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.

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.

Tumor necrosis factor receptor superfamily member 25 (TNFRSF25) agonists in islet transplantation: Endogenous in vivo regulatory T cell expansion promotes prolonged allograft survival.

Regulatory T cells (Tregs) modulate alloimmune responses and may facilitate minimization or withdrawal of immunosuppression posttransplant. Current approaches, however, rely on complex ex vivo Treg expansion protocols. Herein, we explore endogenous in vivo Treg expansion through antibody-mediated agonistic stimulation of the tumor necrosis factor receptor superfamily member 25 (TNFRSF25) pathway and its potential to prolong graft survival in a mouse model of islet allotransplantation. C57BL/6 male mice were treated with a single dose of TNFRSF25 agonistic antibodies (4C12 or mPTX-35) or IgG control. Diabetes was induced using streptozotocin. Four days later, flow cytometry was completed to corroborate Treg expansion, and 500 islets (CBA/J male mice) were transplanted. Glycemia was assessed thrice weekly until rejection/endpoint. Early intra-graft Treg infiltration was assessed 36 h posttransplant. TNFRSF25 antibodies enabled pronounced Treg expansion and treated mice had significantly prolonged graft survival compared with controls (p < .001). Additionally, the degree of Treg expansion significantly correlated with graft survival (p < .001). Immunohistochemistry demonstrated marked Treg infiltration in long-term surviving grafts; intra-graft Treg infiltration occurred early posttransplant. In conclusion, a single dose of TNFRSF25 antibodies enabled in vivo Treg expansion, which promotes prolonged graft survival. TNFRSF25-mediated in vivo Treg expansion could contribute to achieving lasting immunological tolerance in organ transplantation.

Current Status, Barriers, and Future Directions for Humanized Mouse Models to Evaluate Stem Cell-Based Islet Cell Transplant.

Islet cell transplant (ITx) continues to improve, with recently published long-term outcomes suggesting nearly 80% graft survival, leading to improvements in glycemic control, reductions in insulin doses, and near-complete abrogation of severe hypoglycemia. Unfortunately, access to ITx remains limited by immunosuppression requirements and donor supply. Discovery of stem cell-derived functional islet-like clusters with the capacity to reverse diabetes offers a renewable, potentially immunosuppression-free solution for future widespread ITx. Evaluation and optimization of these therapies is ongoing, but may one day provide a realistic cure for type 1 diabetes. However, stem cell-based ITx has unique immunologic questions that remain unanswered. Here, we briefly synthesize current approaches for stem cell-derived ITx, review humanized mice models, and elaborate on the potential of humanized mice models for bridging the gap between current small rodent models and human clinical trials for allogeneic and autologous inducible pluripotent stem cell (iPSC)-based ITx while highlighting limitations and future directions.

Total pancreatectomy with islet cell autotransplantation in a 2-year-old child with hereditary pancreatitis due to a PRSS1 mutation.

Acute recurrent and chronic pancreatitis in children carries high morbidity and burden. Compared to adults, ~75% of the cases of chronic pancreatitis in children are associated with underlying genetic mutations. The decision to intervene and the optimal timing poses unique challenges. Total pancreatectomy and islet cell autotransplantation (TPIAT) provides definitive therapy to relieve pain and improve quality of life while minimizing the risk of pancreatogenic diabetes. Substantial clinical data are available for adults; however, information on clinical outcomes in children remains scarce, particularly for very young children. Herein, we present an unusual, complex case of a 2-year-old child that underwent a successful TPIAT due to hereditary pancreatitis with an underlying mutation in PRSS1 gene, complicated by unremitting pancreatic ascites, hemorrhage, and sepsis. This is the youngest case to be reported in the literature. We provide a comprehensive report of the course and procedures implemented in this patient to guide other teams when considering these extraordinary measures in similar cases.

Frequency of Obliteration of the Dorsal and Ventral Ducts of the Pancreas in Islet Transplantation.

BACKGROUND: Islet isolation is an essential process in every human islet transplantation protocol. Intraductal enzyme delivery followed by adequate distention of the pancreas is the most critical step in islet isolation. Anomalies of the pancreatic duct system can significantly affect this process. Thus, identification and characterization of ductal patency is of paramount importance to achieve optimal islet isolation. AIMS: To investigate the frequency of duct obliteration in the human pancreas and explore donor/patient characteristics associated with specific ductal variations. METHODS: We examined ductal patency of pancreata allocated for islet allotransplantation (n = 597) and autotransplantation (n = 21) after removal of the duodenum during islet isolation procedure. Donor/patient factors were reviewed from the batch files. RESULTS: Among 559 deceased donor pancreata without pancreas divisum (n = 38, 6.4%), both ducts were patent in 50.1%, only ventral duct was patent in 46.7%, and only dorsal duct was patent in 3.2%. Donor age was not associated with the frequency of obliterated dorsal duct. Black race tended to have the higher frequency of patent dorsal duct. As expected, pancreas divisum was more frequent in chronic pancreatitis cases (n = 6, 28.6%). Within 7 cases of chronic pancreatitis with unknown etiology, we encountered one case of ventral duct obliteration. CONCLUSIONS: The minor duodenal papilla and aging do not likely play an important role in the occurrence of dorsal duct obliteration. Although frequency of obliterated ventral duct was low in our population, physicians, including gastroenterologists and endoscopists, as well as islet transplantation researchers should be aware of this possibility.

Update on islet cell transplantation.

PURPOSE OF REVIEW: Chronic diabetes-related complications continue to exert a rapidly growing and unsustainable pressure on healthcare systems worldwide. In type 1 diabetes, glycemic control is particularly challenging, as intensive management substantially increase the risk of severe hypoglycemic episodes. Alternative approaches to address this issue are required. Islet cell transplantation offers the best approach to reduce hypoglycemic risks and glycemic lability, while providing optimal glycemic control. Although ongoing efforts have improved clinical outcomes, the constraints in tissue sources and the need for chronic immunosuppression limit the application of islet cell transplantation as a curative therapy for diabetes. This review provides an update on islet cell transplantation, focusing on recent clinical experience, ongoing research, and future challenges. RECENT FINDINGS: Current evidence demonstrates advances in terms of long-term glycemic control, improved insulin independence rates, and novel approaches to eliminate chronic immunosuppression requirements after islet cell transplantation. Advances in stem cell-based therapies provide a promising path towards truly personalized regenerative therapies, solving both tissue supply shortage and the need for lifelong immunosuppression, enabling widespread use of this potentially curative treatment. However, as these therapies enter the clinical realm, regional access variability and ethical questions regarding commercialization are becoming increasingly important and require a collaborative solution. SUMMARY: In this state-of-the-art review, we discuss current clinical evidence and discuss key aspects on the present and future of islet cell transplantation.

Inactivation of SPAK kinase reduces body weight gain in mice fed a high-fat diet by improving energy expenditure and insulin sensitivity.

The STE20/SPS1-related proline-alanine-rich protein kinase (SPAK) controls the activity of the electroneutral cation-chloride cotransporters (SLC12 family) and thus physiological processes such as modulation of cell volume, intracellular chloride concentration [Cl-]i, and transepithelial salt transport. Modulation of SPAK kinase activity may have an impact on hypertension and obesity, as STK39, the gene encoding SPAK, has been suggested as a hypertension and obesity susceptibility gene. In fact, the absence of SPAK activity in mice in which the activating threonine in the T loop was substituted by alanine (SPAK-KI mice) is associated with decreased blood pressure; however its consequences in metabolism have not been explored. Here, we fed wild-type and homozygous SPAK-KI mice a high-fat diet for 17 wk to evaluate weight gain, circulating substrates and hormones, energy expenditure, glucose tolerance, and insulin sensitivity. SPAK-KI mice exhibit resistance to HFD-induced obesity and hepatic steatosis associated with increased energy expenditure, higher thermogenic activity in brown adipose tissue, increased mitochondrial activity in skeletal muscle, and reduced white adipose tissue hypertrophy mediated by augmented whole body insulin sensitivity and glucose tolerance. Our data reveal a previously unrecognized role for the SPAK kinase in the regulation of energy balance, thermogenesis, and insulin sensitivity, suggesting that this kinase could be a new drug target for the treatment of obesity and the metabolic syndrome.

Circumportal pancreas accompanied with pancreas divisum in a deceased donor for islet transplantation.

Circumportal pancreas is a rare and previously not well-recognized anatomical variant. In contrast, pancreas divisum is the most frequent anatomical variant in the pancreas. We report a case in which circumportal pancreas was accompanied with pancreas divisum in a deceased donor for islet transplantation. A unique aspect of our case is that the connecting portion behind the portal vein between the pancreas head and body originated from the ventral pancreas. This is the first case report describing a ventral embryogenic origin of the connecting portion of a circumportal pancreas accompanied with pancreas divisum.

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.

Scalable Bioreactor-based Suspension Approach to Generate Stem Cell-derived Islets From Healthy Donor-derived iPSCs.

BACKGROUND: Induced pluripotent stem cells (iPSCs) offer the potential to generate autologous iPSC-derived islets (iPSC islets), however, remain limited by scalability and product safety. METHODS: Herein, we report stagewise characterization of cells generated following a bioreactor-based differentiation protocol. Cell characteristics were assessed using flow cytometry, quantitative reverse transcription polymerase chain reaction, patch clamping, functional assessment, and in vivo functional and immunohistochemistry evaluation. Protocol yield and costs are assessed to determine scalability. RESULTS: Differentiation was capable of generating 90.4% PDX1+/NKX6.1+ pancreatic progenitors and 100% C-peptide+/NKX6.1+ iPSC islet cells. However, 82.1%, 49.6%, and 0.9% of the cells expressed SOX9 (duct), SLC18A1 (enterochromaffin cells), and CDX2 (gut cells), respectively. Explanted grafts contained mature monohormonal islet-like cells, however, CK19+ ductal tissues persist. Using this protocol, semi-planar differentiation using 150 mm plates achieved 5.72 × 104 cells/cm2 (total 8.3 × 106 cells), whereas complete suspension differentiation within 100 mL Vertical-Wheel bioreactors significantly increased cell yield to 1.1 × 106 cells/mL (total 105.0 × 106 cells), reducing costs by 88.8%. CONCLUSIONS: This study offers a scalable suspension-based approach for iPSC islet differentiation within Vertical-Wheel bioreactors with thorough characterization of the ensuing product to enable future protocol comparison and evaluation of approaches for off-target cell elimination. Results suggest that bioreactor-based suspension differentiation protocols may facilitate scalability and clinical implementation of iPSC islet therapies.

Human research islet cell culture outcomes at the Alberta Diabetes Institute IsletCore.

Human islets from deceased organ donors have made important contributions to our understanding of pancreatic endocrine function and continue to be an important resource for research studies aimed at understanding, treating, and preventing diabetes. Understanding the impacts of isolation and culture upon the yield of human islets for research is important for planning research studies and islet distribution to distant laboratories. Here, we examine islet isolation and cell culture outcomes at the Alberta Diabetes Institute (ADI) IsletCore (n = 197). Research-focused isolations typically have a lower yield of islet equivalents (IEQ), with a median of 252,876 IEQ, but a higher purity (median 85%) than clinically focused isolations before culture. The median recovery of IEQs after culture was 75%, suggesting some loss. This was associated with a shift toward smaller islet particles, indicating possible islet fragmentation, and occurred within 24 h with no further loss after longer periods of culture (up to 136 h). No overall change in stimulation index as a measure of islet function was seen with culture time. These findings were replicated in a representative cohort of clinical islet preparations from the Clinical Islet Transplant Program at the University of Alberta. Thus, loss of islets occurs within 24 h of isolation, and there is no further impact of extended culture prior to islet distribution for research.

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.

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.

Evaluating the Potential for ABO-incompatible Islet Transplantation: Expression of ABH Antigens on Human Pancreata, Isolated Islets, and Embryonic Stem Cell-derived Islets.

BACKGROUND: ABO-incompatible transplantation has improved accessibility of kidney, heart, and liver transplantation. Pancreatic islet transplantation continues to be ABO-matched, yet ABH antigen expression within isolated human islets or novel human embryonic stem cell (hESC)-derived islets remain uncharacterized. METHODS: We evaluated ABH glycans within human pancreata, isolated islets, hESC-derived pancreatic progenitors, and the ensuing in vivo mature islets following kidney subcapsular transplantation in rats. Analyses include fluorescence immunohistochemistry and single-cell analysis using flow cytometry. RESULTS: Within the pancreas, endocrine and ductal cells do not express ABH antigens. Conversely, pancreatic acinar tissues strongly express these antigens. Acinar tissues are present in a substantial portion of cells within islet preparations obtained for clinical transplantation. The hESC-derived pancreatic progenitors and their ensuing in vivo-matured islet-like clusters do not express ABH antigens. CONCLUSIONS: Clinical pancreatic islet transplantation should remain ABO-matched because of contaminant acinar tissue within islet preparations that express ABH glycans. Alternatively, hESC-derived pancreatic progenitors and the resulting in vivo-matured hESC-derived islets do not express ABH antigens. These findings introduce the potential for ABO-incompatible cell replacement treatment and offer evidence to support scalability of hESC-derived cell therapies in type 1 diabetes.

C-peptide Targets and Patient-centered Outcomes of Relevance to Cellular Transplantation for Diabetes.

BACKGROUND: C-peptide levels are a key measure of beta-cell mass following islet transplantation, but threshold values required to achieve clinically relevant patient-centered outcomes are not yet established. METHODS: We conducted a cross-sectional retrospective cohort study evaluating patients undergoing islet transplantation at a single center from 1999 to 2018. Cohorts included patients achieving insulin independence without hypoglycemia, those with insulin dependence without hypoglycemia, and those with recurrent symptomatic hypoglycemia. Primary outcome was fasting C-peptide levels at 6 to 12 mo postfirst transplant; secondary outcomes included stimulated C-peptide levels and BETA-2 scores. Fasting and stimulated C-peptide and BETA-2 cutoff values for determination of hypoglycemic freedom and insulin independence were evaluated using receiver operating characteristic curves. RESULTS: We analyzed 192 patients, with 122 (63.5%) being insulin independent without hypoglycemia, 61 (31.8%) being insulin dependent without hypoglycemia, and 9 (4.7%) experiencing recurrent symptomatic hypoglycemia. Patients with insulin independence had a median (interquartile range) fasting C-peptide level of 0.66 nmol/L (0.34 nmol/L), compared with 0.49 nmol/L (0.25 nmol/L) for those being insulin dependent without hypoglycemia and 0.07 nmol/L (0.05 nmol/L) for patients experiencing hypoglycemia ( P < 0.001). Optimal fasting C-peptide cutoffs for insulin independence and hypoglycemia were ≥0.50 nmol/L and ≥0.12 nmol/L, respectively. Cutoffs for insulin independence and freedom of hypoglycemia using stimulated C-peptide were ≥1.2 nmol/L and ≥0.68 nmol/L, respectively, whereas optimal cutoff BETA-2 scores were ≥16.4 and ≥5.2. CONCLUSIONS: We define C-peptide levels and BETA-2 scores associated with patient-centered outcomes. Characterizing these values will enable evaluation of ongoing clinical trials with islet or stem cell therapies.

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.

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.

Preclinical systematic review & meta-analysis of cyclosporine for the treatment of myocardial ischemia-reperfusion injury.

Background: Though best known for its immunosuppressant effects, cyclosporine A (CsA) has also been studied as a treatment to mitigate ischemia-reperfusion injury (IRI) by its inhibition of the mitochondria permeability transition pore (mPTP). Despite numerous preclinical studies supporting its benefit in reducing infarct size following myocardial IRI, large randomized controlled clinical trials have been unable to show a beneficial effect. Exploring existing preclinical data can give us the opportunity to revisit some the assumptions that may have led to the failure of these studies to translate clinically. Herein, we present a systematic review of preclinical studies testing CsA to attenuate myocardial IRI (PROSPERO CRD42020159620). Methods: We conducted a systematic search of health research databases Ovid MEDLINE, Ovid EMBASE, Web of Science BIOSIS, and Scopus, as well as Cochrane and PROSPERO systematic review databases, on March 9, 2022 for non-human in vivo animal studies of myocardial IRI, using CsA as a treatment that reported clinically relevant outcomes. Bias was assessed using the Systematic Review Centre for Laboratory Animal Experimentation's risk of bias tool and a modified Collaborative Approach to Meta Analysis and Review of Animal Data from Experimental Studies checklist. Sub-group meta-analyses were conducted to identify potential factors influencing outcomes. Results: We identified 71 studies, 59 of which were studies of coronary occlusion. Overall, 75% of studies reported a clear positive effect of CsA in mitigating myocardial IRI by some clinically relevant parameter (e.g., infarct size). A meta-analysis including 43 coronary occlusion studies showed an overall reduction in infarct size with CsA treatment (16.09%; 95% CI: -18.50% to -13.67%). Subgroup meta-analyses identified species, age, timing of administration, and duration of ischemia as factors potentially affecting the efficacy of CsA in the setting of myocardial IRI. Conclusions: Our systematic review and meta-analysis identifies questions that have yet to be answered by preclinical studies, highlighting important differences between these and clinical studies that should be addressed prior to proceeding with any further clinical studies using CsA to treat IRI in the heart or other organs. We also use the example of CsA to highlight general considerations for researchers attempting to translate animal studies into the clinical setting.