Nutrient-dependent regulation of ß-cell proinsulin content.

Insulin is made from proinsulin, but the extent to which fasting/feeding controls the homeostatically regulated proinsulin pool in pancreatic ß-cells remains largely unknown. Here, we first examined ß-cell lines (INS1E and Min6, which proliferate slowly and are routinely fed fresh medium every 2-3 days) and found that the proinsulin pool size responds to each feeding within 1 to 2 h, affected both by the quantity of fresh nutrients and the frequency with which they are provided. We observed no effect of nutrient feeding on the overall rate of proinsulin turnover as quantified from cycloheximide-chase experiments. We show that nutrient feeding is primarily linked to rapid dephosphorylation of translation initiation factor eIF2α, presaging increased proinsulin levels (and thereafter, insulin levels), followed by its rephosphorylation during the ensuing hours that correspond to a fall in proinsulin levels. The decline of proinsulin levels is blunted by the integrated stress response inhibitor, ISRIB, or by inhibition of eIF2α rephosphorylation with a general control nonderepressible 2 (not PERK) kinase inhibitor. In addition, we demonstrate that amino acids contribute importantly to the proinsulin pool; mass spectrometry shows that ß-cells avidly consume extracellular glutamine, serine, and cysteine. Finally, we show that in both rodent and human pancreatic islets, fresh nutrient availability dynamically increases preproinsulin, which can be quantified without pulse-labeling. Thus, the proinsulin available for insulin biosynthesis is rhythmically controlled by fasting/feeding cycles.

High-throughput identification of RNA localization elements in neuronal cells.

Hundreds of RNAs are enriched in the projections of neuronal cells. For the vast majority of them, though, the sequence elements that regulate their localization are unknown. To identify RNA elements capable of directing transcripts to neurites, we deployed a massively parallel reporter assay that tested the localization regulatory ability of thousands of sequence fragments drawn from endogenous mouse 3' UTRs. We identified peaks of regulatory activity within several 3' UTRs and found that sequences derived from these peaks were both necessary and sufficient for RNA localization to neurites in mouse and human neuronal cells. The localization elements were enriched in adenosine and guanosine residues. They were at least tens to hundreds of nucleotides long as shortening of two identified elements led to significantly reduced activity. Using RNA affinity purification and mass spectrometry, we found that the RNA-binding protein Unk was associated with the localization elements. Depletion of Unk in cells reduced the ability of the elements to drive RNAs to neurites, indicating a functional requirement for Unk in their trafficking. These results provide a framework for the unbiased, high-throughput identification of RNA elements and mechanisms that govern transcript localization in neurons.

Alcohol self-administration and nicotine withdrawal alter biomarkers of stress and inflammation and prefrontal cortex changes in Gß subunits.

Background: Although alcohol and nicotine are often used together, the biological consequences of these substances are not well understood. Identifying shared targets will inform cessation pharmacotherapies and provide a deeper understanding of how co-use of alcohol and nicotine impacts health, including biomarkers of stress and inflammation.Objective: We examined the effects of nicotine exposure and withdrawal on alcohol self-administration (SA), stress and inflammatory biomarkers, and a G-protein coupled receptor subunit (Gß) in brain areas associated with drug use.Methods: Male rats were trained to SA alcohol and then received a nicotine pump (n = 7-8 per group). We assessed alcohol intake for 12 days during nicotine exposure and then following pump removal to elicit withdrawal. After the behavioral studies, we assessed plasma leptin, corticosterone, and interleukin-1ß (IL-1ß), and Gß protein expression in the amygdala, nucleus accumbens (NAc), and prefrontal cortex (PFC).Results: Nicotine exposure or withdrawal did not alter alcohol intake (p > .05). Alcohol and nicotine withdrawal elevated corticosterone levels (p = .015) and decreased Gß levels in the PFC (p = .004). In the absence of nicotine, alcohol SA suppressed IL-1ß levels (p = .039). Chronic exposure to nicotine or withdrawal during alcohol SA did not alter leptin levels or Gß expression in the amygdala or NAc (p's > .05).Conclusions: The combination of alcohol SA and nicotine withdrawal produced a persistent increase in stress biomarkers and a suppression in Gß expression in the PFC, providing an important first step toward understanding the common biological mechanisms of alcohol/nicotine misuse.

Cryopreservation of Stem Cell-Derived ß-Like Cells Enriches for Insulin-Producing Cells With Improved Function.

The generation of stem cell-derived ß-like cells (sBCs) holds promise as not only an abundant insulin-producing cell source for replacement therapy of type 1 diabetes (T1D) but also as an invaluable model system for investigating human ß-cell development, immunogenicity, and function. Several groups have developed methodology to direct differentiate human pluripotent stem cells into pancreatic cell populations that include glucose-responsive sBCs. Nevertheless, the process of generating sBCs poses substantial experimental challenges. It involves lengthy differentiation periods, there is substantial variability in efficiency, and there are inconsistencies in obtaining functional sBCs. Here, we describe a simple and effective cryopreservation approach for sBC cultures that yields homogeneous sBC clusters that are enriched for insulin-expressing cells while simultaneously depleting proliferative progenitors. Thawed sBCs have enhanced glucose-stimulated insulin release compared with controls in vitro and can effectively engraft and function in vivo. Collectively, this approach alleviates current challenges with inefficient and variable sBC generation while improving their functional state. We anticipate that these findings can inform ongoing clinical application of sBCs for the treatment of patients with T1D and serve as an important resource for the wider diabetes field that will allow for accelerated research discoveries.

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.

Stem-Cell-Derived ß-Like Cells with a Functional PTPN2 Knockout Display Increased Immunogenicity.

Type 1 diabetes is a polygenic disease that results in an autoimmune response directed against insulin-producing beta cells. PTPN2 is a known high-risk type 1 diabetes associated gene expressed in both immune- and pancreatic beta cells, but how genes affect the development of autoimmune diabetes is largely unknown. We employed CRISPR/Cas9 technology to generate a functional knockout of PTPN2 in human pluripotent stem cells (hPSC) followed by differentiating stem-cell-derived beta-like cells (sBC) and detailed phenotypical analyses. The differentiation efficiency of PTPN2 knockout (PTPN2 KO) sBC is comparable to wild-type (WT) control sBC. Global transcriptomics and protein assays revealed the increased expression of HLA Class I molecules in PTPN2 KO sBC at a steady state and upon exposure to proinflammatory culture conditions, indicating a potential for the increased immune recognition of human beta cells upon differential PTPN2 expression. sBC co-culture with autoreactive preproinsulin-reactive T cell transductants confirmed increased immune stimulations by PTPN2 KO sBC compared to WT sBC. Taken together, our results suggest that the dysregulation of PTPN2 expression in human beta cell may prime autoimmune T cell reactivity and thereby contribute to the development of type 1 diabetes.

Protecting Stem Cell Derived Pancreatic Beta-Like Cells From Diabetogenic T Cell Recognition.

Type 1 diabetes results from an autoimmune attack directed at pancreatic beta cells predominantly mediated by T cells. Transplantation of stem cell derived beta-like cells (sBC) have been shown to rescue diabetes in preclinical animal models. However, how sBC will respond to an inflammatory environment with diabetogenic T cells in a strict human setting has not been determined. This is due to the lack of model systems that closely recapitulates human T1D. Here, we present a reliable in vitro assay to measure autologous CD8 T cell stimulation against sBC in a human setting. Our data shows that upon pro-inflammatory cytokine exposure, sBC upregulate Human Leukocyte Antigen (HLA) class I molecules which allows for their recognition by diabetogenic CD8 T cells. To protect sBC from this immune recognition, we utilized genome engineering to delete surface expression of HLA class I molecules and to integrate an inducible overexpression system for the immune checkpoint inhibitor Programmed Death Ligand 1 (PD-L1). Genetically engineered sBC that lack HLA surface expression or overexpress PD-L1 showed reduced stimulation of diabetogenic CD8 T cells when compared to unmodified cells. Here, we present evidence that manipulation of HLA class I and PD-L1 receptors on sBC can provide protection from diabetes-specific immune recognition in a human setting.

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.

ENTPD3 Marks Mature Stem Cell-Derived ß-Cells Formed by Self-Aggregation In Vitro.

Stem cell-derived ß-like cells (sBC) carry the promise of providing an abundant source of insulin-producing cells for use in cell replacement therapy for patients with diabetes, potentially allowing widespread implementation of a practical cure. To achieve their clinical promise, sBC need to function comparably with mature adult ß-cells, but as yet they display varying degrees of maturity. Indeed, detailed knowledge of the events resulting in human ß-cell maturation remains obscure. Here we show that sBC spontaneously self-enrich into discreet islet-like cap structures within in vitro cultures, independent of exogenous maturation conditions. Multiple complementary assays demonstrate that this process is accompanied by functional maturation of the self-enriched sBC (seBC); however, the seBC still contain distinct subpopulations displaying different maturation levels. Interestingly, the surface protein ENTPD3 (also known as nucleoside triphosphate diphosphohydrolase-3 [NDPTase3]) is a specific marker of the most mature seBC population and can be used for mature seBC identification and sorting. Our results illuminate critical aspects of in vitro sBC maturation and provide important insights toward developing functionally mature sBC for diabetes cell replacement therapy.

Use of Induced Pluripotent Stem Cells to Build Isogenic Systems and Investigate Type 1 Diabetes.

Type 1 diabetes (T1D) is a disease that arises due to complex immunogenetic mechanisms. Key cell-cell interactions involved in the pathogenesis of T1D are activation of autoreactive T cells by dendritic cells (DC), migration of T cells across endothelial cells (EC) lining capillary walls into the islets of Langerhans, interaction of T cells with macrophages in the islets, and killing of ß-cells by autoreactive CD8+ T cells. Overall, pathogenic cell-cell interactions are likely regulated by the individual's collection of genetic T1D-risk variants. To accurately model the role of genetics, it is essential to build systems to interrogate single candidate genes in isolation during the interactions of cells that are essential for disease development. However, obtaining single-donor matched cells relevant to T1D is a challenge. Sourcing these genetic variants from human induced pluripotent stem cells (iPSC) avoids this limitation. Herein, we have differentiated iPSC from one donor into DC, macrophages, EC, and ß-cells. Additionally, we also engineered T cell avatars from the same donor to provide an in vitro platform to study genetic influences on these critical cellular interactions. This proof of concept demonstrates the ability to derive an isogenic system from a single donor to study these relevant cell-cell interactions. Our system constitutes an interdisciplinary approach with a controlled environment that provides a proof-of-concept for future studies to determine the role of disease alleles (e.g. IFIH1, PTPN22, SH2B3, TYK2) in regulating cell-cell interactions and cell-specific contributions to the pathogenesis of T1D.

ß Cell replacement: improving on the design.

PURPOSE OF REVIEW: Here we summarize recent advancements in ß cell replacement as a therapy for type 1 diabetes. RECENT FINDINGS: ß cell replacement therapy has been proposed as a cure for type 1 diabetes with the introduction of the Edmonton protocol for cadaveric islet transplantation. To allow widespread use of this approach, efforts have focused on establishing an abundant source of insulin-producing ß cells, protecting transplanted cells from ischemia-mediated death, immune rejection, and re-occurring autoimmunity. Recent developments addressing these issues include generation of insulin-producing cells from human pluripotent stem cells, different encapsulation strategies and prevention of ischemia upon transplant. SUMMARY: Despite significant advances in generating functional ß cells from human pluripotent stem cells, several key challenges remain in regard to the survival of ß cell grafts, protection from (auto-) immune destruction and implementation of additional safety mechanisms before a stem cell-based cell replacement therapy approach can be widely applied. Taking current findings into consideration, we outline a multilayered approach to design immune-privileged ß cells from stem cells using state of the art genome editing technologies that if successfully incorporated could result in great benefit for diabetic patients and improve clinical results for cell replacement therapy.