A special population of regulatory T cells potentiates muscle repair.

Long recognized to be potent suppressors of immune responses, Foxp3(+)CD4(+) regulatory T (Treg) cells are being rediscovered as regulators of nonimmunological processes. We describe a phenotypically and functionally distinct population of Treg cells that rapidly accumulated in the acutely injured skeletal muscle of mice, just as invading myeloid-lineage cells switched from a proinflammatory to a proregenerative state. A Treg population of similar phenotype accumulated in muscles of genetically dystrophic mice. Punctual depletion of Treg cells during the repair process prolonged the proinflammatory infiltrate and impaired muscle repair, while treatments that increased or decreased Treg activities diminished or enhanced (respectively) muscle damage in a dystrophy model. Muscle Treg cells expressed the growth factor Amphiregulin, which acted directly on muscle satellite cells in vitro and improved muscle repair in vivo. Thus, Treg cells and their products may provide new therapeutic opportunities for wound repair and muscular dystrophies.

Growth differentiation factor 11 is a circulating factor that reverses age-related cardiac hypertrophy.

The most common form of heart failure occurs with normal systolic function and often involves cardiac hypertrophy in the elderly. To clarify the biological mechanisms that drive cardiac hypertrophy in aging, we tested the influence of circulating factors using heterochronic parabiosis, a surgical technique in which joining of animals of different ages leads to a shared circulation. After 4 weeks of exposure to the circulation of young mice, cardiac hypertrophy in old mice dramatically regressed, accompanied by reduced cardiomyocyte size and molecular remodeling. Reversal of age-related hypertrophy was not attributable to hemodynamic or behavioral effects of parabiosis, implicating a blood-borne factor. Using modified aptamer-based proteomics, we identified the TGF-ß superfamily member GDF11 as a circulating factor in young mice that declines with age. Treatment of old mice to restore GDF11 to youthful levels recapitulated the effects of parabiosis and reversed age-related hypertrophy, revealing a therapeutic opportunity for cardiac aging.

Highly efficient, functional engraftment of skeletal muscle stem cells in dystrophic muscles.

Satellite cells reside beneath the basal lamina of skeletal muscle fibers and include cells that act as precursors for muscle growth and repair. Although they share a common anatomical localization and typically are considered a homogeneous population, satellite cells actually exhibit substantial heterogeneity. We used cell-surface marker expression to purify from the satellite cell pool a distinct population of skeletal muscle precursors (SMPs) that function as muscle stem cells. When engrafted into muscle of dystrophin-deficient mdx mice, purified SMPs contributed to up to 94% of myofibers, restoring dystrophin expression and significantly improving muscle histology and contractile function. Transplanted SMPs also entered the satellite cell compartment, renewing the endogenous stem cell pool and participating in subsequent rounds of injury repair. Together, these studies indicate the presence in adult skeletal muscle of prospectively isolatable muscle-forming stem cells and directly demonstrate the efficacy of myogenic stem cell transplant for treating muscle degenerative disease.

Young, proliferative thymic epithelial cells engraft and function in aging thymuses.

The thymus reaches its maximum size early in life and then begins to shrink, producing fewer T cells with increasing age. This thymic decline is thought to contribute to age-related T cell lymphopenias and hinder T cell recovery after bone marrow transplantation. Although several cellular and molecular processes have been implicated in age-related thymic involution, their relative contributions are not known. Using heterochronic parabiosis, we observe that young circulating factors are not sufficient to drive regeneration of the aged thymus. In contrast, we find that resupplying young, engraftable thymic epithelial cells (TECs) to a middle-aged or defective thymus leads to thymic growth and increased T cell production. Intrathymic transplantation and in vitro colony-forming assays reveal that the engraftment and proliferative capacities of TECs diminish early in life, whereas the receptivity of the thymus to TEC engraftment remains relatively constant with age. These results support a model in which thymic growth and subsequent involution are driven by cell-intrinsic changes in the proliferative capacity of TECs, and further show that young TECs can engraft and directly drive the growth of involuted thymuses.

Systemic signals regulate ageing and rejuvenation of blood stem cell niches.

Ageing in multicellular organisms typically involves a progressive decline in cell replacement and repair processes, resulting in several physiological deficiencies, including inefficient muscle repair, reduced bone mass, and dysregulation of blood formation (haematopoiesis). Although defects in tissue-resident stem cells clearly contribute to these phenotypes, it is unclear to what extent they reflect stem cell intrinsic alterations or age-related changes in the stem cell supportive microenvironment, or niche. Here, using complementary in vivo and in vitro heterochronic models, we show that age-associated changes in stem cell supportive niche cells deregulate normal haematopoiesis by causing haematopoietic stem cell dysfunction. Furthermore, we find that age-dependent defects in niche cells are systemically regulated and can be reversed by exposure to a young circulation or by neutralization of the conserved longevity regulator, insulin-like growth factor-1, in the marrow microenvironment. Together, these results show a new and critical role for local and systemic factors in signalling age-related haematopoietic decline, and highlight a new model in which blood-borne factors in aged animals act through local niche cells to induce age-dependent disruption of stem cell function.

Identification of inducible brown adipocyte progenitors residing in skeletal muscle and white fat.

Brown fat is specialized for energy expenditure and has therefore been proposed to function as a defense against obesity. Despite recent advances in delineating the transcriptional regulation of brown adipocyte differentiation, cellular lineage specification and developmental cues specifying brown-fat cell fate remain poorly understood. In this study, we identify and isolate a subpopulation of adipogenic progenitors (Sca-1(+)/CD45(-)/Mac1(-); referred to as Sca-1(+) progenitor cells, ScaPCs) residing in murine brown fat, white fat, and skeletal muscle. ScaPCs derived from different tissues possess unique molecular expression signatures and adipogenic capacities. Importantly, although the ScaPCs from interscapular brown adipose tissue (BAT) are constitutively committed brown-fat progenitors, Sca-1(+) cells from skeletal muscle and subcutaneous white fat are highly inducible to differentiate into brown-like adipocytes upon stimulation with bone morphogenetic protein 7 (BMP7). Consistent with these findings, human preadipocytes isolated from subcutaneous white fat also exhibit the greatest inducible capacity to become brown adipocytes compared with cells isolated from mesenteric or omental white fat. When muscle-resident ScaPCs are re-engrafted into skeletal muscle of syngeneic mice, BMP7-treated ScaPCs efficiently develop into adipose tissue with brown fat-specific characteristics. Importantly, ScaPCs from obesity-resistant mice exhibit markedly higher thermogenic capacity compared with cells isolated from obesity-prone mice. These data establish the molecular characteristics of tissue-resident adipose progenitors and demonstrate a dynamic interplay between these progenitors and inductive signals that act in concert to specify brown adipocyte development.

DLK signaling in axotomized neurons triggers complement activation and loss of upstream synapses.

Axotomized spinal motoneurons (MNs) lose presynaptic inputs following peripheral nerve injury; however, the cellular mechanisms that lead to this form of synapse loss are currently unknown. Here, we delineate a critical role for neuronal kinase dual leucine zipper kinase (DLK)/MAP3K12, which becomes activated in axotomized neurons. Studies with conditional knockout mice indicate that DLK signaling activation in injured MNs triggers the induction of phagocytic microglia and synapse loss. Aspects of the DLK-regulated response include expression of C1q first from the axotomized MN and then later in surrounding microglia, which subsequently phagocytose presynaptic components of upstream synapses. Pharmacological ablation of microglia inhibits the loss of cholinergic C boutons from axotomized MNs. Together, the observations implicate a neuronal mechanism, governed by the DLK, in the induction of inflammation and the removal of synapses.

Single-cell transcriptomic landscape of the developing human spinal cord.

Understanding spinal cord assembly is essential to elucidate how motor behavior is controlled and how disorders arise. The human spinal cord is exquisitely organized, and this complex organization contributes to the diversity and intricacy of motor behavior and sensory processing. But how this complexity arises at the cellular level in the human spinal cord remains unknown. Here we transcriptomically profiled the midgestation human spinal cord with single-cell resolution and discovered remarkable heterogeneity across and within cell types. Glia displayed diversity related to positional identity along the dorso-ventral and rostro-caudal axes, while astrocytes with specialized transcriptional programs mapped into white and gray matter subtypes. Motor neurons clustered at this stage into groups suggestive of alpha and gamma neurons. We also integrated our data with multiple existing datasets of the developing human spinal cord spanning 22 weeks of gestation to investigate the cell diversity over time. Together with mapping of disease-related genes, this transcriptomic mapping of the developing human spinal cord opens new avenues for interrogating the cellular basis of motor control in humans and guides human stem cell-based models of disease.

Proprioception revisited: where do we stand?

Originally referred to as 'muscle sense', the notion that skeletal muscle held a peripheral sensory function was first described early in the 19th century. Foundational experiments by Sherrington in the early 20th century definitively demonstrated that proprioceptors contained within skeletal muscle, tendons, and joints are innervated by sensory neurons and play an important role in the control of movement. In this review, we will highlight several recent advances in the ongoing effort to further define the molecular diversity underlying the proprioceptive sensorimotor system. Together, the work summarized here represents our current understanding of sensorimotor circuit formation during development and the mechanisms that regulate the integration of proprioceptive feedback into the spinal circuits that control locomotion in both normal and diseased states.

Translatomic analysis of regenerating and degenerating spinal motor neurons in injury and ALS.

The neuromuscular junction is a synapse critical for muscle strength and coordinated motor function. Unlike CNS injuries, motor neurons mount robust regenerative responses after peripheral nerve injuries. Conversely, motor neurons selectively degenerate in diseases such as amyotrophic lateral sclerosis (ALS). To assess how these insults affect motor neurons in vivo, we performed ribosomal profiling of mouse motor neurons. Motor neuron-specific transcripts were isolated from spinal cords following sciatic nerve crush, a model of acute injury and regeneration, and in the SOD1G93A ALS model. Of the 267 transcripts upregulated after nerve crush, 38% were also upregulated in SOD1G93A motor neurons. However, most upregulated genes in injured and ALS motor neurons were context specific. Some of the most significantly upregulated transcripts in both paradigms were chemokines such as Ccl2 and Ccl7, suggesting an important role for neuroimmune modulation. Collectively these data will aid in defining pro-regenerative and pro-degenerative mechanisms in motor neurons.

Single-cell transcriptomic analysis of the adult mouse spinal cord reveals molecular diversity of autonomic and skeletal motor neurons.

The spinal cord is a fascinating structure that is responsible for coordinating movement in vertebrates. Spinal motor neurons control muscle activity by transmitting signals from the spinal cord to diverse peripheral targets. In this study, we profiled 43,890 single-nucleus transcriptomes from the adult mouse spinal cord using fluorescence-activated nuclei sorting to enrich for motor neuron nuclei. We identified 16 sympathetic motor neuron clusters, which are distinguishable by spatial localization and expression of neuromodulatory signaling genes. We found surprising skeletal motor neuron heterogeneity in the adult spinal cord, including transcriptional differences that correlate with electrophysiologically and spatially distinct motor pools. We also provide evidence for a novel transcriptional subpopulation of skeletal motor neuron (γ*). Collectively, these data provide a single-cell transcriptional atlas ( http://spinalcordatlas.org ) for investigating the organizing molecular logic of adult motor neuron diversity, as well as the cellular and molecular basis of motor neuron function in health and disease.

Semaphorin3A Signaling Is Dispensable for Motor Axon Reinnervation of the Adult Neuromuscular Junction.

The neuromuscular junction (NMJ) is a specialized synapse that is formed by motor axon innervation of skeletal muscle fibers. The maintenance of motor-muscle connectivity is critical for the preservation of muscle tone and generation of movement. Injury can induce a robust regenerative response in motor axons, but severe trauma or chronic denervation resulting from neurodegenerative disease typically leads to inefficient repair and poor functional recovery. The axon guidance molecule Semaphorin3A (Sema3A) has been implicated as a negative regulator of motor innervation. Upon binding to a plexinA-neuropilin1 (Npn1) receptor complex, Sema3A initiates a downstream signaling cascade that results in axonal repulsion. Here, we established a reproducible nerve crush model to quantify motor nerve regeneration. We then used that model to investigate the role of Sema3A signaling at the adult NMJ. In contrast to previous findings, we found that Sema3A and Npn1 mRNA decrease in response to denervation, suggesting that Sema3A-Npn1 signaling may regulate NMJ reinnervation. To directly test that hypothesis, we used inducible knockout models to ubiquitously delete Sema3A or Npn1 from adult mice. Despite demonstrating that we could achieve highly efficient gene deletion, disruption of Sema3A-Npn1 signaling did not affect the normal maintenance of the NMJ or disrupt motor axon reinnervation after a denervating injury.

Restoring systemic GDF11 levels reverses age-related dysfunction in mouse skeletal muscle.

Parabiosis experiments indicate that impaired regeneration in aged mice is reversible by exposure to a young circulation, suggesting that young blood contains humoral "rejuvenating" factors that can restore regenerative function. Here, we demonstrate that the circulating protein growth differentiation factor 11 (GDF11) is a rejuvenating factor for skeletal muscle. Supplementation of systemic GDF11 levels, which normally decline with age, by heterochronic parabiosis or systemic delivery of recombinant protein, reversed functional impairments and restored genomic integrity in aged muscle stem cells (satellite cells). Increased GDF11 levels in aged mice also improved muscle structural and functional features and increased strength and endurance exercise capacity. These data indicate that GDF11 systemically regulates muscle aging and may be therapeutically useful for reversing age-related skeletal muscle and stem cell dysfunction.

Liver-derived systemic factors drive ß cell hyperplasia in insulin-resistant states.

Integrative organ crosstalk regulates key aspects of energy homeostasis, and its dysregulation may underlie metabolic disorders such as obesity and diabetes. To test the hypothesis that crosstalk between the liver and pancreatic islets modulates ß cell growth in response to insulin resistance, we used the liver-specific insulin receptor knockout (LIRKO) mouse, a unique model that exhibits dramatic islet hyperplasia. Using complementary in vivo parabiosis and transplantation assays, as well as in vitro islet culture approaches, we demonstrate that humoral, nonneural, non-cell-autonomous factor(s) induces ß cell proliferation in LIRKO mice. Furthermore, we report that a hepatocyte-derived factor(s) stimulates mouse and human ß cell proliferation in ex vivo assays, independent of ambient glucose and insulin levels. These data implicate the liver as a critical source of ß cell growth factor(s) in insulin-resistant states.

M2 microglia and macrophages drive oligodendrocyte differentiation during CNS remyelination.

The lack of therapies for progressive multiple sclerosis highlights the need to understand the regenerative process of remyelination that can follow CNS demyelination. This involves an innate immune response consisting of microglia and macrophages, which can be polarized to distinct functional phenotypes: pro-inflammatory (M1) and anti-inflammatory or immunoregulatory (M2). We found that a switch from an M1- to an M2-dominant response occurred in microglia and peripherally derived macrophages as remyelination started. Oligodendrocyte differentiation was enhanced in vitro with M2 cell conditioned media and impaired in vivo following intra-lesional M2 cell depletion. M2 cell densities were increased in lesions of aged mice in which remyelination was enhanced by parabiotic coupling to a younger mouse and in multiple sclerosis lesions that normally show remyelination. Blocking M2 cell-derived activin-A inhibited oligodendrocyte differentiation during remyelination in cerebellar slice cultures. Thus, our results indicate that M2 cell polarization is essential for efficient remyelination and identify activin-A as a therapeutic target for CNS regeneration.

Rejuvenation of regeneration in the aging central nervous system.

Remyelination is a regenerative process in the central nervous system (CNS) that produces new myelin sheaths from adult stem cells. The decline in remyelination that occurs with advancing age poses a significant barrier to therapy in the CNS, particularly for long-term demyelinating diseases such as multiple sclerosis (MS). Here we show that remyelination of experimentally induced demyelination is enhanced in old mice exposed to a youthful systemic milieu through heterochronic parabiosis. Restored remyelination in old animals involves recruitment to the repairing lesions of blood-derived monocytes from the young parabiotic partner, and preventing this recruitment partially inhibits rejuvenation of remyelination. These data suggest that enhanced remyelinating activity requires both youthful monocytes and other factors, and that remyelination-enhancing therapies targeting endogenous cells can be effective throughout life.

Stem cells for skeletal muscle repair.

Skeletal muscle is a highly specialized tissue composed of non-dividing, multi-nucleated muscle fibres that contract to generate force in a controlled and directed manner. Skeletal muscle is formed during embryogenesis from a subset of muscle precursor cells, which generate both differentiated muscle fibres and specialized muscle-forming stem cells known as satellite cells. Satellite cells remain associated with muscle fibres after birth and are responsible for muscle growth and repair throughout life. Failure in satellite cell function can lead to delayed, impaired or failed recovery after muscle injury, and such failures become increasingly prominent in cases of progressive muscle disease and in old age. Recent progress in the isolation of muscle satellite cells and elucidation of the cellular and molecular mediators controlling their activity indicate that these cells represent promising therapeutic targets. Such satellite cell-based therapies may involve either direct cell replacement or development of drugs that enhance endogenous muscle repair mechanisms. Here, we discuss recent breakthroughs in understanding both the cell intrinsic and extrinsic regulators that determine the formation and function of muscle satellite cells, as well as promising paths forward to realizing their full therapeutic potential.

Id3 is a direct transcriptional target of Pax7 in quiescent satellite cells.

Pax7 is a key regulator of skeletal muscle stem cells and is required along with Pax3 to generate skeletal muscle precursors. We have identified a collection of genes induced by either Pax3 or Pax7 in C2C12 muscle cells. Two notable Pax3/7 targets are the inhibitory helix-loop-helix (HLH) proteins inhibitor of DNA binding (Id) 2 and Id3, both of which are coordinately expressed with Pax7 in quiescent satellite cells and are induced in quiescent C2C12 myogenic cells after ectopic expression of either Pax3 or Pax7. Ectopic Pax7 activates expression of a luciferase reporter driven by the Id3 promoter, and maximal induction of this reporter requires a conserved Pax7 binding site located upstream of the Id3 gene. Chromatin immunoprecipitation indicated that Pax7 is bound upstream of the Id3 promoter in quiescent satellite cells. In addition, short hairpin RNA-mediated knockdown of Pax7 expression in cultured satellite cells coordinately decreased both Id2 and Id3 expression. Together, these findings indicate that Id3 is a direct transcriptional target for Pax7 in quiescent satellite cells, and they suggest that Pax7 acts to block premature differentiation of quiescent satellite cells by inducing the expression of Id2 and Id3, which in turn may act to block either the precocious induction of myogenic basic (b)HLH proteins, the activity of myogenic bHLH proteins, or both.