ABSTRACT
The aim of the research was to establish cultured population of nerve cells reached by cholinergic neurons and their determinative precursors. The most effective combination of neuroinductors which stimulated cholinergic cells differentiation from the nerve stem cells was retinoic acid and acetylcholine. During the period of culturing the amount of ChAT+ cells reliably increased from 5.3 +/- 2.9% to 21.1 +/- 6.2%. At the same time in the control samples their concentration was 9.1 +/- 4.8% of total cell count. Enrichment of cell population by cholinergic neurons and their determinative precursors correlated with increasing of AChE-activity level. So, addition of retinoic acid and acetylcholine stimulate both neurogenesis and cholinogenesis in the culture of human fetal nerve cells.
Subject(s)
Acetylcholinesterase/metabolism , Brain/embryology , Cholinergic Neurons/enzymology , Fetal Stem Cells/enzymology , Neurogenesis , Brain/cytology , Cell Count , Cells, Cultured , Cholinergic Neurons/cytology , Fetal Stem Cells/cytology , HumansABSTRACT
The expansion of Nest-positive nerve stem cells fraction in the culture of human fetal brain was investigated in this work. Our results demonstrated the valuable increase of the percentage of Nest-positive cells from 44.1 to 62.5% of cell population at the response to the presence of EGF and bFGF and using the heat-inactivated serum in the culture medium. Further differentiation in culture tended to decrease of Nest-positive cell amount. Stimulation of the neuroinduction with retinoic acid resulted in decrease of the percentage of these cells in the population to 14.1%. This data allowed obtaining the nerve cell population more effective for using as a graft material in cell therapy.
Subject(s)
Brain/cytology , Neural Stem Cells/cytology , Brain/drug effects , Brain/embryology , Cell Differentiation/drug effects , Cells, Cultured , Culture Media , Fetus , Humans , Neural Stem Cells/drug effects , Tretinoin/pharmacologyABSTRACT
The aim of our experiments was to determine the dofaminergic differentiation potential of human embryonic nerve tissue in culture to obtain cell material for neurotransplantation enriched with determinative dofaminergic cells precursors. During the period of culturing their amount in the experimental samples reliable increased from 5.6 +/- 4.3% dofaminergic neurons cells in the field of vision to 23.5 +/- 2.6%. At the same time in the control samples their concentration was 8.1 +/- 6.5% cells in the field of vision. Dofaminergic differentiation was confirmed by expression analyses of genes associated with this type of neuronal differentiation. We have demonstrated that during the period of culture expression of early transcription factors--Nurr1 and Lmx1b disappears while TH expression level increases. So, using specific culture condition the cell material for neurotransplantation enriched by dofaminergic neuron precursors can be obtained.
Subject(s)
Cell Differentiation , Dopamine/metabolism , Mesencephalon/cytology , Mesencephalon/embryology , Neurons/cytology , Aborted Fetus/cytology , Cell Culture Techniques , Cells, Cultured , Gene Expression Regulation, Developmental , Gestational Age , Homeodomain Proteins/genetics , Humans , LIM-Homeodomain Proteins , Mesencephalon/metabolism , Neurons/metabolism , Neurons/transplantation , Nuclear Receptor Subfamily 4, Group A, Member 2/genetics , Stem Cell Transplantation , Transcription Factors/genetics , Tyrosine 3-Monooxygenase/geneticsABSTRACT
We have examined the mitogenic and differentiation potential and remyelination properties of human embryonic nerve cells in culture. After 1 month of cultivation without differentiation agents CNP-positive cells (the mitotically-active precursors of oligodendrocytes) were expanded at 3,6 times. At the same time the amount of GalC-positive cells (mature oligodendrocytes) remained low. So, the remyelination properties of embryonic nerve cells can be explained by high concentration of oligodendrocytes precursors. Cell population after cultivation maintained the increased remyelination potential by increasing the number of CNP-positive cells that was confirmed by using experimental demyelination.
