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Gene expression profile at single cell level of stromal cells from fresh and cryopreserved tonsils and lymph node

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE224661 | GEO | 2023/05/16

REPOSITORIES: GEO

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Project description:Study question: Does storage time impact on transcriptome of slowly frozen cryopreserved human metaphase II (MII) oocytes? Summary answer: For the first time, we demonstrate that the length of cryostorage has no effect on the gene expression profile of human metaphase II oocytes. What is known already: Oocyte cryopreservation is a largely-used technique in IVF for storage of surplus oocytes, as well as for fertility preservation (i.e., women undergoing gonadotoxic therapies) and oocyte donation programs. Although it is known that cryopreservation negatively impacts on oocyte physiology and it is associated with decrease of transcripts, no experimental data about the effect of storage time on the oocyte molecular profile are available to date. Study design, size, duration: This study included 27 women undergoing IVF treatment, < 38 years aged, without any ovarian pathology. Surplus MII oocytes were donated after written informed consent. A total of 31 non-cryopreserved oocytes and 68 surviving slow-frozen/rapid-thawed oocytes (32 oocytes cryostored for 3 years and 36 cryostored for 6 years) were analyzed. Participants/materials, setting, methods: Pools of ?10 oocytes for each group were prepared. Total RNA was extracted from each pool, amplified, labeled and hybridized on 4x44K v2 microarrays (Agilent). Analyses were performed by R v3.1.3 software using the limma package v3.22.7. Main results and the role of chance: Comparison of gene expression profiles between surviving thawed oocytes after 3 and 6 years of storage in liquid nitrogen found no differently expressed genes. The expression profiles of cryopreserved MII oocytes significantly differed from those of non-cryopreserved oocytes in 107 probe sets corresponding to 73 down-regulated and 29 up-regulated unique transcripts. Gene Ontology analysis by DAVID bioinformatics resource disclosed that cryopreservation deregulates genes involved in oocyte function and early embryo development, such as chromosome organization, RNA splicing and processing, cell cycle process, response to DNA damage stimulus, cellular response to stress and DNA repair, calcium ion binding, malate dehydrogenase activity, mitochondrial membrane respiratory chain. Among the probes significantly up-regulated in cryopreserved oocytes, 2 corresponded to ovary-specific expressed large intergenic noncoding (linc)RNAs. This study included 27 women undergoing IVF treatment, < 38 years aged, without any ovarian pathology. Surplus MII oocytes were donated after written informed consent. A total of 31 non-cryopreserved oocytes and 68 surviving slow-frozen/rapid-thawed oocytes (32 oocytes cryostored for 3 years and 36 cryostored for 6 years) were analyzed.

Project description:Study question: Does storage time impact on transcriptome of slowly frozen cryopreserved human metaphase II (MII) oocytes? Summary answer: For the first time, we demonstrate that the length of cryostorage has no effect on the gene expression profile of human metaphase II oocytes. What is known already: Oocyte cryopreservation is a largely-used technique in IVF for storage of surplus oocytes, as well as for fertility preservation (i.e., women undergoing gonadotoxic therapies) and oocyte donation programs. Although it is known that cryopreservation negatively impacts on oocyte physiology and it is associated with decrease of transcripts, no experimental data about the effect of storage time on the oocyte molecular profile are available to date. Study design, size, duration: This study included 27 women undergoing IVF treatment, < 38 years aged, without any ovarian pathology. Surplus MII oocytes were donated after written informed consent. A total of 31 non-cryopreserved oocytes and 68 surviving slow-frozen/rapid-thawed oocytes (32 oocytes cryostored for 3 years and 36 cryostored for 6 years) were analyzed. Participants/materials, setting, methods: Pools of ≈10 oocytes for each group were prepared. Total RNA was extracted from each pool, amplified, labeled and hybridized on 4x44K v2 microarrays (Agilent). Analyses were performed by R v3.1.3 software using the limma package v3.22.7. Main results and the role of chance: Comparison of gene expression profiles between surviving thawed oocytes after 3 and 6 years of storage in liquid nitrogen found no differently expressed genes. The expression profiles of cryopreserved MII oocytes significantly differed from those of non-cryopreserved oocytes in 107 probe sets corresponding to 73 down-regulated and 29 up-regulated unique transcripts. Gene Ontology analysis by DAVID bioinformatics resource disclosed that cryopreservation deregulates genes involved in oocyte function and early embryo development, such as chromosome organization, RNA splicing and processing, cell cycle process, response to DNA damage stimulus, cellular response to stress and DNA repair, calcium ion binding, malate dehydrogenase activity, mitochondrial membrane respiratory chain. Among the probes significantly up-regulated in cryopreserved oocytes, 2 corresponded to ovary-specific expressed large intergenic noncoding (linc)RNAs.

Project description:Isolated human hepatocytes are used in all facets of liver research, from the clinical management of liver failure to in vitro studies of drug disposition. Cryopreservation provides a reliable source of hepatocytes, but the associated cellular stress causes a highly variable and heterogeneous loss of a differentiated phenotype, manifested by a decreased ability to form cell-matrix and cell-cell interactions. We reasoned that this problem could be mitigated at the post-thawing stage, which would increase the availability of well-functioning human hepatocytes. We applied quantitative global proteomics to analyze the differences between attached and non-attached fractions of cryopreserved human hepatocyte batches. Hepatocytes that were unable to attach to a collagen matrix showed many signs of cellular stress, including a glycolytic phenotype and activation of the heat shock response, with increased apoptosis activation as the ultimate consequence. Further analysis of the activated stress pathways revealed an increase in early apoptosis immediately after thawing, hinting at the possibility of stress reversal. Therefore, we transiently treated the cells with compounds aimed at decreasing cellular stress via different mechanisms. We found that brief exposure to the pan-caspase apoptosis inhibitor Z-VAD-FMK restored the ability to attach to collagen, and promoted a differentiated morphology with increased metabolic function. Further, Z-VAD-FMK treatment did not alter hepatocyte protein expression, suggesting that these ‘rescued’ cells would be suitable for applications where hepatocytes of high quality are required. Conclusion: Cryopreserved human hepatocytes are affected by considerable amounts of cellular stress, often resulting in loss of differentiation with a decreased ability to form cell-matrix and cell-cell interactions. This can be alleviated by brief apoptosis inhibition post-thawing, substantially improving key morphological and functional properties of these cells.

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Gene expression profile at single cell level of stromal cells from fresh and cryopreserved tonsils and lymph node

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