Kevin Ferreri

MESENCHYMAL STEM CELLS SUPPRESS B-CELL TERMINAL DIFFERENTIATION

OBJECTIVE Mesenchymal stem cells (MSCs) have been shown to possess immunomodulatory properties on a diverse array of immune cell lineages. However, their effect on B lymphocytes remains unclear. We investigated the effect of MSCs on B-cell modulation with a special emphasis on gene regulation mediated by MSC humoral factors. MATERIALS AND METHODS MSCs were isolated from C57BL/6 bone marrow and expanded in culture. Splenic B cells were purified using anti-CD43 antibody and immunomagnetic beads. B cells and MSCs were cocultured in separate compartments in a transwell system. For B-cell stimulation, lipopolysaccharide was used in vitro and T-dependent and T-independent antigens were used in vivo. RESULTS In MSC cocultures, lipopolysaccharide-stimulated B-cell proliferation was suppressed, CD138(+) cell percentage decreased, and the number of apoptotic CD138(+) cells decreased. In the B/MSC coculture, the IgM(+) cell percentage was higher and the IgM amount released in the medium was lower than in the control. The B-lymphocyte-induced maturation protein-1 messenger RNA expression in the coculture was suppressed throughout the 3-day culture period. Conditioned media derived from MSC cultures prevented terminal differentiation of B cells in vitro and significantly suppressed the antigen-specific immunoglobulin M and immunoglobulin G1 secretion in mice immunized with T-cell-independent as well as T-cell-dependent antigens in vivo. CONCLUSION Results indicate that humoral factor(s) released by MSCs exert a suppressive effect on the B-cell terminal differentiation. Suppression may be mediated through inhibition of B-lymphocyte-induced maturation protein-1 expression, but the nature of the factor(s) is yet to be determined.

Insulin gene expression is regulated by DNA methylation.

Background Insulin is a critical component of metabolic control, and as such, insulin gene expression has been the focus of extensive study. DNA sequences that regulate transcription of the insulin gene and the majority of regulatory factors have already been identified. However, only recently have other components of insulin gene expression been investigated, and in this study we examine the role of DNA methylation in the regulation of mouse and human insulin gene expression. Methodology/Principal Findings Genomic DNA samples from several tissues were bisulfite-treated and sequenced which revealed that cytosine-guanosine dinucleotide (CpG) sites in both the mouse Ins2 and human INS promoters are uniquely demethylated in insulin-producing pancreatic beta cells. Methylation of these CpG sites suppressed insulin promoter-driven reporter gene activity by almost 90% and specific methylation of the CpG site in the cAMP responsive element (CRE) in the promoter alone suppressed insulin promoter activity by 50%. Methylation did not directly inhibit factor binding to the CRE in vitro, but inhibited ATF2 and CREB binding in vivo and conversely increased the binding of methyl CpG binding protein 2 (MeCP2). Examination of the Ins2 gene in mouse embryonic stem cell cultures revealed that it is fully methylated and becomes demethylated as the cells differentiate into insulin-expressing cells in vitro. Conclusions/Significance Our findings suggest that insulin promoter CpG demethylation may play a crucial role in beta cell maturation and tissue-specific insulin gene expression.

Inhibition of p38 Pathway Suppresses Human Islet Production of Pro-Inflammatory Cytokines and Improves Islet Graft Function

Nonspecific inflammation is associated with primary graft nonfunction (PNF). Inflammatory islet damage is mediated at least partially by pro‐inflammatory cytokines, such as interleukin‐1β (IL‐1β) and tumor necrosis factor‐α (TNF‐α) produced by resident islet macrophages. The p38 pathway is known to be involved in cytokine production in the cells of the monocyte–macrophage lineage. Therefore, inhibition of the p38 pathway may prevent pro‐inflammatory cytokine production by resident islet macrophages and possibly reduce the incidence of PNF. Our present study has demonstrated that inhibition of the p38 pathway by a chemical p38 inhibitor, SB203580, suppresses IL‐1β and TNF‐α production in human islets exposed to lipopolysaccharide (LPS) and/or inflammatory cytokines. Although IL‐1β is predominantly produced by resident macrophages, ductal cells and islet vascular endothelial cells were found to be another cellular source of IL‐1β in isolated human islets. SB203580 also inhibited the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase‐2 (COX‐2) in the treated islets. Furthermore, human islets treated with SB203580 for 1 h prior to transplantation showed significantly improved graft function. These results suggest that inhibition of the p38 pathway may become a new therapeutic strategy to improve graft survival in clinical islet transplantation.

Glucose‐Stimulated Increment in Oxygen Consumption Rate as a Standardized Test of Human Islet Quality

Standardized assessment of islet quality is imperative for clinical islet transplantation. We have previously shown that the increment in oxygen consumption rate stimulated by glucose (ΔOCRglc) can predict in vivo efficacy of islet transplantation in mice. To further evaluate the approach, we studied three factors: islet specificity, islet composition and agreement between results obtained by different groups. Equivalent perifusion systems were set up at the City of Hope and the University of Washington and the values of ΔOCRglc obtained at both institutions were compared. Islet specificity was determined by comparing ΔOCRglc in islet and nonislet tissue. The ΔOCRglc ranged from 0.01 to 0.19 nmol/min/100 islets (n = 14), a wide range in islet quality, but the values obtained by the two centers were similar. The contribution from nonislet impurities was negligible (ΔOCRglc was 0.12 nmol/min/100 islets vs. 0.007 nmol/min/100 nonislet clusters). The ΔOCRglc was statistically independent of percent beta cells, demonstrating that ΔOCRglc is governed more by islet quality than by islet composition. The ΔOCRglc, but not the absolute level of OCR, was predictive of reversal of hyperglycemia in diabetic mice. These demonstrations lay the foundation for testing ΔOCRglc as a measurement of islet quality for human islet transplantation.

Generation of human islets through expansion and differentiation of non-islet pancreatic cells discarded (pancreatic discard) after islet isolation.

Objectives: Islet transplantation is hampered by the shortage of donor tissues. Our objective was to generate islet-like cell clusters (ICCs) from cultures of non-islet pancreatic cells. Methods: The starting cultured cells came from the non-islet fractions of human pancreases after enzymatic digestion and purification for the purpose of islet isolation. Initially, these cells expanded in monolayer cultures and became confluent on collagen-coated flasks. After trypsination and suspension of these cells in a defined islet differentiation medium, the cells aggregated to form ICCs. Results: The initial cell population consisted of less than 1% of insulin-positive cells, 44% amylase-positive cells, and 41% cytokeratin (CK) 7-positive, or CK19+ cells, but PDX-1+ cells were absent. Cells from later stages of the monolayer cultures showed signs of dedifferentiation/transdifferentiation. At the time of harvesting, more than 90% of the cells were positive for CK 7/19 and PDX-1, but less than 1% of the cells were insulin-positive. After aggregation, the ICCs appeared redifferentiated, and contained glucose-responsive, insulin-secreting cells with an insulin content measuring 20% of that found in freshly isolated islets isolated from the same pancreas. ICCs transplanted into athymic mice and removed after 4 months did acquire the morphology of mature islets, indicating further maturation of the ICCs in vivo after transplantation. Human C-peptide was detected in recipient animal sera. Conclusion: Using the specified culture methods, non-islet pancreas cells can generate cell clusters resembling islets. These ICCs, obtained from fractions of the pancreas that are otherwise discarded, continue to differentiate after transplantation to become mature islets.

Quantitative assessment of β-cell apoptosis and cell composition of isolated, undisrupted human islets by laser scanning cytometry.

Background. Assays for assessing human islet cell quality, which provide results before transplantation, would be beneficial to improve the outcomes for islet transplantation therapy. Parameters such as percent &bgr;-cell apoptosis and cell composition are found to vary markedly between different islet preparations and may serve as markers of islet quality. We have developed fluorescence-based assays using laser scanning cytometry for assessing &bgr;-cell apoptosis and islet cell composition on serial sections of intact isolated islets. Methods. Isolated human islets were fixed in formalin and embedded in paraffin. Serial sections were immunostained for the pancreatic hormones and acinar and ductal cell markers. DNA fragmentation was used to label apoptotic cells. Stained cells were quantified using an iCys laser scanning cytometer. Results. Islet preparations from 102 human pancreatic islet isolations were analyzed. For the whole set of islet preparations, we found a mean islet cell composition of 54.5%±1.2% insulin-positive, 33.9%±1.2% glucagon, 12.1%±0.7% somatostatin, and 1.5%±0.2% pancreatic polypeptide-positive cells. The apoptotic &bgr; cells were 2.85%±0.4% with a range of 0.27% to 18.3%. The percentage of apoptotic &bgr; cells correlated well (P<0.0001, n=59) with results obtained in vivo by transplantation of the corresponding islets in diabetic NODscid mice. Conclusions. The analysis of whole, nondissociated islets for cell composition and &bgr;-cell apoptosis using laser scanning cytometry gives reliable and reproducible results and could be performed both before islet transplantation and on preserved cell blocks at any time in future. Thus, they can be a powerful tool for islet quality assessment.

Improvement of human islet cryopreservation by a p38 MAPK inhibitor.

The activation of p38 mitogen‐activated protein kinase (MAPK) has been shown to cause ischemia/reperfusion injury of several organs used for transplantation and also to play a significant role in primary islet graft nonfunction. Activation of p38 MAPK may also occur during islet cryopreservation and thawing. In this study, a p38 MAPK inhibitor (p38IH) was applied to human islet cryopreservation to improve islet yield and quality after thawing. Under serum‐free conditions, human islets were cryopreserved, thawed and cultured using our standard procedures. Three types of solutions were tested: conventional RPMI1640 medium (RPMI), a newly developed islet cryopreservation solution (ICS), and ICS supplemented with a p38IH, SD‐282 (ICS‐p38IH). Activation or inhibition of p38 MAPK was demonstrated by the diminished phosphorylation of HSP27 substrate. Islet recovery on day 2 after thawing was highest with ICS‐p38IH and islet viability was not significantly different in the three groups. β Cell numbers and function were the highest in islets cryopreserved with ICS‐p38IH. Glucose‐stimulated human C‐peptide levels were 86% of that of the nonfrozen islets when measured 4 weeks after transplantation into NODscid mice. This improvement may provide an opportunity to establish islet banks and allow the use of cryopreserved islets for clinical transplantation.

Improvement of canine islet yield by donor pancreas infusion with a p38MAPK inhibitor.

Background. The activation of p38 mitogen-activated protein kinases (MAPK) is implicated in cold ischemia-reperfusion injury of donor organs. The islet isolation process, from pancreas procurement through islet collection, may activate p38MAPK leading to cytokine release and islet damage. This damage may be prevented by treating pancreata with a p38MAPK inhibitor (p38IH) before cold preservation. Methods. Pancreata removed from Beagle dogs were infused with University of Wisconsin solution containing the p38IH, SB203580, and Pefabloc (n=6) or vehicle (dimethyl sulfoxide and Pefabloc) alone (n=7), through the pancreatic duct and preserved using the two-layer method. After 20 to 22 hr, islets were isolated and 3000 IEQ/kg were autotransplanted into the corresponding dog to monitor glucose metabolism. Results. p38IH-treated pancreata yielded significantly more islets than control pancreata (IEQ/g: 2134±297 vs. 1477±145 IEQ/g or 65,012±9385 vs. 45,700±5103 IEQ/pancreas; P<0.05). Apoptotic &bgr;-cell percentages assessed by laser scanning cytometry were lower in p38IH-treated than the controls (44%±9.4% vs. 61.6%±4.8%, P<0.05). Tumor necrosis factor-&agr; expression assessed by real-time reverse transcription polymerase chain reaction was significantly lower in the p38IH-treated group than controls. All dogs (3000 IEQ/kg) transplanted with p38IH-treated islets (n=5) became euglycemic versus four of five dogs that received untreated islets. Plasma C-peptide levels after glucagon challenge were higher in animals receiving p38IH-treated islets (n=5) versus untreated islets (n=4) (0.40±0.78 vs. 0.21±0.05 ng/mL, P<0.05). Conclusions. Infusion of pancreata with University of Wisconsin solution containing p38IH through the duct before preservation suppresses cytokine release, prevents &bgr;-cell apoptosis, and improves islet yield significantly with no adverse effect on islet function after transplantation. p38IH treatment of human pancreata may improve islet yield for use in clinical transplantation.

Cell type specific targeted intracellular delivery into muscle of a monoclonal antibody that binds myosin IIb

Methods for cell type specific targeted intracellular delivery of proteins in vivo remain limited. A murine monoclonal anti-dsDNA antibody, mAb 3E10, was selectively transported into skeletal muscle cells in vivo. The antibody bound a 200 kDa protein only found in lysates of skeletal muscle by Western blotting. The 200 kDa protein was purified from muscle lysate by antibody affinity chromatography and identified as the skeletal muscle specific heavy chain of myosin IIb by electrospray mass spectrometry. Antibody binding specificity for myosin IIb was demonstrated in Western blots by binding myosin in skeletal muscle lysates from mice null for myosin IId but not in mice null for myosin IIb. Myosin IIb is implicated in the specific targeting of mAb 3E10 to skeletal muscle.

The Choice of Enzyme for Human Pancreas Digestion Is a Critical Factor for Increasing the Success of Islet Isolation

Background We evaluated 3 commercially available enzymes for pancreatic digestion by comparing key parameters during the islet isolation process, as well as islet quality after isolation. Methods Retrospectively compared and analyzed islet isolations from pancreata using 3 different enzyme groups: liberase HI (n = 63), collagenase NB1/neutral protease (NP) (n = 43), and liberase mammalian tissue-free collagenase/thermolysin (MTF C/T) (n = 115). A standardized islet isolation and purification method was used. Islet quality assessment was carried out using islet count, viability, in vitro glucose-stimulated insulin secretion (GSIS), glucose-stimulated oxygen consumption rate, and in vivo transplantation model in mice. Results Donor characteristics were not significantly different among the 3 enzyme groups used in terms of age, sex, hospital stay duration, cause of death, body mass index, hemoglobin A1c, cold ischemia time, and pancreas weight. Digestion efficacy (percentage of digested tissue by weight) was significantly higher in the liberase MTF C/T group (73.5 ± 1.5 %) when compared to the liberase HI group (63.6 ± 2.3 %) (P < 0.001) and the collagenase NB1/NP group (61.7 ± 2.9%) (P < 0.001). The stimulation index for GSIS was significantly higher in the liberase MTF C/T group (5.3 ± 0.5) as compared to the liberase HI (2.9 ± 0.2) (P < 0.0001) and the collagenase NB1/NP (3.6 ± 2.9) (P = 0.012) groups. Furthermore, the liberase MTF C/T enzymes showed the highest success rate of transplantation in diabetic non-obese diabetic severe combined immunodeficiency mice (65%), which was significantly higher than the liberase HI (42%, P = 0.001) and the collagenase NB1/NP enzymes (41%, P < 0.001). Conclusions Liberase MTF C/T is superior to liberase HI and collagenase NB1/NP in terms of digestion efficacy and GSIS in vitro. Moreover, liberase MTF C/T had a significantly higher success rate of transplantation in diabetic NOD Scid mice compared to liberase HI and collagenase NB1/NP enzymes.