-estradiol treatments on the total yield of neurons, as well as, neurite growth and branching. Here, we show that combination of fibronectin coating with 17beta-estradiol increased number of generated neurons over 50%. Poly-l-ornithine/fibronectin increased the percent of neurons in all cultures, suggesting its direct influence on neurogenesis. Addition of 17beta-estradiol reduced mean neurite length in culture, but significantly increased branching. Our results indicate a substrate-dependent regulation of estrogen-induced ES cells differentiation into neuronal cells.
