Peripheral Nerve Single-Cell Analysis Identifies Mesenchymal Ligands that Promote Axonal Growth.

Peripheral nerves provide a supportive growth environment for developing and regenerating axons and are essential for maintenance and repair of many non-neural tissues. This capacity has largely been ascribed to paracrine factors secreted by nerve-resident Schwann cells. Here, we used single-cell transcriptional profiling to identify ligands made by different injured rodent nerve cell types and have combined this with cell-surface mass spectrometry to computationally model potential paracrine interactions with peripheral neurons. These analyses show that peripheral nerves make many ligands predicted to act on peripheral and CNS neurons, including known and previously uncharacterized ligands. While Schwann cells are an important ligand source within injured nerves, more than half of the predicted ligands are made by nerve-resident mesenchymal cells, including the endoneurial cells most closely associated with peripheral axons. At least three of these mesenchymal ligands, ANGPT1, CCL11, and VEGFC, promote growth when locally applied on sympathetic axons. These data therefore identify an unexpected paracrine role for nerve mesenchymal cells and suggest that multiple cell types contribute to creating a highly pro-growth environment for peripheral axons.

Mesenchymal Precursor Cells in Adult Nerves Contribute to Mammalian Tissue Repair and Regeneration.

Peripheral innervation plays an important role in regulating tissue repair and regeneration. Here we provide evidence that injured peripheral nerves provide a reservoir of mesenchymal precursor cells that can directly contribute to murine digit tip regeneration and skin repair. In particular, using single-cell RNA sequencing and lineage tracing, we identify transcriptionally distinct mesenchymal cell populations within the control and injured adult nerve, including neural crest-derived cells in the endoneurium with characteristics of mesenchymal precursor cells. Culture and transplantation studies show that these nerve-derived mesenchymal cells have the potential to differentiate into non-nerve lineages. Moreover, following digit tip amputation, neural crest-derived nerve mesenchymal cells contribute to the regenerative blastema and, ultimately, to the regenerated bone. Similarly, neural crest-derived nerve mesenchymal cells contribute to the dermis during skin wound healing. These findings support a model where peripheral nerves directly contribute mesenchymal precursor cells to promote repair and regeneration of injured mammalian tissues.

Motoneurons derived from induced pluripotent stem cells develop mature phenotypes typical of endogenous spinal motoneurons.

Induced pluripotent cell-derived motoneurons (iPSCMNs) are sought for use in cell replacement therapies and treatment strategies for motoneuron diseases such as amyotrophic lateral sclerosis (ALS). However, much remains unknown about the physiological properties of iPSCMNs and how they compare with endogenous spinal motoneurons or embryonic stem cell-derived motoneurons (ESCMNs). In the present study, we first used a proteomic approach and compared protein expression profiles between iPSCMNs and ESCMNs to show that <4% of the proteins identified were differentially regulated. Like ESCs, we found that mouse iPSCs treated with retinoic acid and a smoothened agonist differentiated into motoneurons expressing the LIM homeodomain protein Lhx3. When transplanted into the neural tube of developing chick embryos, iPSCMNs selectively targeted muscles normally innervated by Lhx3 motoneurons. In vitro studies showed that iPSCMNs form anatomically mature and functional neuromuscular junctions (NMJs) when cocultured with chick myofibers for several weeks. Electrophysiologically, iPSCMNs developed passive membrane and firing characteristic typical of postnatal motoneurons after several weeks in culture. Finally, iPSCMNs grafted into transected mouse tibial nerve projected axons to denervated gastrocnemius muscle fibers, where they formed functional NMJs, restored contractile force. and attenuated denervation atrophy. Together, iPSCMNs possess many of the same cellular and physiological characteristics as ESCMNs and endogenous spinal motoneurons. These results further justify using iPSCMNs as a source of motoneurons for cell replacement therapies and to study motoneuron diseases such as ALS.

Generation of motor neurons from pluripotent stem cells.

Alpha motor neurons (also known as lower or skeletal motor neurons) have been studied extensively for over 100 years. Motor neurons control the contraction of skeletal muscles and thus are the final common pathway in the nervous system responsible for motor behavior. Muscles become paralyzed when their innervating motor neurons die because of injury or disease. Motor neuron diseases (MNDs), such as Amyotrophic Lateral Sclerosis, progressively destroy motor neurons until those inflicted succumb to the illness due to respiratory failure. One strategy being explored to study and treat muscle paralysis due to motor neuron loss involves deriving surrogate motor neurons from pluripotent stem cells. Guided by decades of research on the development of the spinal cord, recent advances in neurobiology have shown that functional motor neurons can be derived from mouse and human embryonic stem (ES) cells. Furthermore, ES cell-derived motor neurons restore motor behavior when transplanted into animal models of motor dysfunction. The recent discovery that mouse and human motor neurons can be derived from induced pluripotent stem (iPS) cells (i.e., somatic cells converted to pluripotency) has set the stage for the development of patient-specific therapies designed to treat movement disorders. Indeed, there is now hope within the scientific community that motor neurons derived from pluripotent stem cells will be used to treat MNDs through cell transplantation and/or to screen molecules that will prevent motor neuron death. In this chapter, we review the journey that led to the generation of motor neurons from ES and iPS cells, how stem cell-derived motor neurons have been used to treat/study motor dysfunction, and where the technology will likely lead to in the future.

Conversion of mouse and human fibroblasts into functional spinal motor neurons.

The mammalian nervous system comprises many distinct neuronal subtypes, each with its own phenotype and differential sensitivity to degenerative disease. Although specific neuronal types can be isolated from rodent embryos or engineered from stem cells for translational studies, transcription factor-mediated reprogramming might provide a more direct route to their generation. Here we report that the forced expression of select transcription factors is sufficient to convert mouse and human fibroblasts into induced motor neurons (iMNs). iMNs displayed a morphology, gene expression signature, electrophysiology, synaptic functionality, in vivo engraftment capacity, and sensitivity to degenerative stimuli similar to those of embryo-derived motor neurons. We show that the converting fibroblasts do not transit through a proliferative neural progenitor state, and thus form bona fide motor neurons via a route distinct from embryonic development. Our findings demonstrate that fibroblasts can be converted directly into a specific differentiated and functional neural subtype, the spinal motor neuron.

Differential RIP antigen (CNPase) expression in peripheral ensheathing glia.

The RIP monoclonal antibody is commonly used to identify oligodendrocytes. Recently, the RIP antigen was identified as 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase), a known non-compact myelin protein [Watanabe, M., Sakurai, Y., Ichinose, T., Aikawa, Y., Kotani, M., Itoh, K., 2006. Monoclonal antibody Rip specifically recognizes 2',3'-cyclic nucleotide 3'-phosphodiesterase in oligodendrocytes. J. Neurosci. Res. 84, 525-533]. In the present study we characterize normal and axotomy-induced changes in RIP immunoreactivity in peripheral glia. In myelinating Schwann cells, RIP demarcated paranodal regions of myelinated axons and clearly defined Schmidt-Lantermann incisures. Surprisingly, RIP immunoreactivity was not confined to myelinating glia. Robust RIP immunoreactivity was present in Remak bundles in mixed nerves and in sympathetic ganglia and grey rami. Following peripheral nerve injury, RIP immunoreactivity was redistributed diffusely throughout de-differentiating Schwann cell cytoplasm. In uninjured rats, low levels of RIP immunoreactivity were detectable in satellite cells surrounding dorsal root ganglion (DRG) neurons and in terminal Schwann cells at neuromuscular junctions. This pattern suggested a correlation between RIP immunoreactivity and the amount of axon-glial contact. We therefore injured the L5 spinal nerve to induce sympathetic sprouting and pericellular basket formation in the DRG, and asked whether relatively RIP-negative satellite glia, which normally contact only neuronal somata, would upregulate the RIP antigen upon contact with sprouting sympathetic axons. All perineuronal sympathetic sprouts infiltrated heavily RIP-immunoreactive satellite cell sheaths. RIP immunoreactivity was absent from placode-derived olfactory ensheathing glia, indicating that the relationship between axon-glial contact and RIP-immunoreactivity is restricted to peripheral ensheathing glia of the neural crest-derived Schwann cell lineage.

Comparative postnatal development of spinal, trigeminal and vagal sensory root entry zones.

Somatic and visceral sensory information enters the central nervous system (CNS) via root entry zones where sensory axons span an environment consisting of Schwann cells in the peripheral nervous system (PNS) and astrocytes and oligodendrocytes in the CNS. While the embryonic extension of these sensory axons into the CNS has been well-characterized, little is known about the subsequent, largely postnatal development of the glial elements of the root entry zones. Here we sought to establish a comparative developmental timecourse of the glial elements in the postnatal (P0, P3, P7, P14) and adult rat of three root entry zones: the spinal nerve dorsal root entry zone, the trigeminal root entry zone, and the vagal dorsal root entry zone. We compared entry zone development based on the expression of antigens known to be expressed in astrocytes, oligodendrocytes, oligodendrocyte precursor cells, Schwann cells, radial glial fibres and the PNS extracellular matrix. These studies revealed an unexpected distribution among glial cells of several antigens. In particular, antibodies used to label mature oligodendrocytes (RIP) transiently labelled immature Schwann cell cytoplasm, and a radial glial antigen (recognized by the 3CB2 antibody) initially decreased, and then increased in postnatal astrocytes. While all three root entry zones had reached morphological and antigenic maturity by P14, the glial elements comprising the PNS-CNS interface of cranial root entry zones (the trigeminal root entry zone and the vagal dorsal root entry zone) matured earlier than those of the spinal nerve dorsal root entry zone.