SUMMARY
The mammalian nervous system is composed of a multitude of 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 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.
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.
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 Ͻ4% 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.
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.
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