Single-cell transcriptome conservation in a multispecies comparative analysis of fresh and cryopreserved insulinoma cell lines
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ABSTRACT:
PROVIDER: PRJNA1199559 | ENA |
REPOSITORIES: ENA
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| SRR31752387.fastq.gz | Fastqsanger.gz | |||
| SRR31752389.fastq.gz | Fastqsanger.gz | |||
| SRR31752390.fastq.gz | Fastqsanger.gz |
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Project description:Background Insulinoma is the most common pancreatic neuroendocrine tumour in dogs and humans. The understanding of driving factors and critical survival genes in insulinomas is limited and overall survival is poor for canine and human malignant insulinoma. This study aimed to use single-cell RNA-sequencing to conduct a multispecies analysis of insulinoma cell lines to understand their single-cell transcriptomic landscape. Secondly, the impact of freeze-thawing on the pancreatic beta single-cell transcriptome was investigated, to determine whether cryoarchiving of primary insulinoma samples may be feasible in future studies. Methods Single-cell transcriptomic analysis was performed using fresh and cryopreserved multispecies insulinoma cell lines (canINS, CM, INS-1 and MIN6). R and Seurat were used to perform cell clustering and specific cluster marker genes were identified by the FindMarkers function. Metascape was used to identify statistically enriched pathways for specific cell clusters. Differentially expressed genes between fresh and cryopreserved single-cell transcriptome profiles, were defined as genes with a log2 fold change >0.25 and a Bonferroni-adjusted P<0.05, based on the Wilcoxon rank sum test. Results Based on the specific cell line single-cell transcriptome profiles, five or six cell clusters were constructed per cell line. All cell lines expressed neuroendocrine markers and additionally INS-1 and MIN6 displayed a gene signature indicative of mature/functional pancreatic islet/beta-cells. DEPTOR, BICC1, GHR, CCNB2, CENPA, LMO4, VANGL1, and L1CAM were identified as cross-species conserved insulinoma cluster marker genes. Little effect was found of cryopreservation and thawing on overall gene expression at the single-cell level in insulinoma cell lines: only 6 and 29 genes had a log2 fold change > 1 in cryopreserved versus fresh canINS and CM, respectively. Conclusions canINS, CM, INS-1 and MIN6 are all principally relevant as insulinoma models and the demonstrated differences in their single-cell transcriptomic profiles could aid researchers in selecting the appropriate cell lines for specific study objectives. Cross-species conserved insulinoma cluster marker genes were identified that harbour oncogenes and their involvement in insulinoma tumourigenesis should be investigated in future studies. The good comparability between cryopreserved and fresh insulinoma cells allows for inclusion of cryopreserved insulinoma patient samples in future studies, which allows for reduced assay-based variability.
2025-06-04 | GSE284573 | GEO
Project description:Single Cell Transcriptome Conservation in Cryopreserved Cells and Tissues
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Project description:Single-cell transcriptome conservation in a comparative analysis of fresh and cryopreserved human skin tissue: pilot in localized scleroderma
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Project description:Non-hematopoietic lymph node stromal cells (LNSCs) regulate lymphocyte trafficking, survival, and function for key roles in host defense, autoimmunity, alloimmunity, and lymphoproliferative disorders. However, study of LNSCs in human diseases is complicated by a dependence on viable lymphoid tissues, which are most often excised prior to establishment of a specific diagnosis. Here, we demonstrate that cryopreservation can be used to bank lymphoid tissue for the study of LNSCs in human disease. Using human tonsils, lymphoid tissue fragments were cryopreserved for subsequent enzymatic digestion and recovery of viable non-hematopoietic cells. Flow cytometry and single-cell transcriptomics identified comparable proportions of LNSC cell types in fresh and cryopreserved tissue. Moreover, cryopreservation had little effect on transcriptional profiles, which showed significant overlap between tonsils and lymph nodes. The presence and spatial distribution of transcriptionally defined cell types was confirmed by in situ analyses. Our broadly applicable approach promises to greatly enable research into the roles of LNSC in human disease.
2023-05-16 | GSE224661 | GEO
Project description:Cryoinjury and protein changes are a consequence of cryopreservation and may have a negative impact on sperm quality regarding motility, viability and fertilizing ability. However, potential proteomic changes in rabbit semen throughout the cryopreservation process have never been investigated previously. The aim of the present study was to compare the whole proteome of fresh and cryopreserved rabbit extracellular fluid of semen. Comparative analysis and identification of proteins were performed using 2-dimensional difference in-gel electrophoresis (2D-DIGE) coupled with matrix-assisted laser desorption/ionization mass (MALDI TOF/TOF) spectrometry. We found that 28 proteins differed in abundance between fresh and frozen extracellular fluid.
2022-10-13 | PXD034853 | Pride
Project description:Cryoinjury and protein changes are a consequence of cryopreservation and may have a negative impact on sperm quality regarding motility, viability and fertilizing ability. However, potential proteomic changes in rabbit semen throughout the cryopreservation process have never been investigated previously. The aim of the present study was to compare the whole proteome of fresh and cryopreserved rabbit spermatozoa. Comparative analysis and identification of proteins were performed using 2-dimensional difference in-gel electrophoresis (2D-DIGE) coupled with matrix-assisted laser desorption/ionization mass (MALDI TOF/TOF) spectrometry. We found that 108 proteins differed in abundance in spermatozoa between fresh and frozen semen. Most of these proteins was involved in pathways related to energy metabolism and protein quality control under stress conditions, reproductive processes and mechanisms of cell death/survival regulation, resulting in a significant decrease of motility and viability of post-thawing rabbit sperm and its potential fertilizing ability.
2022-10-14 | PXD034832 | Pride
Project description:Cryopreservation process deregulates the expression of pathways important for fertility in human spermatozoa Study design, size, duration (max. 75 words): Semen samples were obtained from 20 normospermic men from April to May 2022. Each sample was divided in two aliquots. From one aliquot total RNA was immediately extracted. The second aliquot was slowly cryopreserved and after a week of storage in liquid nitrogen total RNA was extracted. A total of 13 paired RNA samples passed quality control and were analyzed after randomization into 4 pools, each of 6 patients. Participants/materials, setting, methods (max. 75 words): The men who participated in this study had a median age of 35.0 years (range: 29.0-46.0). RNA was extracted by miRNeasy Micro Kit (Qiagen), analyzed by High Sensitivity RNA on 2200 Tape Station system (Agilent), amplified by Ovation Pico WTA SystemV2 (Nugen), labeled and hybridized on GE 4x44K v2 microarrays (Agilent). The paired Significance Analysis of Microarray (SAM) was performed using the limma, hgug4112a.db and samr packages in R/BioConductor. Main results and the role of chance (max. 200 words): The expression profiles of cryopreserved sperm significantly differed from those of fresh sperm in 219 down-regulated and 28 up-regulated unique transcripts. The gene ontology analysis disclosed that cryopreservation downregulates genes involved in sperm motility by the mitochondria function (CABS1, SPATA19) and the correct organization of the sperm midpiece and flagellum cytoskeleton (ACTL7A, AKAP4, C9ORF24, CAPZB, CCIN, IQCG, ODF2, SPATA6, SPATA6L, TBC1D21), in fertilization (ACTL7A, AKAP4, PLCZ1, PRSS37, TUBGCP3), early embryo development (AGT, CLMN, DCC, DHRS3, EGFLAM, FAM20A, FREM1, GPI, HEMGN, KRTDAP, IFT172, MICAL2, MLF1, NF2, PGRMC2, PHACTR1, POPDC3, RO60, ROBO1, RORA, SGCA, SPRR2D, TCF4, TNC, TNNI3), oxidant detoxification (TXNDC29) and DNA repair (BRIP1, ERCC6, TRIP12), calcium ion binding and homeostasis (AMY1C, ANO1, ANO2, ASGR1, CABS1, CPNE9, FREM1, KCNIP2, ITPR3, PKD2L1, PLCZ1, SELENOK, SYN3, TNNI3). Upregulated genes were enriched in pathways related to the negative regulation of DNA damage response (ALOX15B, CD74, RPL10, SNAI1, THBS1), cellular response to calcium ion (ALOX15B, FOSB, HLA-DQB1, S100A4, THBS1), regulation of transcription (CD74, FOSB, HLA-DRB1, MAMLD1, RASD1, RPL10, SNAI1, TCF15, TLE1), protein stabilization (CD74), protein ubiquitination (BAG1, DCAF6, ERCC6, HERPUD2, KLHL7, MARCHF8, NF2, PSMA6, RNF133, SERGEF, TRIP12, UBE2DNL, UBL3, UBQLN3, ZNRF3) and ubiquitin protein ligase binding (GPI, HSPA1L, PRKAR2A, STX8). The storage time of cryopreserved human spermatozoa does not affect pathways involved in fertility. Study design, size, duration This study included 24 normospermic men after their written informed consent, of whom 13 had cryostored semen for a short-time (1 week) and 11 had cryostored semen for a long-time (median 9 years, range 7-10). RNA was extracted from each frozen-thawed sperm sample and randomized into pools (4 pools for short-time samples and 3 pools for long-time samples), each of 5-6 patients. Wide-transcriptome comparison of short- and long-time pools were analyzed. Participants/materials, setting, methods The study population had a median age of 34.5 years (range: 22-46), without significant difference between the two groups (p-value = 0.147). RNA was extracted by miRNeasy Micro Kit (Qiagen), analyzed by High Sensitivity RNA on 2200 Tape Station system (Agilent), amplified by Ovation Pico WTA SystemV2 (Nugen), labeled and hybridized on GE 4x44K v2 microarrays (Agilent). The unpaired Significance Analysis of Microarray (SAM) was performed using the limma, hgug4112a.db and samr packages in R/BioConductor. Main results and the role of chance Comparison of gene expression profiles between thawed sperm after a short or long storage period in liquid nitrogen identified 5 unique transcripts significantly upregulated in the latter group (DCP1A, FAM217A, HUWE1, MSI2, PCSKIN). Functional annotation enrichment has shown that the dysregulated transcripts are involved in methylation and protein binding. In particular, HUWE1 encodes an E3 ubiquitin protein ligase required for protein ubiquitination. The protein encoded by DCP1A is a decapping enzyme, necessary for the degradation of mRNAs. The RNA binding protein MSI2 regulates the expression of target mRNAs at the translation level. Interestingly, HUWE1 serves as a central node in cellular stress responses, cell growth and death, signal transduction, etc. During early embryonic development, siRNA-mediated knockdown of HUWE1 induced apoptotic cell death and inhibited normal embryonic development. Consistent with this finding, decreased HUWE1 staining in human embryos was found to be associated with poor embryo quality, suggesting that HUWE1 plays important roles in promoting embryonic development. DCP1A is also essential for embryonic development. Overall, no critical pathways involved in fertility are dysregulated during storage of sperm in liquid nitrogen and upregulation of genes important for early embryonic development may compensate for sperm cryoinjury.
2024-04-09 | GSE225320 | GEO
Project description:BackgroundThe purpose of this study was to assess variability in cell composition and cell-specific gene expression in the skin of patients with localized scleroderma (LS) utilizing CryoStor® CS10 in comparison to RPMI to produce adequate preservation of tissue samples and cell types of interest for use in large-scale multi-institutional collaborations studying localized scleroderma and other skin disorders.MethodsWe performed single-cell RNA sequencing on paired skin biopsy specimens from 3 patients with LS. Each patient with one sample cryopreserved in CryoStor® CS10 and one fresh in RPMI media using 10× Genomics sequencing.ResultsLevels of cell viability and yield were comparable between CryoStor® CS10 (frozen) and RPMI (fresh) preserved cells. Furthermore, gene expression between preservation methods was collectively significantly correlated and conserved across all 18 identified cell cluster populations.ConclusionComparable cell population and transcript expression yields between CryoStor® CS10 and RPMI preserved cells support the utilization of cryopreserved skin tissue in single-cell analysis. This suggests that employing standardized cryopreservation protocols for the skin tissue will help facilitate multi-site collaborations looking to identify mechanisms of disease in disorders characterized by cutaneous pathology.
| S-EPMC7654179 | biostudies-literature