Restoration of FVIII expression by targeted gene insertion in the FVIII locus in hemophilia A patient-derived iPSCs.

Target-specific genome editing, using engineered nucleases zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), and type II clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9), is considered a promising approach to correct disease-causing mutations in various human diseases. In particular, hemophilia A can be considered an ideal target for gene modification via engineered nucleases because it is a monogenic disease caused by a mutation in coagulation factor VIII (FVIII), and a mild restoration of FVIII levels in plasma can prevent disease symptoms in patients with severe hemophilia A. In this study, we describe a universal genome correction strategy to restore FVIII expression in induced pluripotent stem cells (iPSCs) derived from a patient with hemophilia A by the human elongation factor 1 alpha (EF1α)-mediated normal FVIII gene expression in the FVIII locus of the patient.

Pain-Relieving Effects of mTOR Inhibitor in the Anterior Cingulate Cortex of Neuropathic Rats.

The anterior cingulate cortex (ACC) is a well-known brain area that is associated with pain perception. Previous studies reported that the ACC has a specific role in the emotional processing of pain. Chronic pain is characterized by long-term potentiation that is induced in pain pathways and contributes to hyperalgesia caused by peripheral nerve injury.

Neuronal-Glial Interactions Maintain Chronic Neuropathic Pain after Spinal Cord Injury.

The hyperactive state of sensory neurons in the spinal cord enhances pain transmission. Spinal glial cells have also been implicated in enhanced excitability of spinal dorsal horn neurons, resulting in pain amplification and distortions. Traumatic injuries of the neural system such as spinal cord injury (SCI) induce neuronal hyperactivity and glial activation, causing maladaptive synaptic plasticity in the spinal cord.

Rapid generation of OPC-like cells from human pluripotent stem cells for treating spinal cord injury.

Remyelination via the transplantation of oligodendrocyte precursor cells (OPCs) has been considered as a strategy to improve the locomotor deficits caused by traumatic spinal cord injury (SCI). To date, enormous efforts have been made to derive OPCs from human pluripotent stem cells (hPSCs), and significant progress in the transplantation of such cells in SCI animal models has been reported. The current methods generally require a long period of time (>2 months) to obtain transplantable OPCs, which hampers their clinical utility for patients with SCI.

Reactive oxygen species affect spinal cell type-specific synaptic plasticity in a model of neuropathic pain.

Spinal synaptic plasticity is believed to drive central sensitization that underlies the persistent nature of neuropathic pain. Our recent data showed that synaptic plasticity in the dorsal horn is cell type specific: intense afferent stimulation produced long-term potentiation (LTP) in excitatory spinothalamic tract neurons (STTn), whereas it produced long-term depression (LTD) in inhibitory GABAergic interneurons (GABAn). In addition, reactive oxygen species (ROS) were shown to be involved in LTP in STTn (STTn-LTP) and in LTD in GABAn (GABAn-LTD).

Peripheral NMDA Receptors Mediate Antidromic Nerve Stimulation-Induced Tactile Hypersensitivity in the Rat.

We investigated the role of peripheral NMDA receptors (NMDARs) in antidromic nerve stimulation-induced tactile hypersensitivity outside the skin area innervated by stimulated nerve. Tetanic electrical stimulation (ES) of the decentralized L5 spinal nerve, which induced enlargement of plasma extravasation, resulted in tactile hypersensitivity in the L4 plantar dermatome of the hind-paw. When intraplantar (i.

Modulation of Spinal GABAergic Inhibition and Mechanical Hypersensitivity following Chronic Compression of Dorsal Root Ganglion in the Rat.

Chronic compression of dorsal root ganglion (CCD) results in neuropathic pain. We investigated the role of spinal GABA in CCD-induced pain using rats with unilateral CCD. A stereological analysis revealed that the proportion of GABA-immunoreactive neurons to total neurons at L4/5 laminae I-III on the injured side decreased in the early phase of CCD (post-CCD week 1) and then returned to the sham-control level in the late phase (post-CCD week 18).

Different spatial expressions of c-Fos in the nucleus of the solitary tract following taste stimulation with sodium, potassium, and ammonium ions in rats.

Cation-specific epithelial receptors on the tongue have been well demonstrated. However, active regions along the nucleus of the solitary tract (NST) for cations Na(+), K(+), NH4(+) are still unclear, even though the best responses of NST neurons to taste stimuli vary depending on the cell. In the present study, the spatial distribution patterns of cation-specific active regions in the NST are investigated.

Peripheral afferent mechanisms underlying acupuncture inhibition of cocaine behavioral effects in rats.

Administration of cocaine increases locomotor activity by enhancing dopamine transmission. To explore the peripheral mechanisms underlying acupuncture treatment for drug addiction, we developed a novel mechanical acupuncture instrument (MAI) for objective mechanical stimulation. The aim of this study was to evaluate whether acupuncture inhibition of cocaine-induced locomotor activity is mediated through specific peripheral nerves, the afferents from superficial or deep tissues, or specific groups of nerve fibers.

Effects of human mesenchymal stem cell transplantation combined with polymer on functional recovery following spinal cord hemisection in rats.

The spontaneous axon regeneration of damaged neurons is limited after spinal cord injury (SCI). Recently, mesenchymal stem cell (MSC) transplantation was proposed as a potential approach for enhancing nerve regeneration that avoids the ethical issues associated with embryonic stem cell transplantation. As SCI is a complex pathological entity, the treatment of SCI requires a multipronged approach.

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.

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.

Ionotropic glutamate receptors contribute to maintained neuronal hyperexcitability following spinal cord injury in rats.

In this study, we examined whether topical treatment of glutamate receptor antagonists attenuate hyperexcitability of lumbar spinal dorsal horn neurons following low thoracic hemisection spinal cord injury in rats. Four weeks after spinal hemisection, neuronal activity in response to mechanical stimuli applied on the peripheral receptive field was significantly increased in three different phenotypes of lumbar spinal dorsal horn neurons: wide dynamic range (WDR), low threshold (LT) and high threshold (HT). Topical application of MK-801 (NMDA receptor antagonist, 50 microg) significantly attenuated the activity of WDR, but not LT and HT neurons; whereas, NBQX (AMPA receptor antagonist, 0.

Bilateral hyperexcitability of thalamic VPL neurons following unilateral spinal injury in rats.

In the present study, we have examined whether spinal hemisection injury induces changes in the electrophysiological properties of thalamic ventral posteriorlateral (VPL) neurons in rats. Male Sprague-Dawley rats were subjected to unilateral spinal cord injury by transverse hemisection at the T13 spinal segment. Four weeks after the T13 spinal hemisection, the injured rats displayed robust allodynic behaviors on both sides of hindpaws compared to sham controls (P < 0.

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.

Spinal AMPA receptor inhibition attenuates mechanical allodynia and neuronal hyperexcitability following spinal cord injury in rats.

In this study, we examined whether a competitive AMPA receptor antagonist, NBQX, attenuates mechanical allodynia and hyperexcitability of spinal neurons in remote, caudal regions in persistent central neuropathic pain following spinal cord injury in rats. Spinal cord injury was produced by unilateral T13 transverse spinal hemisection, from dorsal to ventral, in male Sprague Dawley rats (200-250 g). Mechanical thresholds were measured behaviorally, and the excitability of wide-dynamic-range (WDR) dorsal horn neurons in the lumbar cord (L4-L5) was measured to assess central neuropathicpain.

The role of uninjured C-afferents and injured afferents in the generation of mechanical hypersensitivity after partial peripheral nerve injury in the rat.

This study was performed to determine which of uninjured lumbar 4 (L4) C-afferents and injured L5 afferents was important for the generation of mechanical hypersensitivity following L5 spinal nerve ligation-and-cut (SNLC, modified spinal nerve ligation) in the rat. The mechanical hypersensitivity established following L5 SNLC was completely abolished 6 weeks after local capsaicin treatment of the sciatic nerve or L4 spinal nerve. At this stage, a substantial number of capsaicin-sensitive C-afferents were eliminated without any loss of A-afferents in the L4 spinal segment, suggesting that the capsaicin-sensitive L4 C-afferents are a major contributor to L5 SNLC-produced mechanical hypersensitivity.

Activation of spinal GABA receptors attenuates chronic central neuropathic pain after spinal cord injury.

In this study, we investigated the role of the spinal GABAergic system in central neuropathic painlike outcomes following spinal cord injury (SCI) produced by a spinal hemitransection at T13 of the rat. After SCI, mechanical allodynia develops bilaterally in both hind paws of the rat, lasting longer than 40 days, as evidenced by an increase in paw withdrawal frequency in response to a weak von Frey filament. In naive rats, intrathecal (i.

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.

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.