Genetic Engineering of Immune Evasive Stem Cell-Derived Islets.

Genome editing has the potential to revolutionize many investigative and therapeutic strategies in biology and medicine. In the field of regenerative medicine, one of the leading applications of genome engineering technology is the generation of immune evasive pluripotent stem cell-derived somatic cells for transplantation. In particular, as more functional and therapeutically relevant human pluripotent stem cell-derived islets (SCDI) are produced in many labs and studied in clinical trials, there is keen interest in studying the immunogenicity of these cells and modulating allogeneic and autoimmune immune responses for therapeutic benefit. Significant experimental work has already suggested that elimination of Human Leukocytes Antigen (HLA) expression and overexpression of immunomodulatory genes can impact survival of a variety of pluripotent stem cell-derived somatic cell types. Limited work published to date focuses on stem cell-derived islets and work in a number of labs is ongoing. Rapid progress is occurring in the genome editing of human pluripotent stem cells and their progeny focused on evading destruction by the immune system in transplantation models, and while much research is still needed, there is no doubt the combined technologies of genome editing and stem cell therapy will profoundly impact transplantation medicine in the future.

Analysis of pancreatic extracellular matrix protein post-translational modifications via electrostatic repulsion-hydrophilic interaction chromatography coupled with mass spectrometry.

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.

Mimicking nature-made beta cells: recent advances towards stem cell-derived 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 ß-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 ß cell differentiation and transplantation will be key to bring this therapy to the clinic. RECENT FINDINGS: The ability to generate stem cell-derived ß-like cells capable of dynamic glucose-responsive insulin secretion, as well as ß-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 ß 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.

Myeloid cells require gp130 signaling for protective anti-inflammatory functions during sepsis.

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.

The Nexus of Stem Cell-Derived Beta-Cells and Genome Engineering.

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.

miR-142-3p prevents macrophage differentiation during cancer-induced myelopoiesis.

Tumor 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/EBPß transcription factor, promoting macrophage generation. miR-142-3p downregulated gp130 by canonical binding to its messenger RNA (mRNA) 3' UTR and repressed C/EBPß 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.

Differential role of gp130-dependent STAT and Ras signalling for haematopoiesis following bone-marrow transplantation.

INTRODUCTION: Bone marrow transplantation (BMT) is a complex process regulated by different cytokines and growth factors. The pleiotropic cytokine IL-6 (Interleukin-6) and related cytokines of the same family acting on the common signal transducer gp130 are known to play a key role in bone marrow (BM) engraftment. In contrast, the exact signalling events that control IL-6/gp130-driven haematopoietic stem cell development during BMT remain unresolved. METHODS: Conditional gp130 knockout and knockin mice were used to delete gp130 expression (gp130(ΔMx)), or to selectively disrupt gp130-dependent Ras (gp130(ΔMxRas)) or STAT signalling (gp130(ΔMxSTAT)) in BM cells. BM derived from the respective strains was transplanted into irradiated wildtype hosts and repopulation of various haematopoietic lineages was monitored by flow cytometry. RESULTS: BM derived from gp130 deficient donor mice (gp130(ΔMx)) displayed a delayed engraftment, as evidenced by reduced total white blood cells (WBC), marked thrombocytopenia and anaemia in the early phase after BMT. Lineage analysis unravelled a restricted development of CD4(+) and CD8(+) T-cells, CD19(+) B-cells and CD11b(+) myeloid cells after transplantation of gp130-deficient BM grafts. To further delineate the two major gp130-induced signalling cascades, Ras-MAPK and STAT1/3-signalling respectively, we used gp130(ΔMxRas) and gp130(ΔMxSTAT) donor BM. BMT of gp130(ΔMxSTAT) cells significantly impaired engraftment of CD4(+), CD8(+), CD19(+) and CD11b(+) cells, whereas gp130(ΔMxRas) BM displayed a selective impairment in early thrombopoiesis. Importantly, gp130-STAT1/3 signalling deficiency in BM grafts severely impaired survival of transplanted mice, thus demonstrating a pivotal role for this pathway in BM graft survival and function. CONCLUSION: Our data unravel a vital function of IL-6/gp130-STAT1/3 signals for BM engraftment and haematopoiesis, as well as for host survival after transplantation. STAT1/3 and ras-dependent pathways thereby exert distinct functions on individual bone-marrow-lineages.

Glucocorticoids increase interleukin-6-dependent gene induction by interfering with the expression of the suppressor of cytokine signaling 3 feedback inhibitor.

UNLABELLED: 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.

Glucagon counteracts interleukin-6-dependent gene expression by redundant action of Epac and PKA.

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.

Hepatic acute-phase proteins control innate immune responses during infection by promoting myeloid-derived suppressor cell function.

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.

Foxl1 promotes liver repair following cholestatic injury in mice.

Cholangiocyte proliferation is one of the hallmarks of the response to cholestatic injury. We previously reported that the winged helix transcription factor Foxl1 is dramatically induced in cholangiocytes following bile duct ligation. In this study, we investigated the function of Foxl1 in the bile duct ligation model of cholestatic liver injury in Foxl1(-/-) and control mice. We found that Foxl1(-/-) livers exhibit an increase in parenchymal necrosis, significantly impaired cholangiocyte and hepatocyte proliferation, and failure to expand bile ductular mass. Wnt3a and Wnt7b expression was decreased in the livers of Foxl1(-/-) mice along with reduced expression of the beta-catenin target gene Cyclin D1 in Foxl1(-/-) cholangiocytes. These results show that Foxl1 promotes liver repair after bile-duct-ligation-induced liver injury through activation of the canonical wnt/beta-catenin pathway.

Foxa1 and Foxa2 regulate bile duct development in mice.

The forkhead box proteins A1 and A2 (Foxa1 and Foxa2) are transcription factors with critical roles in establishing the developmental competence of the foregut endoderm and in initiating liver specification. Using conditional gene ablation during a later phase of liver development, we show here that deletion of both Foxa1 and Foxa2 (Foxa1/2) in the embryonic liver caused hyperplasia of the biliary tree. Abnormal bile duct formation in Foxa1/2-deficient liver was due, at least in part, to activation of IL-6 expression, a proliferative signal for cholangiocytes. The glucocorticoid receptor is a negative regulator of IL-6 transcription; in the absence of Foxa1/2, the glucocorticoid receptor failed to bind to the IL-6 promoter, causing enhanced IL-6 expression. Thus, after liver specification, Foxa1/2 are required for normal bile duct development through prevention of excess cholangiocyte proliferation. Our data suggest that Foxa1/2 function as terminators of bile duct expansion in the adult liver through inhibition of IL-6 expression.

Foxl1 is a marker of bipotential hepatic progenitor cells in mice.

UNLABELLED: The liver contains a population of small bipotential facultative progenitor cells that reconstitute liver function when mature hepatocytes or cholangiocytes are unable to proliferate. Mesenchymal markers, including members of the forkhead transcription factor gene family, have been detected in hepatic progenitor cells. The winged helix transcription factor Foxl1 localizes to mesenchymal cells in the intestine; however, its expression in the liver has not been reported. We found that Foxl1 is expressed in rare cells in the normal liver but is dramatically induced in the livers of mice that have undergone bile duct ligation or were fed a 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-containing or choline-deficient, ethionine-supplemented diet. In addition, we employed genetic lineage tracing using a Foxl1-Cre transgenic mouse crossed with the Rosa26R lacZ reporter line to demonstrate that Foxl1-Cre-expressing cells are present within the periportal region shortly after injury. These cells give rise to both hepatocytes [marked by hepatocyte nuclear factor 4 alpha (HNF-4alpha) expression] and cholangiocytes (marked by CK19 expression), indicating that these cells are derived from Foxl1-Cre-expressing cells. Foxl1-Cre-expressing cells are distinct from hepatic stellate cells, portal fibroblasts, and myofibroblasts, although they are located in close proximity to portal fibroblasts. These results demonstrate that the early Foxl1-Cre lineage cell gives rise to both cholangiocytes and hepatocytes after liver injury and suggest the potential for progenitor-portal fibroblast cell interactions. CONCLUSION: We propose that Foxl1 is a bona fide marker of the facultative progenitor cell in the mouse liver.

Impaired male fertility and atrophy of seminiferous tubules caused by haploinsufficiency for Foxa3.

Foxa1, 2 and 3 (formerly HNF-3alpha, -beta and -gamma) constitute a sub-family of winged helix transcription factors with multiple roles in mammalian organ development. While all three Foxa mRNAs are present in endoderm derivatives including liver and pancreas, only Foxa3 is expressed in the testis. Here we demonstrate by genetic lineage tracing that Foxa3 is expressed in postmeiotic germ and interstitial Leydig cells. The germinal epithelium of Foxa3-deficient testes is characterized by a loss of germ cells secondary to an increase in germ cell apoptosis that ultimately leads to a Sertoli cell-only syndrome. Remarkably, not only the Foxa3(-/-) mice but also Foxa3(+/-) mice exhibited loss of germ cells. This cellular phenotype caused significantly reduced fertility and testis weight of both Foxa3(-/-) and Foxa3(+/-) mice. Using microarray analysis, we found a dramatic downregulation of the zinc finger protein 93 and the testicular tumor-associated paraneoplastic Ma antigen (PNMA) and increased expression of a number of genes including zinc finger protein 94 and several kallikrein 1-related peptidases which could account for at least part of the observed phenotype. In summary, we have identified Foxa3 as a transcriptional regulator with a dominant phenotype in germ cell maintenance and suggest FOXA3 as a potential candidate gene for subfertility in man.

Foxl1 is a mesenchymal Modifier of Min in carcinogenesis of stomach and colon.

Constitutive activation of the Wnt/APC/beta-catenin pathway is a frequent initiating event in gastrointestinal carcinogenesis. Mutations in the Adenomatous Polyposis Coli (APC) gene up-regulate Wnt signaling by stabilizing beta-catenin and causing activation of targets important in proliferation control. Here we show that loss of the mesenchymal transcription factor Foxl1 leads to a marked increase in tumor multiplicity in the colon of Apc(Min) mice. Apc(Min/+);Foxl1-/- mice also develop gastric tumors not observed in Apc(Min) mice. These effects are caused by earlier tumor initiation due to accelerated loss of heterozygosity (LOH) at the Apc locus. Foxl1 is the first mesenchymal Modifier of Min and plays a key role in gastrointestinal tumorigenesis.

Cdx2 ectopic expression induces gastric intestinal metaplasia in transgenic mice.

BACKGROUND & AIMS: Intestinal-type gastric cancer is often preceded by intestinal metaplasia in humans. The genetic events responsible for the transdifferentiation that occurs in intestinal metaplasia are not well understood. Cdx2, a transcription factor whose expression is normally limited to the intestine, has been detected in gastric intestinal metaplasia. Cdx2 induces differentiation of intestinal epithelial cells in vitro; therefore, we sought to establish whether a causal relationship exists between Cdx2 activation and intestinal metaplasia. METHODS: Cdx2 expression was directed to the gastric mucosa in transgenic mice using cis-regulatory elements of Foxa3 (Hnf3gamma). Transgenic mice were analyzed for histologic and gene expression changes. RESULTS: Histologic examination of the gastric mucosa of the Foxa3/Cdx2 mice revealed the presence of alcian blue-positive intestinal-type goblet cells, a hallmark of intestinal metaplasia. In addition, Cdx2 induced the expression of intestine-specific genes. CONCLUSIONS: Gastric expression of Cdx2 alone was sufficient to induce intestinal metaplasia in mice. These mice represent a powerful tool to investigate the molecular mechanisms that promote intestinal metaplasia. Moreover, as gastric cancer in humans is often preceded by intestinal metaplasia, the phenotype described here strongly suggests involvement of Cdx2 in the initiation of the process leading to intestinal neoplasia of the gastric mucosa.