pax7 concentration  (Developmental Studies Hybridoma Bank)

Journal: bioRxiv

Article Title: Human engineered skeletal muscle of hypaxial origin from pluripotent stem cells with advanced function and regenerative capacity

doi: 10.1101/2021.07.12.452030

Figure Lengend Snippet: (A) Summary of the protocol for directed skeletal muscle differentiation from pluripotent stem cells (PSC) indicating the sequence and the timing of factor addition to modulate specific signaling pathways involved in skeletal myogenesis. Reads Per Kilobase Million (RPKM) of signature genes for (B) pluripotency ( POU5F1 ), naïve mesoderm (TBXT), paraxial mesoderm ( MSGN1 ), and lateral plate mesoderm ( MESP1 ); (C) dermomyotome formation ( PAX3 ), hypaxial ( SIM1, LBX1 ) and epaxial ( EN1 ) dermomyotome, and (D) , myogenic regulatory factors ( PAX7 , MYOD1 and MYOG ) and structural assembly ( ACTN2 ), during skeletal muscle differentiation from human PSCs; n = 2-4/time point. ( E) Immunostaining of OCT4, PAX3, PAX7, MYOD1, MYOGENIN, sarcomeric ACTININ (in gray), and nuclei (blue) at indicated time points of skeletal muscle differentiation. Scale bar: 500 µm.

Article Snippet: The following antibodies were applied for primary staining at 20-22°C for 4h: Oct4 (1:500, Abcam), Pax3-concentrate (1:100, DSHB), Pax7-concentrate (1:100, DSHB), MyoD (1:100, Dako) and Myogenin-concentrate (1:10, DSHB), Sarcomeric α-actinin (1:500, Sigma-Aldrich), Laminin (1:50, Sigma-Aldrich), neurofilament H, SMI32 (1:20000, Biolegend), β-dystroglycan (1:50, LCL-b-DG, Leica Biosystem) and Ki67 (1:100, Abcam).

Techniques: Sequencing, Protein-Protein interactions, Immunostaining

Journal: bioRxiv

Article Title: Human engineered skeletal muscle of hypaxial origin from pluripotent stem cells with advanced function and regenerative capacity

doi: 10.1101/2021.07.12.452030

Figure Lengend Snippet: (A) Representative immunostaining of myogenic regulatory factors (MRF): PAX7, MYOD1 and MYOGENIN (gray), f-ACTIN (green) and nuclei (blue) in 22 days old skeletal muscle cultures from TC1133 (iPSC 1) line; Scale bars: 50 µm. (B) Quantification of nuclei positive for PAX7, MYOD1 and MYOGENIN in 22 days old myogenic cultures from HES2 and from TC1133 (iPSC 1) lines; n = 9 -13 differentiations. (C) Unsupervised clustering (UMAP) of single nuclei transcriptomes from a day 22 skeletal muscle culture. (D) A muscle gene panel identifies 3 myogenic cell clusters (skeletal muscle cells, SkM). (E) Quantification of skeletal muscle cells (SkM), neuroectodermal progenitor cells (NPC), neurons, and mesenchymal progenitor cells (limb mesenchyme) of a total of 2,671 nuclei analyzed. (F) Expression levels of representative muscle-related genes and non-muscle genes.

Article Snippet: The following antibodies were applied for primary staining at 20-22°C for 4h: Oct4 (1:500, Abcam), Pax3-concentrate (1:100, DSHB), Pax7-concentrate (1:100, DSHB), MyoD (1:100, Dako) and Myogenin-concentrate (1:10, DSHB), Sarcomeric α-actinin (1:500, Sigma-Aldrich), Laminin (1:50, Sigma-Aldrich), neurofilament H, SMI32 (1:20000, Biolegend), β-dystroglycan (1:50, LCL-b-DG, Leica Biosystem) and Ki67 (1:100, Abcam).

Techniques: Immunostaining, Expressing

Journal: bioRxiv

Article Title: Human engineered skeletal muscle of hypaxial origin from pluripotent stem cells with advanced function and regenerative capacity

doi: 10.1101/2021.07.12.452030

Figure Lengend Snippet: (A) RNA transcript (Reads per Kilobase Million, RPKM) of indicated muscle stem cell markers in 2D monolayer cells at day 22 and day 60, plus day 60 ESM; n = 3-4/group, *p<0.05 by 1-way ANOVA and Tukey’s multiple comparison test. (B) Immunostaining of longitudinal sections of day 60 ESM for LAMININ (magenta), KI67 (magenta), ACTIN (green), PAX7 (gray), and nuclei (blue). Scale bars: 10 µm. Immunostaining of LAMININ (magenta), PAX7 (gray), ACTIN (green), and nuclei (blue) in 2D monolayer cultures at day 60. Scale bar: 50 µm. (C) Experimental design of cardiotoxin (CTX) injury model. ESM were incubated with 25 µg/ml CTX for 24 hrs. (D) Tetanic twitch tension at 100 Hz stimulation frequency of ESM at indicated time points after CTX (25 µg/ml) injury or control (Ctrl) condition; n=7-8/group, *p<0.05 vs. the respective Ctrl day +2, by 1-way ANOVA and Tukey’s multiple comparison test, #*p<0.05 CTX day +2 vs. CTX day +21. (F) RNA transcript for indicated genes at early (d+2) and late (day+21) time points after CTX (25 µg/ml) injury or control (Ctrl) conditions; n=3, *p<0.05 by 1-way ANOVA and Tukey’s multiple comparison test. (F) Immunostaining of sarcomeric a: -TININ (green), PAX7 (gray), and nuclei (blue) in ESM at indicated time points. Scale bars: 50 µm.

Article Snippet: The following antibodies were applied for primary staining at 20-22°C for 4h: Oct4 (1:500, Abcam), Pax3-concentrate (1:100, DSHB), Pax7-concentrate (1:100, DSHB), MyoD (1:100, Dako) and Myogenin-concentrate (1:10, DSHB), Sarcomeric α-actinin (1:500, Sigma-Aldrich), Laminin (1:50, Sigma-Aldrich), neurofilament H, SMI32 (1:20000, Biolegend), β-dystroglycan (1:50, LCL-b-DG, Leica Biosystem) and Ki67 (1:100, Abcam).

Techniques: Comparison, Immunostaining, Incubation, Control