Eva Hedlund

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.

The homeodomain transcription factor Pitx3 facilitates differentiation of mouse embryonic stem cells into AHD2-expressing dopaminergic neurons

The A9 dopaminergic (DA) neuronal group projecting to the dorsal striatum is the most vulnerable in Parkinsons disease (PD). We genetically engineered mouse embryonic stem (ES) cells to express the transcription factors Nurr1 or Pitx3. After in vitro differentiation of Pitx3-expressing ES cells, the proportion of DA neurons expressing aldehyde dehydrogenase 2 (AHD2) increased, while the total number of DA neurons remained the same. The highest levels of AHD2 expression were observed in mouse A9 DA neurons projecting to the dorsal striatum. Furthermore, real-time PCR analyses of in vitro differentiated Pitx3-expressing ES cells revealed that genes highly expressed in A9 DA neurons were up-regulated. When transplanted into the mouse striatum, Pitx3-expressing cells generated an increased proportion of AHD2-expressing DA neurons. Contrastingly, in Nurr1-expressing ES cells, increases of all midbrain DA markers were observed, resulting in a higher total number of DA neurons in vitro and in vivo, whereas the proportion of AHD2-expressing DA neurons was not changed. Our data, using gain-of-function analysis of ES cells, suggest that Pitx3 may be important for specification and/or maintenance of A9-like neuronal properties, while Nurr1 influences overall midbrain DA specification. These findings may be important for modifying ES cells to generate an optimal cell source for transplantation therapy of PD.

Genetic selection of sox1GFP-expressing neural precursors removes residual tumorigenic pluripotent stem cells and attenuates tumor formation after transplantation.

Because of their ability to proliferate and to differentiate into diverse cell types, embryonic stem (ES) cells are a potential source of cells for transplantation therapy of various diseases, including Parkinsons disease. A critical issue for this potential therapy is the elimination of undifferentiated cells that, even in low numbers, could result in teratoma formation in the host brain. We hypothesize that an efficient solution would consist of purifying the desired cell types, such as neural precursors, prior to transplantation. To test this hypothesis, we differentiated sox1‐green fluorescent protein (GFP) knock‐in ES cells in vitro, purified neural precursor cells by fluorescence‐activated cell sorting (FACS), and characterized the purified cells in vitro as well as in vivo. Immunocytofluorescence and RT‐PCR analyses showed that this genetic purification procedure efficiently removed undifferentiated pluripotent stem cells. Furthermore, when differentiated into mature neurons in vitro, the purified GFP+ cell population generated enriched neuronal populations, whereas the GFP– population generated much fewer neurons. When treated with dopaminergic inducing signals such as sonic hedgehog (SHH) and fibroblast growth factor‐8 (FGF8), FACS‐purified neural precursor cells responded to these molecules and generated dopaminergic neurons as well as other neural subtypes. When transplanted, the GFP+ cell population generated well contained grafts containing dopaminergic neurons, whereas the GFP– population generated significantly larger grafts (about 20‐fold) and frequent tumor‐related deaths in the transplanted animals. Taken together, our results demonstrate that genetic purification of neural precursor cells using FACS isolation can effectively remove unwanted proliferating cell types and avoid tumor formation after transplantation.

Cytochrome P450 in the Brain ; A Review

After many frustrating decades of unsuccessful attempts to characterize the isoforms of P450 in the brain, several scientific breakthroughs in the 80s and 90s have resulted in major advances in our understanding of cytochromes P450 (CYP) in brain. We now know that classical CYP inducers, e.g. phenobarbital and pregnenolone 16alpha-carbonitrile, which regulate drug-metabolizing enzymes in the liver, are specific ligands for ligand-activated transcription factors, and that the brain content of many of these transcription factors is low. This explains why these inducers have little effect on brain CYP content. The most effective inducers of brain P450 are some of the CNS active drugs and solvents. The level of CYPs in brain, approximately 0.5-2% of that in liver, is too low to significantly influence the overall pharmacokinetics of drugs and hormones in the body. Instead CYPs appear to have specific functions in brain, e.g. regulation of the levels of endogenous GABAA receptor agonists maintenance of brain cholesterol homeostasis and elimination of retinoids The novel CYPs which catalyse these reactions have recently been characterized. They are abundantly expressed in the brain confirming what has been previously found, i.e. that the major hepatic, adrenal and gonadal CYP isozymes contribute very little to the overall content of CYP in brain. It is not clear what fraction of brain CYP has been characterized, although a complete characterization of constitutive and induced CYPs in brain is essential for understanding the role of these enzymes in brain physiology as well as in age-related and xenobiotic-induced neurotoxicity.

Neurosteroid Hydroxylase CYP7B VIVID REPORTER ACTIVITY IN DENTATE GYRUS OF GENE-TARGETED MICE AND ABOLITION OF A WIDESPREAD PATHWAY OF STEROID AND OXYSTEROL HYDROXYLATION

The major adrenal steroid dehydroepiandrosterone (DHEA) enhances memory and immune function but has no known dedicated receptor; local metabolism may govern its activity. We described a cytochrome P450 expressed in brain and other tissues, CYP7B, that catalyzes the 7α-hydroxylation of oxysterols and 3β-hydroxysteroids including DHEA. We report here that CYP7B mRNA and 7α-hydroxylation activity are widespread in rat tissues. However, steroids related to DHEA are reported to be modified at positions other than 7α, exemplified by prominent 6α-hydroxylation of 5α-androstane-3β,17β-diol (A/anediol) in some rodent tissues including brain. To determine whether CYP7B is responsible for these and other activities we disrupted the mouse Cyp7b gene by targeted insertion of an IRES-lacZ reporter cassette, placing reporter enzyme activity (β-galactosidase) underCyp7b promoter control. In heterozygous mouse brain, chromogenic detection of reporter activity was strikingly restricted to the dentate gyrus. Staining did not exactly reproduce the in situ hybridization expression pattern; post-transcriptional control is inferred. Lower level staining was detected in cerebellum, liver, and kidney, and which largely paralleled mRNA distribution. Liver and kidney expression was sexually dimorphic. Mice homozygous for the insertion are viable and superficially normal, but ex vivo metabolism of DHEA to 7α-hydroxy-DHEA was abolished in brain, spleen, thymus, heart, lung, prostate, uterus, and mammary gland; lower abundance metabolites were also eliminated. 7α-Hydroxylation of 25-hydroxycholesterol and related substrates was also abolished, as was presumed 6α-hydroxylation of A/anediol. These different enzyme activities therefore derive from the Cyp7bgene. CYP7B is thus a major extrahepatic steroid and oxysterol hydroxylase and provides the predominant route for local metabolism of DHEA and related molecules in brain and other tissues.

Specific and integrated roles of Lmx1a, Lmx1b and Phox2a in ventral midbrain development

The severe disorders associated with a loss or dysfunction of midbrain dopamine neurons (DNs) have intensified research aimed at deciphering developmental programs controlling midbrain development. The homeodomain proteins Lmx1a and Lmx1b are important for the specification of DNs during embryogenesis, but it is unclear to what degree they may mediate redundant or specific functions. Here, we provide evidence showing that DN progenitors in the ventral midbrain can be subdivided into molecularly distinct medial and lateral domains, and these subgroups show different sensitivity to the loss of Lmx1a and Lmx1b. Lmx1a is specifically required for converting non-neuronal floor-plate cells into neuronal DN progenitors, a process that involves the establishment of Notch signaling in ventral midline cells. On the other hand, lateral DN progenitors that do not appear to originate from the floor plate are selectively ablated in Lmx1b mutants. In addition, we also reveal an unanticipated role for Lmx1b in regulating Phox2a expression and the sequential specification of ocular motor neurons (OMNs) and red nucleus neurons (RNNs) from progenitors located lateral to DNs in the midbrain. Our data therefore establish that Lmx1b influences the differentiation of multiple neuronal subtypes in the ventral midbrain, whereas Lmx1a appears to be exclusively devoted to the differentiation of the DN lineage.

Selection of Embryonic Stem Cell-Derived Enhanced Green Fluorescent Protein-Positive Dopamine Neurons Using the Tyrosine Hydroxylase Promoter Is Confounded by Reporter Gene Expression in Immature Cell Populations

Transplantation of mouse embryonic stem (mES) cells can restore function in Parkinson disease models, but can generate teratomas. Purification of dopamine neurons derived from embryonic stem cells by fluorescence‐activated cell sorting (FACS) could provide a functional cell population for transplantation while eliminating the risk of teratoma formation. Here we used the tyrosine hydroxylase (TH) promoter to drive enhanced green fluorescent protein (eGFP) expression in mES cells. First, we evaluated 2.5‐kilobase (kb) and 9‐kb TH promoter fragments and showed that clones generated using the 9‐kb fragment produced significantly more eGFP+/TH+ neurons. We selected the 9‐kb TH clone with the highest eGFP/TH overlap for further differentiation, FACS, and transplantation experiments. Grafts contained large numbers of eGFP+ dopamine neurons of an appropriate phenotype. However, there were also numerous eGFP+ cells that did not express TH and did not have a neuronal morphology. In addition, we found cells in the grafts representing all three germ layers. Based on these findings, we examined the expression of stem cell markers in our eGFP+ population. We found that a majority of eGFP+ cells were stage‐specific embryonic antigen‐positive (SSEA‐1+) and that the genetically engineered clones contained more SSEA‐1+ cells after differentiation than the original D3 mES cells. By negative selection of SSEA‐1, we could isolate a neuronal eGFP+ population of high purity. These results illustrate the complexity of using genetic selection to purify mES cell‐derived dopamine neurons and provide a comprehensive analysis of cell selection strategies based on tyrosine hydroxylase expression.

Cell therapy and stem cells in animal models of motor neuron disorders.

Amyotrophic lateral sclerosis (ALS), spinal bulbar muscular atrophy (or Kennedys disease), spinal muscular atrophy and spinal muscular atrophy with respiratory distress 1 are neurodegenerative disorders mainly affecting motor neurons and which currently lack effective therapies. Recent studies in animal models as well as primary and embryonic stem cell models of ALS, utilizing over‐expression of mutated forms of Cu/Zn superoxide dismutase 1, have shown that motor neuron degeneration in these models is in part a non cell‐autonomous event and that by providing genetically non‐compromised supporting cells such as microglia or growth factor‐excreting cells, onset can be delayed and survival increased. Using models of acute motor neuron injury it has been shown that embryonic stem cell‐derived motor neurons implanted into the spinal cord can innervate muscle targets and improve functional recovery. Thus, a rationale exists for the development of cell therapies in motor neuron diseases aimed at either protecting and/or replacing lost motor neurons, interneurons as well as non‐neuronal cells. This review evaluates approaches used in animal models of motor neuron disorders and their therapeutic relevance.

Cellular therapy to target neuroinflammation in amyotrophic lateral sclerosis

Neurodegenerative disorders are characterized by the selective vulnerability and progressive loss of discrete neuronal populations. Non-neuronal cells appear to significantly contribute to neuronal loss in diseases such as amyotrophic lateral sclerosis (ALS), Parkinson, and Alzheimer’s disease. In ALS, there is deterioration of motor neurons in the cortex, brainstem, and spinal cord, which control voluntary muscle groups. This results in muscle wasting, paralysis, and death. Neuroinflammation, characterized by the appearance of reactive astrocytes and microglia as well as macrophage and T-lymphocyte infiltration, appears to be highly involved in the disease pathogenesis, highlighting the involvement of non-neuronal cells in neurodegeneration. There appears to be cross-talk between motor neurons, astrocytes, and immune cells, including microglia and T-lymphocytes, which are subsequently activated. Currently, effective therapies for ALS are lacking; however, the non-cell autonomous nature of ALS may indicate potential therapeutic targets. Here, we review the mechanisms of action of astrocytes, microglia, and T-lymphocytes in the nervous system in health and during the pathogenesis of ALS. We also evaluate the therapeutic potential of these cellular populations, after transplantation into ALS patients and animal models of the disease, in modulating the environment surrounding motor neurons from pro-inflammatory to neuroprotective. We also thoroughly discuss the recent advances made in the field and caveats that need to be overcome for clinical translation of cell therapies aimed at modulating non-cell autonomous events to preserve remaining motor neurons in patients.

Antiangiogenic agents significantly improve survival in tumor-bearing mice by increasing tolerance to chemotherapy-induced toxicity

Chemotherapy-induced broad toxicities are the leading cause of the drug-induced mortality in cancer patients. Antiangiogenic drugs (ADs) in combination with chemotherapy are widely used as front-line therapy for the treatment of various human cancers. However, the beneficial mechanisms underlying combination therapy are poorly understood. Here we show that, in several murine tumor models, administration of sunitinib markedly reduced chemotherapy-induced bone marrow toxicity. Intriguingly, in a sequential treatment regimen, delivery of ADs followed by chemotherapy demonstrated superior survival benefits compared with simultaneous administration of two drugs. In murine tumor models, we show that VEGF increased chemotoxicity by synergistically suppressing bone marrow hematopoiesis with cytostatic drugs. These findings shed light on molecular mechanisms by which ADs in combination with chemotherapy produce survival benefits in cancer patients and provide conceptual information guiding future designs of clinical trials, current practice, and optimization of ADs for the treatment of cancer.