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.

Recent studies have provided important insight into the homeoprotein LIM homeobox transcription factor 1␣ (Lmx1a) and its role in the commitment of cells to a midbrain dopamine (mDA) fate in the developing mouse. We show here that Lmx1a also plays a pivotal role in the mDA differentiation of human embryonic stem (hES) cells. Thus, as indicated by small interfering RNA experiments, the transient early expression of Lmx1a is necessary for the coordinated expression of all other dopamine (DA)-specific phenotypic traits as hES cells move from multipotent human neural progenitor cells (hNPs) to more restricted precursor cells in vitro. Moreover, only Lmx1a-specified hNPs have the potential to differentiate into bona fide mDA neurons after transplantation into the 6-hydroxydopamine-treated rat striatum. In contrast, cortical human neuronal precursor cells (HNPCs) and mouse subventricular zone cells do not express Lmx1a or become mDA neurons even when placed in an environment that fosters their DA differentiation in vitro or in vivo. These findings suggest that Lmx1a may be critical to the development of mDA neurons from hES cells and that, along with other key early DA markers (i.e., Aldh1a1), may prove to be extremely useful for the selection of appropriately staged and suitably mDA-specified hES cells for cell replacement in Parkinson's disease. STEM

The development of cell replacement therapies for the treatment of neurodegenerative disorders such as Parkinson's disease (PD) may depend upon the successful differentiation of human neural stem/progenitor cells into dopamine (DA) neurons. We show here that primary human neural progenitors (HNPs) can be expanded and maintained in culture both as neurospheres (NSPs) and attached monolayers where they develop into neurons and glia. When transplanted into the 6-hydroxydopamine-lesioned rat striatum, undifferentiated NSPs survive longer (60% graft survival at 8-16 weeks vs. 30% graft survival at 8-13 weeks) and migrate farther than their attached counterparts. While both NSP and attached cells continue to express neuronal traits after transplantation, the spontaneous expression of differentiated transmitter-related traits is not observed in either cell type. However, following predifferentiation in culture using a previously described cocktail of reagents, approximately 25% of HNPs can permanently express the DA enzyme tyrosine hydroxylase (TH), even following replating and removal of the DA differentiation cocktail. When these predifferentiated HNPs are transplanted into the brain, however, TH staining is not observed, either because expression is lost or TH-expressing cells preferentially die. Consistent with the latter view is a decrease in total cell survival and migration, and an enhanced glial response in these grafts. In contrast, we found that the overall survival of HNPs is improved when cells engraft near blood vessels or CSF compartments or when they are placed into an intact unlesioned brain, suggesting that there are factors, as yet unidentified, that can better support the development of engrafted HNPs.

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.