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Patent - TH induction in NSC

The therapeutic potential of fetal VM neuron transplantation in patients with Parkinson¡¦s disease (PD) has been demonstrated in the clinic. Immature dopamine (DA) neurons originating from aborted human fetuses, implanted in the brains of patients with end stage PD, have successfully restored function (Lindvall, O. et al, Progress in Brain Research 2000, 127: 299-320.). Despite the positive proof-of-principle, neural transplantation has not become widely applied, primarily due to the limited availability and ethical concerns of the use of fetal VM tissue. In addition, poor survival and heterogeneous and poorly characterized primary cell materials have made the analysis of experimental data difficult, and results and complications have varied greatly between different transplantation centers (Brundin, P. et al, Brain, 2000, 123: 1380-90.3, Freed, C. R. et al, New England Journal of Medicine 2001, 344: 710-9. Piccini, P. et al, Nature Neuroscience 1999, 2: 1137-40). Therefore, there is currently an extensive search for alternative sources of transplantable cells capable of reproducing the positive results seen with primary fetal VM while being better characterized and less heterogeneous.

Multipotent stem/progenitor cells derived from human first trimester forebrain can be expanded as free-floating aggregates. In vitro, cells can differentiate to neurons, astrocytes and oligodendrocytes. Using previously described protocols for in vitro differentiation, a large fraction of the neurons become ’×-aminobutyric acid (GABA)-immunoreactive, whereas only rare tyrosine hydroxylase (TH) expressing neurons are found. The present report describes conditions under which 4-10 % of the cells in the culture become immunoreactive (ir) for TH within 24 h. Factors including acidic Fibroblast Growth Factor (aFGF) in combination with agents that increase intracellular cyclic AMP and activate protein kinase C (PKC) in addition to a substrate that promotes neuronal differentiation appear critical for efficient TH induction. The cells remain THir after trypsinization and replating and in the absence of inducing factors. Consistent with a dopaminergic phenotype, mRNAs encoding aromatic acid decarboxylase (AADC) but not dopamine-’Ò-hydroxylase (DBH) was detected by Q-PCR. Although, differentiated human neurons were demonstrated 10 weeks after implantation into the striatum of adult rats with unilateral dopaminergic lesions, only few THir neurons could be observed.

The method involves plating the cells on a substrate known to promote neuronal differentiation, preserving cell-cell interaction, and exposing the cells to a cocktail consisting of trophic factors and agents that affect intracellular signaling. The ability to induce TH expression is not developmentally dependent but appears to be a general phenomenon of human neurosphere cultures established from first trimester forebrain.

In the present project, we have shown that, under certain defined conditions, human forebrain neurosphere cultures can generate a significant number of neurons expressing dopaminergic neuronal markers in a similar fraction of cells as compared to fetal VM cultures (4-10 %). Based on these findings we have developed a method for the in vitro production of a population of neural cells wherein a significant percentage of those cells express tyrosine hydroxylase (TH). The relative importance of several parameters including substrate, growth factors, metabolic agents, and plating density and dissociation has been extensively studied to generate an optimized protocol. This protocol comprises expanding neural progenitor cells using growth factors and for by plating the cells on a substrate, introducing a defined culture medium containing one or more growth factors belonging to the FGF family, a molecule which gives to an increase in intracellular cyclic AMP (CAMP), and an agent stimulating or capable of activating protein kinase C (PKC). The method provides TH expressing cells in significant numbers, similar to that observed in fetal ventral mesencephalon cultures (5-20%). It also provides cells in which the expression of TH is stable after removal of the induction medium. The method provides a means for generating large numbers of TH expressing neural cells for neurotransplantation into a host in the treatment of CNS disorders, for example, neurodegenerative disease, neurological trauma, stroke, other neurodegenerative diseases, neurological trauma, stroke, and other diseases of the nervous system involving loss of neural cells, particularly Parkinson's disease. However, additional experimental work is required that will improve the maturation and survival of the TH expressing neurons after implantation in vivo before these cultures can become useful in a cell replacement therapy for PD. Additionally, the TH expressing cells may be used for drug screening or gene expression analysis.

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