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The pancreas is a vital organ with digestive and endocrine roles, and diseases of the pancreas affect millions of people yearly. A better understanding of the pancreas proteome and its dynamic post-translational modifications (PTMs) is necessary to engineer higher fidelity tissue analogues for use in transplantation. The extracellular matrix (ECM) has major roles in binding and signaling essential to the viability of insulin-producing islets of Langerhans. To characterize PTMs in the pancreas, native and decellularized tissues from four donors were analyzed. N-Glycosylated and phosphorylated peptides were simultaneously enriched via electrostatic repulsion-hydrophilic interaction chromatography and analyzed with mass spectrometry, maximizing PTM information from one workflow. A modified surfactant and chaotropic agent assisted sequential extraction/on-pellet digestion was used to maximize solubility of the ECM. The analysis resulted in the confident identification of 3650 proteins, including 517 N-glycoproteins and 148 phosphoproteins. We identified 214 ECM proteins, of which 99 were N-glycosylated, 18 were phosphorylated, and 9 were found to have both modifications. Collagens, a major component of the ECM, were the most highly glycosylated of the ECM proteins and several were also heavily phosphorylated, raising the possibility of structural and thus functional changes resulting from these modifications. To our knowledge, this work represents the first characterization of PTMs in pancreatic ECM proteins. This work provides a basal profile of PTMs in the healthy human pancreatic ECM, laying the foundation for future investigations to determine disease-specific changes such as in diabetes and pancreatic cancer, and potentially helping to guide the development of tissue replacement constructs. Data are available via ProteomeXchange with identifier PXD025048.

Extracellular matrix (ECM) plays a multitude of roles, including supporting cells through structural and biochemical interactions. ECM is damaged in the process of isolating human islets for clinical transplantation and basic research. A platform in which islets can be cultured in contact with natural pancreatic ECM is desirable to better understand and support islet health, and to recapitulate the native islet environment. Our study demonstrates the derivation of a practical and durable hydrogel from decellularized human pancreas that supports human islet survival and function. Islets embedded in this hydrogel show increased glucose- and KCl-stimulated insulin secretion, and improved mitochondrial function compared to islets cultured without pancreatic matrix. In extended culture, hydrogel co-culture significantly reduced levels of apoptosis compared to suspension culture and preserved controlled glucose-responsive function. Isolated islets displayed altered endocrine and non-endocrine cell arrangement compared to in situ islets; hydrogel preserved an islet architecture more similar to that observed in situ. RNA sequencing confirmed that gene expression differences between islets cultured in suspension and hydrogel largely fell within gene ontology terms related to extracellular signaling and adhesion. Natural pancreatic ECM improves the survival and physiology of isolated human islets.

Purpose of review Stem cell-derived islets are likely to be useful as a future treatment for diabetes. However, the field has been limited in the ability to generate β-like cells with both phenotypic maturation and functional glucose-stimulated insulin secretion that is similar to primary human islets. The field must also establish a reliable method of delivering the cells to patients while promoting rapid in-vivo engraftment and function. Overcoming these barriers to β cell differentiation and transplantation will be key to bring this therapy to the clinic. Recent findings The ability to generate stem cell-derived β-like cells capable of dynamic glucose-responsive insulin secretion, as well as β-like cells expressing key maturation genes has recently been demonstrated by several groups. Other groups have explored the potential of vascularized subcutaneous transplant sites, as well as endothelial cell co-transplant to support β cell survival and function following transplantation. Summary The generation of stem cell-derived islets with dynamic glucose-responsive insulin secretion has brought the field closer to clinical translation, but there is still need for improving insulin content and secretory capacity, as well as understanding the factors affecting variable consistency and heterogeneity of the islet-like clusters. Other questions remain regarding how to address safety, immunogenicity and transplantation site moving forward.

Extracellular matrix (ECM) is an important component of the pancreatic microenvironment which regulates β cell proliferation, differentiation, and insulin secretion. Protocols have recently been developed for the decellularization of the human pancreas to generate functional scaffolds and hydrogels. In this work, we characterized human pancreatic ECM composition before and after decellularization using isobaric dimethylated leucine (DiLeu) labeling for relative quantification of ECM proteins. A novel correction factor was employed in the study to eliminate the bias introduced during sample preparation. In comparison to the commonly employed sample preparation methods (urea and FASP) for proteomic analysis, a recently developed surfactant and chaotropic agent assisted sequential extraction/on pellet digestion (SCAD) protocol has provided an improved strategy for ECM protein extraction of human pancreatic ECM matrix. The quantitative proteomic results revealed the preservation of matrisome proteins while most of the cellular proteins were removed. This method was compared with a well-established label-free quantification (LFQ) approach which rendered similar expressions of different categories of proteins (collagens, ECM glycoproteins, proteoglycans, etc.). The distinct expression of ECM proteins was quantified comparing adult and fetal pancreas ECM, shedding light on the correlation between matrix composition and postnatal β cell maturation. Despite the distinct profiles of different subcategories in the native pancreas, the distribution of matrisome proteins exhibited similar trends after the decellularization process. Our method generated a large data set of matrisome proteins from a single tissue type. These results provide valuable insight into the possibilities of constructing a bioengineered pancreas. It may also facilitate better understanding of the potential roles that matrisome proteins play in postnatal β cell maturation.

The extracellular matrix (ECM) is unique to each tissue and capable of guiding cell differentiation, migration, morphology, and function. The ECM proteome of different developmental stages has not been systematically studied in the human pancreas. In this study, we apply mass spectrometry-based quantitative proteomics strategies using N,N-dimethyl leucine isobaric tags to delineate proteome-wide and ECM-specific alterations in four age groups: fetal (18-20 weeks gestation), juvenile (5-16 years old), young adults (21-29 years old) and older adults (50-61 years old). We identify 3,523 proteins including 185 ECM proteins and quantify 117 of them. We detect previously unknown proteome and matrisome features during pancreas development and maturation. We also visualize specific ECM proteins of interest using immunofluorescent staining and investigate changes in ECM localization within islet or acinar compartments. This comprehensive proteomics analysis contributes to an improved understanding of the critical roles that ECM plays throughout human pancreas development and maturation., The pancreatic extracellular matrix (ECM) is known to differ between species, age groups and physiological states, but its compositional changes throughout human life are not well understood. Here, the authors study how the proteome of pancreatic ECM changes during human development and maturation.

Purpose of review: Pancreas and islet transplantation provide proof-of-principle that replacing deficient β cell function in patients with insulin-dependent diabetes can restore normoglycemia. The dearth of suitable cadaver organs and need for lifelong immunosuppression, however, limit this therapy to only a few thousand individuals. Therapeutic strategies involving human pluripotent stem cell-derived insulin-producing cells (IPCs) and tissues have long been envisioned as a potential solution to this clinical problem. Recent technical advances in deriving IPCs with improved functionality are bringing this vision closer to reality. Transplanting endocrine progenitors contained in macroencapsulation devices is a strategy that has already entered clinical trials. Many questions still need to be answered related to cellular properties, composition and immunogenicity of the graft and how to protect the graft from immune destruction, as well as transplant site and the role of other tissue components such as extracellular matrix. Exciting new technologies such as tissue engineering, genome editing, and interspecies organogenesis are already impacting this field and will likely contribute to the ultimate delivery of stem cell-derived IPCs to patients. This chapter will review the current state of the preclinical technology and review results of a recent industry-sponsored first-in-human clinical trial.

Extracellular matrix (ECM) plays an important developmental role by regulating cell behaviour through structural and biochemical stimulation. Tissue-specific ECM, attained through decellularization, has been proposed in several strategies for tissue and organ replacement. Decellularization of animal pancreata has been reported, but the same methods applied to human pancreas are less effective due to higher lipid content. Moreover, ECM-derived hydrogels can be obtained from many decellularized tissues, but methods have not been reported to obtain human pancreas-derived hydrogel. Using novel decellularization methods with human pancreas we produced an acellular, 3D biological scaffold (hP-ECM) and hydrogel (hP-HG) amenable to tissue culture, transplantation and proteomic applications. The inclusion of a homogenization step in the decellularization protocol significantly improved lipid removal and gelation capability of the resulting ECM, which was capable of gelation at 37 °C in vitro and in vivo, and is cytocompatible with a variety of cell types and islet-like tissues in vitro. Overall, this study demonstrates the characterisation of a novel protocol for the decellularization and delipidization of human pancreatic tissue for the production of acellular ECM and ECM hydrogel suitable for cell culture and transplantation applications. We also report a list of 120 proteins present within the human pancreatic matrisome.

Pancreatic steatosis is thought to be a negative risk factor for pancreas transplantation outcomes. Despite considering donor body mass index (BMI) and the visualization of intercalated fat as indicators of donor pancreas lipid content, transplant surgeons do not use a quantitative method to directly measure steatosis when deciding to transplant a pancreas. In this study, we utilized non-diabetic human pancreata donated for research to measure the pancreatic and islet-specific lipid content in order to determine which clinical markers correlate best with lipid content. Interestingly, we found that BMI and age correlate with increased pancreatic lipid content (Panc-LC) in men, but not women. Our findings further suggest that total Panc-LC correlates with an increase in islet lipid content (Islet-LC) for both men and women. We noted that pancreata donated from individuals with a history of hypertension have increased Panc-LC independent of donor BMI or sex. Moreover, we identify hypertension as a risk factor for reduced islet function following islet isolation. Together, our findings emphasize differences in pancreas graft quality related to pancreatic and islet lipid content, which may not be predicted by assessing BMI alone, but may be influenced by a donor history of hypertension.

Sepsis represents a major health problem worldwide because of high mortality rates and cost-intensive therapy. Immunomodulatory strategies as a means of controlling overshooting inflammatory responses during sepsis have thus far not been effective, and there is a general paucity of new therapies. Regulatory immune cells have been shown to play important roles in limiting systemic inflammation. However, the signals inducing a regulatory phenotype in myeloid cells during infection are unknown. Here, we report that myeloid cell-intrinsic glycoprotein 130 (gp130) signals constitute a critical element for immune homeostasis during polymicrobial sepsis. We identify an essential role for gp130 signaling in myeloid cells during M2 macrophage polarization in vitro and in vivo. Myeloid cell-specific deletion of gp130 signaling leads to a defective M2 macrophage polarization followed by exacerbated inflammatory responses and increased mortality during sepsis. These data provide new insights into the molecular basis of M1 and M2 phenotypic dichotomy and identify gp130 as a key regulator of immune homeostasis during sepsis. Our study highlights the Janus-faced role of IL-6 family cytokines during inflammation, which may explain the failure of IL-6-targeted anti-inflammatory approaches in the treatment of sepsis.-Sackett, S. D., Otto, T., Mohs, A., Sander, L. E., Strauch, S., Streetz, K. L., Kroy, D. C., Trautwein, C. Myeloid cells require gp130 signaling for protective anti-inflammatory functions during sepsis.

3- and 4- hydroxyprolines (HyP) are regioisomers that play different roles in various species and organs. Despite their distinct functions inside cells, they are generally considered indistinguishable using mass spectrometry due to their identical masses. Here, we demonstrate, for the first time, that characteristic w ions can be produced by electron-transfer/higher energy collision dissociation (EThcD) dual fragmentation technique to confidently discriminate 3-HyP/ 4-HyP isomers. An integrated and high throughput strategy was developed which combined online LC separation with EThcD for large-scale differentiation of 3-HyP/4-HyP in complex samples. An automated algorithm was developed for charge state dependent characterization of 3-HyP/ 4-HyP isomers. Using this combined discrimination approach, we identified 108 3-HyP sites and 530 4-HyP sites from decellularized pancreas, allowing more than fivefold increase of both 3-HyP and 4-HyP identifications compared to previous reports. This approach outperformed ETD and HCD in the analysis of HyP-containing peptides with unique capacity to generate w ions for HyP discrimination, improved fragmentation of precursor ions, as well as unambiguous localization of modifications. A high content of 3-HyP was observed in the C-terminal (GPP)(n) domain of human CO1A1, which was previously only identified in vertebrate fibrillar collagens from tendon. Unexpectedly, some unusual HyP sites at Xaa position in Gly-HyP-Ala, Gly-HyP-Val, Gly-HyP-Gln, Gly-HyP-Ser and Gly-HyP-Arg were also confirmed to be 3-hydroxylated, whose functions and enzymes are yet to be discovered. Overall, this novel discrimination strategy can be readily implemented into de novo sequencing or other proteomic search engines.

Diabetes, type 1 and type 2 (T1D and T2D), are diseases of epidemic proportions, which are complicated and defined by genetics, epigenetics, environment, and lifestyle choices. Current therapies consist of whole pancreas or islet transplantation. However, these approaches require life-time immunosuppression, and are compounded by the paucity of available donors. Pluripotent stem cells have advanced research in the fields of stem cell biology, drug development, disease modeling, and regenerative medicine, and importantly allows for the interrogation of therapeutic interventions. Recent developments in beta-cell differentiation and genomic modifications are now propelling investigations into the mechanisms behind beta-cell failure and autoimmunity, and offer new strategies for reducing the propensity for immunogenicity. This review discusses the derivation of endocrine lineage cells from human pluripotent stem cells for the treatment of diabetes, and how the editing or manipulation of their genomes can transcend many of the remaining challenges of stem cell technologies, leading to superior transplantation and diabetes drug discovery platforms.

IL-6/gp130 dependent signaling plays an important role in modulating inflammation in acute and chronic diseases. The course of Concanavalin A- (Con A) induced hepatitis can be modulated by different immune-mediated mechanisms. IL-6/gp130-dependent signaling has been shown to be protective in hepatocytes. However, the role of this pathway in myeloid cells has not yet been studied. In our present study we used macrophage/neutrophil-specific gp130 knockout (gp130(ΔLys), KO) animals and analyzed its relevance in modulating Con A-induced hepatitis. Additionally, we performed in vitro studies with gp130(ΔLys)-macrophages. We demonstrate that gp130(ΔLys) animals are more susceptible to Con A-induced hepatitis. This is reflected by higher transaminases, higher lethality and more severe liver injury as shown by histological staining. Using flow cytometry analysis we further could show that increased liver injury of gp130(ΔLys) animals is associated with a stronger infiltration of CD11b/F4/80 double-positive cells compared to wild-type (gp130(flox/flox), WT) controls. To further characterize our observations we studied thioglycolate-elicited peritoneal macrophages from gp130(ΔLys) animals. Interestingly, the LPS-dependent IL-6 release in gp130(ΔLys) macrophages is significantly reduced (p

Glucocorticoids are known to be potent regulators of inflammation and have been used pharmacologically against inflammatory, immune, and lymphoproliferative diseases for more than 50 years. Due to their possible and well-documented side effects, it is crucial to understand the molecular mechanisms and targets of glucocorticoid action in detail. Several modes of action have been discussed; nevertheless, none of them fully explain all the functions of glucocorticoids. Therefore, we analyzed the cross-talk between glucocorticoids and interleukin-6 (IL-6) in the liver. IL-6 exerts pro-inflammatory as well as anti-inflammatory properties and is a main inducer of the acute-phase response. The balance between the proinflammatory and anti-inflammatory activities of IL-6 is tightly regulated by suppressor of cytokine signaling 3 (SOCS3), a well-known feedback inhibitor of IL-6 signaling. Here, it is demonstrated that glucocorticoids enhance IL-6–dependent γ-fibrinogen expression. Studying of the underlying mechanism revealed prolonged activation of signal transducer and activator of transcription 3 (STAT3) caused by down-regulation of SOCS3 protein expression. Consequently, in SOCS3-deficient cells glucocorticoids do not affect IL-6–induced signal transduction. Moreover, in hepatocytes lacking the SOCS3 recruiting motif within gp130, IL-6–dependent γ-fibrinogen expression is not influenced by glucocorticoid treatment. Conclusion: Glucocorticoids interfere with IL-6–induced expression of the feedback inhibitor SOCS3, thereby leading to enhanced expression of acute-phase genes in hepatocytes. This mechanism contributes to the explanation of how glucocorticoids affect inflammation and acute-phase gene induction. (HEPATOLOGY 2012;55:256–266)

Inflammation is the biological response to injurious stimuli. In the initial phase of the inflammatory process, interleukin-6 (IL-6) is the main inducer of acute phase protein expression in the liver. A prolonged acute phase response is characterised by a disturbed glucose homeostasis and elevated levels of IL-6, insulin, and counterregulatory hormones such as glucagon. Several studies deal with the impact of IL-6 on glucagon-dependent gene expression. In contrast, only very little is known about the influence of G-protein-coupled receptors on IL-6 signalling. Therefore, the aim of this study is to elucidate the regulation of IL-6-induced gene expression by glucagon. We could reveal a novel mechanism of negative regulation of IL-6-induced MAP kinase activation by glucagon in primary murine hepatocytes. IL-6-dependent induction of the ERK-dependent target gene Tfpi2, coding for a Kunitz-type serine protease inhibitor, was strongly down-regulated by glucagon treatment. Studying the underlying mechanism revealed a redundant action of the signalling molecules exchange protein activated by cyclic AMP (Epac) and protein kinase A. The metabolic hormone glucagon interferes in IL-6-induced gene expression. This observation is indicative for a regulatory role of G-protein-coupled receptors in the IL-6-dependent inflammatory response.

Acute-phase proteins (APPs) are an evolutionarily conserved family of proteins produced mainly in the liver in response to infection and inflammation. Despite vast pro- and antiinflammatory properties ascribed to individual APPs, their collective function during infections remains poorly defined. Using a mouse model of polymicrobial sepsis, we show that abrogation of APP production by hepatocyte-specific gp130 deletion, the signaling receptor shared by IL-6 family cytokines, strongly increased mortality despite normal bacterial clearance. Hepatic gp130 signaling through STAT3 was required to control systemic inflammation. Notably, hepatic gp130–STAT3 activation was also essential for mobilization and tissue accumulation of myeloid-derived suppressor cells (MDSCs), a cell population mainly known for antiinflammatory properties in cancer. MDSCs were critical to regulate innate inflammation, and their adoptive transfer efficiently protected gp130-deficient mice from sepsis-associated mortality. The hepatic APPs serum amyloid A and Cxcl1/KC cooperatively promoted MDSC mobilization, accumulation, and survival, and reversed dysregulated inflammation and restored survival of gp130-deficient mice. Thus, gp130-dependent communication between the liver and MDSCs through APPs controls inflammatory responses during infection.

Tissues derived from induced pluripotent stem cells (iPSCs) are a promising source of cells for building various regenerative medicine therapies; from simply transplanting cells to reseeding decellularized organs to reconstructing multicellular tissues. Although reprogramming strategies for producing iPSCs have improved, the clinical use of iPSCs is limited by the presence of unique human leukocyte antigen (HLA) genes, the main immunologic barrier to transplantation. In order to overcome the immunological hurdles associated with allogeneic tissues and organs, the generation of patient-histocompatible iPSCs (autologous or HLA-matched cells) provides an attractive platform for personalized medicine. However, concerns have been raised as to the fitness, safety and immunogenicity of iPSC derivatives because of variable differentiation potential of different lines and the identification of genetic and epigenetic aberrations that can occur during the reprogramming process. In addition, significant cost and regulatory barriers may deter commercialization of patient specific therapies in the short-term. Nonetheless, recent studies provide some evidence of immunological benefit for using autologous iPSCs. Yet, more studies are needed to evaluate the immunogenicity of various autologous and allogeneic human iPSC-derived cell types as well as test various methods to abrogate rejection. Here, we present perspectives of using allogeneic vs. autologous iPSCs for transplantation therapies and the advantages and disadvantages of each related to differentiation potential, immunogenicity, genetic stability and tumorigenicity. We also review the current literature on the immunogenicity of syngeneic iPSCs and discuss evidence that questions the feasibility of HLA-matched iPSC banks. Finally, we will discuss emerging methods of abrogating or reducing host immune responses to PSC derivatives.

SummaryTumor progression is accompanied by an altered myelopoiesis causing the accumulation of immunosuppressive cells. Here, we showed that miR-142-3p downregulation promoted macrophage differentiation and determined the acquisition of their immunosuppressive function in tumor. Tumor-released cytokines signaling through gp130, the common subunit of the interleukin-6 cytokine receptor family, induced the LAP∗ isoform of C/EBPβ transcription factor, promoting macrophage generation. miR-142-3p downregulated gp130 by canonical binding to its messenger RNA (mRNA) 3' UTR and repressed C/EBPβ LAP∗ by noncanonical binding to its 5' mRNA coding sequence. Enforced miR expression impaired macrophage differentiation both in vitro and in vivo. Mice constitutively expressing miR-142-3p in the bone marrow showed a marked increase in survival following immunotherapy with tumor-specific T lymphocytes. By modulating a specific miR in bone marrow precursors, we thus demonstrated the feasibility of altering tumor-induced macrophage differentiation as a potent tool to improve the efficacy of cancer immunotherapy.

PLoS one 7(6), e39728 (2012). doi:10.1371/journal.pone.0039728, Published by PLoS [u.a.], Lawrence, Kan.