Journal: Stem Cell Research & Therapy

Article Title: Myostatin genetic inactivation inhibits myogenesis by muscle-derived stem cells in vitro but not when implanted in the mdx mouse muscle

doi: 10.1186/scrt152

Figure Lengend Snippet: Effect of genetic inactivation of myostatin on the expression of key stem cell marker genes in MDSC . (A) Flow cytometry (no gate) was conducted for Sca1 (red), CD34 (black), CD44 (green), and Oct 4 (blue) in WT MDSCs (blue) and Mst KO MDSCs (purple), against the respective isotypes (not shown). Top panels: Left: WT MDSCs; Right: Mst KO MDSCs. Bottom panels: each antigen is compared separately for WT (blue) and Mst KO (purple), with the corresponding isotypes (WT, dark gray; Mst KO, light gray). (B) Representative pictures of proliferating MDSCs that were subjected to immunocytochemistry for Oct 4, showing nuclear location in most cells (200×). (C) Proliferating MDSCs that were subjected to cytochemistry for alkaline phosphatase (200×). (D) Homogenates from the same cell cultures that were subjected to Western blot for Oct 4 (nuclear Oct 4a, 45 kDa; cytoplasmic Oct 4 b, 33 kDa). WT, wild type; MDSC, muscle-derived stem cell; Mst KO, myostatin knockout.

Article Snippet: For the tests on the modulation of MDSCs skeletal myotube formation by various factors, cells were allowed to reach confluence, switched to GM-HC, and incubated for 2 weeks with 2 μg/ml recombinant 113-amino acid myostatin protein (R-Mst), a biologically active recombinant 16-kDa protein containing 113 amino acid residues of the processed human myostatin protein (BioVendor Laboratory Medicine Inc., Palackeho, Czech Republic) [ , , ], or with a recombinant mouse follistatin protein (RD Systems, Minneapolis, MN, USA) at 0.2 μg/ml [ , ], changing medium twice a week.

Techniques: Expressing, Marker, Flow Cytometry, Immunocytochemistry, Western Blot, Derivative Assay, Knock-Out

Journal: Stem Cell Research & Therapy

Article Title: Myostatin genetic inactivation inhibits myogenesis by muscle-derived stem cells in vitro but not when implanted in the mdx mouse muscle

doi: 10.1186/scrt152

Figure Lengend Snippet: Some stem cell-related genes are transcribed similarly in MDSCs, irrespective of myostatin inactivation

Article Snippet: For the tests on the modulation of MDSCs skeletal myotube formation by various factors, cells were allowed to reach confluence, switched to GM-HC, and incubated for 2 weeks with 2 μg/ml recombinant 113-amino acid myostatin protein (R-Mst), a biologically active recombinant 16-kDa protein containing 113 amino acid residues of the processed human myostatin protein (BioVendor Laboratory Medicine Inc., Palackeho, Czech Republic) [ , , ], or with a recombinant mouse follistatin protein (RD Systems, Minneapolis, MN, USA) at 0.2 μg/ml [ , ], changing medium twice a week.

Techniques:

Journal: Stem Cell Research & Therapy

Article Title: Myostatin genetic inactivation inhibits myogenesis by muscle-derived stem cells in vitro but not when implanted in the mdx mouse muscle

doi: 10.1186/scrt152

Figure Lengend Snippet: Myostatin genetic inactivation does not block the multipotent nonmyogenic differentiation capacity of MDSCs . Representative pictures of proliferating MDSCs treated for 2 weeks in differentiation media and subjected to immunocytochemistry for NF-70 (A) and ASMA (B) to detect marker expression of neural cells and myofibroblasts (200×). MDSC, muscle-derived stem cell; WT, wild type (muscle-derived stem cells); KO, myostatin knockout (muscle-derived stem cells); ASMA, α-smooth muscle actin.

Article Snippet: For the tests on the modulation of MDSCs skeletal myotube formation by various factors, cells were allowed to reach confluence, switched to GM-HC, and incubated for 2 weeks with 2 μg/ml recombinant 113-amino acid myostatin protein (R-Mst), a biologically active recombinant 16-kDa protein containing 113 amino acid residues of the processed human myostatin protein (BioVendor Laboratory Medicine Inc., Palackeho, Czech Republic) [ , , ], or with a recombinant mouse follistatin protein (RD Systems, Minneapolis, MN, USA) at 0.2 μg/ml [ , ], changing medium twice a week.

Techniques: Blocking Assay, Immunocytochemistry, Marker, Expressing, Derivative Assay, Knock-Out

Journal: Stem Cell Research & Therapy

Article Title: Myostatin genetic inactivation inhibits myogenesis by muscle-derived stem cells in vitro but not when implanted in the mdx mouse muscle

doi: 10.1186/scrt152

Figure Lengend Snippet: Myostatin genetic inactivation blocks the myogenic differentiation capacity of MDSCs . (A-C) Representative pictures of confluent MDSCs from the WT MDSCs and Mst KOs maintained for 2 weeks in myogenic medium and subjected to immunocytochemistry for MHC II to detect differentiation into polynucleated myotubes (magnifications as indicated). (D) Blue/red merge of confluent MDSCs in myogenic medium that were labeled with DAPI and submitted to immunofluorescent detection of MHC (200×); (E) Western blot for MHC II (210 kDa) in the confluent cultures undergoing myogenesis, and (F) for MyoD (45 kDa) in the nonconfluent cultures in nonmyogenic medium. MDSC, muscle-derived stem cell; WT, wild type; Mst KO, myostatin knockout; MHC, myosin heavy chain; DAPI: 4', 6-diaminido-phenylindole.

Article Snippet: For the tests on the modulation of MDSCs skeletal myotube formation by various factors, cells were allowed to reach confluence, switched to GM-HC, and incubated for 2 weeks with 2 μg/ml recombinant 113-amino acid myostatin protein (R-Mst), a biologically active recombinant 16-kDa protein containing 113 amino acid residues of the processed human myostatin protein (BioVendor Laboratory Medicine Inc., Palackeho, Czech Republic) [ , , ], or with a recombinant mouse follistatin protein (RD Systems, Minneapolis, MN, USA) at 0.2 μg/ml [ , ], changing medium twice a week.

Techniques: Immunocytochemistry, Labeling, Western Blot, Derivative Assay, Knock-Out

Journal: Stem Cell Research & Therapy

Article Title: Myostatin genetic inactivation inhibits myogenesis by muscle-derived stem cells in vitro but not when implanted in the mdx mouse muscle

doi: 10.1186/scrt152

Figure Lengend Snippet: Some skeletal myogenesis-related genes are downregulated in MDSCs by myostatin genetic inactivation, whereas others remain unchanged

Article Snippet: For the tests on the modulation of MDSCs skeletal myotube formation by various factors, cells were allowed to reach confluence, switched to GM-HC, and incubated for 2 weeks with 2 μg/ml recombinant 113-amino acid myostatin protein (R-Mst), a biologically active recombinant 16-kDa protein containing 113 amino acid residues of the processed human myostatin protein (BioVendor Laboratory Medicine Inc., Palackeho, Czech Republic) [ , , ], or with a recombinant mouse follistatin protein (RD Systems, Minneapolis, MN, USA) at 0.2 μg/ml [ , ], changing medium twice a week.

Techniques:

Journal: Stem Cell Research & Therapy

Article Title: Myostatin genetic inactivation inhibits myogenesis by muscle-derived stem cells in vitro but not when implanted in the mdx mouse muscle

doi: 10.1186/scrt152

Figure Lengend Snippet: RT-PCR confirmation of selected differences in the transcriptional expression of undifferentiated WT and Mst KO MDSCs, detected with DNA microarrays . RNAs obtained from triplicate cultures of proliferating MDSCs, consecutive to those used for DNA microarrays in Tables 1 and 2, were subjected to RT-PCR with specific primers spanning an intron for the number of PCR cycles stated in parenthesis, as follows: Actc1 (30), Acta1 (30), MyoD1 (33), Pax3 (28), and GAPDH (26). (A) Ethidium bromide-stained agarose gels; (B) densitometry of relative band intensities referred to housekeeping gene for the indicated numbers of PCR cycles. Controls without cDNA were blank. * P < 0.05; *** P < 0.001. RT-PCR, reverse transcription polymerase chain reaction; WT, wild type; Mst KO, myostatin knockout; MDSC, muscle-derived stem cell; DNA, deoxyribonucleic acid; RNA, ribonucleic acid; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Article Snippet: For the tests on the modulation of MDSCs skeletal myotube formation by various factors, cells were allowed to reach confluence, switched to GM-HC, and incubated for 2 weeks with 2 μg/ml recombinant 113-amino acid myostatin protein (R-Mst), a biologically active recombinant 16-kDa protein containing 113 amino acid residues of the processed human myostatin protein (BioVendor Laboratory Medicine Inc., Palackeho, Czech Republic) [ , , ], or with a recombinant mouse follistatin protein (RD Systems, Minneapolis, MN, USA) at 0.2 μg/ml [ , ], changing medium twice a week.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Staining, Knock-Out, Derivative Assay

Journal: Stem Cell Research & Therapy

Article Title: Myostatin genetic inactivation inhibits myogenesis by muscle-derived stem cells in vitro but not when implanted in the mdx mouse muscle

doi: 10.1186/scrt152

Figure Lengend Snippet: Myostatin and follistatin fail to modulate the myogenic differentiation of MDSCs, although the myostatin receptor is expressed . (A-D) Confluent WT MDSCs in myogenic medium were incubated in triplicate on six-well plates for 1 week with recombinant myostatin (B) or follistatin (C) or with no addition (A) , and subjected to immunocytochemistry for MHC II (40×). The relative area occupied by the MHC II + myotubes was estimated by quantitative image analysis (15 fields/well/three wells) (D) . Cont, control; Mst, myostatin; Fst, follistatin. No myotubes were formed in confluent Mst KO under any treatment (not shown). (E) Western blot detection in confluent MDSCs from both mice strains of the expression of the ActRIIb and TGF-β1, in two successive passages for each cell line. Myostatin was not detected. MDSCs, muscle-derived stem cells; WT, wild type; Mst KO, myostatin knockout; MHC, myosin heavy chain.

Article Snippet: For the tests on the modulation of MDSCs skeletal myotube formation by various factors, cells were allowed to reach confluence, switched to GM-HC, and incubated for 2 weeks with 2 μg/ml recombinant 113-amino acid myostatin protein (R-Mst), a biologically active recombinant 16-kDa protein containing 113 amino acid residues of the processed human myostatin protein (BioVendor Laboratory Medicine Inc., Palackeho, Czech Republic) [ , , ], or with a recombinant mouse follistatin protein (RD Systems, Minneapolis, MN, USA) at 0.2 μg/ml [ , ], changing medium twice a week.

Techniques: Incubation, Recombinant, Immunocytochemistry, Western Blot, Expressing, Derivative Assay, Knock-Out

Journal: Stem Cell Research & Therapy

Article Title: Myostatin genetic inactivation inhibits myogenesis by muscle-derived stem cells in vitro but not when implanted in the mdx mouse muscle

doi: 10.1186/scrt152

Figure Lengend Snippet: Mst KO MDSCs failed to generate myotubes in vitro , but in vivo stimulate tissue repair comparable to the WT MDSCs . Aged (10-month-old) mdx mice were used to maximize myofiber loss and lipofibrotic degeneration in the gastrocnemius. (A) Muscles were cryoinjured, implanted with 0.5 × 10 6 DAPI-labeled WT MDSCs, and allowed to undergo repair for 10 days. Frozen muscle sections were stained for MHC-II with Texas red streptavidin, and merging of blue and red fluorescence was obtained (200×). MDSC nuclei centrally located within myofibers are indicated with yellow arrows. (B ) Similar picture, but for Mst KO MDSCs. (C) Gastrocnemius injury in the aged mdx mice was performed in the two apexes of the muscle with notexin, and muscles were injected 4 days later with saline or with 1.0 × 10 6 WT MDSCs or (D) Mst KO MDSCs in saline ( n = 5/group). Repair was allowed to proceed for 3 weeks. Hematoxylin/eosin staining was performed in frozen sections, and a representative picture for each case shows myofibers from the gastrocnemius implanted with MDSCs, with arrows pointing to abundant central nuclei (200×). (E ) Quantitative image analysis of these tissue sections (WT), in comparison to tissue sections from Mst KO MDSC-implanted mice (KO) and saline-injected controls, based on 12 fields per section, three sections per animal. *** P < 0.001. Mst KO, myostatin knockout; MDSC, muscle-derived stem cell; WT, wild type; mdx, X chromosome-linked muscular dystrophy; DAPI, 4', 6-diaminido-phenylindole; MHC, myosin heavy chain; KO, knockout.

Article Snippet: For the tests on the modulation of MDSCs skeletal myotube formation by various factors, cells were allowed to reach confluence, switched to GM-HC, and incubated for 2 weeks with 2 μg/ml recombinant 113-amino acid myostatin protein (R-Mst), a biologically active recombinant 16-kDa protein containing 113 amino acid residues of the processed human myostatin protein (BioVendor Laboratory Medicine Inc., Palackeho, Czech Republic) [ , , ], or with a recombinant mouse follistatin protein (RD Systems, Minneapolis, MN, USA) at 0.2 μg/ml [ , ], changing medium twice a week.

Techniques: In Vitro, In Vivo, Labeling, Staining, Fluorescence, Injection, Knock-Out, Derivative Assay

Journal: Stem Cell Research & Therapy

Article Title: Myostatin genetic inactivation inhibits myogenesis by muscle-derived stem cells in vitro but not when implanted in the mdx mouse muscle

doi: 10.1186/scrt152

Figure Lengend Snippet: Implanted Mst KO MDSCs stimulate more effectively than do WT MDSCs the expression of MHC-II in the muscle, but do not reduce ASMA . (A) Western blot analysis for MHC II, ASMA, and GAPDH (reference gene) in homogenates of skeletal muscle tissue from the central region adjacent to area examined histochemically in Figure 7C. Each lane corresponds to an individual mouse homogenate ( n = 5/group), and the three gels were run simultaneously. (B) Densitometric evaluation of the relative intensity expressed as ratios of the MCH-II or ASMA and GAPDH bands. * P < 0.05. Mst KO, myostatin knockout; MDSC, muscle-derived stem cell; WT, wild type; MHC, myosin heavy chain; ASMA, α-smooth muscle actin; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Article Snippet: For the tests on the modulation of MDSCs skeletal myotube formation by various factors, cells were allowed to reach confluence, switched to GM-HC, and incubated for 2 weeks with 2 μg/ml recombinant 113-amino acid myostatin protein (R-Mst), a biologically active recombinant 16-kDa protein containing 113 amino acid residues of the processed human myostatin protein (BioVendor Laboratory Medicine Inc., Palackeho, Czech Republic) [ , , ], or with a recombinant mouse follistatin protein (RD Systems, Minneapolis, MN, USA) at 0.2 μg/ml [ , ], changing medium twice a week.

Techniques: Expressing, Western Blot, Knock-Out, Derivative Assay

Journal: Stem Cell Research & Therapy

Article Title: Myostatin genetic inactivation inhibits myogenesis by muscle-derived stem cells in vitro but not when implanted in the mdx mouse muscle

doi: 10.1186/scrt152

Figure Lengend Snippet: The dystrophin + MDSCs restore some dystrophin expression in the injured mdx gastrocnemius . (A) Myofibers from the intact gastrocnemius from the WT mouse, the source of WT MDSCs, show in a dual immunofluorescence merge all myofibers stained for dystrophin, and nuclei stained with DAPI (Vectashield mounting medium; Vector Laboratories, Burlingame, CA, USA) (200×). (B) In other tissue sections, DAPI-labeled implanted Mst KO MDSCs appear to have fused with the mdx myofibers, showing dystrophin + staining in a small area. (C) A similar picture but with WT MDSCs. (D) The same field as in (C), examined under visible light, confirming the integrity of the myofibers, including the dystrophin - area. MDSC, muscle-derived stem cell; mdx, X chromosome-linked muscular dystrophy; WT, wild type; DAPI, 4', 6-diaminido-phenylindole; Mst KO, myostatin knockout.

Article Snippet: For the tests on the modulation of MDSCs skeletal myotube formation by various factors, cells were allowed to reach confluence, switched to GM-HC, and incubated for 2 weeks with 2 μg/ml recombinant 113-amino acid myostatin protein (R-Mst), a biologically active recombinant 16-kDa protein containing 113 amino acid residues of the processed human myostatin protein (BioVendor Laboratory Medicine Inc., Palackeho, Czech Republic) [ , , ], or with a recombinant mouse follistatin protein (RD Systems, Minneapolis, MN, USA) at 0.2 μg/ml [ , ], changing medium twice a week.

Techniques: Expressing, Immunofluorescence, Staining, Plasmid Preparation, Labeling, Derivative Assay, Knock-Out

Journal: Stem Cell Research & Therapy

Article Title: Myostatin genetic inactivation inhibits myogenesis by muscle-derived stem cells in vitro but not when implanted in the mdx mouse muscle

doi: 10.1186/scrt152

Figure Lengend Snippet: The Mst KO MDSCs are less effective than the WT MDSCs in reducing fat deposits in the injured mdx mouse gastrocnemius . (A) Representative picture of a positive field from frozen-tissue sections from the untreated mdx-injured gastrocnemius, adjacent to those shown in Figure 8D, fixed in formalin and stained with Oil Red O, showing mostly interstitial fat and occasional myofiber fat infiltration (200×). (B) Staining of a representative field from sections from the muscle implanted with WT MDSCs; the Mst KO pictures were similar, but the reduction in staining was less marked. (C) Quantitative image analysis of the tissue sections from the three rat groups, based on 12 fields per tissue section and the total positive area per section (percentage), calculated as a mean for three adjacent sections per rat, and five mdx mice/group. * P < 0.05. Mst KO, myostatin knockout; MDSC, muscle-derived stem cell; WT, wild type; mdx, X chromosome-linked muscular dystrophy.

Article Snippet: For the tests on the modulation of MDSCs skeletal myotube formation by various factors, cells were allowed to reach confluence, switched to GM-HC, and incubated for 2 weeks with 2 μg/ml recombinant 113-amino acid myostatin protein (R-Mst), a biologically active recombinant 16-kDa protein containing 113 amino acid residues of the processed human myostatin protein (BioVendor Laboratory Medicine Inc., Palackeho, Czech Republic) [ , , ], or with a recombinant mouse follistatin protein (RD Systems, Minneapolis, MN, USA) at 0.2 μg/ml [ , ], changing medium twice a week.

Techniques: Staining, Knock-Out, Derivative Assay