Transplantation of neural stem cells co-transfected with Nurr1 and Brn4 for treatment of Parkinsonian rats

Neural stem cells (NSCs) tranplantation has great potential for the treatment of neurodegenerative disease such as Parkinson's disease (PD). However, the usage of NSCs is limited because the differentiation of NSCs into specific dopaminergic neurons has proven difficult. We have recently demonstrated that transgenic expression of Nurr1 could induce the differentiation of NSCs into tyrosine hydroxylase (TH) immunoreactive dopaminergic neurons, and forced co-expression of Nurr1 with Brn4 caused a dramatic increase in morphological and phenotypical maturity of these neurons. In this study, we investigated the effect of transplanted NSCs in PD model rats. The results showed that overexpression of Nurr1 promoted NSCs to differentiate into dopaminergic neurons in vivo, increased the level of dopamine (DA) neurotransmitter in the striatum, resulting in behavioral improvement of PD rats. Importantly, co-expression of Nurr1 and Brn4 in NSCs significantly increased the maturity and viability of dopaminergic neurons, further raised the DA amount in the striatum and reversed the behavioral deficit of the PD rats. Our findings provide a new potential and strategy for the use of NSCs in cell replacement therapy for PD.     

Fibroblast growth factor-20 promotes the differentiation of Nurr1-overexpressing neural stem cells into tyrosine hydroxylase-positive neurons

Stem cells are currently considered as alternative cell resources for restorative transplantation strategies in Parkinson's disease. However, the mechanisms that induce differentiation of a stem cell toward the dopaminergic phenotype are still partly unknown thus hampering the production of dopaminergic neurons from stem cells. In the past, FGF-20 has been found to promote the survival of ventral mesencephalic (VM) dopaminergic (DA) neurons in culture. We hereby provide evidence that FGF-20, a growth factor of the FGF family, is expressed in the adult and 6-OHDA-lesioned striatum and substantia nigra, but is not expressed by VM glia or DA neurons, suggesting that FGF-20 may work on DA neurons in a paracrine- or target-derived manner. We also found that co-culture of Nurr1-NSCs with Schwann cells overexpressing FGF-20 induced the acquisition of a neuronal morphology by the NSCs and the expression of tyrosine hydroxylase (TH) as assessed by immunocytochemistry, cell ELISA, and Western blot analysis. RT-PCR showed, that both, Schwann cells and Nurr1-NSCs (differentiated or not), expressed the FGF-1 receptor suggesting that both direct and indirect actions of FGF-20 are possible. We show that differentiated Nurr1 cells retained both neuronal morphology and TH expression after transplantation into the striatum of 6-OHDA-lesioned postnatal or adult rats, but that neuritogenesis was only observed after postnatal grafts. Thus, our results suggest that FGF-20 promotes the differentiation of Nurr1-NSCs into TH-positive neurons and that additional factors are required for the efficient differentiation of DA neurons in the adult brain.     

A simple method for large-scale generation of dopamine neurons from human embryonic stem cells

Dopamine (DA) neurons derived from human embryonic stem cells (hESCs) are potentially valuable in drug screening and as a possible source of donor tissue for transplantation in Parkinson's disease. However, existing culture protocols that promote the differentiation of DA neurons from hESCs are complex, involving multiple steps and having unreliable results between cultures. Here we report a simple and highly reproducible culture protocol that induces expandable DA neuron progenitors from hESCs in attached cultures. We found that the hESC-derived neuronal progenitors retain their full capacity to generate DA neurons after repeated passaging in the presence of basic fibroblast growth factor (bFGF) and medium conditioned with PA6 stromal cells. Using immunocytochemistry and RT-PCR, we found that the differentiated DA neurons exhibit a midbrain phenotype and express, e.g., Aldh1a, Ptx3, Nurr1, and Lmx1a. Using HPLC, we monitored their production of DA. We then demonstrated that the expanded progenitors are possible to cryopreserve without loosing the dopaminergic phenotype. With our protocol, we obtained large and homogeneous populations of dopaminergic progenitors and neurons. We conclude that our protocol can be used to generate human DA neurons suitable for the study of disease mechanisms, toxicology, drug screening, and intracerebral transplantation.     

Redefining Parkinson’s Disease Research Using Induced Pluripotent Stem Cells

Parkinson's disease (PD) is a movement disorder associated with the degeneration of nigral dopaminergic (DA) neurons. One of the greatest obstacles for PD research is the lack of patient-specific nigral DA neurons for mechanistic studies and drug discovery. The advent of induced pluripotent stem cells (iPSCs) has overcome this seemingly intractable problem and changed PD research in many profound ways. In this review, we discuss recent development in the generation and analyses of patient-specific iPSC-derived midbrain DA neurons. Results from this novel platform of human cellular models of PD have offered a tantalizing glimpse of the promising future of PD research. With the development of the latest genomic modification technologies, dopaminergic neuron differentiation methodologies, and cell transplantation studies, PD research is poised to enter a new phase that utilizes the human model system to identify the unique vulnerabilities of human nigral DA neurons and disease-modifying therapies based on such mechanistic studies.     

Molecular manipulation targeting regulation of dopaminergic differentiation and proliferation of neural stem cells or pluripotent stem cells

Parkinson's disease (PD) is a severe deliberating neurological disease caused by progressive degenerative death of dopaminergic neurons in the substantia nigra of midbrain. While cell replacement strategy by transplantation of neural stem cells and inducement of dopaminergic neurons is recommended for the treatment of PD, understanding the differentiation mechanism and controlled proliferation of grafted stem cells remain major concerns in their clinical application. Here we review recent studies on molecular signaling pathways in regulation of dopaminergic differentiation and proliferation of stem cells, particularly Wnt/beta-catenin signaling in stimulating formation of the dopaminergic phenotype, Notch signaling in inhibiting stem cell differentiation, and Sonic hedgehog functioning in neural stem cell proliferation and neuronal cell production. Activation of oncogenes involved in uncontrolled proliferation or tumorigenicity of stem cells is also discussed. It is proposed that a selective molecular manipulation targeting strategy will greatly benefit cell replacement therapy for PD by effectively promoting dopaminergic neuronal cell generation and reducing risk of tumorigenicity of in vivo stem cell applications.     

Toward full restoration of synaptic and terminal function of the dopaminergic system in Parkinson's disease by stem cells

New therapeutic nonpharmacological methodology in Parkinson's 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.     

Neuromodulation for Parkinson's disease]

Parkinson's disease (PD) is a neurodegenerative disorder characterized by a progressive loss of midbrain dopaminergic (DA) neurons and a subsequent reduction in striatal dopamine. As a treatment for advanced Parkinson's disease, deep brain stimulation (DBS) of the thalamus was introduced in 1987 to treat tremor, and was applied in 1993 to the subthalamic nucleus. Now high-frequency stimulation of the subthalamic nucleus has become a surgical therapy of choice. Another surgical treatment is a cell replacement therapy. Transplantation of fetal dopaminergic (DA) neurons can produce symptomatic relief, however, the technical and ethical difficulties in obtaining sufficient and appropriate donor fetal brain tissue have limited the application of this therapy. Then, neural precursor cells and embryonic stem (ES) cells are expected to be candidates of potential donor cells for transplantation. We induced DA neurons from monkey ES cells, and analyzed the effect of transplantation of the DA neurons into MPTP-treated monkeys as a primate model of Parkinson's disease. Behavioral studies and functional imaging revealed that the transplanted cells functioned as DA neurons, attenuating the MPTP-induced neurological symptoms. DA neurons have also been generated from several human ES cell lines. Furthermore, functional recovery of rat PD models after transplantation was observed. One of the major problems in ES cell transplantation is tumor formation, which is caused by a small fraction of undifferentiated ES cells in the graft. So, it is essential for undifferentiated ES cells to be eliminated from the graft in order for transplantation to be feasible. These efforts will lead to clinical application of ES cell transplantation to the patients with PD.     

Cell implantation therapies for Parkinson's disease using neural stem, transgenic or xenogeneic donor cells

A new therapeutic neurological and neurosurgical methodology involves cell implantation into the living brain in order to replace intrinsic neuronal systems, that do not spontaneously regenerate after injury, such as the dopaminergic (DA) system affected in Parkinson's disease (PD) and aging. Current clinical data indicate proof of principle for this cell implantation therapy for PD. Furthermore, the disease process does not appear to negatively affect the transplanted cells, although the patient's endogenous DA system degeneration continues. However, the optimal cells for replacement, such as highly specialized human fetal dopaminergic cells capable of repairing an entire degenerated nigro-striatal system, cannot be reliably obtained or generated in sufficient numbers for a standardized medically effective intervention. Xenogeneic and transgenic cell sources of analogous DA cells have shown great utility in animal models and some promise in early pilot studies in PD patients. The cell implantation treatment discipline, using cell fate committed fetal allo- or xenogeneic dopamine neurons and glia, is currently complemented by research on potential stem cell derived DA neurons. Understanding the cell biological principles and developing methodology necessary to generate functional DA progenitors is currently our focus for obtaining DA cells in sufficient quantities for the unmet cell transplantation need for patients with PD and related disorders.     

Nurr1‐Based Therapies for Parkinson's Disease

Previous studies have documented that orphan nuclear receptor Nurr1 (also known as NR4A2) plays important roles in the midbrain dopamine (DA) neuron development, differentiation, and survival. Furthermore, it has been reported that the defects in Nurr1 are associated with Parkinson's disease (PD). Thus, Nurr1 might be a potential therapeutic target for PD. Emerging evidence from in vitro and in vivo studies has recently demonstrated that Nurr1‐activating compounds and Nurr1 gene therapy are able not only to enhance DA neurotransmission but also to protect DA neurons from cell injury induced by environmental toxin or microglia‐mediated neuroinflammation. Moreover, modulators that interact with Nurr1 or regulate its function, such as retinoid X receptor, cyclic AMP‐responsive element‐binding protein, glial cell line‐derived neurotrophic factor, and Wnt/β‐catenin pathway, have the potential to enhance the effects of Nurr1‐based therapies in PD. This review highlights the recent progress in preclinical studies of Nurr1‐based therapies and discusses the outlook of this emerging therapy as a promising new generation of PD medication.