“The preadipocyte factor” DLK1 marks adult mouse adipose tissue residing vascular cells that lack in vitro adipogenic differentiation potential

Delta-like 1 (Dlk1) is expressed in 3T3-L1 preadipocytes and has frequently been referred to as “the” preadipocyte marker, yet the phenotype of DLK1⁺ cells in adipose tissue remains undetermined. Herein, we demonstrate that DLK1⁺ cells encompass around 1-2% of the adult mouse adipose stromal vascular fraction (SVF). Unexpectedly, the DLK1⁺SVF population was enriched for cells expressing genes generally ascribed to the vascular lineage and did not possess any adipogenic differentiation potential in vitro. Instead, DLK1⁺ cells comprised an immediate ability for cobblestone formation, generation of tube-like structures on matrigel, and uptake of Acetylated Low Density-Lipoprotein, all characteristics of endothelial cells. We therefore suggest that DLK1⁺SVF cells are of a vascular origin and not them-selves committed preadipocytes as assumed hitherto.     

Identification and characterization of PDGFRα+ mesenchymal progenitors in human skeletal muscle

Fatty and fibrous connective tissue formation is a hallmark of diseased skeletal muscle and deteriorates muscle function. We previously identified non-myogenic mesenchymal progenitors that contribute to adipogenesis and fibrogenesis in mouse skeletal muscle. In this study, we report the identification and characterization of a human counterpart to these progenitors. By using PDGFRα as a specific marker, mesenchymal progenitors can be identified in the interstitium and isolated from human skeletal muscle. PDGFRα⁺ cells represent a cell population distinct from CD56⁺ myogenic cells, and adipogenic and fibrogenic potentials were highly enriched in the PDGFRα⁺ population. Activation of PDGFRα stimulates proliferation of PDGFRα⁺ cells through PI3K-Akt and MEK2-MAPK signaling pathways, and aberrant accumulation of PDGFRα⁺ cells was conspicuous in muscles of patients with both genetic and non-genetic muscle diseases. Our results revealed the pathological relevance of PDGFRα⁺ mesenchymal progenitors to human muscle diseases and provide a basis for developing therapeutic strategy to treat muscle diseases.     

Sca-1 expression is required for efficient remodeling of the extracellular matrix during skeletal muscle regeneration

Sca-1 (Stem Cell Antigen-1) is a member of the Ly-6 family proteins that functions in cell growth, differentiation, and self-renewal in multiple tissues. In skeletal muscle Sca-1 negatively regulates myoblast proliferation and differentiation, and may function in the maintenance of progenitor cells. We investigated the role of Sca-1 in skeletal muscle regeneration and show here that Sca-1 expression is upregulated in a subset of myogenic cells upon muscle injury. We demonstrate that extract from crushed muscle upregulates Sca-1 expression in myoblasts in vitro, and that this effect is reversible and independent of cell proliferation. Sca-1^−/− mice exhibit defects in muscle regeneration, with the development of fibrosis following injury. Sca-1^−/− muscle displays reduced activity of matrix metalloproteinases, critical regulators of extracellular matrix remodeling. Interestingly, we show that the number of satellite cells is similar in wild-type and Sca-1^−/− muscle, suggesting that in satellite cells Sca-1 does not play a role in self-renewal. We hypothesize that Sca-1 upregulates, directly or indirectly, the activity of matrix metalloproteinases, leading to matrix breakdown and efficient muscle regeneration. Further elucidation of the role of Sca-1 in matrix remodeling may aid in the development of novel therapeutic strategies for the treatment of fibrotic diseases.     

TGF-β mediated FGF10 signaling in cranial neural crest cells controls development of myogenic progenitor cells through tissue-tissue interactions during tongue morphogenesis

Skeletal muscles are formed from two cell lineages, myogenic and fibroblastic. Mesoderm-derived myogenic progenitors form muscle cells whereas fibroblastic cells give rise to the supportive connective tissue of skeletal muscles, such as the tendons and perimysium. It remains unknown how myogenic and fibroblastic cell-cell interactions affect cell fate determination and the organization of skeletal muscle. In the present study, we investigated the functional significance of cell-cell interactions in regulating skeletal muscle development. Our study shows that cranial neural crest (CNC) cells give rise to the fibroblastic cells of the tongue skeletal muscle in mice. Loss of Tgfbr2 in CNC cells (Wnt1-Cre;Tgfbr2^flox/flox) results in microglossia with reduced Scleraxis and Fgf10 expression as well as decreased myogenic cell proliferation, reduced cell number and disorganized tongue muscles. Furthermore, TGF-β2 beads induced the expression of Scleraxis in tongue explant cultures. The addition of FGF10 rescued the muscle cell number in Wnt1-Cre;Tgfbr2^flox/flox mice. Thus, TGF-β induced FGF10 signaling has a critical function in regulating tissue-tissue interaction during tongue skeletal muscle development.     

Target gene selectivity of the myogenic basic helix-loop-helix transcription factor myogenin in embryonic muscle

The myogenic regulatory factors MyoD and myogenin are crucial for skeletal muscle development. Despite their importance, the mechanisms by which these factors selectively regulate different target genes are unclear. The purpose of the present investigation was to compare embryonic skeletal muscle from myogenin^+/+ and myogenin^−/− mice to identify genes whose expression was dependent on the presence of myogenin but not MyoD and to determine whether myogenin-binding sites could be found within regulatory regions of myogenin-dependent genes independent of MyoD. We identified a set of 140 muscle-expressed genes whose expression in embryonic tongue muscle of myogenin^−/− mice was downregulated in the absence of myogenin, but in the presence of MyoD. Myogenin bound within conserved regulatory regions of several of the downregulated genes, but MyoD bound only to a subset of these same regions, suggesting that many downregulated genes were selective targets of myogenin. The regulatory regions activated gene expression in cultured myoblasts and fibroblasts overexpressing myogenin or MyoD, indicating that expression from exogenously introduced DNA could not recapitulate the selectivity for myogenin observed in vivo. The results identify new target genes for myogenin and show that myogenin's target gene selectivity is not based solely on binding site sequences.     

Identification of tissue-specific vasculogenic cells originating from murine uterus

Endometrium is a highly regenerative adult tissue that undergoes repeated degeneration and regeneration following menarche. Therefore, it is believed that endometrium contains stem and/or progenitor cells in order to compensate for the regeneration of tissue components. We report here that stem-like cells having vasculogenic potential are present in the uterus. Enzymatically extracted cells from murine uteri were characterized and fractionated into four subpopulations by flowcytometry; CD34⁺/45⁻ (Ut-34), CD34⁻/45⁻ (Ut-DN) and the remaining CD45⁺ cell fractions (CD34⁺/45⁺ and CD34⁻/45⁺ cells). The Ut-34 and Ut-DN fractions were mostly negative for putative endothelial cell (EC) markers, such as CD31, Flk-1, c-kit and VE-cadherin, although the Ut-DN fraction contained 2.8% CD31⁺ cells. Ut-DN cells were further divided into CD31⁺ and CD31⁻ fractions. Three cell populations were obtained from green fluorescence protein (GFP) transgenic mice and were transplanted into injured wild-type mouse skeletal muscle. At 4 weeks after cell transplantation, donor-derived vascular smooth muscle and ECs were observed in the injured recipient muscle. A similar trend was observed in the Ut-34 group, but differentiation into vascular smooth muscle was predominant. In contrast, the Ut-DN/31⁺ cell-transplanted group showed preferential differentiation into vascular ECs, thus suggesting that they were relatively committed preexisting ECs. These characteristics were also seen in vitro, in clonal cell cultures. Interestingly, donor derived Ut-DN/31⁺, Ut-DN/31⁻ and Ut-34 cells could not be identified after bone marrow (BM) transplantation, thus confirming that they are not derived from BM. It therefore appeared that tissue-specific vasculogenic cells are present in the murine uterus and that they exhibit vascular formation, even in different tissue microenvironments.     

FOXP3+ T Cells Recruited to Sites of Sterile Skeletal Muscle Injury Regulate the Fate of Satellite Cells and Guide Effective Tissue Regeneration

Muscle injury induces a classical inflammatory response in which cells of the innate immune system rapidly invade the tissue. Macrophages are prominently involved in this response and required for proper healing, as they are known to be important for clearing cellular debris and supporting satellite cell differentiation. Here, we sought to assess the role of the adaptive immune system in muscle regeneration after acute damage. We show that T lymphocytes are transiently recruited into the muscle after damage and appear to exert a pro-myogenic effect on muscle repair. We observed a decrease in the cross-sectional area of regenerating myofibers after injury in Rag2^-/- γ-chain^-/- mice, as compared to WT controls, suggesting that T cell recruitment promotes muscle regeneration. Skeletal muscle infiltrating T lymphocytes were enriched in CD4⁺CD25⁺FOXP3⁺ cells. Direct exposure of muscle satellite cells to in vitro induced Treg cells effectively enhanced their expansion, and concurrently inhibited their myogenic differentiation. In vivo, the recruitment of Tregs to acutely injured muscle was limited to the time period of satellite expansion, with possibly important implications for situations in which inflammatory conditions persist, such as muscular dystrophies and inflammatory myopathies. We conclude that the adaptive immune system, in particular T regulatory cells, is critically involved in effective skeletal muscle regeneration. Thus, in addition to their well-established role as regulators of the immune/inflammatory response, T regulatory cells also regulate the activity of skeletal muscle precursor cells, and are instrumental for the proper regeneration of this tissue.     

Divergent and conserved roles of Dll1 signaling in development of craniofacial and trunk muscle

Craniofacial and trunk skeletal muscles are evolutionarily distinct and derive from cranial and somitic mesoderm, respectively. Different regulatory hierarchies act upstream of myogenic regulatory factors in cranial and somitic mesoderm, but the same core regulatory network – MyoD, Myf5 and Mrf4 – executes the myogenic differentiation program. Notch signaling controls self-renewal of myogenic progenitors as well as satellite cell homing during formation of trunk muscle, but its role in craniofacial muscles has been little investigated. We show here that the pool of myogenic progenitor cells in craniofacial muscle of Dll1^LacZ/Ki mutant mice is depleted in early fetal development, which is accompanied by a major deficit in muscle growth. At the expense of progenitor cells, supernumerary differentiating myoblasts appear transiently and these express MyoD. The progenitor pool in craniofacial muscle of Dll1^LacZ/Ki mutants is largely rescued by an additional mutation of MyoD. We conclude from this that Notch exerts its decisive role in craniofacial myogenesis by repression of MyoD. This function is similar to the one previously observed in trunk myogenesis, and is thus conserved in cranial and trunk muscle. However, in cranial mesoderm-derived progenitors, Notch signaling is not required for Pax7 expression and impinges little on the homing of satellite cells. Thus, Dll1 functions in satellite cell homing and Pax7 expression diverge in cranial- and somite-derived muscle.     

The AMPK-SIRT signaling network regulates glucose tolerance under calorie restriction conditions

Aims

SIRT1 and AMP-activated protein kinase (AMPK) share common activators, actions and target molecules. Previous studies have suggested that a putative SIRT1-AMPK regulatory network could act as the prime initial sensor for calorie restriction-induced adaptations in skeletal muscle—the major site of insulin-stimulated glucose disposal. Our study aimed to investigate whether a feedback loop exists between AMPK and SIRT1 in skeletal muscle and how this may be involved glucose tolerance.

Main methods

To investigate this, we used skeletal muscle-specific AMPKα1/2 knockout mice (AMPKα1/2^−/−) fed ad libitum (AL) or a 30% calorie restricted (CR) diet and L6 rat myoblasts incubated with SIRT1 inhibitor (EX527).

Key findings

CR-AMPKα1/2^−/− displayed impaired glucose tolerance (*p < 0.05), in association with down-regulated SIRT1 and PGC-1α expression (< 300% vs. CR-WT, ^±±p < 0.01). Moreover, AMPK activity was decreased following SIRT1 inhibition in L6 cells (~ 0.5-fold vs. control, *p < 0.05).

Significance

This study demonstrates that skeletal muscle-specific AMPK deficiency impairs the beneficial effects of CR on glucose tolerance and that these effects may be dependent on reduced SIRT1 levels.     

Excitation-contraction coupling in resistance mesenteric arteries: Evidence for NKCC1-mediated pathway

Bumetanide and other high-ceiling diuretics (HCD) attenuate myogenic tone and contractions of vascular smooth muscle cells (VSMC) triggered by diverse stimuli. HCD outcome may be mediated by their interaction with NKCC1, the only isoform of Na⁺, K⁺, 2Cl⁻ cotransporter expressed in VSMC as well as with targets distinct from this carrier. To examine these hypotheses, we compared the effect of bumetanide on contractions of mesenteric arteries from wild-type and NKCC1 knockout mice. In mesenteric arteries from wild-type controls, 100 μM bumetanide evoked a decrease of up to 4-fold in myogenic tone and contractions triggered by modest [K⁺]_o-induced depolarization, phenylephrine and UTP. These actions of bumetanide were preserved after inhibition of nitric oxide synthase with NG-nitro-l-arginine methyl ester, but were absent in mesenteric arteries from NKCC1^-/- mice. The data show that bumetanide inhibits VSMC contractile responses via its interaction with NKCC1 and independently of nitric oxide production by endothelial cells.     

Directed In Vitro Myogenesis of Human Embryonic Stem Cells and Their In Vivo Engraftment

Development of human embryonic stem cell (hESC)-based therapy requires derivation of in vitro expandable cell populations that can readily differentiate to specified cell types and engraft upon transplantation. Here, we report that hESCs can differentiate into skeletal muscle cells without genetic manipulation. This is achieved through the isolation of cells expressing a mesodermal marker, platelet-derived growth factor receptor-α (PDGFRA), following embryoid body (EB) formation. The ESC-derived cells differentiated into myoblasts in vitro as evident by upregulation of various myogenic genes, irrespective of the presence of serum in the medium. This result is further corroborated by the presence of sarcomeric myosin and desmin, markers for terminally differentiated cells. When transplanted in vivo, these pre-myogenically committed cells were viable in tibialis anterior muscles 14 days post-implantation. These hESC-derived cells, which readily undergo myogenic differentiation in culture medium containing serum, could be a viable cell source for skeletal muscle repair and tissue engineering to ameliorate various muscle wasting diseases.     

Synchronized reconstitution of muscle fibers,peripheral nerves and blood vessels by murine skeletal muscle-derived CD34<Superscript>−</Superscript>/45<Superscript>−</Superscript> cells

In order to establish the practical isolation and usage of skeletal muscle-derived stem cells (MDSCs), we determined reconstitution capacity of CD34⁻/CD45⁻ (Sk-DN) cells as a candidate somatic stem cell source for transplantation. Sk-DN cells were enzymatically isolated from GFP transgenic mice (C57/BL6N) skeletal muscle and sorted using fluorescence activated cell sorting (FACS), and expanded by collagen gel-based cell culture with bFGF and EGF. The number of Sk-DN cells was small after sorting (2–8 × 10⁴); however, the number increased 10–20 fold (2–16 × 10⁵) after 6 days of expansion culture, and the cells maintained immature state and multipotency, expressing mRNAs for mesodermal and ectodermal cell lineages. Transplantation of expanded Sk-DN cells into the severe muscle damage model (C57/BL6N wild-type) resulted in the synchronized reconstitution of blood vessels, peripheral nerves and muscle fibers following significant recovery of total muscle mass (57%) and contractile function (55%), whereas the non-cell-transplanted control group showed around 20% recovery in both factors. These reconstitution capacities were supported by the intrinsic plasticity of Sk-DN cells that can differentiate into muscular (skeletal muscle), vascular (pericyte, endothelial cell and smooth muscle) and peripheral nerve (Schwann cells and perineurium) cell lineages that was revealed by transplantation to non-muscle tissue (beneath renal capsule) and fluorescence in situ hybridization (FISH) analysis.     

Activation of AKT signaling promotes cell growth and survival in α7β1 integrin-mediated alleviation of muscular dystrophy

Transgenic expression of the α7 integrin can ameliorate muscle pathology in a mouse model of Duchenne muscular dystrophy (mdx/utr^−/−) and thus can compensate for the loss of dystrophin in diseased mice. In spite of the beneficial effects of the α7 integrin in protecting mice from dystrophy, identification of molecular signaling events responsible for these changes remains to be established. The purpose of this study was to determine a role for signaling in the amelioration of muscular dystrophy by α7 integrin. Activation of PI3K, ILK, AKT, mTOR, p70S6K, BAD, ERK, and p38 was measured in the muscle from wild type (WT), mdx/utr^−/− and α7BX2-mdx/utr^−/− mice using in vitro activity assays or phosphospecific antibodies and western blotting. Significant increases in PI3K activity (47%), ILK activity (2.0-fold), mTOR (Ser2448) (57%), p70S6K (Thr389) (11.7-fold), and ERK (Thr202/Tyr204) (66%) were demonstrated in dystrophic mdx/utr^−/− muscle compared to WT. A significant decrease in p38 phosphorylation (2.9-fold) was also observed. Although most of these signaling events were similar in dystrophic mdx/utr^−/− mice overexpressing the α7 integrin, the AKT (Ser473):AKT ratio (2-fold vs. WT) and p70S6K phosphorylation (18-fold vs. WT) were higher in α7BX2-mdx/utr^−/− compared to mdx/utr^−/− mice. In addition, increased phosphorylation of BAD Serine 112 may contribute to the significant reduction in TUNEL⁺ cells observed in α7BX2-mdx/utr^−/− mice. We conclude that the α7β1 integrin confers a protective effect in dystrophic muscle through the activation of the ILK, AKT, p70S6K and BAD signaling to promote muscle cell survival.     

A subpopulation of murine bone marrow cells fully differentiates along the myogenic pathway and participates in muscle repair in the mdx dystrophic mouse

Bone marrow (BM) transplantation in mice suggests the existence of pluripotent cells able to differentiate into skeletal muscle tissue, although sustained myofiber reconstitution has not yet been achieved. We investigated the myogenic potential of mouse BM cells and evaluated whether a BM fraction enriched for cells expressing skeletal muscle markers would ameliorate muscle repair, when compared to whole BM, into the dystrophic mdx mouse. We demonstrate that cells expressing striated-muscle-specific proteins are already present in the BM independently from experimentally forced myogenic conversion. We observed the presence of both markers of early myogenic program such as Pax3, Myf5, MyoD, desmin, and late myogenesis such as myosin heavy chain and alpha-sarcomeric actin. These myogenic cells are more represented in the early nonadherent BM fraction, which generates clones able to fully differentiate into myotubes. Transplantation in mdx mice by intravenous injection of whole BM and a tenfold BM myogenic enriched fraction resulted in BM reconstitution and limited dystrophin restoration. Taken together, these data show that a fraction of BM cells have a definite potential for differentiation along the skeletal muscle pathway and can be recruited by muscle repair mechanisms. They also indicate that factors limiting the degree of muscle recruitment and the host stem cell competition should be assessed in order to evaluate the usefulness of BM-derived myogenic cells into the context of cell-mediated gene therapy of inherited muscle diseases.