Lars M. Bjorklund

Embryonic stem cells develop into functional dopaminergic neurons after transplantation in a Parkinson rat model

Although implantation of fetal dopamine (DA) neurons can reduce parkinsonism in patients, current methods are rudimentary, and a reliable donor cell source is lacking. We show that transplanting low doses of undifferentiated mouse embryonic stem (ES) cells into the rat striatum results in a proliferation of ES cells into fully differentiated DA neurons. ES cell-derived DA neurons caused gradual and sustained behavioral restoration of DA-mediated motor asymmetry. Behavioral recovery paralleled in vivo positron emission tomography and functional magnetic resonance imaging data demonstrating DA-mediated hemodynamic changes in the striatum and associated brain circuitry. These results demonstrate that transplanted ES cells can develop spontaneously into DA neurons. Such DA neurons can restore cerebral function and behavior in an animal model of Parkinsons disease.

Genetic engineering of mouse embryonic stem cells by Nurr1 enhances differentiation and maturation into dopaminergic neurons

Nurr1 is a transcription factor critical for the development of midbrain dopaminergic (DA) neurons. This study modified mouse embryonic stem (ES) cells to constitutively express Nurr1 under the elongation factor‐1α promoter. The Nurr1‐expression in ES cells lead to up‐regulation of all DA neuronal markers tested, resulting in about a 4‐ to 5‐fold increase in the proportion of DA neurons. In contrast, other neuronal and glial markers were not significantly changed by Nurr1 expression. It was also observed that there was an additional 4‐fold increase in the number of DA neurons in Nurr1‐expressing clones following treatment with Shh, FGF8 and ascorbic acid. Several lines of evidence suggest that these neurons may represent midbrain DA neuronal phenotypes; firstly, they coexpress midbrain DA markers such as aromatic l‐amino acid decarboxylase, calretinin, and dopamine transporter, in addition to tyrosine hydroxylase and secondly, they do not coexpress other neurotransmitters such as GABA or serotonin. Finally, consistent with an increased number of DA neurons, the Nurr1 transduction enhanced the ability of these neurons to produce and release DA in response to membrane depolarization. This study demonstrates an efficient genetic manipulation of ES cells that facilitates differentiation to midbrain DA neurons, and it will serve as a framework of genetic engineering of ES cells by key transcription factor to regulate their cell fate.

Proteasome inhibition causes nigral degeneration with inclusion bodies in rats

Structural and functional defects in 26/20S proteasomes occur in the substantia nigra pars compacta and may underlie protein accumulation, Lewy body formation and dopaminergic neuronal death in Parkinsons disease. We therefore determined the pathogenicity of proteasomal impairment following stereotaxic unilateral infusion of lactacystin, a selective proteasome inhibitor, into the substantia nigra pars compacta of rats. These animals became progressively bradykinetic, adopted a stooped posture and displayed contralateral head tilting. Administration of apomorphine to lactacystin-treated rats reversed behavioral abnormalities and induced contralateral rotations. Lactacystin caused dose-dependent degeneration of dopaminergic cell bodies and processes with the cytoplasmic accumulation and aggregation of &agr;-synuclein to form inclusion bodies. These findings support the notion that failure of the ubiquitin-proteasome system to degrade and clear unwanted proteins is an important etiopathogenic factor in Parkinsons disease.

Analysis of Different Promoter Systems for Efficient Transgene Expression in Mouse Embryonic Stem Cell Lines

Mouse embryonic stem (ES) cells are derived from the inner cell mass of the preimplantation embryo and have the developmental capacity to generate all cell types of the body. Combined with efficient genetic manipulation and in vitro differentiation procedures, ES cells are a useful system for the molecular analysis of developmental pathways. We analyzed and compared the transcriptional activities of a cellular polypeptide chain elongation factor 1 alpha (EF), a cellular‐virus hybrid (cytomegalo‐virus [CMV] immediate early enhancer fused to chicken β‐actin [CBA]), and a viral CMV promoter system in two ES cell lines. When transiently transfected, the EF and CBA promoters robustly drove reporter gene expression, while the CMV promoter was inactive. We also demonstrated that the EF and CBA promoters effectively drove gene expression in different stages of cell development: naïve ES cells, embryoid bodies (EBs), and neuronal precursor cells. In contrast, the CMV promoter did not have transcriptional activity in either ES cells or EB but had significant activity once ES cells differentiated into neuronal precursors. Our data show that individual promoters have different abilities to express reporter gene expression in the ES and other cell types tested.

Toward Full Restoration of Synaptic and Terminal Function of the Dopaminergic System in Parkinson's Disease by Stem Cells

New therapeutic nonpharmacological methodology in Parkinsons disease (PD) involves cell and synaptic renewal or replacement to restore function of neuronal systems, including the dopaminergic (DA) system. Using fetal DA cell therapy in PD patients and laboratory models, it has been demonstrated that functional motor deficits associated with parkinsonism can be reduced. Similar results have been observed in animal models with stem cell‐derived DA neurons. Evidence obtained from transplanted PD patients further shows that the underlying disease process does not destroy transplanted fetal DA cells, although degeneration of the host nigrostriatal system continues. The optimal DA cell regeneration system would reconstitute a normal neuronal network capable of restoring feedback‐controlled release of DA in the nigrostriatal system. The success of cell therapy for PD is limited by access to preparation and development of highly specialized dopaminergic neurons found in the A9 and A10 region of the substantia nigra pars compacta as well as the technical and surgical steps associated with the transplantation procedure. Recent laboratory work has focused on using stem cells as a starting point for deriving the optimal DA cells to restore the nigrostriatal system. Ultimately, understanding the cell biological principles necessary for generating functional DA neurons can provide many new avenues for better treatment of patients with PD. Ann Neurol 2003;53 (suppl 3):S135–S148

Context-dependent neuronal differentiation and germ layer induction of Smad4−/− and Cripto−/− embryonic stem cells

Activation of transforming growth factor-beta (TGF-beta) receptors typically elicits mesodermal development, whereas inhibition of this pathway induces neural fates. In vitro differentiated mouse embryonic stem (ES) cells with deletion of the TGF-beta pathway-related factors Smad4 or Cripto exhibited increased numbers of neurons. Cripto-/- ES cells developed into neuroecto-/epidermal cell types, while Smad4-/- cells also displayed mesodermal differentiation. ES cell differentiation into catecholaminergic neurons showed that these ES cells retained their ability to develop into dopaminergic and serotonergic neurons with typical expression patterns of midbrain and hindbrain genes. In vivo, transplanted ES cells to the mouse striatum became small neuronal grafts, or large grafts with cell types from all germ layers independent of their ES cell genotype. This demonstrates that Smad4-/- and Cripto-/- ES cells favor a neural fate in vitro, but also express the mesodermal phenotype, implying that deletion of either Smad4 or Cripto is not sufficient to block nonneuronal tissue formation.

Parkinson's disease: interpretations of transplantation study are erroneous

The recent publication by Freed and colleagues1 of the first double-blind trial of fetal transplantation for Parkinsons disease (PD) has generated controversy in both the scientific and popular press. The results have been widely interpreted as disappointing, but we believe that many of the criticisms are misplaced. Here we present a meta-analysis of several published studies, from which we conclude that the results are in fact quite promising. We also suggest a plausible explanation for the side effects that were seen in the Freed study.

Enhanced axonal growth from fetal human bcl-2 transgenic mouse dopamine neurons transplanted to the adult rat striatum

Embryonic neurons transplanted to the adult CNS extend axons only for a developmentally defined period. There are certain intercellular factors that control the axonal extension, one of which may be the expression of the bcl-2 protein. In this study, rats with complete striatal dopamine fiber denervation received embryonic day 14 mouse ventral mesencephalon cells overexpressing human bcl-2 or control wild-type ventral mesencephalon cells. All rats were treated with cyclosporine to prevent rejection and the surviving grafts were analyzed for cell survival and outgrowth of dopaminergic fibers. The results demonstrate that bcl-2 overexpression does not enhance neuronal graft survival. However, the bcl-2 overexpressing neurons had a higher number of dopaminergic fibers that grew longer distances. These results show that overexpression of bcl-2 can result in longer distance axonal growth of transplanted fetal dopaminergic neurons and that genetic modification of embryonic donor cells may enhance their ability to reinnervate a neuronal target territory.