Iván Velasco

Dopamine neurons derived from embryonic stem cells function in an animal model of Parkinson's disease

Parkinsons disease is a widespread condition caused by the loss of midbrain neurons that synthesize the neurotransmitter dopamine. Cells derived from the fetal midbrain can modify the course of the disease, but they are an inadequate source of dopamine-synthesizing neurons because their ability to generate these neurons is unstable. In contrast, embryonic stem (ES) cells proliferate extensively and can generate dopamine neurons. If ES cells are to become the basis for cell therapies, we must develop methods of enriching for the cell of interest and demonstrate that these cells show functions that will assist in treating the disease. Here we show that a highly enriched population of midbrain neural stem cells can be derived from mouse ES cells. The dopamine neurons generated by these stem cells show electrophysiological and behavioural properties expected of neurons from the midbrain. Our results encourage the use of ES cells in cell-replacement therapy for Parkinsons disease.

Long-Lasting Regeneration After Ischemia in the Cerebral Cortex

Background and Purpose— Because fibroblast growth factor 2 is a mitogen for central nervous system stem cells, we explored whether long-term fibroblast growth factor 2 delivery to the brain can improve functional outcome and induce cortical neurogenesis after ischemia. Methods— Rats underwent permanent distal middle cerebral artery occlusion resulting in an ischemic injury limited to the cortex. We used an adeno-associated virus transfection system to induce long-term fibroblast growth factor 2 expression and monitored behavioral and histological changes. Results— Treatment increased the number of proliferating cells and improved motor behavior. Neurogenesis continued throughout 90 days after the ischemia, and the occurrence of newly generated cells with characteristics of neural precursors and immature neurons was most evident 90 days after treatment. Conclusions— Focal cortical ischemia elicits an ongoing neurogenic response that can be enhanced with fibroblast growth factor 2 leading to improved functional outcome.

Persistent Dopamine Functions of Neurons Derived from Embryonic Stem Cells in a Rodent Model of Parkinson Disease

The derivation of dopamine neurons is one of the best examples of the clinical potential of embryonic stem (ES) cells, but the long‐term function of the grafted neurons has not been established. Here, we show that, after transplantation into an animal model, neurons derived from mouse ES cells survived for over 32 weeks, maintained midbrain markers, and had sustained behavioral effects. Microdialysis in grafted animals showed that dopamine (DA) release was induced by depolarization and pharmacological stimulants. Positron emission tomography measured the expression of presynaptic dopamine transporters in the graft and also showed that the number of postsynaptic DA D2 receptors was normalized in the host striatum. These data suggest that ES cell‐derived neurons show DA release and reuptake and stimulate appropriate postsynaptic responses for long periods after implantation. This work supports continued interest in ES cells as a source of functional DA neurons.

Transient Recovery in a Rat Model of Familial Amyotrophic Lateral Sclerosis after Transplantation of Motor Neurons Derived from Mouse Embryonic Stem Cells

Embryonic stem (ES) cells can be induced to differentiate into motor neurons (MN). Animal models resembling MN degeneration and paralysis observed in familial amyotrophic lateral sclerosis (ALS) have been previously reported. In this work, we aimed to investigate whether transplanted MN could prevent motor deterioration in transgenic rats expressing a mutant form of human superoxide dismutase 1 (hSOD1G93A) associated with inherited ALS. Mouse ES cells were differentiated to neurons that express green fluorescent protein (GFP) under the promoter of the MN-specific gene hb9, as well as molecular markers indicative of MN identity. Cells were grafted into the lumbar spinal cord of adult wild-type (WT) or hSOD1G93A rats at 10 weeks of age, when transgenic animals are presymptomatic. Grafted cells with MN phenotype can survive for at least 1 week in hSOD1G93A animals. To quantitatively evaluate motor performance of WT and transgenic rats, we carried out weekly rotarod tests starting when the animals were 14 weeks old. Sham and grafted WT animals showed no decline in their ability to sustain themselves on the rotating rod. In contrast, sham hSOD1G93A rats decreased in motor performance from week 16 onwards, reaching paralysis by week 19 of age. In grafted transgenic animals, there was a significant improvement in rotarod competence at weeks 16 and 17 when compared to sham hSOD1G93A. However, in the following weeks, transplanted hSOD1G93A rats showed motor deterioration and eventually exhibited paralysis by week 19. At end-stage, we found only a few endogenous MN in sham and grafted hSOD1G93A rats by cresyl violet staining; no choline acetyl transferase-positive nor GFP-positive MN were present in grafted transgenic subjects. In contrast, WT rats analyzed at the same age possessed grafted GFP-positive MN in their spinal cords. These results strongly suggest that the transgenic hSOD1G93A environment is detrimental to grafted MN in the long term.

Histamine induces neural stem cell proliferation and neuronal differentiation by activation of distinct histamine receptors.

Histamine has neurotransmitter/neuromodulator functions in the adult brain, but its role during CNS development has been elusive. We studied histamine effects on proliferation, cell death and differentiation of neuroepithelial stem cells from rat cerebral cortex in vitro. RT‐PCR and Western blot experiments showed that proliferating and differentiated cells express histamine H1, H2 and H3 receptors. Treatments with histamine concentrations (100 nM–1 mM) caused significant increases in cell numbers without affecting Nestin expression. Cell proliferation was evaluated by BrdU incorporation; histamine caused a significant increase dependent on H2 receptor activation. Apoptotic cell death during proliferation was significantly decreased at all histamine concentrations, and cell death was promoted in a concentration‐dependent manner by histamine in differentiated cells. Immunocytochemistry studies showed that histamine increased 3‐fold the number of neurons after differentiation, mainly by activation of H1 receptor, and also significantly decreased the glial (astrocytic) cell proportion, when compared to control conditions. In summary, histamine increases cell number during proliferative conditions, and has a neuronal‐differentiating action on neural stem cells, suggesting that the elevated histamine concentration reported during development might play a role in cerebrocortical neurogenesis, by activation of H2 receptors to promote proliferation of neural precursors, and favoring neuronal fate by H1‐mediated stimulation.

INHIBITION OF GLUTAMATE UPTAKE INDUCES PROGRESSIVE ACCUMULATION OF EXTRACELLULAR GLUTAMATE AND NEURONAL DAMAGE IN RAT CORTICAL CULTURES

It is known that neurons exposed to high concentrations of glutamate degenerate and die. The clearance of this amino acid from the extracellular space depends on their active transport by Na+‐dependent high‐affinity carriers. In the present study we tested whether inhibition of glutamate transport in mixed glial/neuronal cortical cultures induces accumulation of extracellular glutamate and whether such increase results in cell damage. Three inhibitors of glutamate transport were used: L‐trans‐pyrrolidine‐2,4‐dicarboxylate (PDC), DL‐threo‐β‐hydroxyaspartate (THA), and dihydrokainate (DHK). Cell damage was assessed by light microscopy observations, reduction of 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide, and leakage of lactate dehydrogenase. PDC induced a significant concentration‐ and time‐dependent neuronal damage, whereas pure glial cultures were not affected. A good correlation was found between this damage and elevations of glutamate concentration in the medium. These effects of PDC were similar in glutamine‐free medium and in medium supplemented with glutamine. THA induced identical cell damage and elevations of extracellular glutamate to those produced by PDC, while DHK did not affect at all any of these parameters. PDC‐ and THA‐induced toxicity was protected by the N‐methyl‐D‐aspartate receptor antagonist (+)‐5‐methyl‐10,11‐dihydro‐5H‐dibenzo‐(a,d)cyclohepten‐5,10‐imine maleate but not by the non‐N‐methyl‐D‐aspartate receptor antagonist 2,3‐dihydroxy‐6‐nitro‐7‐sulfamoyl‐benzo(f)quinoxaline.

Ruthenium red as a tool to study calcium channels, neuronal death and the function of neural pathways.

The inorganic polycationic dye ruthenium red (RuR) exerts several effects on the nervous system when added in physiological solutions, both in vivo and in vitro. Part of these effects, including the paralysis observed in mammals after the systemic administration of RuR, can be accounted for by the binding of RuR to nerve ending membranes, which results in inhibition of Ca2+ influx through voltage-sensitive calcium channels and the consequent inhibition of neurotransmitter release. On the other hand, the administration of RuR into the cerebrospinal fluid induces intense convulsive activity, and its microinjection into the substantia nigra reticulata or the hippocampus leads to various motor behavior alterations that can be related to hyperexcitability of the neurons of the injected region. In addition, RuR penetrates the neuronal somata present in the area injected and induces cell destruction, which has been interpreted as an excitotoxic action of the dye. The penetration and the toxicity of RuR were also observed in primary neuronal cultures but did not occur in pure glial cultures, suggesting a selective action on neurons. In the present article the in vitro and in vivo effects of RuR are reviewed and discussed in terms of the usefulness of the dye as an interesting tool to study calcium channels linked to transmitter release, neuronal death mechanisms and the function of neural pathways.

Concise Review: Generation of Neurons From Somatic Cells of Healthy Individuals and Neurological Patients Through Induced Pluripotency or Direct Conversion

Access to healthy or diseased human neural tissue is a daunting task and represents a barrier for advancing our understanding about the cellular, genetic, and molecular mechanisms underlying neurogenesis and neurodegeneration. Reprogramming of somatic cells to pluripotency by transient expression of transcription factors was achieved a few years ago. Induced pluripotent stem cells (iPSC) from both healthy individuals and patients suffering from debilitating, life‐threatening neurological diseases have been differentiated into several specific neuronal subtypes. An alternative emerging approach is the direct conversion of somatic cells (i.e., fibroblasts, blood cells, or glial cells) into neuron‐like cells. However, to what extent neuronal direct conversion of diseased somatic cells can be achieved remains an open question. Optimization of current expansion and differentiation approaches is highly demanded to increase the differentiation efficiency of specific phenotypes of functional neurons from iPSCs or through somatic cell direct conversion. The realization of the full potential of iPSCs relies on the ability to precisely modify specific genome sequences. Genome editing technologies including zinc finger nucleases, transcription activator‐like effector nucleases, and clustered regularly interspaced short palindromic repeat/CAS9 RNA‐guided nucleases have progressed very fast over the last years. The combination of genome‐editing strategies and patient‐specific iPSC biology will offer a unique platform for in vitro generation of diseased and corrected neural derivatives for personalized therapies, disease modeling and drug screening. Stem Cells 2014;32:2811–2817

Expression of LPP3 in Bergmann glia is required for proper cerebellar sphingosine‐1‐phosphate metabolism/signaling and development

Bioactive lipids serve as intracellular and extracellular mediators in cell signaling in normal and pathological conditions. Here we describe that an important regulator of some of these lipids, the lipid phosphate phosphatase‐3 (LPP3), is abundantly expressed in specific plasma membrane domains of Bergmann glia (BG), a specialized type of astrocyte with key roles in cerebellum development and physiology. Mice selectively lacking expression of LPP3/Ppap2b in the nervous system are viable and fertile but exhibit defects in postnatal cerebellum development and modifications in the cytoarchitecture and arrangement of BG with a mild non‐progressive motor coordination defect. Lipid and gene profiling studies in combination with pharmacological treatments suggest that most of these effects are associated with alterations in sphingosine‐1‐phosphate (S1P) metabolism and signaling. Altogether our data indicate that LPP3 participates in several aspects of neuron‐glia communication required for proper cerebellum development.

Therapeutic Potential of Motor Neurons Differentiated from Embryonic Stem Cells and Induced Pluripotent Stem Cells

Degeneration of motor neurons (MN) caused by disease or injury leads to paralysis and is fatal in some conditions. To date, there are no effective treatments for MN disorders; therefore, cell therapy is a promising strategy to replace lost MN. Embryonic stem (ES) cells isolated from the inner cell mass of mammalian blastocysts self-renew and are pluripotent because they differentiate into cell types of the three germinal layers. Reprogramming of adult cells to a state similar to ES cells, termed induced pluripotent stem (iPS) cells, has been recently reported. It is well established that pluripotent cell types can give rise to specialized phenotypes, including neurons. Mouse, monkey and human MN can be differentiated from ES and iPS cells using procedures generally involving embryoid bodies formation and stimulation with retinoic acid and Sonic hedgehog. Differentiated MN express characteristic molecular markers such as Islet1, HB9 and Choline acetyltransferase, exhibit electrophysiological maturity and are able to form synaptic contacts similar to neuromuscular junctions in vitro. Furthermore, transplanted MN promote functional recovery in animal models of neurodegenerative diseases and MN injury. The potential clinical applications of stem cell-derived MN was enhanced after iPS cell derivation, which makes possible the generation of patient-specific pluripotent cells for autologous cell replacement therapies and may be used for drug development and disease modeling. This review summarizes MN differentiation protocols from ES and iPS cells in regard to neuronal differentiation efficiency, expression of MN markers and functional properties in vitro, as well as their therapeutic effects after grafting.