Science-based assessment of source materials for cell-based medicines: report of a stakeholders workshop.

Human pluripotent stem cells (hPSCs) have the potential to transform medicine. However, hurdles remain to ensure safety for such cellular products. Science-based understanding of the requirements for source materials is required as are appropriate materials. Leaders in hPSC biology, clinical translation, biomanufacturing and regulatory issues were brought together to define requirements for source materials for the production of hPSC-derived therapies and to identify other key issues for the safety of cell therapy products. While the focus of this meeting was on hPSC-derived cell therapies, many of the issues are generic to all cell-based medicines. The intent of this report is to summarize the key issues discussed and record the consensus reached on each of these by the expert delegates.

Organizational profile: UK regenerative medicine platform immunomodulation hub.

The UK Regenerative Medicine Platform was launched in 2013 as a jointly funded venture by the Biotechnology and Biological Sciences Research Council (BBSRC), Biotechnology and Biological Sciences Research Council, Engineering and Physical Sciences Research Council (EPSRC) and Medical Research Council (MRC) to address the technical and scientific challenges associated with translating promising scientific discoveries into the clinical setting. The first stage of the Platform involved the establishment of five interdisciplinary and cross-institutional research Hubs and the final Hub, the Immunomodulation Hub, was formed in 2014. The Immunomodulation Hub comprises scientists from diverse clinical and nonclinical research backgrounds. Collectively, they provide expertise in tissues for which there is an unmet clinical need for regenerative treatments, in innate and adaptive immunity and in whole organ transplantation. Their vision is that by working together to determine how regenerative medicine cell therapies in a laboratory setting are affected by the immune system, they will make a substantial contribution to long-term clinical deliverables that include improved efficacy of photoreceptor cell therapy to treat blindness; improved repair of damaged heart tissue; and improved survival and functionality of transplanted hepatocytes as an alternative to liver transplantation.

Differential joint-specific corticospinal tract projections within the cervical enlargement.

The motor cortex represents muscle and joint control and projects to spinal cord interneurons and-in many primates, including humans-motoneurons, via the corticospinal tract (CST). To examine these spinal CST anatomical mechanisms, we determined if motor cortex sites controlling individual forelimb joints project differentially to distinct cervical spinal cord territories, defined regionally and by the locations of putative last-order interneurons that were transneuronally labeled by intramuscular injection of pseudorabies virus. Motor cortex joint-specific sites were identified using intracortical-microstimulation. CST segmental termination fields from joint-specific sites, determined using anterograde tracers, comprised a high density core of terminations that was consistent between animals and a surrounding lower density projection that was more variable. Core terminations from shoulder, elbow, and wrist control sites overlapped in the medial dorsal horn and intermediate zone at C5/C6 but were separated at C7/C8. Shoulder sites preferentially terminated dorsally, in the dorsal horn; wrist/digit sites, more ventrally in the intermediate zone; and elbow sites, medially in the dorsal horn and intermediate zone. Pseudorabies virus injected in shoulder, elbow, or wrist muscles labeled overlapping populations of predominantly muscle-specific putative premotor interneurons, at a survival time for disynaptic transfer from muscle. At C5/C6, CST core projections from all joint zones were located medial to regions of densely labeled last-order interneurons, irrespective of injected muscle. At C7/C8 wrist CST core projections overlapped the densest interneuron territory, which was located in the lateral intermediate zone. In contrast, elbow CST core projections were located medial to the densest interneuron territories, and shoulder CST core projections were located dorsally and only partially overlapped the densest interneuron territory. Our findings show a surprising fractionation of CST terminations in the caudal cervical enlargement that may be organized to engage different spinal premotor circuits for distal and proximal joint control.

Developmental plasticity of descending motor pathways.

Juvenile animal brains are highly plastic and thus often achieve better functional recovery after injury compared with adult brains. Recently, Umeda et al. (Umeda T, Takahashi M, Isa K, Isa T. J Neurophysiol 104: 1707-1716, 2010) have shown that the remodeling of both corticospinal and extra-pyramidal pathways can contribute to the recovery of grasping and reaching ability in hemidecorticated juvenile rats. They have further unveiled the strengthening of the cortico-reticulo-spinal pathway after injury, that mediates the fast excitation of ipsilateral motoneurons for functional recovery.

Cortical control of adaptive locomotion in wild-type mice and mutant mice lacking the ephrin-Eph effector protein alpha2-chimaerin.

In voluntary control, supraspinal motor systems select the appropriate response and plan movement mechanics to match task constraints. Spinal circuits translate supraspinal drive into action. We studied the interplay between motor cortex (M1) and spinal circuits during voluntary movements in wild-type (WT) mice and mice lacking the α2-chimaerin gene (Chn1(-/-)), necessary for ephrinB3-EphA4 signaling. Chn1(-/-) mice have aberrant bilateral corticospinal systems, aberrant bilateral-projecting spinal interneurons, and disordered voluntary control because they express a hopping gait, which may be akin to mirror movements. We addressed three issues. First, we determined the role of the corticospinal system in adaptive control. We trained mice to step over obstacles during treadmill locomotion. We compared performance before and after bilateral M1 ablation. WT mice adaptively modified their trajectory to step over obstacles, and M1 ablation increased substantially the incidence of errant steps over the obstacle. Chn1(-/-) mice randomly stepped or hopped during unobstructed locomotion but hopped over the obstacle. Bilateral M1 ablation eliminated this obstacle-dependent hop selection and increased forelimb obstacle contact errors. Second, we characterized the laterality of corticospinal action in Chn1(-/-) mice using pseudorabies virus retrograde transneuronal transport and intracortical microstimulation. We showed bilateral connections between M1 and forelimb muscles in Chn1(-/-) and unilateral connections in WT mice. Third, in Chn1(-/-) mice, we studied adaptive responses before and after unilateral M1 ablation. We identified a more important role for contralateral than ipsilateral M1 in hopping over the obstacle. Our findings suggest an important role for M1 in the mouse in moment-to-moment adaptive control, and further, using Chn1(-/-) mice, a role in mediating task-dependent selection of mirror-like hopping movements over the obstacle. Our findings also stress the importance of subcortical control during adaptive locomotion because key features of the trajectory remained largely intact after M1 ablation.

Descending serotonergic facilitation mediated by spinal 5-HT3 receptors engages spinal rapamycin-sensitive pathways in the rat.

We have recently reported the importance of spinal rapamycin-sensitive pathways in maintaining persistent pain-like states. A descending facilitatory drive mediated through spinal 5-HT3 receptors (5-HT3Rs) originating from superficial dorsal horn NK1-expressing neurons and that relays through the parabrachial nucleus and the rostroventral medial medulla to act on deep dorsal horn neurons is known be important in maintaining these pain-like states. To determine if spinal rapamycin-sensitive pathways are activated by a descending serotonergic drive, we investigated the effects of spinally administered rapamycin on responses of deep dorsal horn neurons that had been pre-treated with the selective 5-HT3R antagonist ondansetron. We also investigated the effects of spinally administered cell cycle inhibitor (CCI)-779 (a rapamycin ester analogue) on deep dorsal horn neurons from rats with carrageenan-induced inflammation of the hind paw. Unlike some other models of persistent pain, this model does not involve an altered 5-HT3R-mediated descending serotonergic drive. We found that the inhibitory effects of rapamycin were significantly reduced for neuronal responses to mechanical and thermal stimuli when the spinal cord was pre-treated with ondansetron. Furthermore, CCI-779 was found to be ineffective in attenuating spinal neuronal responses to peripheral stimuli in carrageenan-treated rats. Therefore, we conclude that 5-HT3R-mediated descending facilitation is one requirement for activation of rapamycin-sensitive pathways that contribute to persistent pain-like states.

Small RNAs control sodium channel expression, nociceptor excitability, and pain thresholds.

To examine the role of small RNAs in peripheral pain pathways, we deleted the enzyme Dicer in mouse postmitotic damage-sensing neurons. We used a Nav1.8-Cre mouse to target those nociceptors important for inflammatory pain. The conditional null mice were healthy with a normal number of sensory neurons and normal acute pain thresholds. Behavioral studies showed that inflammatory pain was attenuated or abolished. Inflammatory mediators failed to enhance excitability of Nav1.8+ sensory neurons from null mutant mice. Acute noxious input into the dorsal horn of the spinal cord was apparently normal, but the increased input associated with inflammatory pain measured using c-Fos staining was diminished. Microarray and quantitative real-time reverse-transcription PCR (qRT-PCR) analysis showed that Dicer deletion lead to the upregulation of many broadly expressed mRNA transcripts in dorsal root ganglia. By contrast, nociceptor-associated mRNA transcripts (e.g., Nav1.8, P2xr3, and Runx-1) were downregulated, resulting in lower levels of protein and functional expression. qRT-PCR analysis also showed lowered levels of expression of nociceptor-specific pre-mRNA transcripts. MicroRNA microarray and deep sequencing identified known and novel nociceptor microRNAs in mouse Nav1.8+ sensory neurons that may regulate nociceptor gene expression.

Mammalian target of rapamycin signaling in the spinal cord is required for neuronal plasticity and behavioral hypersensitivity associated with neuropathy in the rat.

UNLABELLED: The protein kinase mammalian target of rapamycin (mTOR) regulates mRNA translation and is inhibited by rapamycin. Signaling pathways involving mTOR are implicated in physiological and pathophysiological processes. We determined the spinal effects of the rapamycin analogue cell cycle inhibitor (CCI)-779 on neuronal responses and behavioral hypersensitivity in a model of persistent neuropathic pain. We also assessed the anatomical distribution of spinal mTOR signaling pathways. Specifically, we ligated rat spinal nerves L5 and L6 to produce a model of neuropathic pain. After confirming neuropathy with behavioral testing, we obtained in vivo single-unit extracellular stimulus-evoked recordings from deep dorsal horn spinal neurons. We applied CCI-779 spinally in electrophysiological and behavioral studies and assessed its effects accordingly. We also used immunohistochemistry to probe for mTOR signaling pathways in dorsal root ganglia (DRG) and the spinal cord. We found that spinally administered CCI-779 rapidly attenuated calibrated mechanically but not thermally evoked neuronal responses and mechanically evoked behavioral responses. Immunohistochemistry showed presence of mTOR signaling pathways in nociceptive-specific C-fiber DRG and in neurons of inner lamina II of the spinal cord. We conclude that alterations in the activity of spinal mTOR signaling pathways are crucial to the full establishment of spinal neuronal plasticity and behavioral hypersensitivity associated with nerve injury. PERSPECTIVE: This study is consistent with growing evidence implicating mTOR signaling pathways as important modulators of persistent pain, providing novel insights into the molecular mechanisms of pain maintenance and potential for novel approaches into treating chronic pain.

Formalin-induced behavioural hypersensitivity and neuronal hyperexcitability are mediated by rapid protein synthesis at the spinal level.

BACKGROUND: The mammalian target of rapamycin (mTOR) is a key regulator of mRNA translation whose action can be inhibited by the drug rapamycin. Forms of long-term plasticity require protein synthesis and evidence indicates that mRNA in dendrites, axon terminals and cell bodies is essential for long-term synaptic plasticity. Specific to pain, shifts in pain thresholds and responsiveness are an expression of neuronal plasticity and this likely contributes to persistent pain. We investigated this by inhibiting the activity of mTOR with rapamycin at the spinal level, of rats that were subjected to the formalin test, using both behavioural and electrophysiological techniques. RESULTS: For in vivo electrophysiology, Sprague Dawley rats were fully anaesthetised and single-unit extracellular recordings were obtained from lamina V wide dynamic range (WDR) dorsal horn spinal neurones at the region where input is received from the hind paw. Neuronal responses from naive rats showed that rapamycin-sensitive pathways were important in nociceptive-specific C-fibre mediated transmission onto WDR neurones as well mechanically-evoked responses since rapamycin was effective in attenuating these measures. Formalin solution was injected into the hind paw prior to which, rapamycin or vehicle was applied directly onto the exposed spinal cord. When rapamycin was applied to the spinal cord prior to hind paw formalin injection, there was a significant attenuation of the prolonged second phase of the formalin test, which comprises continuing afferent input to the spinal cord, neuronal hyperexcitability and an activated descending facilitatory drive from the brainstem acting on spinal neurones. In accordance with electrophysiological data, behavioural studies showed that rapamycin attenuated behavioural hypersensitivity elicited by formalin injection into the hind paw. CONCLUSION: We conclude that mTOR has a role in maintaining persistent pain states via mRNA translation and thus protein synthesis. We hypothesise that mTOR may be activated by excitatory neurotransmitter release acting on sensory afferent terminals as well as dorsal horn spinal neurones, which may be further amplified by descending facilitatory systems originating from higher centres in the brain.

The cell and molecular basis of mechanical, cold, and inflammatory pain.

Peripheral pain pathways are activated by a range of stimuli. We used diphtheria toxin to kill all mouse postmitotic sensory neurons expressing the sodium channel Nav1.8. Mice showed normal motor activity and low-threshold mechanical and acute noxious heat responses but did not respond to noxious mechanical pressure or cold. They also showed a loss of enhanced pain responses and spontaneous pain behavior upon treatment with inflammatory insults. In contrast, nerve injury led to heightened pain sensitivity to thermal and mechanical stimuli indistinguishable from that seen with normal littermates. Pain behavior correlates well with central input from sensory neurons measured electrophysiologically in vivo. These data demonstrate that Na(v)1.8-expressing neurons are essential for mechanical, cold, and inflammatory pain but not for neuropathic pain or heat sensing.