sarcomeric myosin heavy chain  (Developmental Studies Hybridoma Bank)

Journal: Stem Cell Research & Therapy

Article Title: Human serum and platelet lysate are appropriate xeno-free alternatives for clinical-grade production of human MuStem cell batches

doi: 10.1186/s13287-018-0852-y

Figure Lengend Snippet: Myogenic differentiation potential of hMuStem cells HS and hMuStem cells hPL . a Myogenic differentiation potential of hMuStem cells isolated and expanded either with HS (hMuStem cells HS ) or hPL (hMuStem cells hPL ) determined at P4 ( n = 3, donors #4–#6). Cells expressing myogenic markers MyoD and myogenin counted and fusion index (FI) calculated as percentage of nuclei identified in sarcomeric myosin heavy chain-positive (sMyHC + ) myotubes. b Contribution of hMuStem cells to myogenesis evaluated in coculture experiments with mouse D7 cells. Number of human lamin A/C + nuclei (arrow) counted within hybrid sMyHC + myotubes. Nuclei counterstained with DAPI or DRAQ5 (blue). Donors #4, #5, and #6 represented by squares, triangles, and circles, respectively. Data presented as mean ± SD for each donor. Scale bar, 100 μm. HS human serum, hPL human platelet lysate

Article Snippet: After 4 days, cultures were fixed in 4% PFA, and incubated with 5% Triton X-100 (30 min, 4 °C), 20% goat serum in PBS (20 min, RT), and finally anti-human sarcomeric myosin heavy chain isoform (sMyHC) Ab (1:500; Developmental Studies Hybridoma Bank/DSHB, Iowa City, IA, USA) for 60 min at 37 °C.

Techniques: Isolation, Expressing

Journal: Stem Cell Research & Therapy

Article Title: Human serum and platelet lysate are appropriate xeno-free alternatives for clinical-grade production of human MuStem cell batches

doi: 10.1186/s13287-018-0852-y

Figure Lengend Snippet: Influence of hPL on myogenic differentiation potential of hMuStem cells HS . hMuStem cells HS ( n = 6, donors #7–#12) at P3–4 cultured for two passages with HS (hMuStem cells HS/HS ) or hPL (hMuStem cells HS/hPL ) and resulting myogenic pattern evaluated. Myogenic commitment assessed by determining ( a ) mRNA expression levels of MRFs MYF5 , MYOD1 , and DES , as well as ( b ) proportion of cells positive for each of these markers. Myogenic differentiation potential assessed by quantification of cells expressing ( c ) MyoD and ( d ) myogenin, and by determining ( e ) fusion index (FI), defined as percentage of nuclei identified in sarcomeric myosin heavy chain-positive (sMyHC + ) myotubes. ( f ) Influence of hPL on contribution of hMuStem cells to myogenesis evaluated in coculture experiments with mouse D7 cells by quantification of human lamin A/C + nuclei within hybrid sMyHC + myotubes. Data presented as mean ± SEM (* p < 0.05, Wilcoxon matched-pairs signed rank test). HS human serum, hPL human platelet lysate

Article Snippet: After 4 days, cultures were fixed in 4% PFA, and incubated with 5% Triton X-100 (30 min, 4 °C), 20% goat serum in PBS (20 min, RT), and finally anti-human sarcomeric myosin heavy chain isoform (sMyHC) Ab (1:500; Developmental Studies Hybridoma Bank/DSHB, Iowa City, IA, USA) for 60 min at 37 °C.

Techniques: Cell Culture, Expressing

Journal: Stem Cell Research & Therapy

Article Title: Human serum and platelet lysate are appropriate xeno-free alternatives for clinical-grade production of human MuStem cell batches

doi: 10.1186/s13287-018-0852-y

Figure Lengend Snippet: Influence of heparin and heparin-free hPL-supplemented media on myogenic potential of hMuStem cells. hMuStem cells HS cultured for 6 days in HS-GM or hPL-GM in absence or presence of increasing doses of heparin (0.5–5 IU/ml), or in heparin-free hPL alternatives, and then seeded for evaluation using myogenic differentiation assay. a Immunolabeling of sMyHC performed after 4 days in myogenic differentiation medium DM. Nuclei counterstained with DRAQ5 (blue). Scale bar, 100 μm. Fusion index (FI) determined in ( b ) three independent cell batches cultured with HS or hPL in presence of increasing doses of heparin, or in ( c ) heparin-free hPL alternatives; heparin-free hPL that was allowed to form a nutritive gel over the cell layer (hPL gel ), or heparin-free fibrinogen-depleted hPL (hPL fd ). Data presented as mean ± SEM ( b p < 0.01, c p < 0.001 versus HS-GM; *** p < 0.001; LME model followed by Tukey’s post-hoc test). HS human serum, hPL human platelet lysate, GM growth medium, sMyHC sarcomeric myosin heavy chain

Article Snippet: After 4 days, cultures were fixed in 4% PFA, and incubated with 5% Triton X-100 (30 min, 4 °C), 20% goat serum in PBS (20 min, RT), and finally anti-human sarcomeric myosin heavy chain isoform (sMyHC) Ab (1:500; Developmental Studies Hybridoma Bank/DSHB, Iowa City, IA, USA) for 60 min at 37 °C.

Techniques: Cell Culture, Differentiation Assay, Immunolabeling

Journal: Stem Cell Research & Therapy

Article Title: Human serum and platelet lysate are appropriate xeno-free alternatives for clinical-grade production of human MuStem cell batches

doi: 10.1186/s13287-018-0852-y

Figure Lengend Snippet: Reversal of suppressive effect of hPL on myogenic potential of hMuStem cells. hMuStem cells hPL cultured up to P3 in appropriate GM and then passaged to HS-GM or heparin-free hPL-GM (hPL fd or hPL gel ), then myogenic differentiation assessed. a sMyHC immunolabeling performed after 4 days of culture in fusion-promoting low-serum medium. Nuclei counterstained with DRAQ5 (blue). Scale bar, 100 μm. b Fusion index (FI) in six independent cell batches. c Western blot analyses of sMyHC and GAPDH protein abundance in hMuStem cells HS maintained in HS-GM, as well as hMuStem cells hPL cultured with either hPL-GM or HS-GM. Data presented as mean ± SEM ( a p < 0.05, c p < 0.001 versus HS; * p < 0.05, ** p < 0.01, *** p < 0.001; LME model followed by Tukey’s post-hoc test). HS human serum, hPL human platelet lysate, hPL gel heparin-free hPL that was allowed to form a nutritive gel over the cell layer, hPL fd heparin-free fibrinogen-depleted hPL, sMyHC sarcomeric myosin heavy chain, GAPDH glyceraldehyde 3-phosphate dehydrogenase

Article Snippet: After 4 days, cultures were fixed in 4% PFA, and incubated with 5% Triton X-100 (30 min, 4 °C), 20% goat serum in PBS (20 min, RT), and finally anti-human sarcomeric myosin heavy chain isoform (sMyHC) Ab (1:500; Developmental Studies Hybridoma Bank/DSHB, Iowa City, IA, USA) for 60 min at 37 °C.

Techniques: Cell Culture, Immunolabeling, Western Blot, Quantitative Proteomics

Journal: Frontiers in Physiology

Article Title: Functional Segregation within the Muscles of Aquatic Propulsion in the Asiatic Water Monitor ( Varanus salvator )

doi: 10.3389/fphys.2016.00380

Figure Lengend Snippet: The myosin heavy chain region of a silver-stained gel loaded with homogenates of cardiac and skeletal muscles . The MHC band numbers refer to the MHC isoforms in the skeletal muscle samples.

Article Snippet: An immunoblot with an antibody that recognizes all sarcomeric MHC isoforms (MF 20, diluted 1:50, obtained from the Developmental Studies Hybridoma Bank at the University of Iowa) was run to verify that all of the scanned bands were MHC isoforms.

Techniques: Staining, Muscles