Regenerative potential of human muscle stem cells in chronic inflammation

International audienceABSTRACT: INTRODUCTION: Chronic inflammation is a profound systemic modification of the cellular microenvironment which could affect survival, repair and maintenance of muscle stem cells. The aim of this study was to define the role of chronic inflammation on the regenerative potential of satellite cells in human muscle. METHODS: As a model for chronic inflammation, 11 patients suffering from rheumatoid arthritis (RA) were included together with 16 patients with osteoarthritis (OA) as controls. The mean age of both groups was 64 years, with more females in the RA group compared to the OA group. During elective knee replacement surgery, a muscle biopsy was taken from the distal musculus vastus medialis. Cell populations from four RA and eight OA patients were used for extensive phenotyping because these cell populations showed no spontaneous differentiation and myogenic purity greater than 75% after explantation. RESULTS: After mononuclear cell explantation, myogenic purity, viability, proliferation index, number of colonies, myogenic colonies, growth speed, maximum number of population doublings and fusion index were not different between RA and OA patients. Furthermore, the expression of proteins involved in replicative and stress-induced premature senescence and apoptosis, including p16, p21, p53, hTERT and cleaved caspase-3, was not different between RA and OA patients. Mean telomere length was shorter in the RA group compared to the OA group. CONCLUSIONS: In the present study we found evidence that chronic inflammation in RA does not affect the in vitro regenerative potential of human satellite cells. Identification of mechanisms influencing muscle regeneration by modulation of its microenvironment may, therefore, be more appropriate

Cholesterol depletion by methyl-β-cyclodextrin enhances cell proliferation and increases the number of desmin-positive cells in myoblast cultures

AbstractSkeletal myogenesis comprises myoblast replication and differentiation into striated multinucleated myotubes. Agents that interfere with myoblast replication are important tools for the understanding of myogenesis. Recently, we showed that cholesterol depletion by methyl-β-cyclodextrin (MCD) enhances the differentiation step in chick-cultured myogenic cells, involving the activation of the Wnt/β-catenin signaling pathway. However, the effects of cholesterol depletion on myoblast replication have not been carefully studied. Here we show that MCD treatment increases cell proliferation in primary chick myogenic cell cultures. Treatment of myogenic cells with the anti-mitotic reagent cytosine arabinoside, immediately following cholesterol depletion, blocks the MCD-induced effects on proliferation. Cholesterol depletion induced an increase in the number of desmin-positive mononucleated cells, and an increase in desmin expression. MCD induces an increase in the expression of the cell cycle regulator p53 and the master switch gene MyoD1. Treatment with BIO, a specific inhibitor of GSK3β, induced effects similar to MCD on cell proliferation; while treatment with Dkk1, a specific inhibitor of the Wnt/β-catenin pathway, neutralized the effects of MCD. These findings indicate that rapid changes in the cholesterol content in cell membranes of myoblasts can induce cell proliferation, possibly by the activation of the Wnt/β-catenin signaling pathway

Reverse-transcription is the step affected by PML expression and the required event triggering PML CB formation.

<p>(A) Wt or PML KO MEFs were transduced for different times at MOI 10 with VSV-G pseudotyped HIV-1 derived vector, DNA was extracted and early and late reverse transcript were quantified by qPCR. (B) Wt or PML KO MEFs were treated for 30 min with nevirapine (NVP) before a 2 h transduction at MOI 10 with HIV-1 vector. DNA was extracted and early and late RT products were quantified by qPCR (left panel). PML (in green) and p24 (in red) localization was determined by immunofluorescence (middle panel). For each condition, the number of PML CBs was quantified in 50 cells from 3 independent fields (right panel). Student t-test was performed to determine P values (**P<0.01). (C) The amount of SUMOylated proteins in wt MEFs treated with GA for 24 h was evaluated by western-blot (left panel). In parallel, wt MEFs pretreated with GA for 4 h were transduced for 2 h at MOI 10 with HIV-1 vector and PML (in green) and HIV-1 p24 (in red) localization was determined by immunofluorescence (right panel).</p

Inhibition of retroviral infections and retro-transposition by Daxx.

<p>(A) Wt and Daxx KO MEFs were transduced with increasing doses of HIV-1 vector and the percentage of GFP-expressing cells was determined 48 h later by flow cytometry. Means +/- SD of a typical experiment are shown. Similar results were obtained in three independent experiments. (B) Wt HeLa cells were transfected with an empty (EV) or a Daxx-encoding plasmid. Two days post-transfection, Daxx expression was evaluated by Western-blot analysis (left panel) and the percentage of GFP-expressing cells was determined by flow cytometry 48 h post-transduction with increasing doses of HIV-1 vector (right panel). Graph shows means of triplicate values +/- SD, representative of two independent experiments. (C) PBMCs of healthy adult individuals were activated by anti-CD3/anti-CD28 for 48 h and were subsequently transduced with either a non-coding control (RV_CTR) or a Daxx-expressing (RV_Daxx) MLV-derived retroviral vector. Relative expression of Daxx was estimated by RT-qPCR analysis performed two days post-transduction, and expressed as a ratio to RPL13A mRNA expression. Two days post transduction, activated PBMCs were infected with HIV-1 NL4.3 at a dose corresponding to 0.1 ng of p24 and virus replication was followed at given time points post-infection by measuring the amount of HIV-1 p24 by ELISA on the culture supernatants. Results show the mean of triplicates +/- SD and are representative of two independent experiments. (D) Wt HeLa cells were transfected with an EV or a Daxx-encoding plasmid. Two days later, cells were transduced with VSV-pseudotyped HIV-1 vector for 6 h and early and late reverse transcript were quantified by qPCR. (E) Wt HeLa cells transfected with an EV or a Daxx-encoding plasmid were transduced 48 h later with GFP-expressing VSV-G pseudotyped Mo-MLV or SIVmac vectors. The percentage of GFP-expressing cells was determined by flow cytometry. Graphs show means of triplicate values +/- SD, representative of three independent experiments. (F) Experimental procedure for the detection of retrotransposition. (G) Analysis of the activity of Daxx on IAP and MusD retrotransposons. Retrotranspositions (defined by quantifying G418R clones) are presented as percentages relative to samples containing retroelement alone. Results show the mean of three independent experiments +/- SD.</p

Role of murine PML in HIV-1 transduction.

<p>(A) Wt or PML KO MEFs were transduced at different MOI with HIV-1 derived lentivector expressing GFP during 48h and the percentage of GFP expressing cells was determined by flow cytometry. The graph shows means of duplicate values +/- SD, representative of three independent experiments. (B and C) Wt or PML KO MEFs were treated with 2.5 μM of As₂O₃ during 24h, PML degradation was evaluated by western-blot (B) and cells were infected at different MOI with HIV-1 vector during 48h and the percentage of GFP expressing cells was determined by flow cytometry (C). The graph shows means of duplicate values +/- SD, representative of three independent experiments. (D) Wt MEFs were infected for different times at MOI 10 with HIV-1 derived vector and PML localization was determined by immunofluorescence. For each time-point, the number and perimeter of PML CBs was quantified in 50 cells from 5 independent fields. Student t-test was performed to determine P values. (*P<0.05).</p

PML expression in human cells interferes with HIV infection.

<p>(A and B) HeLa cells were transfected with siRNA directed against all PML isoforms or a non-targeting siRNA (CTR). Knockdown efficiency was estimated by immunofluorescence (A) and Western-blot (B). (C) Cells were then infected with VSV-G pseudotyped HIV-1 vector expressing GFP at increasing MOI during 48h and the percentage of GFP expressing cells was determined by flow cytometry. The graph shows means +/- SD of 3 independent experiments performed in duplicates (D and E) PBMCs of healthy adult individuals were activated by anti-CD3/anti-CD28 for 48 h and were subsequently transduced with a lentiviral vector expressing either a non-targeting control (CTR) or a PML-specific shRNA. (D) Relative expression of PML was estimated by RT-qPCR analyis performed on PBMCs two days post-transduction, and expressed as a ratio to RPL13A mRNA expression. (E) Two days post transduction with the shRNA-expressing vector, activated PBMCs were infected with HIV-1 NL4.3 at a dose corresponding to 0.1 ng of p24 and virus production was measured at given time points by measuring the amount of HIV-1 p24 by ELISA on the culture supernatants. Results show the mean of triplicates +/- SD and are representative of two independent experiments. (F) HeLa wt cells were transduced at MOI 10 with HIV-1 derived vector during 4 h and PML (in green) and HIV-1 p24 (in red) were detected by immunofluorescence.</p

(A) Wt or Daxx KO MEFs were treated with 2.5 μM of As₂O₃ during 24h and cells were infected at different MOI with HIV-1 vector during 48h. The percentage of GFP expressing cells was determined by flow cytometry.

<p>The graph shows means of duplicate values +/- SD, representative of three independent experiments. (B) Wt HeLa cells were transduced for 2 h with HIV-1 vector and PML (in red) and Daxx (in green) localization was analyzed by immunofluorescence. (C) Wt or Daxx KO MEFs were transduced with HIV-1 vector in the presence or not of 100 μM of GA. Six hours post-transduction, early and late reverse transcripts were quantified by qPCR. Graphs show means of triplicate values +/- SD, representative of two independent experiments. Student t-test was performed to determine P values (***P<0.001; ns, not significant). (D) Wt MEFs were treated or not with nevirapine (NVP), transduced with HIV-1 for 2 h and probed with anti-Daxx and anti-HIV-1 p24 antibodies. Representative images of a Duolink experiment are shown.</p

Effect of PML expression on retroviral infection.

<p>(A) Wt or PML KO MEFs were transduced at different MOI with VSV-G pseudotyped EIAV, Moloney-MLV or SIVmac derived vectors expressing GFP during 48h and the percentage of GFP expressing cells was determined by flow cytometry. The graph shows means of triplicate values +/- SD, representative of three independent experiments. (B) Wt MEFs were transduced for 2 h at MOI 5 with EIAV, Moloney-MLV or SIVmac derived vectors and PML localization was determined by immunofluorescence. Arrows point to representative cytoplasmic PML dots. (C) Wt MEFs were infected for 2 h at MOI 10 with SeV, Influenza A virus, VSV or EMCV and PML localization was determined by immunofluorescence.</p