Human stem cell derived beta-like cells engineered to present PD-L1 improve transplant survival in NOD mice carrying human HLA class I.

There is a critical need for therapeutic approaches that combine renewable sources of replacement beta cells with localized immunomodulation to counter recurrence of autoimmunity in type 1 diabetes (T1D). However, there are few examples of animal models to study such approaches that incorporate spontaneous autoimmunity directed against human beta cells rather than allogenic rejection. Here, we address this critical limitation by demonstrating rejection and survival of transplanted human stem cell-derived beta-like cells clusters (sBCs) in a fully immune competent mouse model with matching human HLA class I and spontaneous diabetes development. We engineered localized immune tolerance toward transplanted sBCs via inducible cell surface overexpression of PD-L1 (iP-sBCs) with and without deletion of all HLA class I surface molecules via beta-2 microglobulin knockout (iP-BKO sBCs). NOD.HLA-A2.1 mice, which lack classical murine MHC I and instead express human HLA-A*02:01, underwent transplantation of 1,000 human HLA-A*02:01 sBCs under the kidney capsule and were separated into HLA-A2 positive iP-sBC and HLA-class I negative iP-BKO sBC groups, each with +/- doxycycline (DOX) induced PD-L1 expression. IVIS imaging showed significantly improved graft survival in mice transplanted with PD-L1 expressing iP-sBC at day 3 post transplantation compared to controls. However, luciferase signal dropped below in vivo detection limits by day 14 for all groups in this aggressive immune competent diabetes model. Nonetheless, histological examination revealed significant numbers of surviving insulin+/PD-L1+ sBCs cells for DOX-treated mice at day 16 post-transplant despite extensive infiltration with high numbers of CD3+ and CD45+ immune cells. These results show that T cells rapidly infiltrate and attack sBC grafts in this model but that significant numbers of PD-L1 expressing sBCs manage to survive in this harsh immunological environment. This investigation represents one of the first in vivo studies recapitulating key aspects of human autoimmune diabetes to test immune tolerance approaches with renewable sources of beta cells.

A Novel ex vivo Method for Investigating Vascularization of Transplanted Islets.

Revascularization of transplanted pancreatic islets is critical for survival and treatment of type 1 diabetes. Questions concerning how islets influence local microvascular networks and how networks form connections with islets remain understudied and motivate the need for new models that mimic the complexity of real tissue. Recently, our laboratory established the rat mesentery culture model as a tool to investigate cell dynamics involved in microvascular growth. An advantage is the ability to observe blood vessels, lymphatics, and immune cells. The objective of this study was to establish the rat mesentery tissue culture model as a useful tool to investigate islet tissue integration. DiI-labeled islets were seeded onto adult rat mesentery tissues and cultured for up to 3 days. Live lectin labeling enabled time-lapse observation of vessel growth. During culture, DiI-positive islets remained intact. Radial lectin-positive capillary sprouts with DiI labeling were observed to form from islets and connect to host networks. Lectin-positive vessels from host networks were also seen growing toward islets. PECAM and NG2 labeling confirmed that vessels sprouting from islets contained endothelial cells and pericytes. Our results introduce the rat mesentery culture model as a platform for investigating dynamics associated with the initial revascularization of transplanted islets.

In vitro generation of peri-islet basement membrane-like structures.

The peri-islet extracellular matrix (ECM) is a key component of the microenvironmental niche surrounding pancreatic islets of Langerhans. The cell anchorage and signaling provided by the peri-islet ECM is critical for optimum beta cell glucose responsiveness, but islets lose this important native ECM when isolated for transplantation or in vitro studies. Here, we established a method to construct a peri-islet ECM on the surfaces of isolated rat and human islets by the co-assembly from solution of laminin, nidogen and collagen IV proteins. Successful deposition of contiguous peri-islet ECM networks was confirmed by immunofluorescence, western blot, and transmission electron microscopy. The ECM coatings were disrupted when assembly occurred in Ca2+/Mg2+-free conditions. As laminin network polymerization is divalent cation dependent, our data are consistent with receptor-driven ordered ECM network formation rather than passive protein adsorption. To further illustrate the utility of ECM coatings, we employed stem cell derived beta-like cell clusters (sBCs) as a renewable source of functional beta cells for cell replacement therapy. We observe that sBC pseudo-islets lack an endogenous peri-islet ECM, but successfully applied our approach to construct a de novo ECM coating on the surfaces of sBCs.

Contraction-relaxation coupling is unaltered by exercise training and infarction in isolated canine myocardium.

The two main phases of the mammalian cardiac cycle are contraction and relaxation; however, whether there is a connection between them in humans is not well understood. Routine exercise has been shown to improve cardiac function, morphology, and molecular signatures. Likewise, the acute and chronic changes that occur in the heart in response to injury, disease, and stress are well characterized, albeit not fully understood. In this study, we investigated how exercise and myocardial injury affect contraction-relaxation coupling. We retrospectively analyzed the correlation between the maximal speed of contraction and the maximal speed of relaxation of canine myocardium after receiving surgically induced myocardial infarction, followed by either sedentary recovery or exercise training for 10-12 wk. We used isolated right ventricular trabeculae, which were electrically paced at different lengths, frequencies, and with increasing ß-adrenoceptor stimulation. In all conditions, contraction and relaxation were linearly correlated, irrespective of injury or training history. Based on these results and the available literature, we posit that contraction-relaxation coupling is a fundamental myocardial property that resides in the structural arrangement of proteins at the level of the sarcomere and that this may be regulated by the actions of cardiac myosin binding protein C (cMyBP-C) on actin and myosin.

Effects of sustained GABA releasing implants on pancreatic islets in mice.

Gamma-aminobutyric acid (GABA) is an inhibitory neurotransmitter that is strongly and selectively synthesized in and secreted from pancreatic beta cells. Exogenously delivered GABA has been proposed to induce beta cell regeneration in type 1 diabetes, but these results have been difficult to replicate and may depend on the specifics of the animal model and drug delivery method used. Here, we developed a GABA-releasing ethylene-vinyl acetate polymer implant for sustained GABA delivery to the intraperitoneal space as an alternative to injected or oral GABA. We explored the effect of the GABA-releasing polymer implants compared to implanted osmotic pumps loaded with GABA on islet size in non-diabetic, outbred mice. We also attempted to monitor in vivo GABA release using HPLC on blood samples, but these measurements were confounded by high variability within treatment groups and unexpectedly high serum GABA levels in mice receiving GABA-negative implants. The ethylene-vinyl acetate polymer implants became heavily fibrosed with abdominal adhesion tissue, while the osmotic pumps had no macroscopic fibrosis. Histological analysis showed no significant effect of the sustained GABA delivery polymer or osmotic pumps on islet size, alpha cell to beta cell ratio, or the number of Ki67-positive islet cells. The GABA treatment time course was limited to two weeks due to the drug-release window of the polymer, while others reported islet-trophic effects of GABA after 10 to 12 weeks of treatment. In summary, our study is consistent with the concept that exogenous GABA administration does not significantly alter islet cell mass in non-diabetic CD-1 mice in the short-term. However, more data are needed including higher GABA doses and more prolonged treatment regimens for a better comparison with contrasting reports.

Modelling and molecular docking studies of the cytoplasmic domain of Wsc-family, full-length Ras2p, and therapeutic antifungal compounds.

Saccharomyces cerevisiae, the budding yeast, must remodel initial cell shape and cell wall integrity during vegetative growth and pheromone-induced morphogenesis. The cell wall remodeling is monitored and regulated by the cell wall integrity (CWI) signaling pathway. Wsc1p, together with Wsc2p and Wsc3p, belongs to a family of highly O-glycosylated cell surface proteins that function as stress sensors of the cell wall in S. cerevisiae. These cell surface proteins have the main role of activating the CWI signaling pathway by stimulating the small G-protein Rho1p, which subsequently activates protein kinase C (Pkc1p) and a mitogen activated protein (MAP) kinase cascade that activates downstream transcription factors of stress-response genes. Wsc1p, Wsc2p, and Wsc3p possess a cytoplasmic domain where two conserved regions of the sequence have been assessed to be important for Rom2p interaction. Meanwhile, other research groups have also proposed that these transmembrane proteins could support protein-protein interactions with Ras2p. Molecular structures of the cytoplasmic domain of Wsc1p, Wsc2p and Wsc3p were generated using the standard and fully-automated ORCHESTAR procedures provided by the Sybyl-X 2.1.1 program. The tridimensional structure of full length Ras2p was also generated with Phyre2. These protein models were validated with Procheck-PDBsum and ProSA-web tools and subsequently used in docking-based modeling of protein-protein and protein-compound interfaces for extensive structural and functional characterization of their interaction. The results retrieved from STRING 10.5 suggest that the Wsc-family is involved in protein-protein interactions with each other and with Ras2p. Docking-based studies also validated the existence of protein-protein interactions mainly between Motif I (Wsc3p > Wsc1p > Wsc2p) and Ras2p, in agreement with the data provided by STRING 10.5. Additionally, it has shown that Calcofluor White preferably binds to Wsc1p (-9.5 kcal/mol), meanwhile Caspofungin binds to Wsc3p (-9.1 kcal/mol), Wsc1p (-9.1 kcal/mol) and more weakly Wsc2p (-6.9 kcal/mol). Thus, these data suggests Caspofungin as a common inhibitor for the Wsc-family. MTiOpenScreen database has provided a list of new compounds with energy scores higher than those compounds used in our docking studies, thus suggesting these new compounds have a better affinity towards the cytoplasmic domains and Ras2p. Based on these data, there are new and possibly more effective compounds that should be considered as therapeutic agents against yeast infection.