Preclinical efficacy and safety study of encapsulated human proliferating hepatocyte organoids in the treatment of liver failure

Purpose: The goals of this study are to analyze the NGS-derived transcriptome profiling (RNA-seq) between the normal, control and experimental group after extended hepatectomy. Results: All sequencing reads from RNA-seq were mapped to the mouse reference genome (GRCm38.84) using hisat2-2.10 (Kim et al., 2015). FPKM (fragments per kilobase of exon per million fragments mapped) values were calculated by Cufflinks v.2.2.1 (Trapnell et al., 2012) using default parameters for gene expression levels. We used htseq-count (Anders et al., 2015) to count reads on genes. Differential expression analysis was performed using DESeq2 (R package, (Love et al., 2014)). Genes were considered differentially expressed if FPKM >=1 in at least one sample and |fold change| >= 2, adjusted p-value < 0.05. The gene ontology analyses was performed using Metascape v3.5 (http://metascape.org/gp/index.html, (Zhou et al., 2019)). Principal component analysis was performed with whole genes using the R package (function: prcomp()). Conclusions: Our study represents the first detailed analysis of liver transcriptomes after extended hepatectomy, with biologic replicates, generated by RNA-seq technology. The optimized data analysis workflows reported here should provide a framework for comparative investigations of expression profiles. Our results show that NGS offers a comprehensive and more accurate quantitative and qualitative evaluation of mRNA content within a cell or tissue. We conclude that RNA-seq based transcriptome characterization would expedite genetic network analyses and permit the dissection of complex biologic functions. Overall design: Methods: Liver mRNA profiles from normal control mice (10 weeks old), EMC-treated control mice (2 days after extended hepatectomy) and eLO- treated mice (2 days after extended hepatectomy and 14 days after extended hepatectomy) were generated by deep sequencing, in duplicate using Illumina NovaSeq 6000. The sequence reads that passed quality filters were analyzed at the transcript isoform level with two methods: Burrows–Wheeler Aligner (BWA) followed by ANOVA (ANOVA) and TopHat followed by Cufflinks. qRT–PCR validation was performed using SYBR Green assays. liver tissue mRNA profiles of normal control mice (10 weeks old), EMC-treated control mice (2 days after extended hepatectomy) and eLO- treated mice (2 days after extended hepatectomy and 14 days after extended hepatectomy)