Journal: eLife

Article Title: In vivo transcriptomic profiling using cell encapsulation identifies effector pathways of systemic aging

doi: 10.7554/eLife.57393

Figure Lengend Snippet: ( a ) Flow cytometry strategy used to identify live and apoptotic cells. CytoCalcein is sequestered in the cytoplasm of live cells, while apoptosis is measured by labeling of cell surface phosphatidylserine using Apopxin. ( b ) Quantification of live and apoptotic human (hskMPs) or mouse (mskMPs) skeletal muscle progenitors embedded into different biomaterials and maintained in growth media for 4 or 10 days. A = Growth factor reduced Matrigel, B = Hydrogel, C = MaxGel extracellular matrix. Values were obtained from pooled cells of n = 6 replicates. ( c ) Schematic outlining the construction of the adaptor hub used for mounting and loading of the PES hollow fiber capsules. ( d , e ) Schematics and graphical representations of the parts of the 3D printed capsule cutting and sealing platform. The photograph in the upper left of ( f ) shows the assembled platform loaded with a capsule mounted to the adaptor hub. ( g ) Quantification of Pax7 and MyoD positive hskMPs over a time course of 10 days in 2D culture. Graphs represent means ± s.e.m. n = 6 replicates for each time point. ****p < 0.0001, ***p < 0.001 by one-way ANOVA followed by Bonferroni post hoc test.

Article Snippet: For transcriptomic profiling of hskMPs in vitro, the cells were seeded into human fibronectin coated dishes (Corning, 356008) in human skeletal muscle myoblast growth medium (Zenbio, SKM-M).

Techniques: Flow Cytometry, Labeling

Journal: eLife

Article Title: In vivo transcriptomic profiling using cell encapsulation identifies effector pathways of systemic aging

doi: 10.7554/eLife.57393

Figure Lengend Snippet: ( a , b ) Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-based quantification of apoptosis in encapsulated human (hskMPs) or mouse (mskMPs) skeletal muscle progenitors maintained in growth media for 10 days. ( c , d ) DNA staining-based quantification of hskMP or mskMP numbers in cross sections of capsules maintained for 4, 8, or 10 days in growth media. ( e , f ) Representative DNA stainings of cross sections from a capsule containing hskMPs or mskMPs maintained 10 days in growth media. Scale bars: 75 µm. ( g ) Representative Pax7 immunostainings of cross sections from capsules containing hskMPs or mskMPs maintained 10 days in growth media. Scale bars: 150 µm. ( h , i ) Quantification of Pax7 positive cells in capsules containing hskMPs or mskMPs maintained for 4, 8, or 10 days in growth media. ( j ) Representative MyoD immunostainings of cross sections from capsules containing hskMPs or mskMPs maintained 10 days in growth media. Scale bars: 150 µm. ( k , l ) Quantification of MyoD positive cells in capsules containing hskMPs or mskMPs maintained for 4, 8, or 10 days in growth media. ( m–p ) Quantification of Pax7 and MyoD in cross sections from capsules maintained for 4 days under proliferative (Prolif.) conditions in growth media or in differentiation (Diff.) media. ( q ) Representative myosin heavy chain (MHC) immunostainings of cross sections from capsules containing hskMPs or mskMPs maintained for 4 days in differentiation media. Scale bars: 75 µm. ( r , s ) Quantification of MHC positive cells in capsules containing hskMPs or mskMPs maintained for 4 days in growth media compared to differentiation media. All graphs represent means ± s.e.m. n ≥ 3 cross sections from different capsules were quantified for each experiment and time point. ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05. Two-way comparisons were made with a Student’s t-test and multiple comparisons by one-way ANOVA followed by Bonferroni post hoc test.

Article Snippet: For transcriptomic profiling of hskMPs in vitro, the cells were seeded into human fibronectin coated dishes (Corning, 356008) in human skeletal muscle myoblast growth medium (Zenbio, SKM-M).

Techniques: TUNEL Assay, Staining

Journal: Nature genetics

Article Title: Long-range phasing of dynamic, tissue-specific and allele-specific regulatory elements

doi: 10.1038/s41588-022-01188-8

Figure Lengend Snippet: a, Schematic depicts paternal and maternal alleles of the classical imprinted locus. A canonical enhancer (purple ovals) activates the H19 non-coding RNA on the maternal chromosome (MAT) and the IGF2 growth factor gene on the paternal chromosome (PAT). The differential expression is directed by the ICR, which is bound by the CTCF insulator on the maternal allele, but methylated on the paternal allele. Active and inactive gene alleles are represented by green and grey rectangles, respectively. b, Connecting arrows highlight the positions of RNPs used to excise three overlapping regions for sequencing. NOMe-seq tracks show allele specific CpG methylation (red) and accessibility (green; open runs) in H9 ESCs and myoblasts (HSMM). ChIP-seq tracks for CTCF (ESCs) and H3K27ac (ESCs and HSMM) are also shown. In HSMM, a non-canonical enhancer (dashed box) is strongly marked by accessible chromatin on both parental alleles, and enriched for H3K27ac. c, Expanded view of the ICR shows differential methylation and accessibility on paternal and maternal alleles distinguished from long reads. Also shown are ChIP-seq tracks for CTCF and H3K27ac, LTRs (black bars) and duplicated sequences (grey bars paired by arcs). d, Expanded view shows nanopore DNA sequencing data (top) and RNA-seq data (bottom) for HSMM over a region in the last exon of IGF2 that harbors 3 heterozygous SNPs (chr11:2130822 / rs3802971; chr11:2130876 / rs57156844; chr11:2131037 / rs59196953). The nanopore data identify two SNPs specific to the maternal allele. Their presence in the RNA-seq at ~50% frequency indicates that IGF2 is expressed from both parental alleles in these primary human muscle cells. (Ref = reference; Alt = alternate).

Article Snippet: Cryopreserved human skeletal muscle myoblast (HSMM) cells isolated from a normal donor were obtained from Lonza (CC-2580; 22Y, male).

Techniques: Quantitative Proteomics, Methylation, Sequencing, CpG Methylation Assay, ChIP-sequencing, DNA Sequencing, RNA Sequencing

Journal: Biology

Article Title: Free Fatty Acid Species Differentially Modulate the Inflammatory Gene Response in Primary Human Skeletal Myoblasts

doi: 10.3390/biology10121318

Figure Lengend Snippet: Mitochondrial abundance and activity in human primary myoblasts were differentially affected by FFAs. SkMs were treated with FAs conjugated with BSA or BSA alone (control). After 48 h, SkMs were incubated with MitoTracker Red ( a ) or Orange ( b ). Fluorescence images at identical settings and exposure times from 3 sets of experiments were simultaneously imported into the analysis software, processed to 8-bit images and analyzed. The integrated fluorescence intensity of FFA-treated cells was compared to that of control cells, which was set to 1 (c, dashed gray line). The results are presented as the mean ± SD of the relative fluorescence intensity of MitoTracker Red (white bars) or Orange (black bars). Statistical significance was calculated using one-way ANOVA with Bonferroni’s multiple comparison test. p ≤ 0.05 (*), p ≤ 0.01 (**).

Article Snippet: Primary human skeletal muscle myoblasts (SkMs) were obtained from Lonza, Basel, Switzerland (CC-2561).

Techniques: Activity Assay, Control, Incubation, Fluorescence, Software, Comparison

Journal: Biology

Article Title: Free Fatty Acid Species Differentially Modulate the Inflammatory Gene Response in Primary Human Skeletal Myoblasts

doi: 10.3390/biology10121318

Figure Lengend Snippet: FFAs do not affect the desmin expression pattern in human primary myoblasts. ( a ) SkMs were treated with FAs conjugated with BSA or BSA alone (control) before DAPI and desmin staining. ( b ) Fluorescence images from 3 sets of experiments were analyzed, as described in the legend to . The number of DAPI-positive FFA-treated cells was compared to that of control cells, which was set to 1 (c, dashed gray line). The data are presented as the mean ± SD of cell number (dotted bars). Statistical significance was calculated using one-way ANOVA with Bonferroni’s multiple comparison test. p ≤ 0.05 (*). p ≤ 0.01 (**).

Article Snippet: Primary human skeletal muscle myoblasts (SkMs) were obtained from Lonza, Basel, Switzerland (CC-2561).

Techniques: Expressing, Control, Staining, Fluorescence, Comparison

Journal: Biology

Article Title: Free Fatty Acid Species Differentially Modulate the Inflammatory Gene Response in Primary Human Skeletal Myoblasts

doi: 10.3390/biology10121318

Figure Lengend Snippet: FFAs are differentially utilized in human primary myoblasts. ( a ) SkMs were treated with FAs conjugated with BSA or BSA alone (control) for 48 h and stained using BODIPY. ( b ) Fluorescence images from 3 sets of experiments were analyzed as described in the legend to . The fluorescence intensity of FFA-treated cells was compared to that of control cells, which was set to 1 (c, dashed gray line). The results are presented as the mean ± SD of relative lipid accumulation (hatched gray bars). Statistical significance was calculated using one-way ANOVA with Bonferroni’s multiple comparison test. p ≤ 0.05 (*).

Article Snippet: Primary human skeletal muscle myoblasts (SkMs) were obtained from Lonza, Basel, Switzerland (CC-2561).

Techniques: Control, Staining, Fluorescence, Comparison

Journal: Biology

Article Title: Free Fatty Acid Species Differentially Modulate the Inflammatory Gene Response in Primary Human Skeletal Myoblasts

doi: 10.3390/biology10121318

Figure Lengend Snippet: FFAs selectively activate RTK phosphorylation in human primary myoblasts. SkMs were treated with FAs conjugated with BSA or BSA alone for two hours. Whole-cell lysates were prepared and each incubated with a single RTK array. Following incubation steps, the slides were washed, imaged and analyzed using a laser scanner. Fluorescence images were processed and analyzed simultaneously using analysis software. To obtain the relative phosphorylation (black bars) of individual RTKs indicated at the top of each diagram, integrated signal intensities from FFA-treated cells were compared to that from control cells, which was set to 1 ( a – g , dashed gray line). Data are presented as the mean ± SD of the relative phosphorylation. Statistical significance was calculated using one-way ANOVA with Bonferroni’s multiple comparison test. p ≤ 0.05 (*). p ≤ 0.01 (**).

Article Snippet: Primary human skeletal muscle myoblasts (SkMs) were obtained from Lonza, Basel, Switzerland (CC-2561).

Techniques: Phospho-proteomics, Incubation, Fluorescence, Software, Control, Comparison