DISEASED MUSCLE CELLS IN CULTURE

(1) Cultures of differentiated muscle cells have been grown from diseased human, mouse and chick skeletal muscle, and from cardiac muscle of the myopathic hamster. (2) Methods of culture established for normal embryonic and adult skeletal muscle cells have proved suitable for cultures of diseased muscle cells. (3) Myoblasts obtained from dy^2J mouse muscle crushed in vivo before explanting fuse in culture and form morphologically normal myotubes. Studies of the effects of innervation by dy^2J spinal cord neurones on the differentiation of normal, dy^2J and dy myotubes have been inconclusive but it is probable that innervation does not play a part in the pathogenesis of this disorder. (4) Myoblasts prepared by trypsinization of embryonic dy muscle behave normally in culture and fuse to form myotubes that appear normal. It is not clear if myoblasts that migrate from explants of adult muscle in vitro fuse. Aggregates of non-fusing cells have been described, but under other culture conditions normal and abnormal forms of myotube have been observed. dy muscle fibres fail to regenerate even when cultured with normal spinal cord explants and dy nerves are without effect on regenerating normal muscle fibres. These tissue-culture studies suggest that the dy mouse mutation is a myopathic disorder. (5) Embryonic mdg myoblasts have a normal cell cycle in vitro and fuse to form well-differentiated myotubes with cross-striations. mdg myotubes have normal electro-physiological properties but do not contract spontaneously or on depolarization. The defect in the muscle of the mdg mutant appears to be a failure of excitation-contraction coupling. (6) Cells migrate earlier from explants of adult dystrophic chick muscle than from normal muscle but dystrophic chick myotubes appear morphologically normal. Myotubes prepared from embryonic dystrophic chick muscle become vacuolated and degenerate, changes that can be prevented by anti-proteases such as antipain. Lactic dehydrogenase isozyme subunit M4 is absent from dystrophic muscle in vivo but reappears in cultured myotubes. Dystrophic myotubes innervated in culture by either normal or dystrophic neurones exhibit bi-directional lcoupling and multiple innervation. These results suggest that there are changes in dystrophic myotubes and that chick muscular dystrophy is a myopathy. (7) Cardiac muscle cells from the cardiomyopathic hamster synthesize less actin and myosin than normal cells, and Z lines in dystrophic cells are irregularly arranged. The beat frequency of myopathic cardiac cells is lower than that of normal cells and declines more rapidly. Tissue-culture studies have not been made of hamster skeletal muscle. (8) Human dystrophic myotubes do not show degenerative changes in culture and have normal histochemical reactions. RNA synthesis appears normal in dystrophic myotubes but there may be changes in adenyl-cyclase activity and protein synthesis in dystrophic cells. Morphological and biochemical changes have been found in muscle cells cultured from a case of acid-maltase deficiency but phosphorylase activity re-appeared in myotubes cultured from biopsies of phosphorylase-deficient muscle. Innervation by normal mouse nerves does not induce degenerative changes in dystrophic myotubes. (9) Studies on the origins of myoblasts in explants of muscle fibres in culture suggest that in these conditions myoblasts are derived only from satellite cells and that this process may be the same in normal and diseased muscle.     

Gap Junctions Contribute to the Regulation of Walking-Like Activity in the Adult Mudpuppy (Necturus Maculatus)

Although gap junctions are widely expressed in the developing central nervous system, the role of electrical coupling of neurons and glial cells via gap junctions in the spinal cord in adults is largely unknown. We investigated whether gap junctions are expressed in the mature spinal cord of the mudpuppy and tested the effects of applying gap junction blocker on the walking-like activity induced by NMDA or glutamate in an in vitro mudpuppy preparation. We found that glial and neural cells in the mudpuppy spinal cord expressed different types of connexins that include connexin 32 (Cx32), connexin 36 (Cx36), connexin 37 (Cx37), and connexin 43 (Cx43). Application of a battery of gap junction blockers from three different structural classes (carbenexolone, flufenamic acid, and long chain alcohols) substantially and consistently altered the locomotor-like activity in a dose-dependent manner. In contrast, these blockers did not significantly change the amplitude of the dorsal root reflex, indicating that gap junction blockers did not inhibit neuronal excitability nonselectively in the spinal cord. Taken together, these results suggest that gap junctions play a significant modulatory role in the spinal neural networks responsible for the generation of walking-like activity in the adult mudpuppy.     

Appearance of contractile activity in muscular dysgenesis (mdgmdg) mouse myotubes during coculture with normal spinal cord cells

Muscular dysgenesis (mdg) in the mouse is an autosomal recessive mutation expressed in the homozygous mutant as lack of skeletal muscle contraction. To test the ability of normal neurons to form neuromuscular contacts with, and/or possibly induce contractions in $\text{mdg}\text{mdg}$ muscle, dispersed cell cultures of normal and dysgenic muscle from newborn mice were cocultured with normal embryonic rat, mouse, and chick dissociated spinal cord cells. Contraction was induced in $\text{mdg}\text{mdg}$ muscle 1 to 10 days (depending upon the species of the neuronal source) following establishment of the cocultures. Control experiments indicated that the dispersed spinal cord preparations were free of myoblasts capable of fusing with $\text{mdg}\text{mdg}$ muscle. The establishment of neuromuscular contacts in the rat neuron cocultures was monitored by cytochemical staining of acetylcholinesterase (AChE), autoradiography of ¹²⁵I-α-bungarotoxin-bound acetylcholine receptors (AChR), and electrophysiological study of muscle membrane activity. Patches of high AChE activity were similar in size and distribution to high-density clusters of AChR on both control and $\text{mdg}\text{mdg}$ myotubes cocultured with rat neurons. The resting membrane potentials of normal myotubes and those of $\text{mdg}\text{mdg}$ myotubes in the presence of neurons were similar (? ?52 mV). The mepp frequency and the mepp amplitude distribution were the same for both control and mutant cocultured muscle. Thus, normal rat spinal cord neurons were capable of forming normal, functional neuromuscular junctions with $\text{mdg}\text{mdg}$ myotubes, and contractions were induced under coculture conditions, in otherwise noncontracting mutant muscle.     

Toll-like Receptor 2 Mediates Peripheral Nerve Injury-induced NADPH Oxidase 2 Expression in Spinal Cord Microglia

We have previously reported that NADPH oxidase 2 (Nox2) is up-regulated in spinal cord microglia after spinal nerve injury, demonstrating that it is critical for microglia activation and subsequent pain hypersensitivity. However, the mechanisms and molecules involved in Nox2 induction have not been elucidated. Previous studies have shown that Toll-like receptors (TLRs) are involved in nerve injury-induced spinal cord microglia activation. In this study, we investigated the role of TLR in Nox2 expression in spinal cord microglia after peripheral nerve injury. Studies using TLR knock-out mice have shown that nerve injury-induced microglial Nox2 up-regulation is abrogated in TLR2 but not in TLR3 or -4 knock-out mice. Intrathecal injection of lipoteichoic acid, a TLR2 agonist, induced Nox2 expression in spinal cord microglia both at the mRNA and protein levels. Similarly, lipoteichoic acid stimulation induced Nox2 expression and reactive oxygen species production in primary spinal cord glial cells in vitro. Studies on intracellular signaling pathways indicate that NF-κB and p38 MAP kinase activation is required for TLR2-induced Nox2 expression in glial cells. Conclusively, our data show that TLR2 mediates nerve injury-induced Nox2 gene expression in spinal cord microglia via NF-κB and p38 activation and thereby may contribute to spinal cord microglia activation.     

In vivo and in vitro differentiation of neurons and astrocytes in the rat embryo. Immunofluorescence study with neurofilament and glial filament antisera

Antisera raised against neurofilament (NF) peptides and glial fibrillary acidic protein (GFA) (subunit of glial filaments) have been used to identify neurons and astrocytes in order to study their development and differentiation in rat embryo. In vivo observations showed that NF-positive cells first appeared in 12-day-old embryos, whereas GFA-positive cells appeared in brain and spinal cord on the 18th day. In vitro observations showed that NF-positive cells could be obtained only in cultures from 12-day embryos onwards. The further differentiation of neurons involved neurite elongation, aggregation of cell bodies to form islets, and emergence of very brightly staining prominent neurons with large cell bodies and long neurites which took part in complicate pattern formation. GFA-positive cells appeared in vitro on the 16th day and they could be observed even in cultures obtained from 10-day-old embryos. As the culture aged, the GFA staining became highly fibrillary. There was no physical interaction between neuronal and glial processes.     

Neural and Glial Secretion in the Spinal Cord of the Lamprey (Lampetra planeri)

In the spinal cord of Lampetra planeri neurosecretion and glial secretion could be demonstrated with histochemical methods and the aid of the electron microscope. Neurosecretion is present in either all or some “dorsal cells” which are scattered along the whole spinal cord. These cells have the characteristic fine structure of highly active secretory neurons. The cells produce three types of secretory material. One not bound to vesicles in form of a finely granulated substance, a second one in spherical, uniformely electron-dense elementary vesicles with a diameter of 100–140 nm, and a third one in vesicles of the monoamine type. In the most caudal part of the spinal cord glial secretion is present. Groups of glial cells produce a fine, granulated material and release it into the adjacent intercellular space. From there the material extends into other intercellular spaces of the spinal cord. We are of the opinion that the “dorsal cells” are forerunners of the cells of Dahlgren in higher vertebrates and that the neurosecretion and glial secretion in the spinal cord of lampreys demonstrate very primitive conditions.     

Acetylcholine synthesis by chick spinal cord neurons in dissociated cell culture

Acetylcholine (ACh) synthesis was examined in cultures of chick spinal cord cells to follow the development of the cholinergic neurons. The cells, prepared from 4-day-old embryonic chick spinal cords, were grown either alone in dissociated cell cultures (SC cultures) or with chick myotubes (SC-M cultures). ACh synthesis was measured by incubating the cultures in [³Hcholine and using high-voltage paper electrophoresis to quantitate the amount of [³H]ACh present in cell extracts prepared from the labeled cultures. The amount of [³H]ACh synthesized in SC-M cultures was strictly proportional to the number of spinal cord cells used to prepare the cultures, and was linear with the time of incubation in [³H]choline for periods up to 1 hr. Maximal rates of synthesis were observed with [³H]choline concentrations in excess of 100 μM. Such rates for 1-week-old SC-M cultures were approximately 10–20 pmoles of [³H]ACh/hr/10⁵ spinal cord cells. Studies on the stability of the intracellular [³H]ACh revealed the presence of a major pool with a half-time of 20–30 min. A second, small pool decayed more rapidly. No detectable [³H]ACh was spontaneously released from the cells, suggesting that most of the decay represented intracellular degradation. Development of cholinergic neurons as monitored by [³H]ACh synthesis continued over a 2-week period in SC-M cultures and paralleled general cell growth. When examined at 1 week, SC-M cultures had about a 50% greater capacity for [³H]ACh synthesis and 60% more choline acetyltransferase activity than did SC cultures. No difference was observed in the stability of the [³H]ACh formed for the two types of cultures at 1 week, and no further difference was observed in the rates of [³H]ACh synthesis at 2 weeks. Growth of SC cultures in medium containing different amounts of chick embryo extract (2–10%) or in medium with fetal calf serum (10%) instead of extract produced only small differences in the measured rates of [³H]ACh synthesis. Thus chick spinal cord cells can undergo some of the early stages of cholinergic development in cell culture without sustained contact with skeletal myotubes, one of the normal postsynaptic target cells for the cholinergic neuron population. No absolute requirement for muscle factors was revealed under these conditions, although such factors may have been provided by other cell types in the spinal cord population or may have been present in other additions to the culture medium.     

Analysis of cartilage differentiation from skeletal muscle grown on bone matrix: I. Ultrastructural aspects

Previous studies have demonstrated that embryonic skeletal muscle is competent to form hyaline cartilage when cultured in vitro on demineralized bone matrix (Nogami, H., and Urist, M. R. (1970). Exp. Cell Res.63, 404–410; Nathanson, M. A., et al. (1978). Develop. Biol.64, 99–117). The present experiments were undertaken to determine the nature of the morphological alterations which attend this phenotypic transformation and to investigate the ultrastructural characteristics of the myoblasts and fibroblasts of skeletal muscle during the transformation. Nineteen-day embryonic rat limb muscles were minced and the tissue fragments explanted to bone matrix or collagen gels. The trauma of excision and mincing causes syncytial myotubes to degenerate and the nuclei of mononucleate cells to enter a heterochromatic “resting stage.” In culture, nuclei of mononucleate cells rapidly regain euchromasia. No myoblast or fibroblast cell death can be detected. On bone matrix, the entire mononucleate population transforms into fibroblast-like cells. Myoblasts are the major contributor to this population; they dissociate from the degenerate myotubes and begin to acquire endoplasmic reticulum by 24 h in vitro. The fibroblast-like morphology persists through 4 days in vitro. By 6 days in vitro some of these fibroblast-like cells acquire the phenotypic characteristics of chondrocytes, and by 10 days masses of hyaline cartilage are found. In control explants of skeletal muscle onto collagen gels, the heterochromatic nuclei of the mononucleated cells expand after 24 hr in vitro, but the mononucleated cells remain as myoblasts and fibroblasts and begin to regenerate skeletal muscle by 4 days in vitro. No cartilage forms. The results indicate that both myoblasts and fibroblasts have chondrogenic potential when grown on demineralized bone. It is tempting to conclude that the embryonic mesenchymal cells which give rise to skeletal muscle, cartilage, and other connective tissue of the limb have similar developmental potentials and that local influences, rather than separate cell lineages, account for the final pattern of differentiation.     

Conditioned Medium from Bone Marrow-Derived Mesenchymal Stem Cells Improves Recovery after Spinal Cord Injury in Rats: An Original Strategy to Avoid Cell Transplantation

Spinal cord injury triggers irreversible loss of motor and sensory functions. Numerous strategies aiming at repairing the injured spinal cord have been studied. Among them, the use of bone marrow-derived mesenchymal stem cells (BMSCs) is promising. Indeed, these cells possess interesting properties to modulate CNS environment and allow axon regeneration and functional recovery. Unfortunately, BMSC survival and differentiation within the host spinal cord remain poor, and these cells have been found to have various adverse effects when grafted in other pathological contexts. Moreover, paracrine-mediated actions have been proposed to explain the beneficial effects of BMSC transplantation after spinal cord injury. We thus decided to deliver BMSC-released factors to spinal cord injured rats and to study, in parallel, their properties in vitro. We show that, in vitro, BMSC-conditioned medium (BMSC-CM) protects neurons from apoptosis, activates macrophages and is pro-angiogenic. In vivo, BMSC-CM administered after spinal cord contusion improves motor recovery. Histological analysis confirms the pro-angiogenic action of BMSC-CM, as well as a tissue protection effect. Finally, the characterization of BMSC-CM by cytokine array and ELISA identified trophic factors as well as cytokines likely involved in the beneficial observed effects. In conclusion, our results support the paracrine-mediated mode of action of BMSCs and raise the possibility to develop a cell-free therapeutic approach.     

Characterization of a plasma membrane glycoprotein common to myoblasts, skeletal muscle satellite cells, and glia

A plasma membrane glycoprotein common to embryonic chick myoblasts and adult chicken skeletal muscle satellite cells is the antigen recognized by monoclonal antibody C3/1. Although traces of the same antigen are present on some muscle-derived fibroblasts, the density of antigenic sites on myoblasts and satellite cells is so high that these cell types can be identified in tissues by immunocytochemical techniques. The antigen is exposed on the surfaces of myogenic cells growing in tissue culture and can be solubilized with detergent. This and other criteria establish that the antigen is a plasma membrane protein. The antigen, purified by affinity techniques, consists of a single type of polypeptide chain which migrates as a relatively broad band of apparent molecular weight 38,000 Da in SDS-polyacrylamide gel electrophoresis. It has a very small sedimentation constant, suggesting that the solubilized form is either monomeric or dimeric. The concentration of antigenic sites increases during myogenesis in vitro; but during maturation the antigenic sites are lost from muscle fibers. Electron microscopic autoradiographic study of adult muscle labeled with iodinated monoclonal antibody demonstrated unequivocally that the antigenic sites in adult muscle are concentrated in the satellite cells. Although selective for myoblasts, immature myotubes and satellite cells in the myogenic lineage, the monoclonal antibody also binds at rather high levels to peripheral Schwann cells and teloglia, to some nonneuronal cells in cultures derived from embryonic spinal cord, to some glial elements of adult chicken brain, and to several cell types in the early embryo.     

Cell types required to efficiently innervate human muscle cells in vitro

Previous studies carried out in our laboratory have shown that myofibers formed by fusion of muscle satellite cells from donors with spinal muscular atrophy (SMA) type I or II undergo a characteristic degeneration 1.5-3 weeks after innervation with rat embryonic spinal cord explants. The only cells responsible for degeneration of innervated cocultures are SMA muscle satellite cells. In order to study the kinetics of nerve and muscle cell degeneration in nerve-muscle cocultures implicating SMA muscle cells, we attempted to simplify the nervous component of the coculture and identify the nerve cell types necessary for a successful innervation. We demonstrate here that motoneurons alone were unable to innervate myotubes. However, when three cell types (motoneurons, sensory neurons, and Schwann cells) were added onto a reconstituted muscular component consisting of cloned muscle satellite cells and cloned muscular fibroblasts, myotubes contracted, indicating that functional neuromuscular junctions were formed. We concluded that the three cell types were required for a successful innervation. Moreover, we studied the effects of culture medium conditioned by different combinations of nerve cells on innervation; we observed that physical contacts among sensory neurons, motoneurons, and myotubes are required for a successful innervation; in contrast Schwann cells can be replaced by a Schwann-cell-conditioned medium, indicating that these cells produce a putative soluble "innervation-promoting factor." Obviously such a reconstituted system does not reflect the in vivo situation but it allows the formation of functional motor synapses and could therefore allow us to elucidate neuromuscular disease pathogenesis, especially that of spinal muscular atrophy.     

Human embryonic stem cell-derived neural precursor transplants in collagen scaffolds promote recovery in injured rat spinal cord

Background aimsSeveral studies have reported functional improvement after transplantation of in vivo-derived neural progenitor cells (NPC) into injured spinal cord. However, the potential of human embryonic stem cell-derived NPC (hESC-NPC) as a tool for cell replacement of spinal cord injury (SCI) should be considered.MethodsWe report on the generation of NPC as neural-like tubes in adherent and feeder-free hESC using a defined media supplemented with growth factors, and their transplantation in collagen scaffolds in adult rats subjected to midline lateral hemisection SCI.ResultshESC-NPC were highly expressed molecular features of NPC such as Nestin, Sox1 and Pax6. Furthermore, these cells exhibited the multipotential characteristic of differentiating into neurons and glials in vitro. Implantation of xenografted hESC-NPC into the spinal cord with collagen scaffold improved the recovery of hindlimb locomotor function and sensory responses in an adult rat model of SCI. Analysis of transplanted cells showed migration toward the spinal cord and both neural and glial differentiation in vivo.ConclusionsThese findings show that transplantation of hESC-NPC in collagen scaffolds into an injured spinal cord may provide a new approach to SCI.     

A Functional Motor Unit in the Culture Dish: Co-culture of Spinal Cord Explants and Muscle Cells

Human primary muscle cells cultured aneurally in monolayer rarely contract spontaneously because, in the absence of a nerve component, cell differentiation is limited and motor neuron stimulation is missing¹. These limitations hamper the in vitro study of many neuromuscular diseases in cultured muscle cells. Importantly, the experimental constraints of monolayered, cultured muscle cells can be overcome by functional innervation of myofibers with spinal cord explants in co-cultures.Here, we show the different steps required to achieve an efficient, proper innervation of human primary muscle cells, leading to complete differentiation and fiber contraction according to the method developed by Askanas². To do so, muscle cells are co-cultured with spinal cord explants of rat embryos at ED 13.5, with the dorsal root ganglia still attached to the spinal cord slices. After a few days, the muscle fibers start to contract and eventually become cross-striated through innervation by functional neurites projecting from the spinal cord explants that connecting to the muscle cells. This structure can be maintained for many months, simply by regular exchange of the culture medium.The applications of this invaluable tool are numerous, as it represents a functional model for multidisciplinary analyses of human muscle development and innervation. In fact, a complete de novo neuromuscular junction installation occurs in a culture dish, allowing an easy measurement of many parameters at each step, in a fundamental and physiological context.Just to cite a few examples, genomic and/or proteomic studies can be performed directly on the co-cultures. Furthermore, pre- and post-synaptic effects can be specifically and separately assessed at the neuromuscular junction, because both components come from different species, rat and human, respectively. The nerve-muscle co-culture can also be performed with human muscle cells isolated from patients suffering from muscle or neuromuscular diseases³, and thus can be used as a screening tool for candidate drugs. Finally, no special equipment but a regular BSL2 facility is needed to reproduce a functional motor unit in a culture dish. This method thus is valuable for both the muscle as well as the neuromuscular research communities for physiological and mechanistic studies of neuromuscular function, in a normal and disease context.     

Autoradiographic labeling of spinal cord neurons with high affinity choline uptake in cell culture

Embryonic chick spinal cord neurons grown in dissociated cell culture have a high affinity uptake mechanism for choline. We find that, in addition to acetylcholine synthesis, the accumulated choline is used for the synthesis of metabolites such as lipids that are retained in part by conventional fixation techniques. As a result autoradiographic methods can be used to identify the cells that have the uptake mechanism in spinal cord cultures. About 60% of the neurons are labeled by [³H]choline uptake in cultures prepared with spinal cord cells from 4-day-old embryos, and about 40% are labeled in cultures prepared with cord cells from 7-day-old embryos. Neurons that innervate skeletal myotubes in spinal cord-myotube cultures are consistently labeled by [³H]choline uptake. Neurons unlabeled by the procedure are viable: they exclude the dye trypan blue and accumulate ¹⁴C-amino acids for protein synthesis. Most of the neurons unlabeled by [³H]choline uptake can instead be labeled by uptake of γ-[³H]aminobutyric acid, and vice versa. These results suggest that high affinity choline uptake can be used to label cholinergic neurons in cell culture, and that at least some populations of noncholinergic neurons are not labeled by the procedure. It cannot yet be concluded, however, that all labeled neurons are cholinergic since more labeled neurons are obtained per cord than would be expected from the number of neurons making up identified cholinergic populations in vivo. A three- to fourfold increase in the amount of high affinity choline uptake is observed between Days 3 and 15 in culture for spinal cord cells obtained from 4-day-old embryos. The number of [³H]choline-labeled neurons in such cultures decreases slightly during the same period, suggesting that the increase in uptake reflects neuronal growth or development rather than an increase in population size. Both the magnitude of the uptake and the number of [³H]choline-labeled neurons are the same for spinal cord cells grown with and without skeletal myotubes.     

Synaptic profiles in the outgrowth from chick spinal cord in vitro

Summary Synaptic profiles have been identified in the outgrowth from chick embryo spinal cord maintained in vitro for short periods. Profiles corresponding to types that may be excitatory and inhibitory in the intact central nervous system have been found. Their presence outside expiants, and in occasional relation to glial cells, suggests that neurites themselves may possess a generalised capacity for synapse formation under appropriate circumstances, rather than be limited to specific targets.     

T-system formation in cultured rat skeletal tissue

By using a lanthanum-staining technique which enhances the visualization of the plasma membrane and its derivatives we have studied the formation of the T system in rat muscle cells differentiating in vitro. We have found that: (1) T-system formation normally occurs after myoblast fusion and is especially extensive in mature myotubes; myoblasts grown in calcium-deficient medium to prevent fusion show increased number of sarcolemmal caveolae but rare, short T tubules. (2) T-system formation in vitro differs from that displayed by rat muscle cells in vivo in that it precedes and is independent of junctional SR differentiation; the uncoordinated development of T tubules and junctional SR in vitro leads to the formation of ‘inverted’ triads and labyrinthine T-system networks. (3) Coated vesicles are frequently found either free in the cytoplasm or associated with growing T tubules in rat muscle cells differentiating in vitro. A role of coated vesicles in T-system formation is proposed.     

Uniaxial cyclic strain enhances adipose-derived stem cell fusion with skeletal myocytes

Although adult muscle tissue possesses an exceptional capacity for regeneration, in the case of large defects, the restoration to original state is not possible. A well-known source for the de novo regeneration is the adipose-derived stem cells (ASCs), which can be readily isolated and have been shown to have a broad differentiation and regenerative potential. In this work, we employed uniaxial cyclic tensile strain (CTS), to mechanically stimulate human ASCs to participate in the formation skeletal myotubes in an in vitro model of myogenesis. The application of CTS for 48 h resulted in the formation of a highly ordered array of parallel ASCs, but failed to support skeletal muscle terminal differentiation. When the same stimulation paradigm was applied to cocultures with mouse skeletal muscle myoblasts, the percentage of ASCs contributing to the formation of myotubes significantly exceeded the levels reported in the literature hitherto. In perspective, the mechanical strain may be used to increase the efficiency of incorporation of ASCs in the skeletal muscles, which could be found useful in diverse traumatic or pathologic scenarios.     

Characterization of Proliferating Neural Progenitors after Spinal Cord Injury in Adult Zebrafish

Zebrafish can repair their injured brain and spinal cord after injury unlike adult mammalian central nervous system. Any injury to zebrafish spinal cord would lead to increased proliferation and neurogenesis. There are presences of proliferating progenitors from which both neuronal and glial loss can be reversed by appropriately generating new neurons and glia. We have demonstrated the presence of multiple progenitors, which are different types of proliferating populations like Sox2⁺ neural progenitor, A2B5⁺ astrocyte/ glial progenitor, NG2⁺ oligodendrocyte progenitor, radial glia and Schwann cell like progenitor. We analyzed the expression levels of two common markers of dedifferentiation like msx-b and vimentin during regeneration along with some of the pluripotency associated factors to explore the possible role of these two processes. Among the several key factors related to pluripotency, pou5f1 and sox2 are upregulated during regeneration and associated with activation of neural progenitor cells. Uncovering the molecular mechanism for endogenous regeneration of adult zebrafish spinal cord would give us more clues on important targets for future therapeutic approach in mammalian spinal cord repair and regeneration.     

Versican Processing by a Disintegrin-like and Metalloproteinase Domain with Thrombospondin-1 Repeats Proteinases-5 and -15 Facilitates Myoblast Fusion

Skeletal muscle development and regeneration requires the fusion of myoblasts into multinucleated myotubes. Because the enzymatic proteolysis of a hyaluronan and versican-rich matrix by ADAMTS versicanases is required for developmental morphogenesis, we hypothesized that the clearance of versican may facilitate the fusion of myoblasts during myogenesis. Here, we used transgenic mice and an in vitro model of myoblast fusion, C2C12 cells, to determine a potential role for ADAMTS versicanases. Versican processing was observed during in vivo myogenesis at the time when myoblasts were fusing to form multinucleated myotubes. Relevant ADAMTS genes, chief among them Adamts5 and Adamts15, were expressed both in developing embryonic muscle and differentiating C2C12 cells. Reducing the levels of Adamts5 mRNA in vitro impaired myoblast fusion, which could be rescued with catalytically active but not the inactive forms of ADAMTS5 or ADAMTS15. The addition of inactive ADAMTS5, ADAMTS15, or full-length V1 versican effectively impaired myoblast fusion. Finally, the expansion of a hyaluronan and versican-rich matrix was observed upon reducing the levels of Adamts5 mRNA in myoblasts. These data indicate that these ADAMTS proteinases contribute to the formation of multinucleated myotubes such as is necessary for both skeletal muscle development and during regeneration, by remodeling a versican-rich pericellular matrix of myoblasts. Our study identifies a possible pathway to target for the improvement of myogenesis in a plethora of diseases including cancer cachexia, sarcopenia, and muscular dystrophy.     

Clonal analysis for elucidating the lineage potential of embryonic NG2+ cells

Background aimsThe widespread NG2-expressing neural progenitors in the central nervous system (CNS) are considered to be multifunctional cells with lineage plasticity, thereby possessing the potential for treating CNS diseases. Their lineages and functional characteristics have not been completely unraveled. The present study aimed to disclose the lineage potential of clonal NG2⁺ populations in vitro and in vivo.MethodsTwenty-four clones from embryonic cerebral cortex-derived NG2⁺ cells were induced for oligodendrocyte, astrocyte, neuronal and chondrocyte differentiation. The expression profiles of neural progenitor markers chondroitin sulfate proteoglycan 4 (NG2), platelet-derived growth factor-α receptor (PDGFαR); nestin and neuronal cell surface antigen (A2B5) were subsequently sorted on cells with distinct differentiation capacity. Transplantation of these NG2⁺ clones into the spinal cord was used to examine their lineage potential in vivo.ResultsIn vitro differentiation analysis revealed that all the clones could differentiate into oligodendrocytes, and seven of them were bipotent (oligodendrocytes and astrocytes). Amazingly, one clone exhibited a multipotent capacity of differentiating into not only neuronal–glial lineages but also chondrocytes. These distinct subtypes were further found to exhibit phenotypic heterogeneity based on the examination of a spectrum of neural progenitor markers. Transplanted clones survived, migrated extensively and differentiated into oligodendrocytes, astrocytes or even neurons to integrate with the host spinal cord environmentConclusionsThese results suggest that NG2⁺ cells contain heterogeneous progenitors with distinct differentiation capacities, and the immortalized clonal NG2⁺ cell lines might provide a cell source for treating spinal cord disorders.