scRNA-seq assessment of the human lung, spleen, and esophagus tissue stability after cold preservation.
Author(s): Madissoon E, Wilbrey-Clark A, Miragaia RJ, Saeb-Parsy K, Mahbubani KT, Georgakopoulos N, Harding P, Polanski K, Huang N, Nowicki-Osuch K, Fitzgerald RC, Loudon KW, Ferdinand JR, Clatworthy MR, Tsingene A, van Dongen S, Dabrowska M, Patel M, Stubbington MJT, Teichmann SA, Stegle O, Meyer KB
Publication: Genome Biol, 2019, Vol. 21, Page 1
PubMed ID: 31892341 PubMed Review Paper? No
Purpose of Paper
The purpose of this paper was to determine how 0-72 h of cold ischemia time (at 4°C in HypoThermosol FRS solution) affects single-cell RNA sequencing (scRNAseq) and bulk RNA sequencing (RNAseq) results in healthy lung, spleen, and esophagus tissue specimens procured from deceased organ donors.
Conclusion of Paper
The authors conclude that limiting cold ischemia time to ≤24 h will minimize the effects of cold ischemia detectable by scRNAseq and bulk RNAseq for esophagus, lung, and spleen samples when stored in 4°C HypoThermosol FRS. A cold ischemia of ≤24 h (incubation of postmortem, healthy lung, spleen, and esophagus in 4°C in HypoThermosol FRS solution) did not significantly affect cell yield, RNA integrity number (RIN), the total number of reads per cell, the median number of genes detected per cell, or the percentage of reads mapped to the transcriptome in lung, esophagus, and spleen cells undergoing scRNAseq. However, cells from spleen specimens with a cold ischemia time of 72 h had a lower percentage of reads that mapped to the transcriptome, a significant reduction in the percentage of exonic reads (p=0.009) and an increase in intronic reads (p=0.02) relative to earlier timepoints. The proportion of mitochondrial reads, an indicator of cellular stress (during tissue storage or dissociation), remained stable in all esophagus and lung specimens at all cold ischemia timepoints but increased significantly in spleen cells with a cold ischemia time of 24 h (p=0.02) or 72 h (p=0.03) and also displayed significant differences in the proportion of cells with >10% mitochondrial reads (24 h, p=0.02; 72 h, p=0.03) relative to the 0 h timepoint. While the proportion of droplets containing unique molecular identifiers (UMIs) classified as acellular RNA, debris, or cellular material did not change with cold ischemia in the tissue types examined, the mean number of UMIs was significantly higher in debris droplets (p=0.027) and significantly lower in cellular material droplets (p=0.00012) in, and only in, spleen cells subjected to 72 h cold ischemia. A definitive conclusion was not possible due to the level of variability observed in the staining for cell viability and apoptotic cells, although the authors noted staining by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) tended to increase with progressive ischemia in all three tissue types, with the most extensive TUNEL staining observed in spleen samples from the 72 h timepoint. No significant changes in the proportion of cell types (although a nonsignificant increase in B-cells in spleen and a decrease in T-cells in lung were reported) or the level of variability in the transcriptome were observed over the cold ischemia timecourse for any of the three tissues. When mitochondrial reads were assessed in specific cell types, the highest fold-change (relative to the 0 h ischemia timepoint) in mitochondrial percentages occurred in spleen at the 72 h cold ischemia time timepoint (>6 fold-change), with plasma cells strongly affected. When scRNAseq (data was combined) and bulk RNAseq data (from frozen tissue) were compared on a UMAP plot to assess effects of tissue dissociation, samples did not cluster by cold ischemia time but clustered by sequencing method and tissue of origin Differential analysis of bulk RNAseq data from cold ischemia timecourse samples, relative to the 0 h timepoint (which included unspecified transport delays from the clinic to the processing laboratory), did not reveal any significantly affected genes in any of the three tissues examined; nor were any significant changes observed when the lung sample that was snap-frozen immediately after tissue excision (collected in the clinic) was used for cold ischemia comparisons.
Studies
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Study Purpose
The purpose of this study was to determine how cold ischemia time (at 4°C in HypoThermosol FRS solution; 0-72 h) affects single-cell RNA sequencing (scRNAseq) and bulk RNAseq results in healthy lung, spleen, and esophagus tissue specimens procured from deceased organ donors. Twelve organ donors (6 males, 6 females, 20-70 y) were perfused with cold University of Wisconsin (UW) solution within 12 min of cardiac cessation. Research samples (1.5 cm3) were collected from lung, spleen, and esophagus and placed in HypoThermosol at 4°C within 2 h of cessation of circulation. Fresh tissue was then transported from the clinic to the processing laboratory, dissected/divided into samples that were frozen in pre-cooled isopentane (temperature not specified) for bulk DNA/RNA sequencing and histological review (OCT-embedded and hematoxylin and eosin stained), dissociated immediately (0 h timepoint, a mean duration of 4 h from cessation of circulation), or held at 4°C for an additional 12, 24, or 72 h (from the 0 h timepoint) at which point tissue samples were dissociated to single-cell suspensions. A portion of each lung specimen was snap-frozen in pre-cooled isopentane immediately after excision (in the clinic) to compare bulk RNA sequencing results between the 0 h timepoint (after transport to the clinic and dissection, duration not specified) and the sample reflecting a true ischemic time of 0 h (collected in the clinic). Tissue samples were mechanically dissociated using a cell strainer (spleen), forceps and scissors (esophagus), or scalpels (lung), resuspended, diluted, and cells were counted and viability was determined using a C-chip hemacytometer with trypan blue. Cells were loaded onto a 10X Genomics Chromium Controller using the single-cell 3’ v2 protocol to recover 2,000-5,000 cells. cDNA libraries prepared from single-cell suspensions were sequenced on a HiSeq4000 machine at a targeted depth of 150 M reads/sample. Bulk DNA/RNA was extracted from excised tissue specimens with the Qiagen AllPrep DNA/RNA mini Kit with a TissueLyser II. RNA was quantified with a QuanitFluor RNA system, RNA integrity number (RIN) was determined by Agilent Bioanalyzer, and poly(A) libraries were prepared using the NEW RNA Ultra II Custom Kit on an automated system and then sequencing on an Illumina HiSeq4000 machine at a depth of 35 M reads per sample. DNA from 13 donors was sheared to 450 bp and purified (AMPure XP SPRI beads) before libraries were prepared (NEB Ultra II Custom Kit) and sequenced at 30x on an Illumina HiSeqX. Single-cell RNAseq reads were mapped to the GRCH38 1.2.0 Human Genome reference by the Cell Ranger 2.0.2 pipeline; cells with <300 and > 5,000 (lung and spleen) or 8,000 (esophagus) detected genes, > 20,000 unique molecular identifiers (UMI), and >10% mitochondrial reads were removed. Cell Ranger-generated “gene expression count matrices” were used to determine and annotate cell type identity based on the reported expression of known cell type markers.
Summary of Findings:
Whole genome sequencing (WGS) of snap-frozen tissue specimens confirmed the absence of any large genomic abnormalities in participants, and examination of hematoxylin and eosin staining of OCT-embedded tissue sections by a pathologist confirmed that all tissues appeared healthy. The authors conclude that limiting cold ischemia time to ≤24 h will minimize the effects of cold ischemia detectable by scRNAseq and bulk RNAseq for esophagus, lung, and spleen samples when stored in 4°C HypoThermosol FRS. A cold ischemia time of ≤24 h (incubation of postmortem, healthy lung, spleen, and esophagus in 4°C in HypoThermosol FRS solution) did not significantly affect cell yield, RNA integrity number (RIN), the total number of reads per cell, the median number of genes detected per cell, or the percentage of reads mapped to the transcriptome in lung, esophagus, and spleen cells undergoing scRNAseq. However, cells from spleen specimens with a cold ischemia time of 72 h had a lower percentage of reads that mapped to the transcriptome, a significant reduction in the percent of exonic reads (p=0.009) and an increase in intronic reads (p=0.02) relative to earlier timepoints. The proportion of mitochondrial reads, an indicator of cellular stress (during tissue storage or dissociation), remained stable in all esophagus and lung specimens at all cold ischemia timepoints but increased significantly in spleen cells with a cold ischemia time of 24 h (p=0.02) or 72 h (p=0.03) and also displayed significant differences in the proportion of cells with >10% mitochondrial reads (24 h, p=0.02; 72 h, p=0.03) relative to the 0 h timepoint. The authors normalized the number of UMIs by the read depth and defined thresholds to differentiate between “acellular RNA” (non-cellular RNA; 0-0.25 normalized UMI/drop), “debris” (0.25-5), and “cellular material” (>5). While the proportion of droplets containing UMIs classified as acellular RNA, debris, or cellular material did not change with cold ischemia in the tissue types examined, the mean number of UMIs was significantly higher in debris droplets (p=0.027) and significantly lower in cellular material droplets (p=0.00012) in, and only in, spleen cells subjected to 72 h cold ischemia. A definitive conclusion was not possible due to the level of variability observed in the staining for cell viability and apoptotic cells, although the authors noted staining by TUNEL tended to increase with progressive ischemia in all three tissue types, with the most extensive TUNEL staining observed in spleen samples from the 72 h timepoint. No significant changes in the proportion of cell types (although a nonsignificant increase in B-cells in spleen and a decrease in T-cells in lung were reported) or the level of variability in the transcriptome were observed over the cold ischemia timecourse for any of the three tissues. When mitochondrial reads were assessed in specific cell types, the highest fold-change (relative to the 0 h ischemia timepoint) in mitochondrial percentages occurred in spleen at the 72 h cold ischemia time timepoint (>6 fold-change), with plasma cells strongly affected. When scRNAseq (data was combined) and bulk RNAseq data (from frozen tissue) were compared on a UMAP plot to assess effects of tissue dissociation, samples did not cluster by cold ischemia time but clustered by sequencing method and tissue of origin. Differential analysis of bulk RNAseq data from cold ischemia timecourse samples, relative to the 0 h timepoint (which included unspecified transport delays from the clinic to the processing laboratory), did not reveal any significantly affected genes in any of the three tissues examined; nor were any significant changes observed when the lung sample that was snap-frozen immediately after tissue excision (collected in the clinic) was used for cold ischemia comparisons.
Biospecimens
Preservative Types
- Frozen
- Other Preservative
Diagnoses:
- Normal
- Autopsy
Platform:
Analyte Technology Platform RNA Next generation sequencing RNA Automated electrophoresis/Bioanalyzer DNA Next generation sequencing Cell count/volume Next generation sequencing Cell count/volume Colorimetric assay Morphology H-and-E microscopy Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Cold ischemia time 0 h at 4°C
12 h at 4°C
24 h at 4°C
72 h at 4°C
Next generation sequencing Specific Technology platform Bulk sequencing
scRNAseq