Evaluating Islet Cell Isolation and Transplantation From Donors Following Medical Assistance in Dying.

Background: Limited information is available regarding outcomes of islet cell isolation (ICI) and transplantation (ITx) using medical assistance in dying (MAiD) donors. We aimed to assess the feasibility and outcomes of ICI and ITx in MAiD donors. Methods: ICI and ITx from MAiD were compared with donation after circulatory death (DCD) type III between 2016 and 2023. Differences of isolated islet equivalents (IEQs), numeric viability and other quantitative in vitro metabolic measures were assessed. Results: Overall, 81 ICIs were available of whom 34 (42%) and 47 (58%) from MAiD and DCD-III, respectively. There were no differences of pancreas and digested tissue weight and islets viability among the 2 groups; however, cold ischemic time was longer in MAiD (11.5 versus 9.1 h; P = 0.021). The IEQ (P < 0.001) and percent trapped (P < 0.001) were higher in the DCD-III; however, MAiD islets demonstrated a higher purity (P = 0.020). Overall, 15 ITx were performed of whom 3 (8.8%) and 12 (25.5%) from MAiD and DCD-III, respectively (P = 0.056). Patients had a median fasting C-peptide of 0.51 ng/mL (interquartile range, 0.30-0.76 nmol/L), with no differences between groups (MAiD = 0.52 versus DCD-III = 0.51; P = 0.718). The median HbA1c was 6.2% (interquartile range, 5.7%-7%) (MAiD = 6.3% versus DCD-III = 6.1%; P = 0.815) and BETA2 scores (MAiD = 7.4 versus DCD-III = 12.8; P = 0.229) did not differ. Conclusions: ICI from MAiD donor pancreas may be successfully transplanted with comparable outcomes to DCD-III and may be used for research. These results justify additional efforts to consider MAiD as another valuable source of grafts for ITx. Further multicenter studies and larger clinical experience are needed to validate our findings.

Persistent hyperbilirubinemia following preoperative biliary stenting in patients undergoing anatomic hepatectomy predicts serious complications.

BACKGROUND: Biliary obstruction before liver resection is a known risk factor for post-operative complications. The aim of this study was to determine the impact of persistent hyperbilirubinemia following preoperative biliary drainage before liver resection. METHODS: The ACS-NSQIP (2016-2021) database was used to extract patients with cholangiocarcinoma who underwent anatomic liver resection with preoperative biliary drainage comparing those with persistent hyperbilirubinemia (> 1.2 mg/dL) to those with resolution. Patient characteristics and outcomes were compared with bivariate analysis. Multivariable modeling evaluated factors including persistent hyperbilirubinemia to evaluate their independent effect on serious complications, liver failure, and mortality. RESULTS: We evaluated 463 patients with 217 (46.9%) having hyperbilirubinemia (HB) despite biliary stenting. Bivariate analysis demonstrated that patients with HB had a higher rate of serious complications than those with non-HB (80.7% vs 70.3%; P = 0.010) including bile leak (40.9% vs 31.8%; P = 0.045), liver failure (26.7% vs 17.9%; P = 0.022), and bleeding (48.4% vs 36.6%; P = 0.010). Multivariable analysis demonstrated that persistent HB was independently associated with serious complications (OR 1.88, P = 0.020) and mortality (OR 2.39, P = 0.049) but not post-operative liver failure (OR 1.65, P = 0.082). CONCLUSIONS: Failed preoperative biliary decompression is a predictive factor for post-operative complications and mortality in patients undergoing hepatectomy and may be useful for preoperative risk stratification.

Mitochondrial DNA levels in perfusate and bile during ex vivo normothermic machine correspond with donor liver quality.

Ex vivo normothermic machine perfusion (NMP) preserves donor organs and permits real-time assessment of allograft health, but the most effective indicators of graft viability are uncertain. Mitochondrial DNA (mtDNA), released consequent to traumatic cell injury and death, including the ischemia-reperfusion injury inherent in transplantation, may meet the need for a biomarker in this context. We describe a real time PCR-based approach to assess cell-free mtDNA during NMP as a universal biomarker of allograft quality. Measured in the perfusate fluid of 29 livers, the quantity of mtDNA correlated with metrics of donor liver health including International Normalized Ratio (INR), lactate, and warm ischemia time, and inversely correlated with inferior vena cava (IVC) flow during perfusion. Our findings endorse mtDNA as a simple and rapidly measured feature that can inform donor liver health, opening the possibility to better assess livers acquired from extended criteria donors to improve organ supply.

Islet transplantation outcomes in type 1 diabetes and transplantation of HLA-DQ8/DR4: results of a single-centre retrospective cohort in Canada.

Background: In solid organ transplantation, HLA matching between donor and recipient is associated with superior outcomes. In islet transplantation, an intervention for Type 1 diabetes, HLA matching between donor and recipient is not performed as part of allocation. Susceptibility to Type 1 diabetes is associated with the presence of certain HLA types. This study was conducted to determine the impact of these susceptibility antigens on islet allograft survival. Methods: This is a single-centre retrospective cohort study. This cohort of transplant recipients (n = 268) received islets from 661 donor pancreases between March 11th, 1999 and August 29th, 2018 at the University of Alberta Hospital (Edmonton, AB, Canada). The frequency of the Type 1 diabetes susceptibility HLA antigens (HLA-A24, -B39, -DQ8, -DQ2 and-DQ2-DQA1∗05) in recipients and donors were determined. Recipient and donor HLA antigens were examined in relation to time to first C-peptide negative status/graft failure or last observation point. Taking into account multiple transplants per patient, we fitted a Gaussian frailty survival analysis model with baseline hazard function stratified by transplant number, adjusted for cumulative islet dose and other confounders. Findings: Across all transplants recipients of donors positive for HLA-DQ8 had significantly better graft survival (adjusted HRs 0.33 95% CI 0.17-0.66; p = 0.002). At first transplant only, donors positive for HLA-DQ2-DQA1∗05 had inferior graft survival (adjusted HR 1.96 95% CI 1.10-3.46); p = 0.02), although this was not significant in the frailty analysis taking multiple transplants into account (adjusted HR 1.46 95% CI 0.77-2.78; p = 0.25). Other HLA antigens were not associated with graft survival after adjustment for confounders. Interpretation: Our findings suggest islet transplantation from HLA-DQ8 donors is associated with superior graft outcomes. A donor positive for HLA-DQ2-DQA1∗05 at first transplant was associated with inferior graft survival but not when taking into account multiple transplants per recipient. The relevance of HLA-antigens on organ allocation needs further evaluation and inclusion in islet transplant registries and additional observational and interventional studies to evaluate the role of HLA-DQ8 in islet graft survival are required. Funding: None.

Cyclosporine A Does Not Mitigate Liver Ischemia/Reperfusion Injury in an Ex Vivo Porcine Model of Donation After Circulatory Death.

BACKGROUND Ischemia/reperfusion injury (IRI) is an inherent problem in organ transplantation, owing to the obligate period of ischemia that organs must endure. Cyclosporine A (CsA), though better know as an immunosuppressant, has been shown to mitigate warm IRI in a variety of organ types, including the liver. However, there is little evidence for CsA in preventing hepatic IRI in the transplant setting. MATERIAL AND METHODS In the present study, we tested the effect of CsA on hepatic IRI in a large-animal ex vivo model of donation after circulatory death (DCD). Porcine donors were pre-treated with either normal saline control or 20 mg/kg of CsA. Animals were subject to either 45 or 60 minutes of warm ischemia before hepatectomy, followed by 2 or 4 hours of cold storage prior to reperfusion on an ex vivo circuit. Over the course of a 12-hour perfusion, perfusion parameters were recorded and perfusate samples and biopsies were taken at regular intervals. RESULTS Peak perfusate lactate dehydrogenase was significantly decreased in the lower-ischemia group treated with CsA compared to the untreated group (4220 U/L [3515-5815] vs 11 305 [10 100-11 674]; P=0.023). However, no difference was seen between controls and CsA-treated groups on other parameters in perfusate alanine or asparagine aminotransferase (P=0.912, 0.455, respectively). Correspondingly, we found no difference on midpoint histological injury score (P=0.271). CONCLUSIONS We found minimal evidence that CsA is protective against hepatic IRI in our DCD model.

Inflammation-induced subcutaneous neovascularization for the long-term survival of encapsulated islets without immunosuppression.

Cellular therapies for type-1 diabetes can leverage cell encapsulation to dispense with immunosuppression. However, encapsulated islet cells do not survive long, particularly when implanted in poorly vascularized subcutaneous sites. Here we show that the induction of neovascularization via temporary controlled inflammation through the implantation of a nylon catheter can be used to create a subcutaneous cavity that supports the transplantation and optimal function of a geometrically matching islet-encapsulation device consisting of a twisted nylon surgical thread coated with an islet-seeded alginate hydrogel. The neovascularized cavity led to the sustained reversal of diabetes, as we show in immunocompetent syngeneic, allogeneic and xenogeneic mouse models of diabetes, owing to increased oxygenation, physiological glucose responsiveness and islet survival, as indicated by a computational model of mass transport. The cavity also allowed for the in situ replacement of impaired devices, with prompt return to normoglycemia. Controlled inflammation-induced neovascularization is a scalable approach, as we show with a minipig model, and may facilitate the clinical translation of immunosuppression-free subcutaneous islet transplantation.

Mitochondrial regulation in human pluripotent stem cells during reprogramming and ß cell differentiation.

Mitochondria are the powerhouse of the cell and dynamically control fundamental biological processes including cell reprogramming, pluripotency, and lineage specification. Although remarkable progress in induced pluripotent stem cell (iPSC)-derived cell therapies has been made, very little is known about the role of mitochondria and the mechanisms involved in somatic cell reprogramming into iPSC and directed reprogramming of iPSCs in terminally differentiated cells. Reprogramming requires changes in cellular characteristics, genomic and epigenetic regulation, as well as major mitochondrial metabolic changes to sustain iPSC self-renewal, pluripotency, and proliferation. Differentiation of autologous iPSC into terminally differentiated ß-like cells requires further metabolic adaptation. Many studies have characterized these alterations in signaling pathways required for the generation and differentiation of iPSC; however, very little is known regarding the metabolic shifts that govern pluripotency transition to tissue-specific lineage differentiation. Understanding such metabolic transitions and how to modulate them is essential for the optimization of differentiation processes to ensure safe iPSC-derived cell therapies. In this review, we summarize the current understanding of mitochondrial metabolism during somatic cell reprogramming to iPSCs and the metabolic shift that occurs during directed differentiation into pancreatic ß-like cells.

AT7867 promotes pancreatic progenitor differentiation of human iPSCs.

Generation of pure pancreatic progenitor (PP) cells is critical for clinical translation of stem cell-derived islets. Herein, we performed PP differentiation with and without AKT/P70 inhibitor AT7867 and characterized the resulting cells at protein and transcript level in vitro and in vivo upon transplantation into diabetic mice. AT7867 treatment increased the percentage of PDX1+NKX6.1+ (-AT7867: 50.9% [IQR 48.9%-53.8%]; +AT7867: 90.8% [IQR 88.9%-93.7%]; p = 0.0021) and PDX1+GP2+ PP cells (-AT7867: 39.22% [IQR 36.7%-44.1%]; +AT7867: 90.0% [IQR 88.2%-93.6%]; p = 0.0021). Transcriptionally, AT7867 treatment significantly upregulated PDX1 (p = 0.0001), NKX6.1 (p = 0.0005), and GP2 (p = 0.002) expression compared with controls, while off-target markers PODXL (p < 0.0001) and TBX2 (p < 0.0001) were significantly downregulated. Transplantation of AT7867-treated PPs resulted in faster hyperglycemia reversal in diabetic mice compared with controls (time and group: p < 0.0001). Overall, our data show that AT7867 enhances PP cell differentiation leading to accelerated diabetes reversal.

The Current Status of Allogenic Islet Cell Transplantation.

Type 1 Diabetes (T1D) is an autoimmune destruction of pancreatic beta cells. The development of the Edmonton Protocol for islet transplantation in 2000 revolutionized T1D treatment and offered a glimpse at a cure for the disease. In 2022, the 20-year follow-up findings of islet cell transplantation demonstrated the long-term safety of islet cell transplantation despite chronic immunosuppression. The Edmonton Protocol, however, remains limited by two obstacles: scarce organ donor availability and risks associated with chronic immunosuppression. To overcome these challenges, the search has begun for an alternative cell source. In 2006, pluripotency genomic factors, coined "Yamanaka Factors," were discovered, which reprogram mature somatic cells back to their embryonic, pluripotent form (iPSC). iPSCs can then be differentiated into specialized cell types, including islet cells. This discovery has opened a gateway to a personalized medicine approach to treating diabetes, circumventing the issues of donor supply and immunosuppression. In this review, we present a brief history of allogenic islet cell transplantation from the early days of pancreatic remnant transplantation to present work on encapsulating stem cell-derived cells. We review data on long-term outcomes and the ongoing challenges of allogenic islet cell and stem cell-derived islet cell transplant.

Suspension culture improves iPSC expansion and pluripotency phenotype.

BACKGROUND: Induced pluripotent stem cells (iPSCs) offer potential to revolutionize regenerative medicine as a renewable source for islets, dopaminergic neurons, retinal cells, and cardiomyocytes. However, translation of these regenerative cell therapies requires cost-efficient mass manufacturing of high-quality human iPSCs. This study presents an improved three-dimensional Vertical-Wheel® bioreactor (3D suspension) cell expansion protocol with comparison to a two-dimensional (2D planar) protocol. METHODS: Sendai virus transfection of human peripheral blood mononuclear cells was used to establish mycoplasma and virus free iPSC lines without common genetic duplications or deletions. iPSCs were then expanded under 2D planar and 3D suspension culture conditions. We comparatively evaluated cell expansion capacity, genetic integrity, pluripotency phenotype, and in vitro and in vivo pluripotency potential of iPSCs. RESULTS: Expansion of iPSCs using Vertical-Wheel® bioreactors achieved 93.8-fold (IQR 30.2) growth compared to 19.1 (IQR 4.0) in 2D (p < 0.0022), the largest expansion potential reported to date over 5 days. 0.5 L Vertical-Wheel® bioreactors achieved similar expansion and further reduced iPSC production cost. 3D suspension expanded cells had increased proliferation, measured as Ki67+ expression using flow cytometry (3D: 69.4% [IQR 5.5%] vs. 2D: 57.4% [IQR 10.9%], p = 0.0022), and had a higher frequency of pluripotency marker (Oct4+Nanog+Sox2+) expression (3D: 94.3 [IQR 1.4] vs. 2D: 52.5% [IQR 5.6], p = 0.0079). q-PCR genetic analysis demonstrated a lack of duplications or deletions at the 8 most commonly mutated regions within iPSC lines after long-term passaging (> 25). 2D-cultured cells displayed a primed pluripotency phenotype, which transitioned to naïve after 3D-culture. Both 2D and 3D cells were capable of trilineage differentiation and following teratoma, 2D-expanded cells generated predominantly solid teratomas, while 3D-expanded cells produced more mature and predominantly cystic teratomas with lower Ki67+ expression within teratomas (3D: 16.7% [IQR 3.2%] vs.. 2D: 45.3% [IQR 3.0%], p = 0.002) in keeping with a naïve phenotype. CONCLUSION: This study demonstrates nearly 100-fold iPSC expansion over 5-days using our 3D suspension culture protocol in Vertical-Wheel® bioreactors, the largest cell growth reported to date. 3D expanded cells showed enhanced in vitro and in vivo pluripotency phenotype that may support more efficient scale-up strategies and safer clinical implementation.

Stem Cell-Derived Islet Transplantation in Patients With Type 2 Diabetes: Can Diabetes Subtypes Guide Implementation?

Historically, only patients with brittle diabetes or severe recurrent hypoglycemia have been considered for islet transplantation (ITx). This population has been selected to optimize the risk-benefit profile, considering risks of long-term immunosuppression and limited organ supply. However, with the advent of stem cell (SC)-derived ITx and the potential for immunosuppression-free ITx, consideration of a broader recipient cohort may soon be justified. Simultaneously, the classical categorization of diabetes is being challenged by growing evidence in support of a clustering of disease subtypes that can be better categorized by the All New Diabetics in Scania (ANDIS) classification system. Using the ANDIS classification, 5 subtypes of diabetes have been described, each with unique causes and consequences. We evaluate consideration for ITx in the context of this broader patient population and the new classification of diabetes subtypes. In this review, we evaluate considerations for ITx based on novel diabetes subtypes, including their limitations, and we elaborate on unique transplant features that should now be considered to enable ITx in these "unconventional" patient cohorts. Based on evidence from those receiving whole pancreas transplant and our more than 20-year experience with ITx, we offer recommendations and potential research avenues to justify implementation of SC-derived ITx in broader populations of patients with all types of diabetes.

Long-term outcomes after normothermic machine perfusion in liver transplantation-Experience at a single North American center.

Normothermic machine perfusion (NMP) has emerged as a valuable tool in the preservation of liver allografts before transplantation. Randomized trials have shown that replacing static cold storage (SCS) with NMP reduces allograft injury and improves graft utilization. The University of Alberta's liver transplant program was one of the early adopters of NMP in North America. Herein, we describe our 7-year experience applying NMP to extend preservation time in liver transplantation using a "back-to-base" approach. From 2015 to 2021, 79 livers were transplanted following NMP, compared with 386 after SCS only. NMP livers were preserved for a median time of minutes compared with minutes in the SCS cohort (P < .0001). Despite this, we observed significantly improved 30-day graft survival (P = .030), although there were no differences in long-term patient survival, major complications, or biliary or vascular complications. We also found that although SCS time was strongly associated with increased graft failure at 1 year in the SCS cohort (P = .006), there was no such association among NMP livers (P = .171). Our experience suggests that NMP can safely extend the total preservation time of liver allografts without increasing complications.

COVID-19 Infection and Acute Pancreas Transplant Graft Thrombosis.

The coronavirus disease 2019 (COVID-19) pandemic created an unprecedented challenge for healthcare, and the world continues to struggle in recovering from its aftermath. COVID-19 has been clearly linked to hypercoagulable states and can lead to end-organ ischemia, morbidity, and mortality. Immunosuppressed solid organ transplant recipients represent a highly vulnerable population for the increased risk of complications and mortality. Early venous or arterial thrombosis with acute graft loss after whole pancreas transplantation is well-described, but late thrombosis is rare. We herein report a case of acute, late pancreas graft thrombosis at 13 years post pancreas-after-kidney (PAK) transplantation coinciding with an acute COVID-19 infection in a previously double-vaccinated recipient.

Successful Liver Transplantation After Neurologically Determined Death Donation Following Sodium Nitrite Poisoning.

Sodium nitrite poisoning has been reported with increasing frequency since 2017 and popularized on social media as an effective means to commit suicide. Though accidental, non-intentional consumption has been reported, it is uncommon. Sodium nitrite is a colorless, odorless, yellowish-white crystalline material that resembles table salt, is easily ingested for self-harm, and is readily accessible through purchase from online portals at low cost. The chemical is used industrially as a curing agent for meat, fish, and cheese, as it inhibits Clostridium botulinum and prevents botulism. We herein report a successful case of liver transplantation from an organ donor who suffered brain death after intentional sodium nitrite consumption. Despite conflicting evidence on sodium nitrite's toxic versus protective effects on the liver, our transplant recipient showed normal graft function in the four months following liver transplantation. It would have been helpful and reassuring to have had access to similar positive case reports when deciding to use such a donor.

Trends in types of graduate degrees and research output for academic general surgeons in Canada.

SummaryThe proportion of general surgeons with graduate degrees in Canada is increasing. We sought to evaluate the types of graduate degree held by surgeons in Canada, and whether differences in publication capacity exist. We evaluated all general surgeons working at English-speaking Canadian academic hospitals to determine the types of degrees achieved, changes over time and research output associated with each degree. We identified 357 surgeons, of whom 163 (45.7 %) had master's degrees and 49 (13.7 %) had PhDs. Achievement of graduate degrees increased over time, with more surgeons earning master's degrees in public health (MPH), clinical epidemiology and education (MEd), and fewer master's degrees in science (MSc) or PhDs. Most publication metrics were similar by degree type, but surgeons with PhDs published more basic science research than those with clinical epidemiology, MEd or MPH degrees (2.0 v. 0.0, p < 0.05); surgeons with clinical epidemiology degrees published more first-author articles than surgeons with MSc degrees (2.0 v. 0.0, p = 0.007). An increasing number of general surgeons hold graduate degrees, with fewer pursuing MSc and PhD degrees, and more holding MPH or clinical epidemiology degrees. Research productivity is similar for all groups. Support to pursue diverse graduate degrees could enable a greater breadth of research.

A DNA methylation atlas of normal human cell types.

DNA methylation is a fundamental epigenetic mark that governs gene expression and chromatin organization, thus providing a window into cellular identity and developmental processes1. Current datasets typically include only a fraction of methylation sites and are often based either on cell lines that underwent massive changes in culture or on tissues containing unspecified mixtures of cells2-5. Here we describe a human methylome atlas, based on deep whole-genome bisulfite sequencing, allowing fragment-level analysis across thousands of unique markers for 39 cell types sorted from 205 healthy tissue samples. Replicates of the same cell type are more than 99.5% identical, demonstrating the robustness of cell identity programmes to environmental perturbation. Unsupervised clustering of the atlas recapitulates key elements of tissue ontogeny and identifies methylation patterns retained since embryonic development. Loci uniquely unmethylated in an individual cell type often reside in transcriptional enhancers and contain DNA binding sites for tissue-specific transcriptional regulators. Uniquely hypermethylated loci are rare and are enriched for CpG islands, Polycomb targets and CTCF binding sites, suggesting a new role in shaping cell-type-specific chromatin looping. The atlas provides an essential resource for study of gene regulation and disease-associated genetic variants, and a wealth of potential tissue-specific biomarkers for use in liquid biopsies.

Pancreas and Islet Transplantation: Comparative Outcome Analysis of a Single-centre Cohort Over 20-years.

OBJECTIVE: To provide the largest single-center analysis of islet (ITx) and pancreas (PTx) transplantation. SUMMARY BACKGROUND DATA: Studies describing long-term outcomes with ITx and PTx are scarce. METHODS: We included adults undergoing ITx (n=266) and PTx (n=146) at the University of Alberta from January 1999 to October 2019. Outcomes include patient and graft survival, insulin independence, glycemic control, procedure-related complications, and hospital readmissions. Data are presented as medians (interquartile ranges, IQR) and absolute numbers (percentages, %) and compared using Mann-Whitney and χ2 tests. Kaplan-Meier estimates, Cox proportional hazard models and mixed main effects models were implemented. RESULTS: Crude mortality was 9.4% and 14.4% after ITx and PTx, respectively ( P= 0.141). Sex-adjusted and age-adjusted hazard-ratio for mortality was 2.08 (95% CI, 1.04-4.17, P= 0.038) for PTx versus ITx. Insulin independence occurred in 78.6% and 92.5% in ITx and PTx recipients, respectively ( P= 0.0003), while the total duration of insulin independence was 2.1 (IQR 0.8-4.6) and 6.7 (IQR 2.9-12.4) year for ITx and PTx, respectively ( P= 2.2×10 -22 ). Graft failure ensued in 34.2% and 19.9% after ITx and PTx, respectively ( P =0.002). Glycemic control improved for up to 20-years post-transplant, particularly for PTx recipients (group, P= 7.4×10 -7 , time, P =4.8×10 -6 , group*time, P= 1.2×10 -7 ). Procedure-related complications and hospital readmissions were higher after PTx ( P =2.5×10 -32 and P= 6.4×10 -112 , respectively). CONCLUSIONS: PTx shows higher sex-adjusted and age-adjusted mortality, procedure-related complications and readmissions compared with ITx. Conversely, insulin independence, graft survival and glycemic control are better with PTx. This study provides data to balance risks and benefits with ITx and PTx, which could improve shared decision-making.