ABSTRACT
Progenitors were discovered in the corpus striatum several years ago, but little is known about their proliferation and differentiation. The aim of this study was to analyze embryonic progenitor cells from the corpus striatum using a bioassay with trophic stimulation. Primary cells obtained from brains of rat embryos at E13-14 were dissected from striatum niches and cultured in stem cell media. These floating dispersed cells clumped together to forming floating bodies like irregular spheres (spheroids), which were placed in type I collagen gel and cultured under basal conditions or with the addition of NGF, NT-3, or NTN. Optimum growth of neurites was obtained, and after 24 and 48 h, they were measured for number and length. The expression of proliferation markers such as PCNA and Ki67, and of neural progenitor markers such as GFAP, nestin, vimentin, O4, A2B5, Pax6, S100, TubIII, and NeuN, was then analyzed. The initial behavior in cell cultures showed distinguishable spheroids that, when placed in 3D gels and with trophic support, generated neurites. A similar effect was observed in glial cell outgrowth from the spheroids. Our assay showed high reproducibility, short culture time, and high resolution for tracing neuron-neurite outgrowth or visualizing glial outgrowth in a few hours.
Subject(s)
Biological Assay/methods , Central Nervous System Agents/pharmacology , Embryonic Stem Cells/drug effects , Neural Stem Cells/physiology , Neurogenesis , Neurons/physiology , Animals , Biological Assay/instrumentation , Cell Culture Techniques , Cell Enlargement , Cells, Cultured , Collagen , Corpus Striatum/cytology , Corpus Striatum/drug effects , Corpus Striatum/embryology , Corpus Striatum/physiology , Embryonic Stem Cells/cytology , Embryonic Stem Cells/physiology , Gels , Nerve Growth Factor/pharmacology , Neural Stem Cells/cytology , Neural Stem Cells/drug effects , Neurites/drug effects , Neurites/physiology , Neurons/cytology , Neurons/drug effects , Neurotrophin 3/pharmacology , Neurturin/pharmacology , Rats, Sprague-DawleyABSTRACT
The in vitro differentiation of embryonic stem cells into glia has received relatively limited attention to date when compared with the interest in the generation of neurons. We are interested in a particular glial phenotype, the aldynoglia, and their differentiation from multipotential neural precursors (MNP), since this type of glia can promote neuronal regeneration. We constructed cDNA libraries from cultures of purified olfactory ensheathing cells (OEC), an aldynoglia cell type, and MNP to perform subtractive hybridization. As a result, we isolated four genes from the OEC: one tenascin C (Tn-C) isoform, Insulin-like growth factor binding protein 5 (Igfbp-5), cytochrome oxidase subunit I (COX1) and a phosphodiesterase for cyclic nucleotides (CNPase). With the exception of CNPase, these genes are expressed more strongly in the OEC than in the MNP and moreover, the expression of all four is induced when MNP were exposed to OEC conditioned media. The data suggest a role for these genes in MNP differentiation, and their products appear to represent characteristic proteins of the aldynoglia phenotype.
Subject(s)
Neuroglia/metabolism , Neurons/metabolism , Stem Cells/metabolism , 2',3'-Cyclic-Nucleotide Phosphodiesterases/genetics , 2',3'-Cyclic-Nucleotide Phosphodiesterases/metabolism , Animals , Cell Differentiation/genetics , Cell Differentiation/physiology , Cells, Cultured , Culture Media, Conditioned , Cyclooxygenase 1/genetics , Cyclooxygenase 1/metabolism , Gene Expression , Gene Library , Genetic Techniques , Glial Fibrillary Acidic Protein/metabolism , Immunohistochemistry , Insulin-Like Growth Factor Binding Protein 5/genetics , Insulin-Like Growth Factor Binding Protein 5/metabolism , Membrane Proteins/genetics , Membrane Proteins/metabolism , Nerve Tissue Proteins , Protein Isoforms/genetics , Protein Isoforms/metabolism , RNA, Messenger/metabolism , Rats , Rats, Wistar , Receptors, Growth Factor , Receptors, Nerve Growth Factor/metabolism , Tenascin/genetics , Tenascin/metabolismABSTRACT
The mammalian central nervous system contains well-defined regions of plasticity in which cells of the aldynoglia phenotype promote neuronal growth and regeneration. Only now are the factors that regulate the production of new cells from multipotential neural precursors (MNP) starting to be identified. We are interested in understanding how differentiation towards the aldynoglia phenotype is controlled, and to study these events we have induced the differentiation of embryonic MNP towards this phenotype in vitro. Accordingly, we have used microarrays to analyze gene expression in three different cell populations: olfactory bulb ensheathing cells (EC), a prototypic aldynoglia cell type; undifferentiated MNP; and MNP differentiated in vitro for 24 hr in EC-conditioned media. The expression profiles identified support the idea that the EC are more closely related to Schwann cells and astrocytes than to oligodendrocytes. Following MNP differentiation, more strongly expressed genes define a neuroglial cell phenotype. RT-PCR confirms that S100a6, Mtmr2, and Col5a were highly expressed by EC, whereas Pou3f3 were more strongly expressed in MNP than in EC, and SafB1 and Mash1 expression were induced in MNP by EC-conditioned media. The profile of gene expression after differentiation suggests that Wnt signaling may be inactivated during this process, while activation of the BMP pathway may be elicited through the BMPr1A. These results provide us with a starting point to study the genes involved in the induction of aldynoglia differentiation from MNP.
