RNA-sequencing of cycling PKH26-Negative cells and PKH26-Positive label retaining quiescent cancer stem cells from colon cancer patient-derived organoids
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ABSTRACT:
PROVIDER: PRJEB41875 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| ERR4975110_1.fastq.gz | Fastqsanger.gz | |||
| ERR4975110_2.fastq.gz | Fastqsanger.gz | |||
| ERR4975120_1.fastq.gz | Fastqsanger.gz | |||
| ERR4975120_2.fastq.gz | Fastqsanger.gz | |||
| ERR4975130_1.fastq.gz | Fastqsanger.gz |
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Project description:Recent data suggests that colon tumors contain a subpopulation of therapy resistant quiescent cancer stem cells (qCSCs) are the source of relapse following treatment. The isolation and molecular characterisation of qCSCs may therefore lead to the identification of novel therapeutic targets for their elimination. Label retention of the lipophilic fluorescent dye PKH26, wherein dividing cells lose the label and quiescent or slow cycling cells retain the label for an extended period of time, was used to isolate qCSCs from a panel of colon cancer patient-derived organoids (PDOs). PDOs were dissociated to single cells, labelled with PKH26 and plated in Matrigel culture. After 12 days, PDOs were processed to single cells, labelled with DAPI (to exclude dead cells) and isolated by fluorescence assisted cell sorting (FACS) into two fractions of PKH26-Negative and PKH26-Positive (label retaining) cells. Functional analyses of these cellular subpopulations demonstrated that PKH26-Positive cells are self-renewing qCSCs. RNA was collected from FAC sorted PKH26-Negative and PKH26-Positive cells and analysed by RNA-sequencing. Cells were lysed in RLTplus buffer and RNA was extracted using Qiagen RNeasy Mini Kit. RNA quality was assessed prior to sequencing. Samples were processed using NuGEN’s Ovation RNA-Seq System V2 and Ultralow V2 Library System and sequenced on an Illumina HiSeq 2500 machine as 2x125nt paired-end reads. Reads were mapped to the human reference genome (assembly hg19) using the STAR aligner (version 2.4.2a). Total read counts per gene were computed using the program “featureCounts” (version 1.4.6-p2) in the “subread” package, with the gene annotation taken from Gencode (version 19).
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Project description:The epithelial lining of the small intestine is continuously renewed from a small number of stem cells including Lgr5 expressing crypt base cells that have been shown to be a rapidly cycling stem cell population in homeostasis. Alternative markers of intestinal stem cells have variously identified populations as rapidly cycling or quiescent with proposed roles for the latter as a parallel or reserve stem cell population. However, the exact nature of quiescent crypt cells remains unknown. Here by applying novel mouse models that permit their isolation and characterisation as label-retaining cells (LRCs), and for the first time performing lineage tracing from them, we show quiescent cells to be committed secretory precursors that are capable of recall to the stem cell state. We reveal LRCs to be a small subset of the rapidly cycling Lgr5 expressing population committed along the Paneth-enteroendocrine lineage. Significantly, after injury and subsequent regeneration they are clonogenic, as shown by both lineage tracing and organoid growth demonstrating that they can be recalled to the stem cell pool. These findings in establishing quiescent cells as an effective clonogenic reserve during injury provides a motivation for investigating their role in the maintenance of cancer growth following adjuvant treatment.
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Project description:Transcriptional profiling of mouse kidney papilla cells comparing control GFP- cells with GFP+ labeled cells. Mouse kidney papillae were dissected. GFP- and GFP+ cell populations were isolated by FACS. The latter represent slow cycling label retaining cells (LRCs). Two-condition experiment, GFP- vs. GFP+ label retaining cells cells. Biological replicates: 5 control GFP- replicates, 5 GFP+ label retaining cell replicates.
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Project description:The epithelial lining of the small intestine is continuously renewed from a small number of stem cells including Lgr5 expressing crypt base cells that have been shown to be a rapidly cycling stem cell population in homeostasis. Alternative markers of intestinal stem cells have variously identified populations as rapidly cycling or quiescent with proposed roles for the latter as a parallel or reserve stem cell population. However, the exact nature of quiescent crypt cells remains unknown. Here by applying novel mouse models that permit their isolation and characterisation as label-retaining cells (LRCs), and for the first time performing lineage tracing from them, we show quiescent cells to be committed secretory precursors that are capable of recall to the stem cell state. We reveal LRCs to be a small subset of the rapidly cycling Lgr5 expressing population committed along the Paneth-enteroendocrine lineage. Significantly, after injury and subsequent regeneration they are clonogenic, as shown by both lineage tracing and organoid growth demonstrating that they can be recalled to the stem cell pool. These findings in establishing quiescent cells as an effective clonogenic reserve during injury provides a motivation for investigating their role in the maintenance of cancer growth following adjuvant treatment. Six age and sex (female) matched Cyp1a1-H2B-YFP mice ten days post βNF induction were used comparing three cell populations from each. Following epithelial isolation, single cell preparation and staining with anti-CD24 anitbody and UEA-1 lectin, 30,000 cells were flow sorted for each population: Paneth cells (Paneth) were defined as CD24+/UEA+, YFP-LRCs (YFPpos) as CD24+/UEA-/YFP+ and LCCs (YFPneg) as CD24+/UEA-/YFP-. Microarray expression comparison was then performed to identify unique markers of each population. RNA amplification and hybridisation was performed at the Paterson Institute, Manchester, UK, Microarray Facility using Nugen Ovation Pico WTA System for amplification and then hybridisation to a Mouse Exon 1.0 ST Array. Arrays were scanned using an Affymetrix GeneChip scanner 3000 running GCOS software.
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Project description:Hierarchically organized tissues, such as hematopoietic systems, muscle, or skin harbor deeply quiescent stem cells which start proliferating in response to external insults. In contrast, it remains obscure whether similar quiescent cells exist in epithelia of digestive organs. Here we identified a deeply quiescent population in gastric corpus but not in other gastrointestinal organs after systematic examination of H2b-GFP label-retaining cells. The label-retaining cells in corpus epithelia belonged to a subpopulation of chief cells that were located near basal layers of corpus and did not overlap with Troyhigh, Lgr5high, or Misthigh cell population. The identified quiescent cells were marked with activation of Atf4 and unfolded protein response. External damages by indomethacin treatment triggered proliferation of the quiescent populations, indicating that chief cells of gastric corpus harbor deeply quiescent reserve cells with high levels of internal stress response activity.
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