Unknown,Transcriptomics,Genomics,Proteomics

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Bulk RNA-seq of Adult and Fetal Enteroids (Control or Treated with DBZ)

ABSTRACT: Bulk RNA sequencing data of both adult and fetal enteroid lines cultured for 1 or 3 days. Conditions include enteroids grown in control basal media and basal media with the notch inhibitor DBZ added.

INSTRUMENT(S): Illumina HiSeq 2500

ORGANISM(S): Homo sapiens

SUBMITTER: Yu-Hwai Tsai

PROVIDER: E-MTAB-11619 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Background & Aims Patient-derived gastrointestinal organoids are powerful tools for studying human physiology and disease. However, generating organoids requires prompt isolation and culture of fresh tissue, which is labor and time intensive, placing restraints on basic and clinical research. For example organoid biobanking efforts, or the ability to obtain specimens from distant locations that lack the expertise to culture organoids, is limited by current approaches. We sought to develop a method by which tissue biopsies from patients could be cryo-preserved, stored, or shipped, and later thawed in order to establish live organoid cultures. Methods A method for freezing and recovery, along with straightforward isolation methods using readily available materials were developed. Epithelial isolation and culture conditions were optimized to promote cell survival and the establishment of long-term culture post-thawing. Results Patient biopsies from stomach, duodenum, ileum, colon and from adenomateous colonic polyps were frozen, subsequently thawed and used to successfully establish patient-specific epithelium-only organoid cultures with 100% efficiency (n=31 independent patient biopsies from different regions of the GI tract). Frozen tissue could be shipped internationally and across the United States and used to establish successful organoid cultures. Organoid cultures could be expanded, passaged and frozen down for long-term storage. RNA-sequencing demonstrates that organoids derived from fresh tissue or from frozen biopsies are >99% transcriptionally identical. Conclusions Cryo-preservation of human tissue biopsies followed by the establishment of long-term, expandable cultures has negligible effect on transcription when compared to freshly prepared organoids, and will be of immense benefit to research and for clinical applications. This technique will allow for the establishment of biobanks of human tissues that can be revived and cultured in the future as well as transported across the globe for research, therapeutic or diagnostic use in remote labs and/or clinics.

Project description:The host response of the primary intestinal epithelium to human astrovirus (HAstV infection has not been elucidated to date. In order to characterize the global effects of AstV infection on human intestinal tissue, we performed transcriptional profiling of VA1-infected human intestinal enteroids (HIE) by RNAseq. We used the D124 line for our studies since AstV infections are typically symptomatic in very young children, and our prior infection studies indicated rapid infection in D124. D124 HIE were mock-infected or infected with VA1 (MOI = 1) and harvested at 0 hpi (i.e., 1h post-adsorption), 12 hpi, and 24 hpi (Fig 4A). To monitor viral replication, VA1 genome copies were detected by RT-qPCR, revealing an approximately 1 and 2 log increase at 12 and 24 hpi, respectively (Fig 4B). Cellular RNA was extracted and analyzed by RNAseq. Genome copies as determined by RT-qPCR were correlated to the proportion of viral transcripts in the pool of sequenced RNA collected from the same HIE cultures, revealing an exceptionally strong correlation between the two measures of viral replication (r2 = 0.98, P = 2.5 x 10^-14. VA1 genome reads contributed 0.05 ± 0.02 x% of total RNAseq reads at 24 hpi, further confirming robust infection. Differential expression analysis was performed to identify genes associated with the HIE host response to VA1 infection. A total of 23,220 genes were detected. Differential expression of genes at each timepoint was graphed in a volcano plot. The log2 fold change in normalized expression (transcripts per million reads [TPM]) of all expressed host genes in VA1-infected HIEs relative to mock-infected HIE is shown on the x-axis. The -log10 transformed P-value is given on the y-axis. Genes that are significantly up-regulated (adjusted P < 0.05) in VA1-infected D124 HIE relative to mock-infected HIE are colored red, while significantly down-regulated genes are colored blue. Overall, after a 1 hour adsorption (0 hpi), 110 genes were significantly upregulated and 136 genes were downregulated , indicating changes due to viral attachment to cells. This number was reduced at 12 hpi, with 8 significantly upregulated and 5 downregulated genes. At 24 hpi, 154 upregulated and 49 downregulated genes compared to the mock-infected control were identified. We next identified the top 15 significantly up- and down-regulated genes at 24 hpi. This group of genes was used to generated a heatmap of the mean scaled fold-change (Z-score) in expression of each of them in virus-infected HIE relative to mock-infected HIE at each timepoint (Fig 4D). Most of the upregulated genes at 24 hpi were involved in type I and type III interferon (IFN) signaling. Of the IFN genes, IFNL1 was highly upregulated, with IFNA1 and IFNB1 upregulation being slightly lower (Fig S4B). No upregulation was observed for the genes encoding IFN-γ, or the type I and III IFN receptors (data not shown). The top 12 IFN-stimulated genes (ISGs) also positively correlated with VA1 infection (Fig S4C). Conversely, the top 12 downregulated genes, including fermitin family member 1 (FERMT1), signal peptide peptidase like 3 (SPPL3), and tetratricopeptide repeat domain 19 (TTC19) negatively correlated with VA1 infection (Fig S4D). Next, we evaluated lists of the top 100 up- and down-regulated genes at 24 hpi using an over-abundance test to identify significantly over-represented REACTOME pathways in these lists. These data revealed that the top four significantly enriched pathways among upregulated genes were all related to innate antiviral signaling (Fig 4E), which will be investigated in more detail below. For downregulated genes, the top two pathways were “neurexins and neuroligins”, which play signaling roles in synapse development, and “protein-protein interactions at synapses”. The biological significance of synapses during astrovirus infection remains to be elucidated. In order to evaluate the potential for coordinated and directional activation of genes in known signaling pathways, we applied gene set enrichment analysis (GSEA) to our RNAseq differential expression data. Based on the strong dominance of IFN signaling pathways, we focused our GSEA analysis on immune signaling (Fig 4F). During the adsorption phase, nucleic acid pattern recognition receptor signaling pathways (TLRs, STING) were upregulated, consistent with their early role in virus recognition and induction of IFN signaling. At 24 hpi, these early signaling events had been largely replaced by the later phase of IFN signaling and expression of ISGs. Taken together, these data indicate that VA1 infection predominantly elicited antiviral IFN signaling in HIE-derived fetal duodenum at the transcript level.

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Bulk RNA-seq of Adult and Fetal Enteroids (Control or Treated with DBZ)

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