Joong Woo Leem

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.

Robust Enhancement of Neural Differentiation from Human ES and iPS Cells Regardless of their Innate Difference in Differentiation Propensity

Our analyses of three human induced pluripotent stem cell (hiPSC) and six human embryonic stem cell (hESC) lines showed marked variability in differentiation potential into specific lineages, which often hampers their differentiation into specific cell types or cell lineages of interest. Simultaneous inhibition of both Activin/Nodal and BMP pathways with small molecules, SB431542 and dorsomorphin (DM), respectively, promoted significant neural differentiation from all human pluripotent stem cell (hPSC) lines tested, regardless of their differentiation propensity. On the contrary, differentiation into other cell lineages and the number of undifferentiated cells were significantly reduced after differentiation by the dual inhibition. These results demonstrate that innate differentiation propensity of hPSCs could be overcome, at least in part, by modulation of intracellular signaling pathways, resulting in efficient generation of desirable cell types, such as neural cells.

Mechanical and heat sensitization of cutaneous nociceptors in rats with experimental peripheral neuropathy.

This study examined whether or not the properties of cutaneous nociceptive fibers are altered in the neuropathic state by comparing lumbars 5 and 6 spinal nerve ligation (SNL) rats with sham-operated controls. The rats with the unilateral SNL developed mechanical allodynia in the ipsilateral hind limb, whereas the sham group did not. Two to 5 weeks after the neuropathic or sham surgery, rats were subjected to single fiber-recording experiments to examine the properties of afferent fibers in the sural and plantar nerves. A total of 224 afferents in the C- and Adelta-ranges were characterized in the neuropathic and sham groups. Spontaneous activity was observed in 16 of 155 fibers in the neuropathic group and one of 69 fibers in the sham group. The response threshold of both the C- and Adelta-fibers to mechanical stimuli was lower in the neuropathic group than the sham group. The afferent fibers responsive to heat stimuli were all C-fibers, and none were Adelta-fibers. The response threshold of the C-fibers to the heat stimuli was lower in the neuropathic group than the sham group. The magnitude of the responses of both C- and Adelta-fibers to the suprathreshold intensity of the mechanical stimulus was greater in the neuropathic group than the sham group. However, the magnitude of the responses of C-fibers to the suprathreshold intensity of the heat stimulus in the neuropathic group was not different from that in the sham group. These results suggest that after a partial peripheral nerve injury, the nociceptors on the skin supplied by an uninjured nerve become sensitized to both mechanical and heat stimuli. This nociceptor sensitization can contribute to neuropathic pain.

Attenuation of mechanical hyperalgesia following spinal cord injury by administration of antibodies to nerve growth factor in the rat

Spinal cord injury (SCI) often leads to central pain syndrome including hyperalgesia to mechanical stimulation. Since there is evidence that nerve growth factor (NGF) contributes to pain-related behaviors, we wished to determine if anti-NGF might inhibit abnormal somatosensory behaviors that develop following SCI in rats. SCI was performed in male Sprague-Dawley rats by T13 spinal hemisection. After spinal hemisection, animals were untreated or treated daily with anti-NGF or saline intraperitoneally for 10 days. In groups of both hemisection only and hemisection with saline treatment, mechanical hyperalgesia developed in both hindlimbs, as evidenced by a decrease in paw withdrawal thresholds. Mechanical responsiveness of wide dynamic range (WDR) neurons on both sides of spinal cord also increased. The anti-NGF treated group demonstrated significant suppression of both mechanical hyperalgesia and increased WDR neuronal responsiveness. These results indicate that anti-NGF prevents the development of abnormal somatosensory behavior and suggest a potential pre-emptive analgesic treatment for central pain.

Electrophysiological evidence for the antinociceptive effect of transcutaneous electrical stimulation on mechanically evoked responsiveness of dorsal horn neurons in neuropathic rats

Using a rat model of peripheral neuropathy induced by a tight ligation of L5-6 spinal nerves, the effects of transcutaneous electrical stimulation on the mechanical responses of wide dynamic range (WDR) dorsal horn neurons were investigated. The responses of the WDR neurons to both the brush and pinch stimuli were found to be enhanced in the neuropathic rats compared to those in the normal rats. These enhanced responses were depressed by low-frequency and high-intensity transcutaneous electrical stimulation (2 Hz, 4-5 mA) applied to the somatic receptive field. The durations of the depressive effects on the brush responses ranged between 30 and 45 min and those on the pinch responses were 60-90 min. These results imply that the transcutaneous electrical stimulation used here produces an antinociceptive effect via a depressive action on the enhanced mechanical responsiveness of the spinal neurons in this rat model of peripheral neuropathy.

Involvement of peripherally released substance P and calcitonin gene-related peptide in mediating mechanical hyperalgesia in a traumatic neuropathy model of the rat.

We hypothesized that neuropeptides released from the peripheral terminals of primary afferents play an important role in mechanical hyperalgesia after peripheral nerve injury. Nerve injury was performed on rats with lumbar 5 spinal nerve lesion (L5 SNL), which was preceded by L5 dorsal rhizotomy (L5 DR) to avoid the potential central effects induced by L5 SNL through the L5 dorsal root. L5 DR produced a short-lasting (<6 days) decrease in paw withdrawal threshold (PWT) while the following L5 SNL produced a persistent (>42 days) PWT decrease. When intraplantar injection to the affected hind paw was given immediately before L5 SNL, antagonists for both neurokinin 1 (NK1) and calcitonin gene-related peptide 1 (CGRP1) receptors delayed the onset of the PWT decrease for 2-4 days. However, when the same injection was given after L5 SNL, CGRP1, but not NK1, receptor antagonist reversed the decreased PWT for 105 min. It is suggested that peripherally released neuropeptides contribute to the generation of neuropathic pain, with substance P and CGRP contributing to its induction phase, but only CGRP to its maintenance phase.

N-Methyl-d-aspartate (NMDA) and non-NMDA glutamate receptor antagonists differentially suppress dorsal horn neuron responses to mechanical stimuli in rats with peripheral nerve injury ☆

The effects of iontophoretically ejected glutamate receptor antagonists on mechanically evoked responsiveness were examined on wide dynamic range (WDR) dorsal horn neurons in anesthetized rats that received a unilateral ligation of the L5 and L6 spinal nerves 10-15 days previously. Both brush- and pinch-evoked responses of dorsal horn neurons on the nerve-injured side were enhanced. N-Methyl-D-aspartate (NMDA) receptor antagonist, 2-amino-5-phosphonopentanoic acid (AP5), preferentially suppressed the enhanced pinch-evoked response, whereas (RS)-a-amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA) receptor selective antagonist, 6-nitro-7-sulphamoylbenzo(f)quinoxaline-2,3-dione (NBQX), preferentially attenuated the enhanced brush-evoked response. The results indicate that the enhanced responses to noxious and non-noxious peripheral inputs induced in WDR dorsal horn neurons following the nerve injury are mediated by activation of NMDA and AMPA receptors, respectively.

Highly pure and expandable PSA-NCAM-positive neural precursors from human ESC and iPSC-derived neural rosettes.

Homogeneous culture of neural precursor cells (NPCs) derived from human pluripotent stem cells (hPSCs) would provide a powerful tool for biomedical applications. However, previous efforts to expand mechanically dissected neural rosettes for cultivation of NPCs remain concerns regarding non-neural cell contamination. In addition, several attempts to purify NPCs using cell surface markers have not demonstrated the expansion capability of the sorted cells. In the present study, we show that polysialic acid-neural cell adhesion molecule (PSA-NCAM) is detected in neural rosette cells derived from hPSCs, and employ PSA-NCAM as a marker for purifying expandable primitive NPCs from the neural rosettes. PSA-NCAM-positive NPCs (termed hNPCPSA-NCAM+) were isolated from the heterogeneous cell population of mechanically harvested neural rosettes using magnetic-based cell sorting. The hNPCPSA-NCAM+ extensively expressed neural markers such as Sox1, Sox2, Nestin, and Musashi-1 (80∼98% of the total cells) and were propagated for multiple passages while retaining their primitive characteristics in our culture condition. Interestingly, PSA-NCAM-negative cells largely exhibited characteristics of neural crest cells. The hNPCPSA-NCAM+ showed multipotency and responsiveness to instructive cues towards region-specific neuronal subtypes in vitro. When transplanted into the rat striatum, hNPCPSA-NCAM+ differentiated into neurons, astrocytes, and oligodendrocytes without particular signs of tumorigenesis. Furthermore, Ki67-positive proliferating cells and non-neural lineage cells were rarely detected in the grafts of hNPCPSA-NCAM+ compared to those of neural rosette cells. Our results suggest that PSA-NCAM-mediated cell isolation provides a highly expandable population of pure primitive NPCs from hPSCs that will lend themselves as a promising strategy for drug screening and cell therapy for neurodegenerative disorders.

The role of peripheral N-methyl-D-aspartate receptors in Freund's complete adjuvant induced mechanical hyperalgesia in rats.

We investigated the role of excitatory amino acid receptors in mechanical hyperalgesia induced by subcutaneous injection of Freunds complete adjuvant (FCA) into the rat hind paw. In normal rats, an intraplantar (i.pl.) injection of L-glutamate, but not of D-glutamate (3 pmol/0.1 ml each) produced a mechanical hyperalgesia in the hind paw with a lowered paw-withdrawal threshold to pressure. In rats that developed mechanical hyperalgesia associated with inflammation in the hind paw following i.pl. injection of FCA (0.15 ml), the injection of N-methyl-D-aspartate (NMDA) receptor antagonist, MK-801 (1 pmol/0.1 ml) into the inflamed paw increased the paw pressure threshold. On the other hand, the injection of non-NMDA receptor antagonist, 6-cyano-7-nitroqiunoxaline-2,3-dione (CNQX, 10 pmol/0.1 ml) into the inflamed paw had no effect on FCA-induced lowering of the paw pressure threshold. The results suggest that NMDA, but not non-NMDA receptors play a substantial role in mediating the development of mechanical hyperalgesia induced in the inflamed paw following i.pl. FCA injection.

Involvement of α2-adrenoceptors in mediating sympathetic excitation of injured dorsal root ganglion neurons in rats with spinal nerve ligation

The present study examined the effects of sympathetic stimulation on the activity of primary afferent neurons that had peripheral axons being injured previously by a spinal nerve ligation. About 22% of afferents with injured fibers that showed spontaneous discharge were excited by sympathetic stimulation or systemic injection of adrenaline. Most sympathetically-excited afferent neurons had axons that conducted in the A-fiber range. This sympathetically-evoked afferent excitation was not affected by cutting the spinal nerve at a place close to the dorsal root ganglion (DRG). Yohimbine, alpha2-antagonist, suppressed sympathetically-evoked afferent excitation which was not affected by alpha1-antagonist prazosin. Clonidine, alpha2-agonist, exerted an excitatory effect, whereas alpha1-agonist phenylephrine had no effect on the activity of afferents with injured fibers. No afferent fibers in control preparations responded to sympathetic stimulation. The results suggest that after a spinal nerve ligation, injured DRG neurons with fast-conducting fibers become sensitive to sympathetic activity via activation of alpha2-adrenoceptors.