Ole Isacson

Parkinson's Disease Patient-Derived Induced Pluripotent Stem Cells Free of Viral Reprogramming Factors

Induced pluripotent stem cells (iPSCs) derived from somatic cells of patients represent a powerful tool for biomedical research and may provide a source for replacement therapies. However, the use of viruses encoding the reprogramming factors represents a major limitation of the current technology since even low vector expression may alter the differentiation potential of the iPSCs or induce malignant transformation. Here, we show that fibroblasts from five patients with idiopathic Parkinsons disease can be efficiently reprogrammed and subsequently differentiated into dopaminergic neurons. Moreover, we derived hiPSCs free of reprogramming factors using Cre-recombinase excisable viruses. Factor-free hiPSCs maintain a pluripotent state and show a global gene expression profile, more closely related to hESCs than to hiPSCs carrying the transgenes. Our results indicate that residual transgene expression in virus-carrying hiPSCs can affect their molecular characteristics and that factor-free hiPSCs therefore represent a more suitable source of cells for modeling of human disease.

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.

Neurons derived from reprogrammed fibroblasts functionally integrate into the fetal brain and improve symptoms of rats with Parkinson's disease

The long-term goal of nuclear transfer or alternative reprogramming approaches is to create patient-specific donor cells for transplantation therapy, avoiding immunorejection, a major complication in current transplantation medicine. It was recently shown that the four transcription factors Oct4, Sox2, Klf4, and c-Myc induce pluripotency in mouse fibroblasts. However, the therapeutic potential of induced pluripotent stem (iPS) cells for neural cell replacement strategies remained unexplored. Here, we show that iPS cells can be efficiently differentiated into neural precursor cells, giving rise to neuronal and glial cell types in culture. Upon transplantation into the fetal mouse brain, the cells migrate into various brain regions and differentiate into glia and neurons, including glutamatergic, GABAergic, and catecholaminergic subtypes. Electrophysiological recordings and morphological analysis demonstrated that the grafted neurons had mature neuronal activity and were functionally integrated in the host brain. Furthermore, iPS cells were induced to differentiate into dopamine neurons of midbrain character and were able to improve behavior in a rat model of Parkinsons disease upon transplantation into the adult brain. We minimized the risk of tumor formation from the grafted cells by separating contaminating pluripotent cells and committed neural cells using fluorescence-activated cell sorting. Our results demonstrate the therapeutic potential of directly reprogrammed fibroblasts for neuronal cell replacement in the animal model.

Specific MicroRNAs Modulate Embryonic Stem Cell–Derived Neurogenesis

MicroRNAs (miRNAs) are recently discovered small non‐coding transcripts with a broad spectrum of functions described mostly in invertebrates. As post‐transcriptional regulators of gene expression, miRNAs trigger target mRNA degradation or translational repression. Although hundreds of miRNAs have been cloned from a variety of mammalian tissues and cells and multiple mRNA targets have been predicted, little is known about their functions. So far, a role of miRNA has only been described in hematopoietic, adipocytic, and muscle differentiation; regulation of insulin secretion; and potentially regulation of cancer growth. Here, we describe miRNA expression profiling in mouse embryonic stem (ES) cell– derived neurogenesis in vitro and show that a number of miRNAs are simultaneously co‐induced during differentiation of neural progenitor cells to neurons and astrocytes. There was a clear correlation between miRNA expression profiles in ES cell– derived neurogenesis in vitro and in embryonal neurogenesis in vivo. Using both gain‐of‐function and loss‐of‐function approaches, we demonstrate that brain‐specific miR‐124a and miR‐9 molecules affect neural lineage differentiation in the ES cell– derived cultures. In addition, we provide evidence that signal transducer and activator of transcription (STAT) 3, a member of the STAT family pathway, is involved in the function of these miRNAs. We conclude that distinct miRNAs play a functional role in the determination of neural fates in ES cell differentiation.

Altered Proteasomal Function in Sporadic Parkinson's Disease

Parkinsons disease (PD) is characterized pathologically by preferential degeneration of the dopaminergic neurons in the substantia nigra pars compacta (SNc). Nigral cell death is accompanied by the accumulation of a wide range of poorly degraded proteins and the formation of proteinaceous inclusions (Lewy bodies) in dopaminergic neurons. Mutations in the genes encoding alpha-synuclein and two enzymes of the ubiquitin-proteasome system, parkin and ubiquitin C-terminal hydrolase L1, are associated with neurodegeneration in some familial forms of PD. We now show that, in comparison to age-matched controls, alpha-subunits (but not beta-subunits) of 26/20S proteasomes are lost within dopaminergic neurons and 20S proteasomal enzymatic activities are impaired in the SNc in sporadic PD. In addition, while the levels of the PA700 proteasome activator are reduced in the SNc in PD, PA700 expression is increased in other brain regions such as the frontal cortex and striatum. We also found that levels of the PA28 proteasome activator are very low to almost undetectable in the SNc compared to other brain areas in both normal and PD subjects. These findings suggest that failure of the ubiquitin-proteasome system to adequately clear unwanted proteins may underlie vulnerability and degeneration of the SNc in both sporadic and familial PD.

Failure of the ubiquitin–proteasome system in Parkinson's disease

Parkinsons disease (PD) is a neurodegenerative movement disorder characterized by degeneration of dopamine-containing neurons in the midbrain. In cases of familial PD, mutations that lead to failure of the ubiquitin–proteasome system (UPS) have been identified. These genetic abnormalities do not occur in sporadic PD, but we propose that impairment of the UPS could also contribute to neurodegeneration in this disorder. We discuss evidence that failure of the UPS is a common aetiopathogenic factor that underlies the development of familial and sporadic PD, an idea that might help to explain clinical and pathological differences and similarities in these disorders.

Differentiated Parkinson patient-derived induced pluripotent stem cells grow in the adult rodent brain and reduce motor asymmetry in Parkinsonian rats

Recent advances in deriving induced pluripotent stem (iPS) cells from patients offer new possibilities for biomedical research and clinical applications, as these cells could be used for autologous transplantation. We differentiated iPS cells from patients with Parkinsons disease (PD) into dopaminergic (DA) neurons and show that these DA neurons can be transplanted without signs of neurodegeneration into the adult rodent striatum. The PD patient iPS (PDiPS) cell-derived DA neurons survived at high numbers, showed arborization, and mediated functional effects in an animal model of PD as determined by reduction of amphetamine- and apomorphine-induced rotational asymmetry, but only a few DA neurons projected into the host striatum at 16 wk after transplantation. We next applied FACS for the neural cell adhesion molecule NCAM on differentiated PDiPS cells before transplantation, which resulted in surviving DA neurons with functional effects on amphetamine-induced rotational asymmetry in a 6-OHDA animal model of PD. Morphologically, we found that PDiPS cell-derived non-DA neurons send axons along white matter tracts into specific close and remote gray matter target areas in the adult brain. Such findings establish the transplantation of human PDiPS cell-derived neurons as a long-term in vivo method to analyze potential disease-related changes in a physiological context. Our data also demonstrate proof of principle of survival and functional effects of PDiPS cell-derived DA neurons in an animal model of PD and encourage further development of differentiation protocols to enhance growth and function of implanted PDiPS cell-derived DA neurons in regard to potential therapeutic applications.

Dopamine neurons implanted into people with Parkinson's disease survive without pathology for 14 years

Postmortem analysis of five subjects with Parkinsons disease 9–14 years after transplantation of fetal midbrain cell suspensions revealed surviving grafts that included dopamine and serotonin neurons without pathology. These findings are important for the understanding of the etiopathogenesis of midbrain dopamine neuron degeneration and future use of cell replacement therapies.

Mechanisms of action of intracerebral neural implants: studies on nigral and striatal grafts to the lesioned striatum

Abstract The ability of intracerebrally implanted grafts of neural tissues to promote functional recovery in brain-damaged recipient animals has raised the question of how such implants exert their functional effects. Non-specific, diffuse release of active compounds may be sufficient to restore defective neurotransmission in a denervated brain region, for example, or to provide trophic support for the survival and regeneration of damaged host neurons. The positive therapeutic effects of adrenal medullary grafts, recently reported in patients with Parkinsons disease, are likely to reflect such nonspecific hormonal or neurohumoral mechanisms. Morphological and electrophysiological studies, on the other hand, have shown that grafted fetal neurons can establish extensive efferent synaptic connections with previously denervated or neuron-depleted host brain regions and become at least partially integrated into the host neuronal circuitry. In the damaged nigrostriatal system, grafts of fetal nigral or striatal neurons can restore normal synaptic transmitter release and can also participate in a partial reconstruction of functional neural circuits in the host brain. This indicates that the potential of intracerebral grafts to induce or improve behavioral recovery in brain-damaged recipients rests on the multitude of trophic, neurohumoral and synaptic mechanisms that may allow the implanted tissue to promote host brain function and repair.

Neuroinflammation of the nigrostriatal pathway during progressive 6-OHDA dopamine degeneration in rats monitored by immunohistochemistry and PET imaging

We investigated the microglial response to progressive dopamine neuron degeneration using in vivo positron emission tomography (PET) imaging and postmortem analyses in a Parkinsons disease (PD) rat model induced by unilateral (right side) intrastriatal administration of 6‐hydroxydopamine (6‐OHDA). Degeneration of the dopamine system was monitored by PET imaging of presynaptic dopamine transporters using a specific ligand 11C‐CFT (2β‐carbomethoxy‐3β‐(4‐fluorophenyl) tropane). Binding of 11C‐CFT was markedly reduced in the striatum indicating dopaminergic degeneration. Parallel PET studies of 11C‐PK11195 (1‐(2‐chlorophenyl)‐N‐methyl‐N‐(1‐methylpropyl)‐3 isoquinoline carboxamide) (specific ligand for activated microglia) showed increased binding in the striatum and substantia nigra indicative of a microglial response. Postmortem immunohistochemical analyses were performed with antibodies against CR3 for microglia/macrophage activation. Using a qualitative postmortem index for microglial activation we found an initially focal, then widespread microglial response at striatal and nigral levels at 4 weeks postlesion. These data support the hypothesis that inflammation is a significant component of progressive dopaminergic degeneration that can be monitored by PET imaging.