RESUMEN
Duchenne muscular dystrophy (DMD) is a severe muscular disorder. It was reported that multiple exon skipping (MES), targeting exon 45-55 of the DMD gene, might improve patients' symptoms because patients who have a genomic deletion of all these exons showed very mild symptoms. Thus, exon 45-55 skipping treatments for DMD have been proposed as a potential clinical cure. Herein, we detected the expression of endogenous exons 44-56 connected mRNA transcript of the DMD using total RNAs derived from human normal skeletal muscle by reverse transcription polymerase chain reaction (RT-PCR), and identified a total of eight types of MES products around the hotspot. Surprisingly, the 5' splice sites of recently reported post-transcriptional introns (remaining introns after co-transcriptional splicing) act as splicing donor sites for MESs. We also tested exon combinations to generate DMD circular RNAs (circRNAs) and determined the preferential splice sites of back-splicing, which are involved not only in circRNA generation, but also in MESs. Our results fit the current circRNA-generation model, suggesting that upstream post-transcriptional introns trigger MES and generate circRNA because its existence is critical for the intra-intronic interaction or for extremely distal splicing.
Asunto(s)
Distrofina/genética , Exones , Distrofia Muscular de Duchenne/genética , Precursores del ARN/genética , Empalme del ARN , Expresión Génica , Humanos , Mutación , ARN Mensajero/genética , Transcripción GenéticaRESUMEN
Satellite cells are muscle stem cells that have important roles in postnatal muscle growth and adult muscle regeneration. Although fast- and slow-dividing populations in activated satellite cells have been observed, the functional differences between them remain unclear. Here we elucidated the relationship between proliferation behaviour and satellite cell function. To assess the frequency of cell division, satellite cells isolated from mouse EDL muscle were labelled with the fluorescent dye PKH26, stimulated to proliferate and then sorted by FACS. The vast majority of activated satellite cells were PKH26(low) fast-dividing cells, whereas PKH26(high) slow-dividing cells were observed as a minority population. The fast-dividing cells generated a higher number of differentiated and self-renewed cells compared with the slow-dividing cells. However, cells derived from the slow-dividing population formed secondary myogenic colonies when passaged, whereas those from the fast-dividing population rapidly underwent myogenic differentiation without producing self-renewing cells after a few rounds of cell division. Furthermore, slow-dividing cells transplanted into injured muscle extensively contributed to muscle regeneration in vivo. Id1, a HLH protein, was expressed by all activated satellite cells, but the expression level varied within the slow-dividing cell population. We show that the slow-dividing cells retaining long-term self-renewal ability are restricted to an undifferentiated population that express high levels of Id1 protein (PKH26(high)Id1(high) population). Finally, genome-wide gene expression analysis described the molecular characteristics of the PKH26(high)Id1(high) population. Taken together, our results indicate that undifferentiated slow-dividing satellite cells retain stemness for generating progeny capable of long-term self-renewal, and so might be essential for muscle homeostasis throughout life.
Asunto(s)
División Celular , Fibras Musculares Esqueléticas/citología , Fibras Musculares Esqueléticas/fisiología , Regeneración , Células Satélite del Músculo Esquelético/citología , Células Satélite del Músculo Esquelético/fisiología , Animales , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Citometría de Flujo , Colorantes Fluorescentes , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones SCID , Ratones Transgénicos , Fibras Musculares Esqueléticas/metabolismoRESUMEN
Exon-skipping therapy mediated by antisense oligonucleotides is expected to provide a therapeutic option for Duchenne muscular dystrophy. Antisense oligonucleotides for exon skipping reported so far target a single continuous sequence in or around the target exon. In the present study, we investigated antisense oligonucleotides for exon 44 skipping (applicable to approximately 6% of all Duchenne muscular dystrophy patients) to improve activity by using a novel antisense oligonucleotide design incorporating two connected sequences. Phosphorodiamidate morpholino oligomers targeting two separate sequences in exon 44 were created to target two splicing regulators in exon 44 simultaneously, and their exon 44 skipping was measured. NS-089/NCNP-02 showed the highest skipping activity among the oligomers. NS-089/NCNP-02 also induced exon 44 skipping and dystrophin protein expression in cells from a Duchenne muscular dystrophy patient to whom exon 44 skipping is applicable. We also assessed the in vivo activity of NS-089/NCNP-02 by intravenous administration to cynomolgus monkeys. NS-089/NCNP-02 induced exon 44 skipping in skeletal and cardiac muscle of cynomolgus monkeys. In conclusion, NS-089/NCNP-02, an antisense oligonucleotide with a novel connected-sequence design, showed highly efficient exon skipping both in vitro and in vivo.
RESUMEN
This study was conducted to evaluate the safety and efficacy of human peripheral blood CD34 positive (CD34+) cells transplanted into a murine chronic stroke model to obtain pre-clinical proof of concept, prior to clinical testing. Granulocyte colony stimulating factor (G-CSF) mobilized human CD34+ cells [1 × 104 cells in 50 µl phosphate-buffered saline (PBS)] were intravenously (iv) or intra-carotid arterially (ia) transplanted 4 weeks after the induction of stroke (chronic stage), and neurological function was evaluated. In this study, severe combined immune deficiency (SCID) mice were used to prevent excessive immune response after cell therapy. Two weeks post cell therapy, the ia CD34+ cells group demonstrated a significant improvement in neurological functions compared to the PBS control. The therapeutic effect was maintained 8 weeks after the treatment. Even after a single administration, ia transplantation of CD34+ cells had a significant therapeutic effect on chronic stroke. Based on the result of this pre-clinical proof of concept study, a future clinical trial of autologous peripheral blood CD34+ cells administration in the intra-carotid artery for chronic stroke patients is planned.
RESUMEN
Muscle satellite cells are essential for muscle growth and regeneration and their morphology, behavior and gene expression have been extensively studied. However, the mechanisms involved in their proliferation and differentiation remain elusive. Six1 and Six4 proteins were expressed in the nuclei of myofibers of adult mice and the numbers of myoblasts positive for Six1 and Six4 increased during regeneration of skeletal muscles. Six1 and Six4 were expressed in quiescent, activated and differentiated muscle satellite cells isolated from adult skeletal muscle. Overexpression of Six4 and Six5 repressed the proliferation and differentiation of satellite cells. Conversely, knockdown of Six5 resulted in augmented proliferation, and that of Six4 inhibited differentiation. Muscle satellite cells isolated from Six4(+/-)Six5(-/-) mice proliferated to higher cell density though their differentiation was not altered. Meanwhile, overproduction of Six1 repressed proliferation and promoted differentiation of satellite cells. In addition, Six4 and Six5 repressed, while Six1 activated myogenin expression, suggesting that the differential regulation of myogenin expression is responsible for the differential effects of Six genes. The results indicated the involvement of Six genes in the behavior of satellite cells and identified Six genes as potential target for manipulation of proliferation and differentiation of muscle satellite cells for therapeutic applications.
Asunto(s)
Diferenciación Celular/genética , Proliferación Celular , Proteínas de Homeodominio/genética , Células Satélite del Músculo Esquelético/citología , Transactivadores/genética , Adulto , Animales , Regulación de la Expresión Génica/fisiología , Proteínas de Homeodominio/fisiología , Humanos , Ratones , Ratones Noqueados , Células Musculares/citología , Miogenina/biosíntesis , Transactivadores/fisiologíaRESUMEN
Objective: We sought to investigate the efficacy of human bone marrow mesenchymal stem/stromal cell (hBM-MSC) in a murine spinal cord ischemia/reperfusion (SCIR) model. Methods: C57BL/6J mice were subjected to SCIR by crossclamping the aortic arch and left subclavian artery for 5.5 minutes. Two hours after reperfusion, hBM-MSCs (hBM-MSC group) or phosphate-buffered saline (control group) were intravenously injected without immunosuppressant. Hindlimb motor function was assessed until day 28 after reperfusion using the Basso Mouse Scale (BMS). The lumbar spinal cord was harvested at hour 24 and day 28, and the histologic number of NeuN-positive motor neurons in 3 cross-sections of each lumbar spinal cord and the gene expression were evaluated. Results: BMS score was 0 throughout the study period in all control mice. BMS score was significantly greater in the hBM-MSC group than the control group from hour 8 (P < .05) to day 28 (P < .01). The numbers of motor neurons at hour 24 (P < .01) and day 28 (P < .05) were significantly preserved in the hBM-MSC group than the control group. mRNA expression levels of proinflammatory cytokines were significantly lower (P < .05), and those of insulin-like growth factor-1 (P < .01) and proangiogenic factors (P < .05) were significantly greater in the hBM-MSC group than the control group at hour 24. Conclusions: hBM-MSC therapy may attenuate SCIR injury by preserving motor neurons, at least in part, through inhibition of proinflammatory cytokines and upregulation of proangiogenic factors in the reperfusion-injured spinal cord.
RESUMEN
Skeletal muscle satellite cells play key roles in postnatal muscle growth and regeneration. To study molecular regulation of satellite cells, we directly prepared satellite cells from 8- to 12-week-old C57BL/6 mice and performed genome-wide gene expression analysis. Compared with activated/cycling satellite cells, 507 genes were highly upregulated in quiescent satellite cells. These included negative regulators of cell cycle and myogenic inhibitors. Gene set enrichment analysis revealed that quiescent satellite cells preferentially express the genes involved in cell-cell adhesion, regulation of cell growth, formation of extracellular matrix, copper and iron homeostasis, and lipid transportation. Furthermore, reverse transcription-polymerase chain reaction on differentially expressed genes confirmed that calcitonin receptor (CTR) was exclusively expressed in dormant satellite cells but not in activated satellite cells. In addition, CTR mRNA is hardly detected in nonmyogenic cells. Therefore, we next examined the expression of CTR in vivo. CTR was specifically expressed on quiescent satellite cells, but the expression was not found on activated/proliferating satellite cells during muscle regeneration. CTR-positive cells reappeared at the rim of regenerating myofibers in later stages of muscle regeneration. Calcitonin stimulation delayed the activation of quiescent satellite cells. Our data provide roles of CTR in quiescent satellite cells and a solid scaffold to further dissect molecular regulation of satellite cells. Disclosure of potential conflicts of interest is found at the end of this article.
Asunto(s)
Perfilación de la Expresión Génica , Desarrollo de Músculos/genética , Proteínas Musculares/análisis , Células Satélite del Músculo Esquelético/química , Animales , Proteínas Reguladoras de la Apoptosis/biosíntesis , Proteínas Reguladoras de la Apoptosis/genética , Biomarcadores , Calcitonina/farmacología , Moléculas de Adhesión Celular/biosíntesis , Moléculas de Adhesión Celular/genética , Proteínas de Ciclo Celular/biosíntesis , Proteínas de Ciclo Celular/genética , Diferenciación Celular , División Celular/genética , Femenino , Regulación del Desarrollo de la Expresión Génica , Ratones , Ratones Endogámicos C57BL , Proteínas Musculares/biosíntesis , Proteínas Musculares/genética , Músculo Esquelético/fisiología , Factores Reguladores Miogénicos/biosíntesis , Factores Reguladores Miogénicos/genética , ARN Mensajero/biosíntesis , Receptores de Calcitonina/biosíntesis , Receptores de Calcitonina/genética , Regeneración/genética , Células Satélite del Músculo Esquelético/efectos de los fármacos , Células Satélite del Músculo Esquelético/metabolismoRESUMEN
Duchenne muscular dystrophy (DMD), the most common lethal heritable childhood disease, is caused by mutations in the DMD gene that result in the absence of functional dystrophin protein. Exon skipping mediated by antisense oligonucleotides has recently emerged as an effective approach for the restoration of dystrophin, and skipping of exon 51 of DMD has received accelerated approval. Identifying antisense sequences that can provide the highest possible skipping efficiency is crucial for future clinical applications. Herein, we systematically tested two-step antisense oligonucleotide walks along human DMD exon 53 in order to define sequence-dependent effects of antisense oligonucleotide binding sites in human rhabdomyosarcoma cell lines. The first rough whole-exon 53 walk enabled the identification of a target region, and a second walk of this region was used to determine an optimal antisense oligonucleotide sequence (NS-065/NCNP-01) for exon 53 skipping. This oligonucleotide strongly promoted exon 53 skipping in a dose-dependent manner during pre-mRNA splicing in rhabdomyosarcoma and DMD patient-derived cells, and it restored dystrophin protein levels in patient-derived cells. NS-065/NCNP-01, a phosphorodiamidate morpholino oligomer, appears to be a promising candidate for treating exon 53 skipping, and it is potentially applicable to 10.1% of patients with DMD.
RESUMEN
Duchenne muscular dystrophy (DMD) is a lethal hereditary muscle disease caused by mutations in the gene encoding the muscle protein dystrophin. These mutations result in a shift in the open reading frame leading to loss of the dystrophin protein. Antisense oligonucleotides (ASOs) that induce exon skipping correct this frame shift during pre-mRNA splicing and partially restore dystrophin expression in mouse and dog models. We conducted a phase 1, open-label, dose-escalation clinical trial to determine the safety, pharmacokinetics, and activity of NS-065/NCNP-01, a morpholino ASO that enables skipping of exon 53. Ten patients with DMD (6 to 16 years old), carrying mutations in the dystrophin gene whose reading frame would be restored by exon 53 skipping, were administered NS-065/NCNP-01 at doses of 1.25, 5, or 20 mg/kg weekly for 12 weeks. The primary endpoint was safety; the secondary endpoints were pharmacokinetics and successful exon skipping. No severe adverse drug reactions were observed, and no treatment discontinuation occurred. Muscle biopsy samples were taken before and after treatment and compared by reverse transcription polymerase chain reaction (RT-PCR), immunofluorescence, and Western blotting to assess the amount of exon 53 skipping and dystrophin expression. NS-065/NCNP-01 induced exon 53 skipping in dystrophin-encoding mRNA in a dose-dependent manner and increased the dystrophin/spectrin ratio in 7 of 10 patients. Furthermore, the amount of exon skipping correlated with the maximum drug concentration in plasma (Cmax) and the area under the concentration-time curve in plasma (AUC0-t ). These results indicate that NS-065/NCNP-01 has a favorable safety profile and promising pharmacokinetics warranting further study in a phase 2 clinical trial.
Asunto(s)
Exones/genética , Distrofia Muscular de Duchenne/tratamiento farmacológico , Distrofia Muscular de Duchenne/genética , Oligonucleótidos Antisentido/uso terapéutico , Administración Intravenosa , Adolescente , Niño , Preescolar , Distrofina/genética , Humanos , Masculino , Morfolinos/administración & dosificación , Morfolinos/uso terapéutico , Oligonucleótidos Antisentido/administración & dosificación , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Human induced pluripotent stem cells (hiPSCs) are a potential source for cell therapy of Duchenne muscular dystrophy. To reliably obtain skeletal muscle progenitors from hiPSCs, we treated hiPS cells with a Wnt activator, CHIR-99021 and a BMP receptor inhibitor, LDN-193189, and then induced skeletal muscle cells using a previously reported sphere-based culture. This protocol greatly improved sphere formation efficiency and stably induced the differentiation of myogenic cells from hiPS cells generated from both healthy donors and a patient with congenital myasthenic syndrome. hiPSC-derived myogenic progenitors were enriched in the CD57(-) CD108(-) CD271(+) ERBB3(+) cell fraction, and their differentiation was greatly promoted by TGF-ß inhibitors. TGF-ß inhibitors down-regulated the NFIX transcription factor, and NFIX short hairpin RNA (shRNA) improved the differentiation of iPS cell-derived myogenic progenitors. These results suggest that NFIX inhibited differentiation of myogenic progenitors. hiPSC-derived myogenic cells differentiated into myofibers in muscles of NSG-mdx 4Cv mice after direct transplantation. Our results indicate that our new muscle induction protocol is useful for cell therapy of muscular dystrophies.
Asunto(s)
Diferenciación Celular , Mioblastos/citología , Mioblastos/metabolismo , Células Madre Pluripotentes/citología , Biomarcadores , Técnicas de Cultivo de Célula , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Técnica del Anticuerpo Fluorescente , Expresión Génica , Humanos , Inmunofenotipificación , Células Madre Pluripotentes Inducidas/citología , Músculo Esquelético/metabolismo , Pirazoles/farmacología , Piridinas/farmacología , Pirimidinas/farmacología , Regeneración/genética , Trasplante de Células Madre , Factor de Crecimiento Transformador beta/metabolismo , Factor de Crecimiento Transformador beta/farmacologíaRESUMEN
Three to eight percent of female carriers of Duchenne muscular dystrophy (DMD) develop dystrophic symptoms ranging from mild muscle weakness to a rapidly progressive DMD-like muscular dystrophy due to skewed inactivation of X chromosomes during early development. Here, we generated human induced pluripotent stem cells (hiPSCs) from a manifesting female carrier using retroviral or Sendai viral (SeV) vectors and determined their X-inactivation status. Although manifesting carrier-derived iPS cells showed normal expression of human embryonic stem cell markers and formed well-differentiated teratomas in vivo, many hiPS clones showed bi-allelic expression of the androgen receptor (AR) gene and loss of X-inactivation-specific transcript and trimethyl-histone H3 (Lys27) signals on X chromosomes, suggesting that both X chromosomes of the hiPS cells are in an active state. Importantly, normal dystrophin was expressed in multinucleated myotubes differentiated from a manifesting carrier of DMD-hiPS cells with XaXa pattern. AR transcripts were also equally transcribed from both alleles in induced myotubes. Our results indicated that the inactivated X chromosome in the patient's fibroblasts was activated during reprogramming, and XCI occurred randomly during differentiation.
RESUMEN
Skeletal muscle contains two distinct stem/progenitor populations. One is the satellite cell, which acts as a muscle stem cell, and the other is the mesenchymal progenitor, which contributes to muscle pathogeneses such as fat infiltration and fibrosis. Detailed and accurate characterization of these progenitors in humans remains elusive. Here, we performed comprehensive cell-surface protein profiling of the two progenitor populations residing in human skeletal muscle and identified three previously unrecognized markers: CD82 and CD318 for satellite cells and CD201 for mesenchymal progenitors. These markers distinguish myogenic and mesenchymal progenitors, and enable efficient isolation of the two types of progenitors. Functional study revealed that CD82 ensures expansion and preservation of myogenic progenitors by suppressing excessive differentiation, and CD201 signaling favors adipogenesis of mesenchymal progenitors. Thus, cell-surface proteins identified here are not only useful markers but also functionally important molecules, and provide valuable insight into human muscle biology and diseases.
Asunto(s)
Membrana Celular/metabolismo , Proteínas de la Membrana/metabolismo , Músculo Esquelético/citología , Proteómica/métodos , Células Madre/metabolismo , Adipogénesis , Anticuerpos/metabolismo , Antígenos CD/metabolismo , Biomarcadores , Separación Celular , HumanosRESUMEN
Induced pluripotent stem cells (iPSCs) hold promise as a potential treatment for Duchenne muscular dystrophy (DMD). To determine the impact of the donor's age on reprogramming, we generated iPSCs from muscle-derived fibroblasts (MuFs) of mdx mice aged 6 weeks, 6 months, and 14 months. MuFs from 14-month-old mdx mice showed lower proliferative activity and lower reprogramming efficiency, compared with those from younger mdx mice. Furthermore, iPSCs derived from 14-month-old mdx mice (14m-MuF-iPSCs) gradually lost Nanog expression, and regressed in conventional ES medium during passages. Interestingly, inhibition of TGF-ß signaling and BMP signaling stabilized Nanog expression and promoted self-renewal of 14m-MuF-iPSCs. Finally, rescued mdx-derived iPSCs efficiently differentiated into the skeletal muscle lineage.
RESUMEN
Duchenne muscular dystrophy (DMD) is a lethal muscle disorder caused by mutations in the dystrophin gene. Transplantation of autologous myogenic cells genetically corrected ex vivo is a possible treatment for this disorder. In order to test the regenerative efficiency of freshly isolated satellite cells, we purified quiescent satellite cells from limb muscles of 8-12-week-old green fluorescent protein-transgenic (GFP-Tg) mice using SM/C-2.6 (a recently developed monoclonal antibody) and flow cytometry. Freshly isolated satellite cells were shown to participate in muscle regeneration more efficiently than satellite cell-derived myoblasts passaged in vitro do, when transplanted into tibialis anterior (TA) muscles of 8-12-week-old cardiotoxin-injected C57BL/6 mice and 5-week-old dystrophin-deficient mdx mice, and analyzed at 4 weeks after injection. Importantly, expansion of freshly isolated satellite cells in vitro without passaging had no detrimental effects on their regenerative capacity. Therefore we directly isolated satellite cells from 5-week-old mdx mice using SM/C-2.6 antibody and cultured them with lentiviral vectors expressing micro-dystrophin CS1. The transduced cells were injected into TA muscles of 5-week-old mdx mice. At 4 weeks after transplantation, the grafted cells efficiently contributed to regeneration of mdx dystrophic muscles and expressed micro-dystrophin at the sarcolemma. These results suggest that there is potential for lentiviral vector-mediated ex vivo gene therapy for DMD.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Trasplante de Células , ADN Complementario/administración & dosificación , Distrofina/genética , Vectores Genéticos , Transducción Genética , Animales , Línea Celular , ADN Complementario/genética , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos mdx , TransfecciónRESUMEN
Skeletal muscle regeneration has been exclusively attributed to myogenic precursors, satellite cells. A stem cell-rich fraction referred to as side population (SP) cells also resides in skeletal muscle, but its roles in muscle regeneration remain unclear. We found that muscle SP cells could be subdivided into three sub-fractions using CD31 and CD45 markers. The majority of SP cells in normal non-regenerating muscle expressed CD31 and had endothelial characteristics. However, CD31(-)CD45(-) SP cells, which are a minor subpopulation in normal muscle, actively proliferated upon muscle injury and expressed not only several regulatory genes for muscle regeneration but also some mesenchymal lineage markers. CD31(-)CD45(-) SP cells showed the greatest myogenic potential among three SP sub-fractions, but indeed revealed mesenchymal potentials in vitro. These SP cells preferentially differentiated into myofibers after intramuscular transplantation in vivo. Our results revealed the heterogeneity of muscle SP cells and suggest that CD31(-)CD45(-) SP cells participate in muscle regeneration.
Asunto(s)
Músculo Esquelético/citología , Músculo Esquelético/fisiología , Animales , Diferenciación Celular , Linaje de la Célula , Proliferación Celular , Células Cultivadas , Células Endoteliales/citología , Regulación de la Expresión Génica , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Ratones , Ratones Transgénicos , Desarrollo de Músculos , Músculo Esquelético/irrigación sanguínea , Músculo Esquelético/metabolismo , Miofibrillas/metabolismo , RegeneraciónRESUMEN
In denervated skeletal muscle, mononuclear interstitial cells accumulate in the perisynaptic regions before fibrotic change occurs. These cells are currently considered to be fibroblasts that originate from muscle tissue. However, when we denervated hind limbs of GFP-bone marrow chimeric mice by excising the sciatic nerve unilaterally, many bone marrow-derived cells (BM-DCs) infiltrated the interstitial spaces and accumulated in the perisynaptic regions, peaking 14 days after denervation. They accounted for nearly one-half of the increase in mononuclear interstitial cells. Although BM-DCs did not incorporate into satellite cells, immunohistochemical and FACS analyses revealed that BM-DCs were both CD45 and CD11b positive, indicating that they were of macrophage/monocyte lineage. BrdU staining showed inactive proliferation of BM-DCs. Reverse transcriptase-polymerase chain reaction of mononuclear cells isolated by FACS revealed that BM-DCs did not express type I collagen or tenascin-C; however, they did express transforming growth factor-beta1, suggesting that they regulate the fibrotic process. In contrast, muscle tissue-derived interstitial cells expressed type I collagen and tenascin-C, suggesting that these populations were the final effectors of fibrosis. These findings identify elementary targets that may regulate the migration, homing, differentiation, and function of BM-DCs, leading to amelioration of the excessive fibrosis of denervated skeletal muscle.
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Células de la Médula Ósea/citología , Células de la Médula Ósea/metabolismo , Fibrosis/patología , Leucocitos Mononucleares/metabolismo , Músculo Esquelético/patología , Animales , Apoptosis/fisiología , Western Blotting , Antígeno CD11b/metabolismo , Linaje de la Célula , Movimiento Celular , Proliferación Celular , Colágeno Tipo I/biosíntesis , Citometría de Flujo , Inmunohistoquímica , Antígenos Comunes de Leucocito/metabolismo , Leucocitos Mononucleares/citología , Masculino , Ratones , Ratones Transgénicos , Desnervación Muscular , Músculo Esquelético/inervación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tenascina/biosíntesis , Factor de Crecimiento Transformador beta/metabolismo , Factor de Crecimiento Transformador beta1RESUMEN
Recent studies have shown that bone marrow (BM) cells, including the BM side population (BM-SP) cells that enrich hematopoietic stem cells (HSCs), are incorporated into skeletal muscle during regeneration, but it is not clear how and what kinds of BM cells contribute to muscle fiber regeneration. We found that a large number of SP cells migrated from BM to muscles following injury in BM-transplanted mice. These BM-derived SP cells in regenerating muscles expressed different surface markers from those of HSCs and could not reconstitute the mouse blood system. BM-derived SP/Mac-1(low) cells increased in number in regenerating muscles following injury. Importantly, our co-culture studies with activated satellite cells revealed that this fraction carried significant potential for myogenic differentiation. By contrast, mature inflammatory (Mac-1(high)) cells showed negligible myogenic activities. Further, these BM-derived SP/Mac-1(low) cells gave rise to mononucleate myocytes, indicating that their myogenesis was not caused by stochastic fusion with host myogenic cells, although they required cell-to-cell contact with myogenic cells for muscle differentiation. Taken together, our data suggest that neither HSCs nor mature inflammatory cells, but Mac-1(low) early myeloid cells in the BM-derived SP fraction, play an important role in regenerating skeletal muscles.
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Antígeno de Macrófago-1/metabolismo , Músculo Esquelético/fisiología , Células Mieloides/inmunología , Células Mieloides/fisiología , Regeneración , Animales , Células de la Médula Ósea/citología , Células de la Médula Ósea/inmunología , Células de la Médula Ósea/fisiología , Diferenciación Celular , Movimiento Celular , Proteínas Fluorescentes Verdes , Hematopoyesis , Proteínas Luminiscentes/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Modelos Biológicos , Fibras Musculares Esqueléticas/fisiología , Músculo Esquelético/citología , Músculo Esquelético/lesiones , Células Mieloides/citologíaRESUMEN
To examine the roles of cytokines in muscle regeneration, we injected cardiotoxin into mouse tibialis anterior muscle and examined the expression profiles of cytokines and related genes in the regeneration process. Expression of 40, 64, and 7 genes among 522 genes spotted on a cytokine expression array were increased more than fivefold at 48 hours, 96 hours, and 7 days after toxin injection, respectively, when compared with those of the control muscle. Especially the levels of mRNA for chemokines and chemokine receptors, many of which are potent regulators of macrophages, were highly elevated 48 hours after injury. The expression of osteopontin (OPN), a versatile regulator of inflammation and tissue repair, was up-regulated more than 118-fold in regenerating muscle at 48 hours after injury. Northern blotting confirmed that the expression of OPN was highest at 48 hours after cardiotoxin injection and declined sharply thereafter. Immunohistochemistry showed that OPN was detected both in the cytoplasm of macrophages and in necrotic muscle infiltrated with macrophages. Our studies suggest OPN may serve as an adhesion molecule that promotes macrophage binding to necrotic fibers and may be an important mediator in the early phase of muscle regeneration.