Transcriptional profiling of stress-response in cultured porcine islets
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ABSTRACT:
ORGANISM(S): Sus scrofa
PROVIDER: GSE4744 | GEO | 2006/05/05
SECONDARY ACCESSION(S): PRJNA95687
REPOSITORIES: GEO
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Project description:An increasing demand for cell-based diabetes therapy could be met through xenotransplantation of adult porcine islets. Use of islet xenotranplantation on a large scale would require rigorous safety and quality control measures to maximize transplant success. Development of molecular tools to monitor porcine islet cellular responses to ischemic, osmotic, mechanical and oxidative stresses during islet cell processing and post-isolation culturing would aid the rational design of cytoprotective strategies aimed at improving transplant outcomes. In addition, gene expression signatures informative for islet quality could serve as an adjunct to physiological testing to establish the suitability of islet products for transplantation. Nine adult Landrace sows (2-3.5 y old, 249±27 kg) were sacrificed, the pancreases were dissected, and islet cells isolated as previously described [8]. Islet preparation purity was assessed by light microscopy after staining with diphenylthiocarbazone and ranged between 90-95% for the preparations used. Islet yield, based on islet equivalents (IEQ, number of islets standardized to 150 µm diameter), was estimated at 1930±520 per gram of pancreas tissue.Profiles of islet cells cultured under standard conditions were compared to islet cells cultured under stress conditions with elevated glucose (16.7 mM) or addition of inflammatory cytokines (IL-1ï¢, TNF-ï¡, and IFN-ï§), or both, for 48 hours.
2010-07-01 | E-GEOD-4744 | biostudies-arrayexpress
Project description:Pig-to-human xenotransplantation is rapidly approaching the clinical arena; however, it is unclear which immunomodulatory regimens will effectively control human immune responses to pig xenografts. Here, we transplant a gene-edited pig kidney into a brain-dead human recipient on pharmacologic immunosuppression and study the human immune response to the xenograft using spatial transcriptomics and single-cell RNA sequencing. Human immune cells are uncommon in the porcine kidney cortex early after xenotransplantation and consist of primarily myeloid cells. Both the porcine resident macrophages and human infiltrating macrophages express genes consistent with an alternatively activated, anti-inflammatory phenotype. No significant infiltration of human B or T cells into the porcine kidney xenograft is detectable. Altogether, these findings provide proof of concept that conventional pharmacologic immunosuppression is sufficient to restrict infiltration of human immune cells into the xenograft early after compatible pig-to-human kidney xenotransplantation.
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Project description:Progress in clinical translation of gene edited porcine kidney xenotransplantation is accelerating. However, kidney function entails more than removal of metabolic waste products and it is unknown if all endocrine functions will be faithfully reproduced by porcine kidneys in primate recipients. Seventeen cynomolgus macaques with survival >60days after life sustaining kidney xenotransplantation (LSKX) from gene edited Yucatan minipigs underwent measurement of xenograft growth and two kidney dependent endocrine pathways: renin-angiotensin-aldosterone-system (RAAS) and calcium-vitamin D-parathyroid-hormone. Clinical chemistries, plasma-renin-activity, Beta-C-terminal-telopeptide (CTx) assay, RNA-seq, single-cell RNA-seq, and serial ultrasonography were conducted and generalized linear models assessed statistical relationships. Xenografts transplanted from minipigs demonstrated only modest growth (estimated ~8.0% per year) and didn’t significantly contribute to recipient RAAS pathway. However, PTH-independent hypercalcemia and hypophosphatemia were observed and might be xenograft intrinsic suggesting close monitoring and timely intervention during the first month post-transplant. Further study of these phenotypes is warranted in designing prospective clinical trials.
2023-04-25 | GSE216033 | GEO
Project description:Progress in clinical translation of gene edited porcine kidney xenotransplantation is accelerating. However, kidney function entails more than removal of metabolic waste products and it is unknown if all endocrine functions will be faithfully reproduced by porcine kidneys in primate recipients. Seventeen cynomolgus macaques with survival >60days after life sustaining kidney xenotransplantation (LSKX) from gene edited Yucatan minipigs underwent measurement of xenograft growth and two kidney dependent endocrine pathways: renin-angiotensin-aldosterone-system (RAAS) and calcium-vitamin D-parathyroid-hormone. Clinical chemistries, plasma-renin-activity, Beta-C-terminal-telopeptide (CTx) assay, RNA-seq, single-cell RNA-seq, and serial ultrasonography were conducted and generalized linear models assessed statistical relationships. Xenografts transplanted from minipigs demonstrated only modest growth (estimated ~8.0% per year) and didn’t significantly contribute to recipient RAAS pathway. However, PTH-independent hypercalcemia and hypophosphatemia were observed and might be xenograft intrinsic suggesting close monitoring and timely intervention during the first month post-transplant. Further study of these phenotypes is warranted in designing prospective clinical trials.
2023-04-25 | GSE216029 | GEO
Project description:There is growing evidence that transplantation of cadaveric human islets is an effective therapy for type 1 diabetes. However, gauging the suitability of islet samples for clinical use remains a challenge. We hypothesized that islet quality is reflected in the expression of specific genes. Therefore, gene expression in 59 human islet preparations was analyzed and correlated with diabetes reversal after transplantation in diabetic mice. Analysis yielded 262 differentially expressed probesets, which together predict islet quality with 83% accuracy. Pathway analysis revealed that failing islet preparations activated inflammatory pathways, while functional islets showed increased regeneration pathway gene expression. Gene expression associated with apoptosis and oxygen consumption showed little overlap with each other or with the 262 probeset classifier, indicating that the three tests are measuring different aspects of islet cell biology. A subset of 36 probesets surpassed the predictive accuracy of the entire set for reversal of diabetes, and was further reduced by logistic regression to sets of 14 and 5 without losing accuracy. These genes were further validated with an independent cohort of 16 samples. We believe this limited number of gene classifiers in combination with other tests may provide complementary verification of islet quality prior to their clinical use.
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Project description:In transplantation, there is a critical need for non-invasive biomarker platforms for monitoring immunologic rejection. We hypothesized that transplanted tissues release donor specific exosomes into recipient circulation/ bodily fluids, and that the quantitation and profiling of their intra-exosomal cargoes would constitute a novel biomarker platform for monitoring rejection. We tested this hypothesis in a human into mouse xenogeneic islet transplant model, and validated the concept in clinical settings of islet and renal transplantation. In the xenogeneic model, islet transplant exosomes in recipient blood were quantified over long-term follow-up using anti-human leukocyte antigen (HLA) antibody that is only expressed on human islets (p=1.6x10-14). Transplant islet exosomes were purified using anti-HLA antibody conjugated beads and their cargoes contained bona fide islet endocrine hormone markers insulin, glucagon, and somatostatin. Rejection led to significant decrease in transplant islet exosome signal (p=4x10-15), along with distinct changes in its microRNA and proteomic profiles prior to appearance of hyperglycemia. In the clinical settings of islet (n=5) and renal (n=5) transplantation, donor exosomes with respective tissue specificity for islet β cells and renal epithelial cells were reliably characterized in recipient plasma over follow-up (up to 5 years; p=0.0001). Collectively, these findings demonstrate the biomarker potential of transplant exosome characterization for providing a non-invasive window into the conditional state of the transplant tissue.
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Project description:In transplantation, there is a critical need for non-invasive biomarker platforms for monitoring immunologic rejection. We hypothesized that transplanted tissues release donor specific exosomes into recipient circulation/ bodily fluids, and that the quantitation and profiling of their intra-exosomal cargoes would constitute a novel biomarker platform for monitoring rejection. We tested this hypothesis in a human into mouse xenogeneic islet transplant model, and validated the concept in clinical settings of islet and renal transplantation. In the xenogeneic model, islet transplant exosomes in recipient blood were quantified over long-term follow-up using anti-human leukocyte antigen (HLA) antibody that is only expressed on human islets (p=1.6x10-14). Transplant islet exosomes were purified using anti-HLA antibody conjugated beads and their cargoes contained bona fide islet endocrine hormone markers insulin, glucagon, and somatostatin. Rejection led to significant decrease in transplant islet exosome signal (p=4x10-15), along with distinct changes in its microRNA and proteomic profiles prior to appearance of hyperglycemia. In the clinical settings of islet (n=5) and renal (n=5) transplantation, donor exosomes with respective tissue specificity for islet β cells and renal epithelial cells were reliably characterized in recipient plasma over follow-up (up to 5 years; p=0.0001). Collectively, these findings demonstrate the biomarker potential of transplant exosome characterization for providing a non-invasive window into the conditional state of the transplant tissue.
2016-10-18 | GSE88844 | GEO
Project description:Gene expression patterns that form the basis for the observed variation in islet density among porcine pancreata were examined. RNA-Seq analysis was performed to investigate differential gene expression of intact porcine pancreas samples and pure cultured islet samples according to a density of islets. Most prominently upregulated genes in the islet-rich pancreas samples included GP2 and GCG. The mRNA expression of pure islet cell samples from both groups were statistically identical. This transcriptome RNA-Seq analysis showed that pancreatic islet density is associated with GP2 expression.
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Project description:The pig is a promising candidate for xenotransplantation. Understanding the differences between human and swine immune systems is critical for addressing xeno-transplant rejection and hematopoietic reconstitution. We performed single-cell RNA-seq on human and pig immune cells. High oxidative phosphorylation, HIF-1, glycolysis, and lysosome-related gene expressions in pig CD14+ monocytes were observed, whereas pig CD14+ monocytes exhibited lower levels of cytokine receptors and JAK-STAT-related genes. Pig activated CD4+T cells decreased cell adhesion and inflammation, while enriched for migration and activation processes. Porcine GNLY+CD8+T cells reduced cytotoxicity and increased proliferation compared with human GNLY+CD8+T cells. Pig CD2+CD8+γδT cells were functionally homologous to human CD2+CD4+ γδT cells. Pig CD2-CD8-γδT cells expressed genes with quiescent and precursor characteristics, while CD2-CD8+γδT cells expressed migration and memory-related molecules. Pig CD24+ and CD5+B cells are associated with inflammatory responses. Our research with integrated scRNA-seq enables a deeper understanding of the development and function of porcine immune cells.
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Project description:Type 1 diabetes (T1D) results from insulin insufficiency due to the loss or dysfunction of pancreatic beta cells following T cell-mediated autoimmune attack. Currently, the only long-term therapy is daily exogenous insulin replacement. The ideal curative approach is the durable restoration of functional islets via transplantation with or without immunosuppression or immune modulation. To date, the limiting factors impeding realization of this goal is the lack of a cost effective and limitless source of high-quality islets suitable for transplantation and the ability to provide long-term islet graft acceptance without prolonged need for deleterious immunosuppression. Ongoing clinical trials are testing the suitability of islets derived from human induced pluripotent stem cells (iPSC); however, long-term acceptance of these or any other islet graft will require a clinically effective therapeutic strategy to prevent engrafted islet destruction by pre-existing islet-antigen specific T cells. While current immunomodulatory strategies focus on autologous T regulatory (Treg) cell expansion or the use of broad immunosuppression, here we demonstrate in the NOD mouse model for T1D that autologous islet graft acceptance can be achieved by the targeted elimination of (re)-activated islet-reactive CD4+ and CD8+ T effector (Teff) cells in the initial post-transplantation period by using a short-acting, combination therapy that results in the elimination of islet-reactive Teff cells by exacerbation of their natural DNA damage response (DDR) to drive apoptosis while at the same time maintaining endogenous Treg cell function and reducing the effector function of residual islet-reactive T cells.
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