Cell-Surface Protein Profiling Identifies Distinctive Markers of Progenitor Cells in Human Skeletal Muscle

SummarySkeletal 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

Measurement of Lateral Transmission of Force in the Extensor Digitorum Longus Muscle of Young and Old Mice

The main function of skeletal muscles is to generate force. The force developed by myofiber contraction is transmitted to the tendon. There are two pathways of force transmission from myofibers to tendons: longitudinal transmission that depends on tension elicited via the myotendinous junction and lateral transmission that depends on shear elicited via the interface between the myofiber surface and surrounding connective tissue. Experiments using animal muscle and mathematical models indicated that lateral transmission is the dominant pathway in muscle force transmission. Studies using rat muscle showed that the efficiency of lateral force transmission declines with age. Here, the lateral transmission of force was measured using the extensor digitorum longus muscle from young and old mice. Dependence on longitudinal transmission increased in the old muscle, and there was a trend for lower efficiency of lateral force transmission in the old muscle compared to the young muscle. There was a noticeable increase in the connective tissue volume in the old muscle; however, there was no significant change in the expression of dystrophin, a critical molecule for the link between the myofiber cytoskeleton and extracellular matrix. This study demonstrates the measurement of lateral force transmission in mouse muscles and that alteration in force transmission property may underlie age-related muscle weakness

Transcriptome analysis of mesenchymal stromal cells of the large and small intestinal smooth muscle layers reveals a unique gastrontestinal stromal signature

Mesenchymal stromal cells in the muscle layer of the large intestine are essential for the regulation of intestinal motility. They form electrogenic syncytia with the smooth muscle and interstitial cells of Cajal (ICCs) to regulate smooth muscle contraction. Mesenchymal stromal cells are present in the muscle layer throughout the gastrointestinal tract. However, their area-specific characteristics remain ambiguous.In this study, we compared mesenchymal stromal cells from the large and small intestinal muscle layers. Histological analysis using immunostaining showed that the cells in the large and small intestines were morphologically distinct. We established a method to isolate mesenchymal stromal cells from wild-type mice with platelet-derived growth factor receptor-alpha (PDGFRα) as a marker on the cell surface and performed RNAseq. Transcriptome analysis revealed that PDGFRα+ cells in the large intestine exhibited increased expression levels of collagen-related genes, whereas PDGFRα+ cells in the small intestine exhibited increased expression levels of channel/transporter genes, including Kcn genes. These results suggest that mesenchymal stromal cells differ morphologically and functionally depending on gastrointestinal tract. Further investigations of the cellular properties of mesenchymal stromal cells in the gastrointestinal tract will aid in optimizing methods for the prevention and treatment of gastrointestinal diseases

Interleukin-1beta (IL-1β)-induced Notch ligand Jagged1 suppresses mitogenic action of IL-1β on human dystrophic myogenic cells

<div><p>Duchenne muscular dystrophy (DMD) is a severe X-linked recessive muscle disorder caused by mutations in the dystrophin gene. Nonetheless, secondary processes involving perturbation of muscle regeneration probably exacerbate disease progression, resulting in the fatal loss of muscle in DMD patients. A dysfunction of undifferentiated myogenic cells is the most likely cause for the reduction of regenerative capacity of muscle. To clarify molecular mechanisms in perturbation of the regenerative capacity of DMD muscle, we have established several NCAM (CD56)-positive immortalized human dystrophic and non-dystrophic myogenic cell lines from DMD and healthy muscles. A pro-inflammatory cytokine, IL-1β, promoted cell cycle progression of non-dystrophic myogenic cells but not DMD myogenic cells. In contrast, IL-1β upregulated the Notch ligand Jagged1 gene in DMD myogenic cells but not in non-dystrophic myogenic cells. Knockdown of Jagged1 in DMD myogenic cells restored the IL-1β-promoted cell cycle progression. Conversely, enforced expression of Jagged1-blocked IL-1β promoted proliferation of non-dystrophic myogenic cells. In addition, IL-1β prevented myogenic differentiation of DMD myogenic cells depending on Jagged1 but not of non-dystrophic myogenic cells. These results demonstrate that Jagged1 induced by IL-1β in DMD myogenic cells modified the action of IL-1β and reduced the ability to proliferate and differentiate. IL-1β induced Jagged1 gene expression may be a feedback response to excess stimulation with this cytokine because high IL-1β (200–1000 pg/ml) induced Jagged1 gene expression even in non-dystrophic myogenic cells. DMD myogenic cells are likely to acquire the susceptibility of the Jagged1 gene to IL-1β under the microcircumstances in DMD muscles. The present results suggest that Jagged1 induced by IL-1β plays a crucial role in the loss of muscle regeneration capacity of DMD muscles. The IL-1β/Jagged1 pathway may be a new therapeutic target to ameliorate exacerbation of muscular dystrophy in a dystrophin-independent manner.</p></div

IL-1β inhibited myogenesis of dystrophic myogenic cells in a Jagged1-dependent manner.

<p>(A-R) Human non-dystrophic (Hu37KD5, A-C) and dystrophic (D4P4, D-F) myogenic cell clones and derivatives of D4P4 were stimulated with (B, C, E, F, H, I, K, and L) or without (A, D, G, and J) IL-1β (500 pg/ml) under the differentiation-inducing condition for 3 d. D4P4 cells were transduced by a lentivirus encoding random shRNA (D4shCTR, G-I) or shJagged1 (D4shJ1, J-L). Myosin heavy chain and troponin T were visualized using an inverted microscope (red). Nuclei were stained with DAPI (blue). Scale bar, 100 μm. (M) Hu37KD5, D4P4, and derivatives of D4P4 were cultured for up to 3 or 4 d under the differentiation-inducing condition with (+) or without (-) IL-1β (500 pg/ml). Twenty micrograms of total proteins was subjected to immunoblot analysis with antibodies against MyHC and β-tubulin.</p

Human myogenic cells derived from non-dystrophic and dystrophic muscles were immortalized and isolated from non-myogenic cells.

<p>(A-D) Human muscle satellite cells are identified by their M-cadherin⁺/NCAM⁺ status and position under the basal lamina. DAPI, laminin α2, M-cadherin (Mcad), and NCAM were pseudocolored with different colors as indicated at the tops of the panels and then merged. Scale bar, 10 μm. (E and F) NCAM⁺ cells (red squares) were isolated from human immortalized muscle cell cultures using flow cytometry. Hu37KD is immortalized cells derived from a non-dystrophic muscle cell culture (E), whereas DMD1cmv is derived from dystrophic muscle cell culture (F). ALP, alkaline phosphatase. (G and H) Myogenic cells in the NCAM⁺ fraction were isolated. DMD3cmv is immortalized cells derived from a dystrophic muscle cell culture, and then separated into NCAM^- (G) and (H) cells. The cells were cultured for 7 d under the differentiation-inducing condition. Phase contrast images are shown. Arrows represent myotubes. NCAM⁺ cells were designated D3P in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188821#pone.0188821.g004" target="_blank">Fig 4</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188821#pone.0188821.s006" target="_blank">S2 Table</a>. Scale bar, 50 μm. (I) Procedure of isolation of human myogenic cells from primary cultured muscle cell cultures. (J) Purification and expansion of immortalized human myogenic cells. Primary cultured cells DMD1 derived from dystrophic muscle of a one-year-old boy were immortalized by the three-factor method, and 0.1% of NCAM+ cells were isolated by flow cytometry and expanded in culture. Then, 47% of NCAM+ cells in the primary sorted cell culture were isolated again and expanded. Finally, 96% of secondary sorted cells were NCAM⁺. (K-N) NCAM⁺ cells are desmin-positive myogenic cells. A single cell-derived clone D1P1 was isolated from immortalized dystrophic myogenic cell D1P derived from DMD1 cells. D1P1 expressed desmin (M) and gave rise to prominent myotubes expressing myosin heavy chain (MyHC in N) under the differentiation-inducing condition. Scale bar, 100 μm.</p

Suppression of Jagged1 expression restored mitogenic action of IL-1β in dystrophic myogenic cells.

<p>(A) The expression of Jagged1 in random shRNA (shCTR)- and shJagged1 (shJ1)-expressing D4P4-derived clones, designated D4shCTR and D4shJ1, was analyzed by qRT-PCR. The amounts of mRNA were normalized to the control POLR2a mRNA value. The means and standard deviations were estimated from three independent experiments. Data were statistically analyzed using Student’s <i>t</i>-test. The statistical significance of the difference between D4shCTR and D4shJ1cells was less than 0.01. (B and C) D4shCTR (B) and D4shJ1 (C) cells were treated with IL-1β for 24 h. The cells were incubated with EdU (10 μM) for further 6 h and then subjected to fluorescence analysis. EdU-positive nuclei were counted in nine areas of each culture and the means and standard deviations were estimated. Similar results were obtained from three independent experiments, and representative data are shown. The statistical significance of difference between the unstimulated control (0 pg/ml) and IL-1β-stimulated cells (30–500 pg/ml) was analyzed using one-way ANOVA. *, p<0.05; **, p<0.01.</p

Enforced expression of jagged1 interfered with cell cycle progression and antagonized IL-1β.

<p>(A) The non-dystrophic myogenic cell clone Hu37KD5 (white column) was transduced with a control lentivirus vector (Hu37CTR, gray column) or a Jagged1-expression lentivirus (Hu37rJ1, filled column). The amounts of transcripts of Jagged1, Notch1, Notch2, and Notch3 were determined by qRT-PCR. The amounts of mRNA were normalized to control the POLR2a mRNA value. The means and standard deviations were estimated from three independent experiments. (B-C) Twenty micrograms of total proteins from Hu37CTR (CTR) and Hu37rJ1 (J1) were subjected to immunoblotting with antibodies against Jagged1 (B) and Notch3 (C). F, Unprocessed full-length protein; CTF, cytoplasmic fragment; asterisks, non-specific bands. Numbers represent the positions of protein size markers. (D) Hu37CTR (white circles) and Hu37rJ1 (filed circles) cell clones were treated with IL-1β (0–250 pg/ml) for 24 h. The cells were incubated with EdU (10 μM) for a further 6 h culture, and then subjected to fluorescence analysis. EdU-positive nuclei were counted in nine areas of each culture and the means and standard deviation were estimated. Data were statistically analyzed using one-way ANOVA. The statistical significance of the difference between the unstimulated control (0 pg/ml) and cytokine-stimulated cells (60–250 pg/ml) was less than 0.01 in both Hu37CTR and Hu37rJ1. The statistical significance of the difference between Hu37CTR and Hu37rJ1 was less than 0.01 at each dosage of IL-1β. (E) The expression of CSF2 in Hu37CTR (white column) and Hu37rJ1 (gray column) cells was analyzed by qRT-PCR after 24 h of exposure to IL-1β. The amounts of mRNA were normalized to the control POLR2a mRNA value. The means and standard deviations were estimated from three independent experiments. Data were statistically analyzed using Student’s <i>t</i>-test. *, p<0.05.</p

IL-1β promoted cell cycle progression of non-dystrophic myogenic cells.

<p>(A) Non-dystrophic cells (Hu37KD5) were stimulated with TNFα (500 pg/ml), IL-1β (500 pg/ml), or IL-6 (100 ng/ml) and cultured for six days. The nuclei were counted. The means and standard deviations were estimated from four independent cultures for each treatment. The statistical significance (P-value) of the difference between samples was analyzed using one-way ANOVA. ND, p>0.05; **, p<0.01. (B and C) Non-dystrophic (Hu37KD5, B) and dystrophic (D4P4, C) myogenic cell clones were treated with various concentrations of IL-1β (0–500 pg/ml) for 24 h. The cells were incubated with EdU (10 μM) for further 6 h, and the Click-iT reaction was carried out; then the cells were subjected to fluorescent image analysis. EdU-positive nuclei were counted in nine areas of each culture and the means and standard deviations were estimated. Similar results were obtained from two independent experiments, and representative data are shown. The statistical significance of the difference between the unstimulated control (0 pg/ml) and cytokine-stimulated cells were analyzed using one-way ANOVA. ND, p>0.05; *, p<0.05; **, p<0.01.</p

IL-1β promoted expression of NF-κB downstream genes in both human dystrophic and non-dystrophic myogenic cells.

<p>(A and B) The expressions of IL-1β (A) and IL-1 receptor (IL1R1) (B) in non-dystrophic (Hu37KD5) and dystrophic (D4P4) myogenic cell clones were analyzed by qRT-PCR. The amounts of mRNA were normalized to control POLR2a mRNA value. The means and standard deviations were estimated from four independent experiments. The statistical significance of the difference between non-dystrophic and dystrophic myogenic cells was analyzed using Student’s <i>t</i>-test. P-values were larger than 0.1 for both genes. (C) Expression profiles of members of the NF-κB signaling pathway in Hu37KD5 (a) and D4P4 (b) were determined using RT-PCR arrays after 24 h of exposure to IL-1β (500 pg/ml). Changes in transcript abundance are expressed as log2 ratio to untreated control mean and designated “Fold regulation”. (D) The expressions of CSF2, CSF3, IL-8, and IL-1β in Hu37KD5 (white column) and D4P4 (black column) were analyzed by qRT-PCR after 24 h exposure to IL-1β (500 pg/ml). The amounts of mRNA were normalized to control POLR2a mRNA values. The means and standard deviations were estimated from at least three independent experiments. Data were statistically analyzed using Student’s <i>t</i>-test. *, p<0.05.</p