Our ability to use human embryonic stem (hES) cells in cell replacement therapy for Parkinson's disease depends on the discovery of ways to simply and reliably differentiate a dopaminergic (DA) phenotype in these cells. Although several protocols exist for the differentiation of DA traits in hES, they involve the prolonged use of complex media with undefined components, cell conditioned media and/or co-culture with various cells, usually of animal origin. In this study, several well-characterized (H9, BG01) and several new uncharacterized (HUES7, HUES8) hES cell lines were studied for their capacity to differentiate into DA neurons in culture using a novel rapid protocol which uses only chemically-defined human-derived media additives and substrata. Within 3 weeks, cells from all 4 cell lines progressed from the undifferentiated state to β-tubulin III positive cells expressing DA markers in vitro. Moreover, transplantation of these cells into the striata of 6-hydroxydopaminetreated rats at the neuronal progenitor stage resulted in the appearance of differentiated DA traits in vivo 2-3 weeks later.

A DNA fragment of about 13 kb containing the human tyrosine hydroxylase (TH) promoter was previously isolated from a genomic DNA library and sequenced. The 11 kb from the transcription start of the human TH promoter was successively joined to the green fluorescent protein (GFP) to generate a transgenic mouse model. High levels of GFP expression could be observed in TH-positive cells of the Substantia nigra of embryonic and adult mice. Intriguingly, the sequence of the human TH promoter showed a low degree of homology with the mouse and rat TH promoters. In fact, comparative analysis of the sequences of human, rat, and mouse TH promoters revealed only five small regions of high homology. These five evolutionarily conserved regions were numbered in numeric progression from the 5' end of human TH promoter. In the present study, a panel of minimal human TH promoters was generated to analyze the transcriptional activity and specificity of gene expression conferred by the five conserved regions (CRs). The series of constructs was termed 250 bp and contained the first -194 bp of the human TH promoter immediately upstream of the transcription start, the first 35 bp the human TH messenger RNA leader, plus one or more of the five CRs. All the constructs were assembled in a self-inactivating form of the latest series of lentiviral vector system based on the human immunodeficiency virus type 1 (HIV-1). Lentiviral-mediated gene transfer was highly efficient for the in vitro transduction of human neuronal progenitor cells (hNPCs). Since a subset of hNPCs express TH following in vitro treatment with a mixture of differentiating agents, it was possible to assess specificity of expression for all the minimal human TH promoters. Overall, the successive addition of the five conserved regions produced a greater degree of specificity in induced TH-positive hNPCs, in particular after the addition of CRI (-8,917, -8,876). However, the human TH minimal promoters did not show any specificity for TH-positive differentiated mouse primary striatal and S. nigra cells, indicating a difference of TH gene regulation between the human and mouse systems. The human TH minimal promoters may provide the opportunity for the selection of TH-positive human embryonic and adult stem cells for brain transplantation experiments in animal models for Parkinson's disease.

Studies on rodent bone marrow stromal cells (MSCs) have revealed a capacity, for at least a portion of cells, to express neuron-like traits after differentiation in culture. Little, however, is known about the ability of human MSCs in this regard. We show here that incubation with certain differentiation cocktails, particularly those that include reagents that increase cellular cAMP levels, produces a rapid (1-4 h) and transient (24-48 h) transformation of nearly all hMSCs into neuron-like cells displaying a complex network of processes using phase or scanning electron microscopic optics. In addition, differentiated human (h) MSCs express increased quantities of neuron-[β-tubulin III, neurofilament (NF), neuronal-specific enolase (NSE)] and glial-[glial fibrillary acidic protein (GFAP)] specific proteins and mRNAs, which are also expressed in low levels in undifferentiated MSCs. In contrast, the mesenchymal marker, fibronectin, which is highly expressed in the undifferentiated state, is reduced following differentiation. These biochemical changes, but not the acquisition of a neuronlike appearance, are partially inhibited by incubation of hMSCs with protein (cycloheximide) and mRNA (actinomycin D) synthesis inhibitors with differentiating reagents. Only incubation with 100 ng/ml colchicine, which disrupts the microtubular cytoskeleton, prevents the conversion of hMSCs into neuron-like cells. These results demonstrate that hMSCs acquire the morphological appearance and the biochemical makeup typical of neurons by independently regulated mechanisms.