Seungsoo Chung

Highly efficient and large-scale generation of functional dopamine neurons from human embryonic stem cells

We developed a method for the efficient generation of functional dopaminergic (DA) neurons from human embryonic stem cells (hESCs) on a large scale. The most unique feature of this method is the generation of homogeneous spherical neural masses (SNMs) from the hESC-derived neural precursors. These SNMs provide several advantages: (i) they can be passaged for a long time without losing their differentiation capability into DA neurons; (ii) they can be coaxed into DA neurons at much higher efficiency than that from previous reports (86% tyrosine hydroxylase-positive neurons/total neurons); (iii) the induction of DA neurons from SNMs only takes 14 days; and (iv) no feeder cells are required during differentiation. These advantages allowed us to obtain a large number of DA neurons within a short time period and minimized potential contamination of unwanted cells or pathogens coming from the feeder layer. The highly efficient differentiation may not only enhance the efficacy of the cell therapy but also reduce the potential tumor formation from the undifferentiated residual hESCs. In line with this effect, we have never observed any tumor formation from the transplanted animals used in our study. When grafted into a parkinsonian rat model, the hESC-derived DA neurons elicited clear behavioral recovery in three behavioral tests. In summary, our study paves the way for the large-scale generation of purer and functional DA neurons for future clinical applications.

Stromal Cell–Derived Inducing Activity, Nurr1, and Signaling Molecules Synergistically Induce Dopaminergic Neurons from Mouse Embryonic Stem Cells

To induce differentiation of embryonic stem cells (ESCs) into specialized cell types for therapeutic purposes, it may be desirable to combine genetic manipulation and appropriate differentiation signals. We studied the induction of dopaminergic (DA) neurons from mouse ESCs by overexpressing the transcription factor Nurr1 and coculturing with PA6 stromal cells. Nurr1‐expressing ESCs (N2 and N5) differentiated into a higher number of neurons (∼twofold) than the naïve ESCs (D3). In addition, N2/N5‐derived cells contained a significantly higher proportion (>50%) of tyrosine hydroxylase (TH)+ neurons than D3 (<30%) and an even greater proportion of TH+ neurons (∼90%) when treated with the signaling molecules sonic hedgehog, fibroblast growth factor 8, and ascorbic acid. N2/N5‐derived cells express much higher levels of DA markers (e.g., TH, dopamine transporter, aromatic amino acid decarboxylase, and G protein–regulated inwardly rectifying K+ channel 2) and produce and release a higher level of dopamine, compared with D3‐derived cells. Furthermore, the majority of generated neurons exhibited electrophysiological properties characteristic of midbrain DA neurons. Finally, transplantation experiments showed efficient in vivo integration/generation of TH+ neurons after implantation into mouse striatum. Taken together, our results show that the combination of genetic manipulation(s) and in vitro cell differentiation conditions offers a reliable and effective induction of DA neurons from ESCs and may pave the way for future cell transplantation therapy in Parkinsons disease.

Generation of functional dopamine neurons from neural precursor cells isolated from the subventricular zone and white matter of the adult rat brain using nurr1 overexpression

Neural precursor (NP) cells from adult mammalian brains can be isolated, expanded in vitro, and potentially used as cell replacement source material for treatment of intractable brain disorders. Reduced ethical concerns, lack of teratoma formation, and possible ex vivo autologous transplantation are critical advantages to using adult NP donor cells over cells from fetal brain tissue or embryonic stem cells. However, the usage of adult NP cells is limited by the ability to induce specific neurochemical phenotypes in these cells. Here, we demonstrate induction of a dopaminergic phenotype in NP cells isolated from the subventricular zone (SVZ) and white matter of rodent adult brains using overexpression of the nuclear receptor Nurr1 in vitro. Forced expression of Nurr1, a transcriptional factor specific to midbrain dopamine (DA) neuron development, caused in the adult cells an acquisition of the DA neurotransmitter phenotype and sufficient differentiation toward morphologically, phenotypically, and ultrastructurally mature DA neurons. Co‐expression of neurogenic factor Mash1 and treatment with neurogenic cytokines brain‐derived neurotrophic factor and neurotrophin‐3 greatly enhanced Nurr1‐induced DA neuron yield. The Nurr1‐induced DA neurons demonstrated in vitro presynaptic DA neuronal functionality, releasing DA neurotransmitter in response to depolarization stimuli and specific DA reuptake. Furthermore, Nurr1‐engineered adult SVZ NP cells survived, integrated, and differentiated into DA neurons in vivo that can reverse the behavioral deficit in the host striatum of parkinsonian rats. These findings open the possibility for the use of precursor cells from adult brains as a cell source for neuronal replacement treatment of Parkinson disease.

Differential actions of the proneural genes encoding Mash1 and neurogenins in Nurr1-induced dopamine neuron differentiation

The steroid receptor-type transcription factor Nurr1 has a crucial role in the development of the mesencephalic dopamine (DA) neurons. Although ectopic expression of Nurr1 in cultured neural precursor cells is sufficient in establishing the DA phenotype, Nurr1-induced DA cells are morphologically and functionally immature, suggesting the necessity of additional factor(s) for full neuronal differentiation. In this study, we demonstrate that neurogenic basic helix-loop-helix (bHLH) factors Mash1, neurogenins (Ngns) and NeuroD play contrasting roles in Nurr1-induced DA neuronal differentiation. Mash1, but not Ngn2, spatially and temporally colocalized with aldehyde dehydrogenase 2 (AHD2), a specific midbrain DA neuronal progenitor marker, in the early embryonic ventral mesencephalon. Forced expression of Mash1 caused immature Nurr1-induced DA cells to differentiate into mature and functional DA neurons as judged by electrophysiological characteristics, release of DA, and expression of presynaptic DA neuronal markers. By contrast, atonal-related bHLHs, represented by Ngn1, Ngn2 and NeuroD, repressed Nurr1-induced expression of DA neuronal markers. Domain-swapping experiments with Mash1 and NeuroD indicated that the helix-loop-helix domain, responsible for mediating dimerization of bHLH transcription factors, imparts the distinct effect. Finally, transient co-transfection of the atonal-related bHLHs with Nurr1 resulted in an E-box-independent repression of Nurr1-induced transcriptional activation of a reporter containing Nurr1-binding element (NL3) as well as a reporter driven by the native tyrosine hydroxylase gene promoter. Taken together, these findings suggest that Mash1 contributes to the generation of DA neurons in cooperation with Nurr1 in the developing midbrain whereas atonal-related bHLH genes inhibit the process.

Acquisition of in vitro and in vivo functionality of Nurr1-induced dopamine neurons

Neural precursor cells provide an expandable source of neurons and glia for basic and translational applications. However, little progress has been made in directing naive neural precursors toward specific neuronal fates such as midbrain dopamine (DA) neurons. We have recently demonstrated that transgenic expression of the nuclear orphan receptor Nurr1 is sufficient to drive dopaminergic differentiation of forebrain embryonic rat neural precursors in vitro. However, Nurr1‐induced DA neurons exhibit immature neuronal morphologies and functional properties and are unable to induce behavioral recovery in rodent models of Parkinsons disease (PD). Here, we report on the identification of key genetic factors that drive morphological and functional differentiation of Nurr1‐derived DA neurons. We show that coexpression of Nurr1, Bcl‐XL, and Sonic hedgehog (SHH) or Nurr1 and the proneural bHLH factor Mash1 is sufficient to drive naive rat forebrain precursors into neurons exhibiting the biochemical, electrophysiological, and functional properties of DA neuron in vitro. On transplantation into the striatum of Parkinsonian rats, precursor cells engineered with Nurr1/SHH/Bcl‐XL or Nurr1/Mash1 survived in vivo and differentiated into mature DA neurons that can reverse the behavioral deficits in the grafted animals.—Park, C.‐H., Kang, J. S., Shin, Y. H., Chang, M.‐Y., Chung, S., Koh, H.‐C., Zhu, M. H., Oh, S. B., Lee, Y.‐S., Panagiotakos, G., Tabar, V., Studer, L., and Lee, S.‐H. Acquisition of in vitro and in vivo functionality of Nurr1‐induced dopamine neurons. FASEB J. 20, E1910‐E1923 (2006)

Ictal electrocorticographic findings related with surgical outcomes in nonlesional neocortical epilepsy.

PURPOSE To characterize ictal electrocorticographic features related to surgical outcomes in nonlesional neocortical epilepsy (NE). METHODS We analyzed 187 ictal electrocorticograms (ECoG) obtained from 18 patients who had undergone presurgical evaluation and subsequent neocortical resections (frontal: seven, parietal: one, occipital: four, multilobar: six). None of them had any MRI-detectable lesions. Various ECoG data sets recorded from eight patients who achieved a favorable surgical outcome (either seizure free or more than 90% reduction of seizure frequencies) were compared with that from ten patients with unfavorable outcome (less than 90% reduction of seizure frequencies) (follow up duration: 47+/-11 months). RESULTS Reproducible ictal onset zone (IOZ) in recurrent seizures (P=0.013) and persistent ictal discharges in IOZ from the onset to the end of seizure (P=0.004) were found more frequently in the patients with good outcome. Ictal onset patterns consisting of low voltage fast or high amplitude beta spikes predicted a good surgical outcome while rhythmic sinusoidal activity or rhythmic spike/sharp waves of slow frequency were predictive of poor outcome (P=0.01). The ictal onset rhythm consisting of gamma or beta frequencies was more prevalent in the favorable group (P=0.015). CONCLUSIONS The presence of stable ictal circuit suggested by the consistent earliest activation of specific electrodes in the repetitive seizures (reproducible IOZ) and the active participation of IOZ throughout the attack were valuable prognostic factors in addition to the morphology and frequency of ictal onset rhythm.

Modulation of N-type Ca2+ currents by moxonidine via imidazoline I1 receptor activation in rat superior cervical ganglion neurons

Moxonidine, an imidazoline deriviatives, suppress the vasopressor sympathetic outflow to produce hypotension. This effect has been known to be mediated in part by suppressing sympathetic outflow via acting imidazoline I(1) receptors (IR(1)) at postganglionic sympathetic neurons. But, the cellular mechanism of IR(1)-induced inhibition of noradrenaline (NA) release is still unknown. We therefore, investigated the effect of IR(1) activation on voltage-dependent Ca(2+) channels which is known to play an pivotal role in regulating NA in rat superior cervical ganglion (SCG) neurons, using the conventional whole-cell patch-clamp method. In the presence of rauwolscine (3 μΜ), which blocks α(2)-adrenoceptor (R(α2)), moxonidine inhibited voltage-dependent Ca(2+) current (I(Ca)) by about 30%. This moxonidine-induced inhibition was almost completely prevented by efaroxan (10 μΜ) which blocks IR(1) as well as R(α2). In addition, ω-conotoxin (CgTx) GVIA (1 μΜ) occluded moxonidine-induced inhibition of I(Ca), but, moxonidine-induced I(Ca) inhibition was not affected by pertussis toxin (PTX) nor shows any characteristics of voltage-dependent inhibition. These data suggest that moxonidine inhibit voltage-dependent N-type Ca(2+) current (I(Ca-N)) via activating IR(1). Finally, moxonidine significantly decreased the frequency of AP firing in a partially reversible manner. This inhibition of AP firing was almost completely occluded in the presence of ω-CgTx. Taken together, our results suggest that activation of IR(1) in SCG neurons reduced I(Ca-N) in a PTX-and voltage-insensitive pathway, and this inhibition attenuated repetitive AP firing in SCG neurons.

Experimental Physiology –Research Paper: Modulation of N‐type calcium currents by presynaptic imidazoline receptor activation in rat superior cervical ganglion neurons

Presynaptic imidazoline receptors (Ri‐pre) are found in the sympathetic axon terminals of animal and human cardiovascular systems, and they regulate blood pressure by modulating the release of peripheral noradrenaline (NA). The cellular mechanism of Ri‐pre‐induced inhibition of NA release is unknown. We, therefore, investigated the effect of Ri‐pre activation on voltage‐dependent Ca2+ channels in rat superior cervical ganglion (SCG) neurons, using the conventional whole‐cell patch‐clamp method. Cirazoline (30 μm), an Ri‐pre agonist as well as an α‐adrenoceptor (Rα) agonist, decreased Ca2+ currents (ICa) by about 50% in a voltage‐dependent manner with prepulse facilitation. In the presence of low‐dose rauwolscine (3 μm), which blocks the α2‐adrenoceptor (Rα2), cirazoline still inhibited ICa by about 30%, but prepulse facilitation was significantly attenuated. This inhibitory action of cirazoline was almost completely prevented by high‐dose rauwolscine (30 μm), which blocks Ri‐pre as well as Rα2. In addition, pretreatment with LY320135 (10 μm), another Ri‐pre antagonist, in combination with low‐dose rauwolscine (3 μm), also blocked the Rα2‐resistant effect of cirazoline. Addition of guanosine‐5′‐O‐(2‐thiodiphosphate) (2 mm) to the internal solutions significantly attenuated the action of cirazoline. However, pertussis toxin (500 ng ml−1) did not significantly influence the inhibitory effect of cirazoline. Moreover, cirazoline (30 μm) suppressed M current in SCG neurons cultured overnight. Finally, ω‐conotoxin (ω‐CgTx) GVIA (1 μm) obstructed cirazoline‐induced current inhibition, and cirazoline (30 μm) significantly decreased the frequency of action potential firing in a partly reversible manner. This cirazoline‐induced inhibition of action potential firing was almost completely occluded in the presence of ω‐CgTx. Taken together, our results suggest that activation of Ri‐pre in SCG neurons reduced N‐type ICa in a pertussis toxin‐ and voltage‐insensitive pathway, and this inhibition attenuated repetitive action potential firing in SCG neurons.

Intracellular acidification evoked by moderate extracellular acidosis attenuates transient receptor potential V1 (TRPV1) channel activity in rat dorsal root ganglion neurons

Transient receptor potential V1 (TRPV1) has been suggested to play an important role in detecting decreases in extracellular pH (pHo). Results from recent in vivo studies, however, have suggested that TRPV1 channels play less of a role in sensing a moderately acidic pHo (6.0 < pH < 7.0) than predicted from the in vitro experiments. A clear explanation for this discrepancy between the in vitro and in vivo data has not yet been provided. We report here that intracellular acidification induced by a moderately low pHo (6.4) almost completely inhibited the effect of extracellular acidosis on TRPV1 activity. In our experiments, sodium acetate (20 mm), which was used to induce intracellular acidosis, attenuated the capsaicin‐evoked TRPV1 current (ICAP) in a reversible manner in whole‐cell patch‐clamp mode and shifted the concentration–response curve to the right. Likewise, the concentration–response curve was significantly shifted to the right by lowering the pH of the pipette solution from 7.2 to 6.5. In addition, application of an acidic bath solution (pH 6.4) to the intracellular side also significantly suppressed ICAP in inside‐out patch mode. In cell‐attached patch mode, the single‐channel activity of iCAP was significantly attenuated by intracellular acidosis that was induced by a decrease in pHo (6.4). These results suggested that intracellular acidification induced by a low pHo inhibited TRPV1 activity. When studied in perforated patch mode or by acidifying the intracellular pipette solution, potentiation or activation of TRPV1 by extracellular acidosis (pH 6.4) at 37°C was almost completely inhibited. Likewise, enhancement of neuronal excitability by a moderately acidic pHo (6.4) at a physiological temperature (37°C) was attenuated by lowering the pH of the pipette solution to 6.5 or using perforated patch mode. Taken together, these results suggest that extracellular acidosis of moderate intensity may not significantly modulate TRPV1 activity in physiological conditions at which intracellular pH can be readily affected by pHo, and this phenomenon is due to attenuation of TRPV1 channel activity by low‐pHo‐induced intracellular acidification.

Suppression of Peripheral Sympathetic Activity Underlies Protease-Activated Receptor 2-Mediated Hypotension

Protease-activated receptor (PAR)-2 is expressed in endothelial cells and vascular smooth muscle cells. It plays a crucial role in regulating blood pressure via the modulation of peripheral vascular tone. Although some reports have suggested involvement of a neurogenic mechanism in PAR-2-induced hypotension, the accurate mechanism remains to be elucidated. To examine this possibility, we investigated the effect of PAR-2 activation on smooth muscle contraction evoked by electrical field stimulation (EFS) in the superior mesenteric artery. In the present study, PAR-2 agonists suppressed neurogenic contractions evoked by EFS in endothelium-denuded superior mesenteric arterial strips but did not affect contraction elicited by the external application of noradrenaline (NA). However, thrombin, a potent PAR-1 agonist, had no effect on EFS-evoked contraction. Additionally, ω-conotoxin GVIA (CgTx), a selective N-type Ca2+ channel (ICa-N) blocker, significantly inhibited EFS-evoked contraction, and this blockade almost completely occluded the suppression of EFS-evoked contraction by PAR-2 agonists. Finally, PAR-2 agonists suppressed the EFS-evoked overflow of NA in endothelium-denuded rat superior mesenteric arterial strips and this suppression was nearly completely occluded by ω-CgTx. These results suggest that activation of PAR-2 may suppress peripheral sympathetic outflow by modulating activity of ICa-N which are located in peripheral sympathetic nerve terminals, which results in PAR-2-induced hypotension.