Spatially patterned scaffolds enhances vascular organization and functional integration in volumetrc muscle loss. Spatially patterned scaffolds enhances vascular organization and functional integration in volumetrc muscle loss

We report the application of bioengineered skeletal muscle composed of murine skeletal myoblasts interspersed with human vascular endothelial cells (ECs) using spatially patterned scaffolds for the treatment of volumetric muscle loss. To gain insights into the molecular mechanisms by which spatial patterning promoted myo-angiogenesis, we performed mRNA sequencing of engineered muscle and endothelialized engineered muscle on either randomly-oriented or aligned scaffolds. The goals of this study are to compare NGS-derived transcriptome profiling (RNA-seq) from four groups: 1) randomly-oriented scaffold seeded with myoblasts; 2) randomly-oriented scaffolds seeded with myoblasts + ECs; 3) aligned scaffold seeded with myoblasts only; 4) aligned scaffold seeded with myoblasts + ECs. Samples were generated in triplicate and the NovaSeq 6000 (Illumina) sequencing platform was utilized. 150-bp paired-end reads were generated (20-30 million reads per sample). The raw data were checked for quality with FastQC (Version 0.11.7) and results were aggregated with MultiQC and were aligned to the mouse genome (GRCm38) using STAR (Version 2.5.3a) with ENCODE options for long RNA-seq pipeline. The alignment results were assessed using Samtools and aggregated with MultiQC (Version 1.5) and the differential gene expression analysis of the uniquely mapped reads/raw counts wwas performed using the DESeq2 package (Version 1.20.0). Each DE analysis was composed of a pairwise comparison between an experimental group and the control group. Differentially expressed genes were identified after false discovery rate (FDR = 0.05) correction. Overall design: Examination of differences in transcriptome profiling between 4 different bioengineered skeletal muscle

本研究报道了采用空间图案化支架（spatially patterned scaffolds），构建由小鼠成肌细胞（murine skeletal myoblasts）与人类血管内皮细胞（ECs）混合组成的生物工程化骨骼肌（bioengineered skeletal muscle），用于治疗体积性肌肉缺损（volumetric muscle loss）。为阐明空间图案化调控肌血管生成（myo-angiogenesis）的分子机制，我们分别对随机取向支架和对齐取向支架上构建的工程化骨骼肌、内皮化工程化骨骼肌进行了mRNA测序。本研究的目标是比较4组样本的下一代测序（NGS）来源转录组谱分析（RNA-seq）结果，4组分别为：1）仅接种成肌细胞的随机取向支架；2）接种成肌细胞+ECs的随机取向支架；3）仅接种成肌细胞的对齐取向支架；4）接种成肌细胞+ECs的对齐取向支架。所有样本均设置三次生物学重复，测序采用Illumina公司的NovaSeq 6000测序平台，生成150bp双端读段，每个样本产出2000万至3000万条读段。原始数据使用FastQC（版本0.11.7）进行质量评估，结果通过MultiQC进行整合；随后采用STAR（版本2.5.3a）搭配ENCODE长RNA-seq流程参数，将测序读段比对至小鼠基因组（GRCm38）。比对结果通过Samtools进行质控，并再次通过MultiQC（版本1.5）完成整合；针对唯一比对读段/原始计数，采用DESeq2软件包（版本1.20.0）进行差异基因表达分析。每一组差异表达分析均为实验组与对照组的两两比较；经错误发现率（FDR=0.05）校正后，筛选得到差异表达基因。整体实验设计：分析4组不同生物工程化骨骼肌样本的转录组谱差异。