Satellite glial cells (SGCs) are homeostatic cells enveloping the somata of peripheral sensory and autonomic neurons. A wide variety of neuronal stressors trigger activation of SGCs, contributing to, for example, neuropathic pain through modulation of neuronal activity. However, compared to neurons and other glial cells of the nervous system, SGCs have received modest scientific attention and very little is known about SGC biology, possibly due to the experimental challenges associated with studying them in vivo and in vitro. Utilizing a recently developed method to obtain SGC RNA from dorsal root ganglia (DRG), we took a systematic approach to characterize the SGC transcriptional fingerprint by using next-generation sequencing and, for the first time, obtain an overview of the SGC injury response. Our RNA sequencing data are easily accessible in supporting information in Excel format. They reveal that SGCs are enriched in genes related to the immune system and cell-to-cell communication. Analysis of SGC transcriptional changes in a nerve injury-paradigm reveal a differential response at 3 days versus 14 days postinjury, suggesting dynamic modulation of SGC function over time. Significant downregulation of several genes linked to cholesterol synthesis was observed at both time points. In contrast, regulation of gene clusters linked to the immune system (MHC protein complex and leukocyte migration) was mainly observed after 14 days. Finally, we demonstrate that, after nerve injury, macrophages are in closer physical proximity to both small and large DRG neurons, and that previously reported injury-induced proliferation of SGCs may, in fact, be proliferating macrophages. K E Y W O R D Sdorsal root ganglion, macrophages, nerve injury, nociceptors, pain, peripheral nervous system, satellite glial cells, transcriptome

Motor neurons project axons from the hindbrain and spinal cord to muscle, where they induce myofibre contractions through neurotransmitter release at neuromuscular junctions. Studies of neuromuscular junction formation and homeostasis have been largely confined to in vivo models. In this study, three powerful tools have been merged—pluripotent stem cells, optogenetics, and microfabrication—and an open microdevice is designed in which motor axons grow from a neural compartment containing embryonic stem cell‐derived motor neurons and astrocytes through microchannels to form functional neuromuscular junctions with contractile myofibres in a separate compartment. Optogenetic entrainment of motor neurons in this reductionist neuromuscular circuit enhances neuromuscular junction formation more than twofold, mirroring the activity‐dependence of synapse development in vivo. An established motor neuron disease model is incorporated into the system and it is found that coculture of motor neurons with SOD1G93A astrocytes results in denervation of the central compartment and diminishes myofibre contractions, a phenotype which is rescued by the receptor interacting serine/threonine kinase 1 inhibitor necrostatin. This coculture system replicates key aspects of nerve–muscle connectivity in vivo and represents a rapid and scalable alternative to animal models of neuromuscular function and disease.

Number-average molecular weight; b) Weight-average molecular weight; c) Molecular weight of highest peak; d) Dispersion index.

Correct communication between immune cells and peripheral neurons is crucial for the protection of our bodies. Its breakdown is observed in many common, often painful conditions, including arthritis, neuropathies, and inflammatory bowel or bladder disease. Here, we have characterised the immune response in a mouse model of neuropathic pain using flow cytometry and cell-type-specific RNA sequencing (RNA-seq). We found few striking sex differences, but a very persistent inflammatory response, with increased numbers of monocytes and macrophages up to 3 1/2 months after the initial injury. This raises the question of whether the commonly used categorisation of pain into “inflammatory” and “neuropathic” is one that is mechanistically appropriate. Finally, we collated our data with other published RNA-seq data sets on neurons, macrophages, and Schwann cells in naive and nerve injury states. The result is a practical web-based tool for the transcriptional data mining of peripheral neuroimmune interactions. http://rna-seq-browser.herokuapp.com/

Duchenne muscular dystrophy (DMD) is caused by dystrophin gene mutations leading to skeletal muscle weakness and wasting. Dystrophin is enriched at the neuromuscular junction (NMJ), but how NMJ abnormalities contribute to DMD pathogenesis remains unclear. Here, we combine transcriptome analysis and modeling of DMD patientderived neuromuscular circuits with CRISPR-corrected isogenic controls in compartmentalized microdevices. We show that NMJ volumes and optogenetic motor neuron-stimulated myofiber contraction are compromised in DMD neuromuscular circuits, which can be rescued by pharmacological inhibition of TGF signaling, an observation validated in a 96-well human neuromuscular circuit coculture assay. These beneficial effects are associated with normalization of dysregulated gene expression in DMD myogenic transcriptomes affecting NMJ assembly (e.g., MUSK) and axon guidance (e.g., SLIT2 and SLIT3). Our study provides a new human microphysiological model for investigating NMJ defects in DMD and assessing candidate drugs and suggests that enhancing neuromuscular connectivity may be an effective therapeutic strategy.

Correct communication between immune cells and peripheral neurons is crucial for the protection of our bodies. Its breakdown is observed in many common, often painful conditions, including arthritis, neuropathies and inflammatory bowel or bladder disease. Here, we have characterised the immune response in a mouse model of neuropathic pain using flow cytometry and cell-type specific RNA sequencing (RNA-seq). We found few striking sex differences, but a very persistent inflammatory response, with increased numbers of monocytes and macrophages up to 3½ months after the initial injury. This raises the question of whether the commonly used categorisation of pain into "inflammatory" and "neuropathic" is one that is mechanistically appropriate. Finally, we collated our data with other published RNA-seq datasets on neurons, satellite glial cells, macrophages and Schwann cells in naïve and nerve injury states. The result is a practical web-based tool for the transcriptional data-mining of peripheral neuroimmune interactions. Figure 8: At one week post PSNL, MHCII+/Ly6C-myeloid cells from sciatic nerve upregulate functions relating to interactions with other immune cells, in favour of more generic pro-inflammatory and homeostatic activities.A) STRING network analysis reveals that 77 of 186 genes upregulated in ipsilateral MHCII+ macrophages at adj. p < 0.05 are likely to be functionally connected with overrepresented processes including antigen presentation, regulation of lymphocytes and myeloid cell activation. B) Conversely, 41 of 93 significantly downregulated genes formed a network that includes transcripts relating to pro-inflammatory function (TNF, complements), regulation of angiogenesis and canonical macrophage markers, like CD163 typically found in resident macrophages. See Suppl. Table 5 for differential expression tables.

Dysregulated neuronal excitability is a hallmark of amyotrophic lateral sclerosis (ALS). We sought to investigate how functional changes to the axon initial segment (AIS), the site of action potential generation, could impact neuronal excitability in a human iPSC model of ALS. We found that early (6-week) ALS-related TDP-43G298S motor neurons showed an increase in the length of the AIS, relative to CRISPR-corrected controls. This was linked to neuronal hyperexcitability and increased spontaneous contractions of hiPSC-myofibers in compartmentalised neuromuscular co-cultures. In contrast late (10-week) TDP-43G298S motor neurons showed reduced AIS length and hypoexcitability. At a molecular level aberrant expression of the AIS master scaffolding protein Ankyrin-G, and the AIS-specific voltage-gated ion channels SCN1A (Nav1.1) and SCN8A (Nav1.6) mirrored these dynamic changes in excitability. Finally, at all stages, TDP-43G298S motor neurons showed compromised activity-dependent plasticity of the AIS, further contributing to abnormal excitability. Our results point toward the AIS as an important subcellular target driving changes to neuronal excitability in ALS.

Regenerative medicine is a diverse and rapidly evolving field, employing core expertise from biologists, engineers, and clinicians. Recently the field has made significant progress towards regenerating or replacing tissues lost to age, disease or injury. Current strategies include transplantation of adult or pluripotent stem cells to replace tissue or support tissue healing. Promising approaches for the future of regenerative medicine include stimulating endogenous stem cells for in situ repair, transplantation of organoids to repair minor tissue injury, and the use of interspecies chimerism to produce functional metabolic organs for transplantation. In our review we focus on these emerging strategies, paying particular attention to their current and prospective translational impacts and challenges.