Islet transplantation (ITx), a form of cell-based insulin replacement therapy, is an attractive approach for the treatment of uncontrolled or “brittle” type 1 diabetes. However, large scale application of ITx is limited by the need for lifelong immunosuppression and a shortage of human cadaveric pancreas donors, which is exacerbated by the need for islets from multiple pancreata per patient to achieve insulin independence.1,2 Encapsulation (immunoisolation) devices have the potential to address these critical limitations. They have demonstrated efficacy in protecting transplanted islets even in the absence of immunosuppression, and may allow for alternative cell-based insulin replacement therapies for type 1 diabetes, for example, by enabling the use of stem cell-derived β cells (SC-βs) which can be sourced on a virtually unlimited scale.3-10
However, encapsulating therapeutic β cell doses into reasonably sized (postage stamp) immunoisolation devices creates a high-density environment in which the availability of nutrients (especially glucose and oxygen) is limited, leading to ischemia.7 This has ultimately hindered the clinical application of macroencapsulation in β cell replacement therapies. Insufficient oxygen is a particular concern for the insulin secreting β cells within islets. β cells lack sufficient levels of the enzyme lactate dehydrogenase α to generate ATP using anaerobic glycolysis, and overexpression of lactate dehydrogenase α in β cells diminishes their glucose responsiveness.11,12 Moreover, once separated from their native vasculature, islets depend solely on diffusion for the delivery of oxygen and energetic substrates. Deprivation of these critical nutrients and the accumulation of toxic metabolites lead to impaired insulin secretion and eventual cell death.13-19 These effects are even more pronounced in islets with large diameters, and clinical ITx outcomes are improved for patients receiving preparations composed of smaller average diameter islets.20-22
Considering the recent surge of interest in immunoisolation devices and the benefits that their application can offer, it will be of critical importance to understand how these devices impact β cell physiology to facilitate successful clinical outcomes. Therefore, we investigated the viability, insulin secretion, and transcriptional adaptations of a highly purified human islets with an emphasis on β cell function, stress, and inflammation after acute ischemic exposure in high-density “pellet” model. This model has been characterized previously in the context of islet shipping, and is used here to simulate and study the effects of the high density environments created by delivery of therapeutic β cell doses in reasonably sized immunoisolation devices.23,24
MATERIALS AND METHODS
Islet Source and Maintenance
Human islets (n = 11 independent preparations) were obtained from the Integrated Islet Distribution Program, the University of Minnesota, the University of Arizona, the McGill University Health Centre, and the University of California-San Francisco. Islets were cultured in oxygen permeable silicone rubber membrane GRex vessels (Wilson Wolf, St Paul, MN). Islet culture media consisted of CMRL 1066 (Mediatech, Inc., Manassas, VA) supplemented with 0.5% human serum albumin ( BioChemed Services, Winchester, VA), 1% heparin (SAGENT, Schaumburg, IL), 1% l-glutamine (Mediatech, Inc.), and 1% penicillin/streptomycin (GE Healthcare Life Sciences, Logan, UT). Before experiments, islets were maintained at 25°C and ambient pO2 supplemented with 5% CO2.
Induction of Pelletized Culture
After standard culture, islets from each donor were divided into normoxic and ischemic conditions. Ischemia was modeled through high-density “pelletized” culture in which 10000 islet equivalents (IE) as quantified by DNA were allowed to settle in the bottom of a 1.5-mL centrifuge tube.25 This condition was selected because it mimics both traditional islet shipping conditions as well as the high-density environments that can result from encapsulation. For the normoxic condition, an equal number of islets were cultured in a 10-cm2 GRex vessel (Wilson Wolf). Normoxic and ischemic islets were cultured for 12 hours at 37°C, ambient O2, and 5% CO2.
To test the effects of high density encapsulation on human islets and verify comparable effects to pelletized culture, 8000 IE as quantified by DNA were loaded into 4.5 μL (0.34 cm2) TheraCyte (TheraCyte, Laguna Hills, CA) devices using a Hamilton syringe. Devices were then placed into oxygen permeable, 10 cm2 GRex vessels (Wilson Wolf) filled with culture media and maintained for 12 hours at 37°C, ambient O2, and 5% CO2. Encapsulated islets were compared with matched pellets and controls prepared as described above.
Measurement of DNA
Islets were suspended in 1 mL of AT Buffer (1 M solution of ammonium hydroxide in nanopure water, supplemented with 0.2% Triton X-100) and sonicated using a Sonic Dismembrator Model 500 (Fisher Scientific, Waltham, MA) for 30 seconds at 11% amplitude. DNA was assessed using a Quant-iT PicoGreen dsDNA kit (Life Technologies, Carlsbad, CA) according to manufacturer instructions. 96 well plates were read using a SpectraMax M5 plate reader and SoftMax Pro software (Molecular Devices, Sunnyvale, CA).
Measurement of Oxygen Consumption Rate Normalized to DNA
After 12 hours, islets and media were mixed to ensure homogenous sampling. Oxygen consumption rate (OCR) was conducted as previously described.26 Briefly, islets were resuspended in Media 199 (Mediatech, Inc.) warmed to 37°C and divided evenly between 3 OCR chambers of known volumes, or conducted on whole devices as in the case of encapsulated islets (Instech Laboratories, Inc., Plymouth Meeting, PA). Measurements of pO2 in each chamber over time were recorded using fiber optic sensors and NeoFox viewer software (Ocean Optics, Inc., Dunedin, FL). The oxygen consumption rate in nanomoles of O2 per minute was then estimated from the slope of the decline in pO2 over time. This value was normalized to the DNA content of each chamber (as described above) to give a final measurement of OCR/DNA (nmol O2/min*mg DNA).
Measurement of % Viability by Fluorescein Diacetate/Propidium Iodide Staining
From each condition, 100-μL samples of islets were combined with 377.6 μL of dithizone (Sigma-Aldrich, St. Louis, MO). To this, 1.39 μL of fluorescein diacetate (Sigma-Aldrich) and 21.05 μL of propidium iodide (Sigma-Aldrich) were added for final concentrations of 0.067 and 4.0 μM, respectively. Islets were incubated in the dark for 20 minutes, after which they were allowed to settle and 100 μL of islets were placed on a slide for imaging. To determine the proportion of live versus dead cells, islets were imaged on a Zeiss Observer.Z1 (Zeiss, Oberkochen, Germany) using the 10× objective and an AxioCam MRm camera with ZEN 2012 (blue addition) software (Zeiss).
β cell Function
The Biorep Technologies Peri-4.2 Perifusion System (Biorep Techonologies, Inc., Miami Lakes, FL) was used to measure dynamic glucose-stimulated insulin secretion (GSIS). Triplicate measurements using 100 IE each underwent baseline stimulation with 2.8 mM glucose in oxygen-saturated (95% O2) Krebs-Ringer Bicarbonate buffer for 20 minutes before and after a 40-minute stimulation period with 16.7-mM glucose. Perfusate was collected at a rate of 100 μL/min in a 96-well plate. Perfusate was tested for insulin content using an insulin enzyme linked immunosorbent assay (ELISA, Mercodia, Winston-Salem, NC). ELISA plates were read using Softmax Pro software and a SpectraMax M5 plate reader (Molecular Devices). Insulin content was normalized to DNA as described above.
RNA Library Preparation
Paired normoxic and ischemic islets from 4 donors were selected at random for high throughput RNA sequencing (RNAseq). For RNA samples, islets were collected in microcentrifuge tubes and washed twice 1× Dulbecco's phosphate buffered saline to remove serum. RNA was isolated using the RNeasy Mini Kit (Qiagen, Valencia, CA) according to the manufacturer's instructions. RNA quality was assessed by Agilent Bioanalyzer (Agilent Technologies, Santa Clara, CA), and a minimum RIN of 7.0 was required for inclusion in the study. Libraries were prepared using the Illumina TruSeq Stranded mRNA Sample Preparation Kit (Illumina, San Diego, CA). mRNA was sequenced using Illumina HiSeq 2000 sequencer and quantified into transcripts using EA- Quintiles mRNAv8 pipeline.
Briefly, 50 × 50 base, paired-end reads were checked for quality by comparison to intergenic and ribosomal sequences. Sequencing reads were aligned to the human University of California Santa Cruz known gene transcriptome using RNA-Seq by Expectation Maximization v1.2.0 and transcript abundance was quantified. An average of 97.4 ± 0.4% of total reads mapped to reference genomes. The aligned reads for each gene were summarized as described in Li and Dewey.27 All genes and isoforms have been assigned a normalized coverage rate in fragments per kilobase per million mapped reads (FPKM). Differential gene expression was performed using the edgeR R package scripts (bioconductor.org) using paired sample statistical procedures.28,29 A paired-sample (generalized paired t test) analysis was conducted to investigate the effects of ischemia while adjusting for baseline differences between patients. Genes with a P value less than 0.05 following Benjamini-Hochberg correction were considered to be statistically significant.
Functional Analysis of Differentially Expressed Genes
Significantly upregulated or downregulated genes were submitted to KOBAS 2.0 to determine which canonical pathways were enriched by ischemia, as well as to determine associated gene ontology (GO) terms.30 Significant enrichments were defined as a corrected P value less than 0.05 following a Fisher exact test with Benjamini Hochberg correction in KOBAS 2.0. To summarize GO enrichment and reduce redundant terms, ReviGO was used to cluster similar GO terms using small (0.5) SimRel similarity and the whole Uniprot as the database for categorical GO term sizes.31 Significant pathways were manually curated to remove pathways containing redundant or nonspecific gene findings. Preferential association between the lists of upregulated and downregulated genes was also evaluated.
RT qPCR Array
Results from RNAseq were compared with quantitative reverse transcriptase polymerase chain reaction results for a single islet preparation. Purified islet total RNA was run on RT2 Profiler PCR Array Human Hypoxia Signaling Pathway plates (Qiagen) using iQ5 Real-Time PCR Detection System (Bio-Rad Laboratories, Irvine, CA). Fold changes were determined according to manufactures instructions. Gene expression was normalized to housekeeping genes RPLP0, B2M, and HPRT1.
Statistical analysis of physiological parameters was conducted in SAS 9.4 (SAS Institute Inc., Cary, NC). OCR/DNA data were analyzed using Wilcoxon Signed Rank Test. Matched fluorescein diacetate/propidium iodide and OCR/DNA data were analyzed using a Student t test. Perifusion data were analyzed using a generalized linear mixed model for repeated measurements. Fold changes from the RT qPCR array were correlated to fold changes from RNAseq using linear regression analysis in GraphPad Prism version 6.07 (GraphPad Software, Inc., La Jolla, CA). Unless otherwise indicated, data represent mean ± standard deviation.
Individual donor characteristics and islet quality parameters (% viability measured by membrane integrity staining, purity, and cold ischemia time) as reported by the distributing isolation center are presented in Table 1.
Pellet Model Characterization and Comparison to Encapsulation
The viability of islets from n = 2 human islet preparations was compared under matched normoxic, pelletized, and encapsulated conditions to confirm that the ischemic damage caused by the pellet condition is similar to what is caused in devices loaded at high densities.7 Pelletized islets and encapsulated islets showed similar % reductions in OCR/DNA values versus control (57.7 ± 19.5% and 80.3 ± 21.1%, respectively).
Islet viability was determined in n = 8 independent islet preparations. Average OCR/DNA values for normoxic islets align closely with clinical averages observed for both islet auto and allotransplantation.32,33 Islet OCR/DNA values are reduced after warm ischemic exposure (P < 0.01, Figure 1), which correlates to lower, although not entirely diminished, viability. In a subcohort of n = 4 independent islet preparations, OCR/DNA results were compared with fluorescein diacetate/propidium iodide staining. Staining revealed a significant loss of viability for control versus pelletized islets (% 87.3 ± 1.0% vs 8.8 ± 5.5%, respectively, P < 0.01). OCR/DNA measurements for matched samples were similarly reduced for control vs. pelletized islets (122.9 ± 53.3 and 53.4 ± 12.0 nmol O2/min*mg DNA, respectively, P < 0.05).
B Cell Function
GSIS was measured in a subset of control and ischemic islet pairs (n = 4 independent preparations, Figure 2). GSIS of ischemic islets is virtually absent compared with control islets. Basal secretion was not different between control and ischemic islets (Figure 3A). However, under stimulatory glucose concentrations (Figure 3B), ischemic islets secrete less insulin than control islets (P < 0.01). Normoxic islets show elevated (P < 0.01) insulin secretion in response to stimulatory glucose concentrations, whereas ischemic islets are unresponsive.
Global Gene Expression Changes After Ischemia
An average of 19 932 ± 138 genes were detected with RNAseq. Of these, 657 genes were differentially expressed in ischemic versus control islets (P < 0.05) (Figure 4A). Genes most upregulated include immune molecules interleukins 6 and 8 (IL6, IL8), chemokine 3 (CCL3), and chemokine 3 like (CXCL3). Genes most downregulated include the inward rectifier K+ channel Kir 3.1 (KCNJ3) and Na+-coupled neutral amino acid transporter 4 (SLC38A4) (Figure 4B).
Fold changes calculated from RNAseq correlated (P < 0.0001, r2 = 0.75) to fold changes calculated by RT qPCR array (Figure S1, SDC,http://links.lww.com/TP/B421). The high correlation between independent measures of gene expression corroborates findings from RNAseq model.
Functional Analysis of Differentially Expressed Genes
Canonical pathways significantly associated with differentially expressed genes were identified (Table 2). Specific differentially expressed genes associated with inflammation and cytokine signaling, and also associated with pathways for the HIF-1α response, AP-1 (FOS) signaling, and ion transport are detailed in Figure 5. Enriched GO terms were in agreement with pathway findings, showing enhancement of cytokine signaling and inflammation as well as downregulation of cellular ion transport (Table 3). Genes associated with hypoxia and nutrient deprivation (including SLC2A1, HK1, HMOX1, ARG1, and UPP1) were also upregulated (P < 0.05).
Ischemia remains a pervasive issue that directly influences the outcomes of cell-based insulin replacement therapies, such as ITx. In this study, we implement an ischemic model that has previously been used to study islet shipping and captures the conditions seen in high density islet encapsulation, including a significant reduction of islet viability which is associated with oxygen and nutrient deprivation, and consistent with results from the transcriptome analysis.19,23,24 Furthermore, we demonstrate that the viable population of islets following acute ischemia exhibits significant β cell dysfunction corresponding to global alterations in gene expression. Inflammatory responses were evident in the gene signatures of islets after ischemia, with concurrent downregulation of genes involved in nutrient and ion transport. The persistence of β cell dysfunction due to ischemic stress has been previously demonstrated, whether due to cellular reprogramming or ischemia-reperfusion injury, and the endurance of a concurrent proinflammatory signature is likely, given the present work.19,34,35 Interestingly, kidney capsule islet transplants represent a commonly used in vivo correlate to our high-density in vitro model in which islets are transplanted as a “pellet” below the renal capsule.36 Although this method induces less ischemic stress than what might be experienced in an immunoisolation device, when these islets are exposed to nerve growth factor (which has an anti-apoptotic effect), graft function is improved.37-39 This supports our finding that even acute ischemia is detrimental to islet viability and function and indicates that early mitigation of these factors is beneficial to transplant outcomes.
The differential gene expression profile caused by acute ischemia was predominantly increased inflammatory and stress response signaling. More specifically, there was increased expression of key inflammatory genes (including TNF and IL6), which are known to contribute to islet death in the peritransplant period by the instant blood mediated inflammatory reaction.40 The link between hypoxia and this proinflammatory profile has been demonstrated previously in rat islets.41 This increased inflammatory signaling may be important not only from an immunological standpoint, but also in the context of β cell function. TNFα has previously been shown to inhibit GSIS in β cell lines as well as downstream insulin signaling in adipocytes.42-44 The potential damage to insulin signaling is compelling, as autocrine insulin signaling in β cells has been demonstrated to influence insulin expression.45 Key genes involved in the coupling of glucose sensing to insulin secretion (for example KCNJ11, which encodes the major subunit of the ATP-sensitive K+ channel) are also significantly downregulated. Together with the effects of inflammatory factors, these changes may underlie the significant loss of β cell function. By mitigating these damaging adaptations, loss of islets in the peritransplant period may be minimized and ultimately help to facilitate lower curative doses of islets for diabetes reversal.46
Lifelong immunosuppression remains a critical barrier which limits the application of β cell replacement therapies. Implantable macroencapsulation devices are a possible solution and have been demonstrated to be alloprotective in animal models as well as in humans.3,47 However, packing therapeutic β cell doses into practically sized devices results in a high-density environment in which diffusion of oxygen and nutrients is severely limited. This study demonstrates that such high-density environments are damaging to β cell function and may ultimately affect transplant outcomes.5,48 Addressing the effects if ischemia by ensuring adequate nutrient delivery will be critical to the success of cell-based insulin replacement therapies going forward. With respect to maintaining oxygenation, numerous advances have been made with regard to supplying oxygen to the pancreas during the preservation period as well as to isolated islets during culture.49,50 Attempts have also been made to supply islets in encapsulation devices with oxygen, and this remains an active area of research although a permanent solution has yet to be achieved.3
The authors would like to acknowledge the members of the Papas laboratory and collaborators, including William Purivs, Nicholas Price, Ivan Georgiev, Anna Maria Burachek, Alexandra Hoeger, Diana Molano, Jana Jandova, Kristina Rivera, Aikaterina Assimacopoulos, and Breonna Smith for their support. The authors would like to thank our collaborators at the University of California-San Francisco, McGill University Health Centre, and the University of Minnesota as well as the Integrated Islet Distribution Program for supplying islets used in these studies, as well as Dr. Linda Tempelman and Dr. Jan Burt for their advisement and mentorship. The authors would additionally like the ARCS Foundation Phoenix Chapter for their generous support.
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