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The ability to preserve metabolically active kidneys ex vivo for multiple days may permit reconditioning, repair and regeneration of deceased donor kidneys. However, the kidneys high metabolic demand limits its functional preservation. Current approaches focus on normothermic machine perfusion (NMP) at 37°C or hypothermic machine perfusion (HMP) at 4-8°C. At normothermia, kidneys are metabolically active butex vivopreservation is limited to hours. During hypothermia kidneys can be preserved up to 24 hours but are metabolically inactive and suffer cold-induced injury. Therefore, we revisited sub normothermic perfusion (at 25°C) as an alternative approach to preserve human kidneys in a metabolically active state for extended periods of time.In a custom-made platform that includes a cell-free perfusate enriched with TCA cycle fuels, urine recirculation, and continuous hemofiltration we perfused discarded human kidneys up to 8 days. Using spatially resolved single cell resolution isotope tracing we demonstrate active metabolism in all the different renal cell types over this period. However, beyond 4 days cell composition of nephron segments assessed with spatial lipidomics changed substantially and injury markers such as NGAL and LDH increased in the perfusate. Up to 4 days, perfused human discarded donor kidneys maintained metabolic fluxes, functional parameters and allow for reperfusion using a porcine auto transplantation model. These data underpin that extended multi-day metabolic preservation of human kidneys is achievable using a sub normothermic perfusion platform.

Background. Successful pancreatic islet isolations are a key requirement for islet transplantation in selected patients with type 1 diabetes. However, islet isolation is a technically complex, time-consuming, and manual process. Optimization and simplification of the islet isolation procedure could increase islet yield and quality, require fewer operators, and thus reduce cost. Methods. We developed a new, closed system of tissue collection, washing, buffer change, and islet purification termed PancReatic Islet Separation Method (PRISM). In the developmental phase, pump and centrifuge speed was tested using microspheres with a similar size, shape, and density as digested pancreatic tissue. After optimization, PRISM was used to isolate islets from 10 human pancreases. Results. Islet equivalents viability (fluorescein diacetate/propidium iodide), morphology, and dynamic glucose-stimulated insulin secretion were evaluated. PRISM could be performed by 1 operator in 1 flow cabinet. A similar islet yield was obtained using PRISM compared to the traditional islet isolation method (431 234 +/- 292 833 versus 285 276 +/- 197 392 islet equivalents, P = 0.105). PRISM islets had similar morphology and functionality. Conclusions. PRISM is a novel islet isolation technique that can significantly improve islet isolation efficiency using fewer operators.

Pancreatic beta-cell failure is a critical event in the onset of both main types of diabetes mellitus but underlying mechanisms are not fully understood. beta-cells have low anti-oxidant capacity, making them more susceptible to oxidative stress. In type 1 diabetes (T1D), reactive oxygen species (ROS) are associated with pro-inflammatory conditions at the onset of the disease. Here, we investigated the effects of hydrogen peroxide-induced oxidative stress on human beta-cells. We show that primary human beta-cell function is decreased. This reduced function is associated with an ER stress response and the shuttling of FOXO1 to the nucleus. Furthermore, oxidative stress leads to loss of beta-cell maturity genes MAFA and PDX1, and to a concomitant increase in progenitor marker expression of SOX9 and HES1. Overall, we propose that oxidative stress-induced beta-cell failure may result from partial dedifferentiation. Targeting antioxidant mechanisms may preserve functional beta-cell mass in early stages of development of T1D.

Normothermic machine perfusion (NMP) is emerging as a novel preservation strategy. During NMP, the organ is maintained in a metabolically active state that may not only provide superior organ preservation, but that also facilitates viability testing before transplantation, and ex situ resuscitation of marginal kidney grafts. Although the prevailing perfusion protocols for renal NMP are refined from initial pioneering studies concerning short periods of NMP, it could be argued that these protocols are not optimally tailored to address the putatively compromised metabolic plasticity of marginal donor grafts (i.e., in the context of viability testing and/or preservation), or to meet the metabolic prerequisites associated with prolonged perfusions and the required anabolic state in the context of organ regeneration. Herein, we provide a theoretical framework for the metabolic requirements for renal NMP. Aspects are discussed along the lines of carbohydrates, fatty acids, amino acids, and micronutrients required for optimal NMP of an isolated kidney. In addition, considerations for monitoring aspects of metabolic status during NMP are discussed.

Summary Due to an increasing scarcity of pancreases with optimal donor characteristics, islet isolation centers utilize pancreases from extended criteria donors, such as from donation after circulatory death (DCD) donors, which are particularly susceptible to prolonged cold ischemia time (CIT). We hypothesized that hypothermic machine perfusion (HMP) can safely increase CIT. Five human DCD pancreases were subjected to 6 h of oxygenated HMP. Perfusion parameters, apoptosis, and edema were measured prior to islet isolation. Five human DBD pancreases were evaluated after static cold storage (SCS). Islet viability, and in vitro and in vivo functionality in diabetic mice were analyzed. Islets were isolated from HMP pancreases after 13.4 h [12.9–14.5] CIT and after 9.2 h [6.5–12.5] CIT from SCS pancreases. Histological analysis of the pancreatic tissue showed that HMP did not induce edema nor apoptosis. Islets maintained >90% viable during culture, and an appropriate in vitro and in vivo function in mice was demonstrated after HMP. The current study design does not permit to demonstrate that oxygenated HMP allows for cold ischemia extension; however, the successful isolation of functional islets from discarded human DCD pancreases after performing 6 h of oxygenated HMP indicates that oxygenated HMP may be a useful technology for better preservation of pancreases., Doppenberg and Leemkuil et al. demonstrate clinically relevant human pancreatic islet isolation results, in vitro and in vivo, after six hours oxygenated hypothermic machine perfusion of discarded DCD pancreases during transport.

As a treatment for type I diabetes, clinical islet transplantation (CIT) in which donor islets of Langerhans are transplanted intrahepatically has become a viable option for patients. However, the success of this procedure is limited by factors including ischemia, host immunological factors, and delayed vascularization of the hypoxia-sensitive islets. One solution would be to use a synthetic polymer scaffold as a carrier for the transplanted islets, as it would allow for their transplantation into a more favorable environment and could protect the cells from host immune reactions. To realize this potential solution, it is important that the synthetic polymer used does not interfere with the functionality and survival of the islets. In order to determine which synthetic polymers best meet this requirement, we examined the interactions of human islets from six donors with four clinically approved materials: polyetheretherketone (PEEK), polyvinylidene fluoride (PVDF), polyphenylsulfone (PPSU) and polysulfone (PSU) in vitro. Human islet morphology, viability, insulin secretion, functionality and gene expression were investigated to assess the suitability of these synthetic polymers as a carrier for transplanted islets. We found three of the synthetic synthetic polymers (PEEK, PPSU and PVDF) showed promise based on their overall performance, while the glucose responsiveness of islets cultured on PSU resulted in significantly reduced insulin secretion from five of six donors. Our findings demonstrate that close examination of human islets and their interaction with synthetic polymers is an important factor to consider when selecting synthetic polymers for engineering islet replacement devices.

Due to a shortage of donation after brain death (DBD) organs, donation after circulatory death (DCD) is increasingly performed. In the field of islet transplantation, there is uncertainty regarding the suitability of DCD pancreas in terms of islet yield and function after islet isolation. The aim of this study was to investigate the potential use of DCD pancreas for islet transplantation. Islet isolation procedures from 126 category 3 DCD and 258 DBD pancreas were performed in a 9-year period. Islet yield after isolation was significantly lower for DCD compared to DBD pancreas (395 515 islet equivalents [IEQ] and 480 017 IEQ, respectively; p = .003). The decrease in IEQ during 2 days of culture was not different between the two groups. Warm ischemia time was not related to DCD islet yield. In vitro insulin secretion after a glucose challenge was similar between DCD and DBD islets. After islet transplantation, DCD islet graft recipients had similar graft function (AUC C-peptide) during mixed meal tolerance tests and Igls score compared to DBD graft recipients. In conclusion, DCD islets can be considered for clinical islet transplantation.

The clinical success rate of islet transplantation, namely independence from insulin injections, is limited by factors that lead to graft failure, including inflammation, acute ischemia, acute phase response, and insufficient vascularization. The ischemia and insufficient vascularization both lead to high levels of oxidative stress, which are further aggravated by islet encapsulation, inflammation, and undesirable cell-biomaterial interactions. To identify biomaterials that would not further increase damaging oxidative stress levels and that are also suitable for manufacturing a beta cell encapsulation device, we studied five clinically approved polymers for their effect on oxidative stress and islet (alpha and beta cell) function. We found that 300 poly(ethylene oxide terephthalate) 55/poly(butylene terephthalate) 45 (PEOT/PBT300) was more resistant to breakage and more elastic than other biomaterials, which is important for its immunoprotective function. In addition, it did not induce oxidative stress or reduce viability in the MIN6 beta cell line, and even promoted protective endogenous antioxidant expression over 7 days. Importantly, PEOT/PBT300 is one of the biomaterials we studied that did not interfere with insulin secretion in human islets.

Macroencapsulation of islets of Langerhans is a promising strategy for transplantation of insulin-producing cells in the absence of immunosuppression to treat type 1 diabetes. Hollow fiber membranes are of interest there because they offer a large surface-to-volume ratio and can potentially be retrieved or refilled. However, current available fibers have limitations in exchange of nutrients, oxygen, and delivery of insulin potentially impacting graft survival. Here, multibore hollow fibers for islets encapsulation are designed and tested. They consist of seven bores and are prepared using nondegradable polymers with high mechanical stability and low cell adhesion properties. Human islets encapsulated there have a glucose induced insulin response (GIIS) similar to nonencapsulated islets. During 7 d of cell culture in vitro, the GIIS increases with graded doses of islets demonstrating the suitability of the microenvironment for islet survival. Moreover, first implantation studies in mice demonstrate device material biocompatibility with minimal tissue responses. Besides, formation of new blood vessels close to the implanted device is observed, an important requirement for maintaining islet viability and fast exchange of glucose and insulin. The results indicate that the developed fibers have high islet bearing capacity and can potentially be applied for a clinically applicable bioartificial pancreas.

In many countries, there are an insufficient number of organs retrieved from donation after brain death (DBD) procedures in order to keep time on deceased donor organ waiting lists acceptable. Organs from donation after circulatory death (DCD) procedures could help to relieve this pressure. In some countries, DCD pancreas is increasingly used for both vascularized pancreas and islet transplantation. With more stringent donor pancreas acceptance criteria, preliminary results indicate that islet isolation from DCD pancreas results in a somewhat reduced number of isolated islets, but no consistent difference in functionality in vitro or clinical outcome in islet transplant recipients. Although more data on clinical outcome are clearly needed, islets from DCD donors can be considered for clinical islet transplantation.

While it is well recognized that obesity is associated with an increased β-cell mass, the association with α-cell mass is less clear. Type 2 diabetes (T2DM) associated with obesity is a bihormonal disease characterized by inadequate insulin secretion and hyperglucagonaemia. We examined β- and α-cell mass throughout the pancreas in obese and lean subjects. Pancreatic tissue of the head, body and tail region of the pancreas was examined from 15 obese subjects (body mass index [BMI] ≥ 27 kg/m2 ) and 15 age-matched lean subjects (BMI ≤ 25 kg/m2 ) without diabetes. In obese subjects both β- and α-cell mass were proportionally higher compared with lean subjects, thereby maintaining the α- to β-cell ratio. The adaptation to obesity occurred preferentially in the head of the pancreas. As data so far have been derived from histological studies of β- and α-cell adaptation, in which the head region of the human pancreas was not included, the adaptive capacity of humans to obesity has previously been underestimated. Obesity is associated with an increased α-cell mass, which could contribute to the hyperglucagonaemia observed in people with T2DM.

The clinical success of islet transplantation is limited by factors including acute ischemia, stress upon transplantation, and delayed vascularization. Islets experience high levels of oxidative stress due to delayed vascularization after transplantation and this can be further aggravated by their encapsulation and undesirable cell-biomaterial interactions. To identify biomaterials that would not further increase oxidative stress levels and that are also suitable for manufacturing a beta cell encapsulation device, we studied five clinically approved polymers for their effect on oxidative stress and islet (alpha and beta cell) function. We found that 300 poly(ethylene oxide terephthalate) 55/poly(butylene terephthalate) 45 (PEOT/PBT300) was more resistant to breakage and more elastic than other biomaterials, which is important for its immunoprotective function. In addition, PEOT/PBT300 did not induce oxidative stress or reduce viability in MIN6 beta cells, and even promoted protective endogenous antioxidant expression over 7 days. Importantly, PEOT/PBT300 is one of the biomaterials we studied that did not interfere with insulin secretion in human islets. These data indicate that PEOT/PBT300 may be a suitable biomaterial for an islet encapsulation device.

Intrahepatic islet transplantation is a promising therapy for treatment of type 1 diabetes. During islet isolation, collagenase is used to extract islets from the pancreas, leading to loss of important cell-matrix interactions. Loss of the native pancreatic microenvironment is associated with apoptosis of islet cells, early graft failure, and poor islet function. The islet extracellular matrix (ECM) is composed of a specific combination of collagen (Col), laminin (LN), and fibronectin (FN) molecules. Reintroducing these molecules has been shown to boost the function, viability, morphology, and proliferation of beta-cells. In this research, the effect of combinatorial ECM on islet function and survival was investigated. Specifically, thin-film microwell array scaffolds made from two distinct biomaterials were coated with FN, collagen type IV (Col4), LN111, LN332, or a combination thereof. We found that coatings containing a single type of ECM molecule, for example, FN or Col, can improve short-term islet function. However, these single proteins do not prevent loss of morphology and subsequent loss of islet function afterward. In contrast, combining Col4 with LN111 at a ratio of 8:2 not only improved short-term islet function but also preserved islet structure and islet function on a longer term. This effect was reproducibly shown on poly(ester-urethane) and poly(ethylene-glycol-terephthalate-poly(butylene-terephthalate) microwell islet delivery devices as well as tissue culture polystyrene. We concluded that biofunctionalization of inert biomaterials regardless of their molecular composition with a specific combination of islet ECM molecules can support and improve islet function over longer time periods. Our data suggested that creating a biomimetic islet niche by biofunctionalization of biomaterials can significantly improve the endocrine function of beta-cells. The creation of islet mimicking niches in islet delivery devices leads to an improvement of islet function by restoring part of the islet's ECM in these devices. Impact Statement This research deals with finding a proper bioengineering strategy to improve the outcome of islets transplantation for treatment of type 1 diabetes. It is focused on the mimicking of islet extracellular matrix niche in microwell islet delivery devices to improve their endocrine function.

The bioengineering of a replacement kidney has been proposed as an approach to address the growing shortage of donor kidneys for the treatment of chronic kidney disease. One approach being investigated is the recellularization of kidney scaffolds. In this study, we present several key advances toward successful re‐endothelialization of whole kidney matrix scaffolds from both rodents and humans. Based on the presence of preserved glycosoaminoglycans within the decelullarized kidney scaffold, we show improved localization of delivered endothelial cells after preloading of the vascular matrix with vascular endothelial growth factor and angiopoietin 1. Using a novel simultaneous arteriovenous delivery system, we report the complete re‐endothelialization of the kidney vasculature, including the glomerular and peritubular capillaries, using human inducible pluripotent stem cell –derived endothelial cells. Using this source of endothelial cells, it was possible to generate sufficient endothelial cells to recellularize an entire human kidney scaffold, achieving efficient cell delivery, adherence, and endothelial cell proliferation and survival. Moreover, human re‐endothelialized scaffold could, in contrast to the non‐re‐endothelialized human scaffold, be fully perfused with whole blood. These major advances move the field closer to a human bioengineered kidney., Human decellularized kidney scaffolds can be functionally re‐endothelialized using expanded human pluripotent stem cell–derived endothelial cells.

Simultaneous pancreas-kidney (SPK) transplantation is an important treatment option for patients with type 1 diabetes (T1D) and end-stage renal disease (ESRD). Due to complications, in up to 10% of patients, allograft pancreatectomy is necessary shortly after transplantation. Usually the donor pancreas is discarded. Here, we report on a novel procedure to rescue endocrine tissue after allograft pancreatectomy. A 39-year-old woman with T1D and ESRD who had undergone SPK transplantation required emergency allograft pancreatectomy due to bleeding at the vascular anastomosis. Islets were isolated from the removed pancreas allograft, and almost 480 000 islet equivalents were infused into the portal vein. The patient recovered fully. After 3 months, near-normal mixed meal test (fasting glucose 7.0 mmol/L, 2-hour glucose 7.5 mmol/L, maximal stimulated C-peptide 3.25 nmol/L, without insulin use in the preceding 36 hours) was achieved. Glycated hemoglobin while taking a low dose of long-acting insulin was 32.7 mmol/mol hemoglobin (5.3%). When a donor pancreas is lost after transplantation, rescue β cell therapy by islet alloautotransplantation enables optimal use of scarce donor pancreata to optimize glycemic control without additional HLA alloantigen exposure.

Summary Generating an unlimited source of human insulin-producing cells is a prerequisite to advance β cell replacement therapy for diabetes. Here, we describe a 3D culture system that supports the expansion of adult human pancreatic tissue and the generation of a cell subpopulation with progenitor characteristics. These cells display high aldehyde dehydrogenase activity (ALDHhi), express pancreatic progenitors markers (PDX1, PTF1A, CPA1, and MYC), and can form new organoids in contrast to ALDHlo cells. Interestingly, gene expression profiling revealed that ALDHhi cells are closer to human fetal pancreatic tissue compared with adult pancreatic tissue. Endocrine lineage markers were detected upon in vitro differentiation. Engrafted organoids differentiated toward insulin-positive (INS+) cells, and circulating human C-peptide was detected upon glucose challenge 1 month after transplantation. Engrafted ALDHhi cells formed INS+ cells. We conclude that adult human pancreatic tissue has potential for expansion into 3D structures harboring progenitor cells with endocrine differentiation potential., Highlights • A 3D culture system can support the expansion of adult human pancreatic tissue • An ALDHhi cell subpopulation is identified in these organoids • ALDHhi cells, and not ALDHlo cells, are capable of forming new organoids • ALDHhi cells show endocrine differentiation potential, In the context of β cell replacement therapy for diabetes, de Koning and colleagues describe a 3D culture platform that supports ex vivo expansion of human pancreatic tissue as organoids. These organoids harbor a subpopulation of ALDHhi cells that display proliferative capacity and can differentiate to an endocrine fate.

Background Obesity and type 2 diabetes have not only been linked to fatty liver, but also to fatty kidney and chronic kidney disease. Since non-invasive tools are lacking to study fatty kidney in clinical studies, we explored agreement between proton magnetic resonance spectroscopy (1 H-MRS) and enzymatic assessment of renal triglyceride content (without and with dietary intervention). We further studied the correlation between fatty kidney and fatty liver. Methods Triglyceride content in the renal cortex was measured by 1 H-MRS on a 7-Tesla scanner in 27 pigs, among which 15 minipigs had been randomized to a 7-month control diet, cafeteria diet (CAF) or CAF with low-dose streptozocin (CAF-S) to induce insulin-independent diabetes. Renal biopsies were taken from corresponding MRS-voxel locations. Additionally, liver biopsies were taken and triglyceride content in all biopsies was measured by enzymatic assay. Results Renal triglyceride content measured by 1 H-MRS and enzymatic assay correlated positively (r = 0.86, P < 0.0001). Compared with control diet-fed minipigs, renal triglyceride content was higher in CAF-S-fed minipigs (137 ± 51 nmol/mg protein, mean ± standard error of the mean, P < 0.05), but not in CAF-fed minipigs (60 ± 10 nmol/mg protein) compared with controls (40 ± 6 nmol/mg protein). Triglyceride contents in liver and kidney biopsies were strongly correlated (r = 0.97, P < 0.001). Conclusions Non-invasive measurement of renal triglyceride content by 1 H-MRS closely predicts triglyceride content as measured enzymatically in biopsies, and fatty kidney appears to develop parallel to fatty liver. 1 H-MRS may be a valuable tool to explore the role of fatty kidney in obesity and type 2 diabetic nephropathy in humans in vivo.

Islets of Langerhans need to maintain their round morphology and to be fast revascularized after transplantation to preserve functional insulin secretion in response to glucose stimulation. For this purpose, a non-cell-adhesive environment is preferable for their embedding. Conversely, nutrient and oxygen supply to islets is guaranteed by capillary ingrowth within the construct and this can only be achieved in a matrix that provides adhesion cues for cells. In this study, two different approaches are explored, which are both based on a layered architecture, in order to combine these two opposite requirements. A non-adhesive islet encapsulation layer is based on polyethyleneglycole diacrylate (PEGDA). This first layer is combined with a second hydrogel based on thiolated-gelatin, thiolated-heparin and thiolated-hyaluronic acid providing cues for endothelial cell adhesion and acting as a growth factor releasing matrix. In an alternative approach, a conformal PEGDA coating is covalently applied on the surface of the islets. The coated islets are subsequently embedded in the previously mentioned hydrogel containing thiolated glycosaminoglycans. The suitability of this approach as a matrix for controlled growth factor release has been demonstrated by studying the controlled release of VEGF and bFGF for 14 days. Preliminary tube formation has been quantified on the growth factor loaded hydrogels. This approach should facilitate blood vessel ingrowth towards the embedded islets and maintain islet round morphology and functionality upon implantation. Graphical abstract

We recently demonstrated that the adult human kidney cortex contains a perivascular stromal cell (kPSC) that shows organotypic properties and is important for repair and stabilisation of kidney function. Not only the kidney cortex but also the kidney capsule contains stromal cells that are important for the three dimensional organisation of the kidney during nephrogenesis. They provide the barrier function of the capsule which is critical for homeostatic processes such as pressure natriuresis. We postulated that stromal cells derived from the kidney capsule may therefore also have specific properties and functions. To this end, we isolated these capsule mesenchymal stromal cells (cMSC) from human cadaveric kidneys that were not suitable for transplantation. There were several similarities between cMSCs and kPSCs including support of vascular plexus formation, phenotypic marker expression and resistance against myofibroblast transformation. However, compared to kPSCs, cMSCs showed distinct mRNA and miRNA expression profiles, showed increased immunosuppressive capacity, and displayed strongly reduced HGF production, contributing to the inability to enhance kidney epithelial repair. Therefore cMSCs are a distinct, novel human kidney-derived MSC-population and these data underpin the large functional diversity of phenotypic similar stromal cells in relation to their anatomic site, even within one organ.

Contains fulltext : 177862.pdf (Publisher’s version ) (Open Access) Allogeneic islet transplantation into the liver in combination with immune suppressive drug therapy is widely regarded as a potential cure for type 1 diabetes. However, the intrahepatic system is suboptimal as the concentration of drugs and nutrients there is higher compared to pancreas, which negatively affects islet function. Islet encapsulation within semipermeable membranes is a promising strategy that allows for the islet transplantation outside the suboptimal liver portal system and provides environment, where islets can perform their endocrine function. In this study, we develop a macroencapsulation device based on thin microwell membranes. The islets are seeded in separate microwells to avoid aggregation, whereas the membrane porosity is tailored to achieve sufficient transport of nutrients, glucose and insulin. The non-degradable, microwell membranes are composed of poly (ether sulfone)/polyvinylpyrrolidone and manufactured via phase separation micro molding. Our results show that the device prevents aggregation and preserves the islet's native morphology. Moreover, the encapsulated islets maintain their glucose responsiveness and function after 7 days of culture (stimulation index above 2 for high glucose stimulation), demonstrating the potential of this novel device for islet transplantation.

Mesenchymal Stromal Cells (MSCs) are tissue homeostatic and immune modulatory cells that have shown beneficial effects in kidney diseases and transplantation. Perivascular Stromal Cells (PSCs) share characteristics with bone marrow MSCs (bmMSCs). However, they also possess, most likely due to local imprinting, tissue-specific properties and play a role in local tissue homeostasis. This tissue specificity may result in tissue specific repair, also within the human kidney. We previously showed that human kidney PSCs (kPSCs) have enhanced kidney epithelial wound healing whereas bmMSCs did not have this potential. Moreover, kPSCs can ameliorate kidney injury in vivo. Therefore, kPSCs constitute an interesting source for cell therapy, particularly for kidney diseases and renal transplantation. Here we show the detailed isolation and culture method for kPSCs from transplant-grade human kidneys based on whole-organ perfusion of digestive enzymes via the renal artery and enrichment for the perivascular marker NG2. In this way, large cell quantities can be obtained that are suitable for cellular therapy.

Mesenchymal stromal cells (MSCs) are immunomodulatory and tissue homeostatic cells that have shown beneficial effects in kidney diseases and transplantation. Perivascular stromal cells (PSCs) identified within several different organs share characteristics of bone marrow-derived MSCs (BM-MSCs). These PSCs may also possess tissue-specific properties and play a role in local tissue homeostasis. We hypothesized that human kidney-derived PSCs (hkPSCs) would elicit improved kidney repair in comparison with BM-MSCs. Here we introduce a novel, clinical-grade isolation method of hkPSCs from cadaveric kidneys by enriching for the perivascular marker, NG2. hkPSCs show strong transcriptional similarities to BM-MSCs but also show organotypic expression signatures, including the HoxD10 and HoxD11 nephrogenic transcription factors. Comparable to BM-MSCs, hkPSCs showed immunosuppressive potential and, when cocultured with endothelial cells, vascular plexus formation was supported, which was specifically in the hkPSCs accompanied by an increased NG2 expression. hkPSCs did not undergo myofibroblast transformation after exposure to transforming growth factor-β, further corroborating their potential regulatory role in tissue homeostasis. This was further supported by the observation that hkPSCs induced accelerated repair in a tubular epithelial wound scratch assay, which was mediated through hepatocyte growth factor release. In vivo, in a neonatal kidney injection model, hkPSCs reintegrated and survived in the interstitial compartment, whereas BM-MSCs did not show this potential. Moreover, hkPSCs gave protection against the development of acute kidney injury in vivo in a model of rhabdomyolysis-mediated nephrotoxicity. Overall, this suggests a superior therapeutic potential for the use of hkPSCs and their secretome in the treatment of kidney diseases.

Islet transplantation is a promising therapy for type 1 diabetes, but graft function and survival are compromised by recurrent islet autoimmunity. Immunoprotection of islets will be required to improve clinical outcome. We engineered human beta cells to express herpesvirus-encoded immune-evasion proteins, "immunevasins." The capacity of immunevasins to protect beta cells from autoreactive T-cell killing was evaluated in vitro and in vivo in humanized mice. Lentiviral vectors were used for efficient genetic modification of primary human beta cells without impairing their function. Using a novel beta-cell-specific reporter gene assay, we show that autoreactive cytotoxic CD8(+) T-cell clones isolated from patients with recent onset diabetes selectively destroyed human beta cells, and that coexpression of the human cytomegalovirus-encoded US2 protein and serine proteinase inhibitor 9 offers highly efficient protection in vitro. Moreover, coimplantation of these genetically modified pseudoislets with beta-cell-specific cytotoxic T cells into immunodeficient mice achieves preserved human insulin production and C-peptide secretion. Collectively, our data provide proof of concept that human beta cells can be efficiently genetically modified to provide protection from killing mediated by autoreactive T cells and retain their function in vitro and in vivo.

The liver is currently the site for transplantation of islets in humans. This is not optimal for islets, but alternative sites in humans are not available. Polymeric scaffolds in surgically accessible areas are a solution. As human donors are rare, the polymers should not interfere with functional survival of human-islets. We applied a novel platform to test the adequacy of polymers for application in scaffolds for human-islet transplantation. Viability, functionality, and immune parameters were included to test poly(D,L-lactide-co-ε-caprolactone) (PDLLCL), poly(ethylene oxide terephthalate)/polybutylene terephthalate (PEOT/PBT) block copolymer, and polysulfone. The type of polymer influenced the functional survival of human islets. In islets cultured on PDLLCL the glucagon-producing α-cells and insulin-producing β-cells contained more hormone granules than in islets in contact with PEOT/PBT or polysulfone. This was studied with ultrastructural analysis by electron microscopy (nanotomy) during 7 d of culture. PDLLCL was also associated with statistically significant lower release of double-stranded DNA (dsDNA, a so called danger-associate molecular pattern (DAMP)) from islets on PDLLCL when compared to the other polymers. DAMPs support undesired immune responses. Hydrophilicity of the polymers did not influence dsDNA release. Islets on PDLLCL also showed less cellular outgrowth. These outgrowing cells were mainly fibroblast and some β-cells undergoing epithelial to mesenchymal cell transition. None of the polymers influenced the glucose-stimulated insulin secretion. As PDLLCL was associated with less release of DAMPs, it is a promising candidate for creating a scaffold for human islets. Our study demonstrates that for sensitive, rare cadaveric donor tissue such as pancreatic islets it might be necessary to first select materials that do not influence functionality before proposing the biomaterial for in vivo application. Our presented platform may facilitate this selection of biomaterials.

Contains fulltext : 172329.pdf (Publisher’s version ) (Closed access) Derailment of immune responses can lead to autoimmune type 1 diabetes, and this can be accelerated or even induced by local stress caused by inflammation or infection. Dendritic cells (DCs) shape both innate and adaptive immune responses. Here, we report on the responses of naturally occurring human myeloid BDCA1(+) DCs towards differentially stressed pancreatic beta cells. Our data show that BDCA1(+) DCs in human pancreas-draining lymph node (pdLN) suspensions and blood-derived BDCA1(+) DCs both effectively engulf beta cells, thus mimicking physiological conditions. Upon uptake of enterovirus-infected, but not mock-infected cells, BDCA1(+) DCs induced interferon (IFN)-alpha/beta responses, co-stimulatory molecules and proinflammatory cytokines and chemokines. Notably, induction of stress in beta cells by ultraviolet irradiation, culture in serum-free medium or cytokine-induced stress did not provoke strong DC activation, despite efficient phagocytosis. DC activation correlated with the amount of virus used to infect beta cells and required RNA within virally infected cells. DCs encountering enterovirus-infected beta cells, but not those incubated with mock-infected or stressed beta cells, suppressed T helper type 2 (Th2) cytokines and variably induced IFN-gamma in allogeneic mixed lymphocyte reaction (MLR). Thus, stressed beta cells have little effect on human BDCA1(+) DC activation and function, while enterovirus-infected beta cells impact these cells significantly, which could help to explain their role in development of autoimmune diabetes in individuals at risk.

Although regarded as a promising treatment for type 1 diabetes, clinical islet transplantation in the portal vein is still hindered by a low transplantation outcome. Alternative transplantation sites have been proposed, but the survival of extra-hepatically transplanted islets of Langerhans critically depends on quick revascularization after engraftment. This study aims at developing a new 3D scaffold platform that can actively boost vascularization and may find an application for extra-hepatic islet transplantation. The construct consists of a 3D ring-shaped polycaprolactone (PCL) scaffold with heparinized surface to electrostatically bind vascular endothelial growth factor (VEGF), surrounding a hydrogel core for islets encapsulation. Heparin immobilization improves the amount of VEGF retained by the construct, up to 3.6 fold, compared to untreated PCL scaffolds. In a chicken chorioallanthoic membrane model, VEGF immobilized on the construct enhances angiogenesis in close proximity and on the surface of the scaffolds. After 7 days, islets encapsulated in the alginate core show functional response to glucose stimuli comparable to free-floating islets. Thus, the developed platform has the potential to support rapid vascularization and islet endocrine function.

Pancreatic islet transplantation is performed in a select group of patients with type 1 diabetes mellitus. Immunosuppressive regimens play an important role in long-term islet function. We aimed to investigate the efficacy of islet transplantation in patients with type 1 diabetes and a previous kidney transplantation using an alemtuzumab-based induction regimen and triple maintenance immunosuppression. Patients with type 1 diabetes, who had received a kidney transplant previously, were treated with alemtuzumab as induction therapy for their first islet transplantation and basiliximab induction therapy for subsequent islet transplantations. Maintenance immunosuppression consisted of triple immunosuppression (tacrolimus, mycophenolate mofetil, and prednisolone). Thirteen patients (age 50.9 ± 9.2 years, duration of diabetes 35 ± 9 years) received a total of 22 islet transplantations. One- and 2-year insulin independence was 62% and 42%, respectively; graft function was 100% and 92%, respectively. HbA1c dropped from 57.2 ± 13.1 (7.4 ± 1.2%) to 44.5 ± 11.8 mmol/molHb (6.2 ± 0.9%) (p = 0.003) after 2 years. Six of 13 patients suffered from severe hypoglycemia before islet transplantation. After transplantation, severe hypoglycemia was restricted to the only patient who lost graft function. Creatinine clearance was unchanged. Islet-after-kidney transplantation in patients with type 1 diabetes using an alemtuzumab-based induction regimen leads to considerable islet allograft function and improvement in glycemic control.

While subcutaneous tissue has been proposed as a clinically relevant site for pancreatic islet transplantation, a major issue of concern remains, which is its poor vascular state. In an effort to overcome this limitation, we present an efficient and reproducible method to form human composite islets (CIs) with proangiogenic cell types in a controlled manner using nonadherent agarose microwell templates. In this study, we assessed the three-dimensional structure, function, and angiogenic potential of human CIs with human mesenchymal stromal cells (hMSCs), with or without human umbilical vein endothelial cells (HUVECs), and preconditioned hMSCs (PC-hMSCs) in EGM-2 under shear stress. Distinct cellular rearrangements could be observed in CIs, but islet functionality was maintained. In vitro angiogenesis assays found significantly enhanced sprout formation in case of CIs. In particular, the number of sprouts emanating from CIs with PC-hMSCs was significantly increased compared to other conditions. Subsequent in vivo assessment confirmed the proangiogenic potential of CIs. However, in contrast to our in vitro angiogenesis assays, CIs with hMSCs and HUVECs exhibited a higher in vivo angiogenic potential compared to control islets or islets combined with hMSCs or PC-hMSCs. These findings highlight the importance and necessity of verifying in vitro studies with in vivo models to reliably predict, in this case, revascularization outcomes. Regardless, we demonstrate here the therapeutic potential of CIs with proangiogenic support cells to enhance islet revascularization at a clinically relevant, although poorly vascularized, transplantation site.

Aims: Activin A and transforming growth factor-β1 (TGF-β1) belong to the same family of growth and differentiation factors that modulate vascular lesion formation in distinct ways, which we wish to understand mechanistically. Methods and results: We investigated the expression of cell-surface receptors and activation of Smads in human vascular smooth muscle cells (SMCs) and demonstrated that activin receptor-like kinase-1 (ALK-1), ALK-4, ALK-5 and endoglin are expressed in human SMCs. As expected, TGF-β1 activates Smad1 and Smad2 in these cells. Interestingly, activin A also induces phosphorylation of both Smads, which has not been reported for Smad1 before. Transcriptome analyses of activin A and TGF-β1 treated SMCs with subsequent Gene-Set Enrichment Analyses revealed that many downstream gene networks are induced by both factors. However, the effect of activin A on expression kinetics of individual genes is less pronounced than for TGF-β1, which is explained by a more rapid dephosphorylation of Smads and p38-MAPK in response to activin A. Substantial differences in expression of fibronectin, alpha-V integrin and total extracellular collagen synthesis were observed. Conclusions: Genome-wide mRNA expression analyses clarify the distinct modulation of vascular lesion formation by activin A and TGF-β1, most significantly because activin A is non-fibrotic. © 2010 Elsevier Inc., This work was supported by the Molecular Cardiology Program from the Netherlands Heart Foundation [NHS93.007] and a grant from Junta del Andalucía, Spain [YMP].

The fibrinous exudate of a wound or tumor stroma facilitates angiogenesis. We studied the involvement of RGD-binding integrins during tube formation in human plasma-derived fibrin clots and human purified fibrin matrices. Capillary-like tube formation by human microvascular endothelial cells in a 3D plasma-derived fibrinous matrix was induced by FGF-2 and TNF-α and depended largely on cell-bound u-PA and plasmin activities. While tube formation was minimally affected by the addition of either the αvβ3-integrin inhibiting mAb LM609 or the α5-integrin inhibiting mAb IIA1, the general RGD-antagonist echistatin completely inhibited this process. Remarkably, when αvβ3- and α5β1-integrins were inhibited simultaneously, tube formation was reduced by 78%. It was accompanied by a 44% reduction of u-PA antigen accumulation and 41% less production of fibrin degradation products. αvβ5-integrin-blocking antibodies further enhanced the inhibition by mAb LM609 and mAb IIA1 to 94%, but had no effect by themselves. αv-specific cRGD only inhibited angiogenesis when α5β1-integrin was simultaneously blocked. Endostatin mimicked the effect of α5β1-integrin and inhibited tube formation only in the presence of LM609 or cRGD (73 and 80%, respectively). Comparable results were obtained when purified fibrin matrices were used instead of the plasma-derived fibrinous matrices. These data show that blocking of tube formation in a fibrinous exudate requires the simultaneous inhibition of αvβ3- and α5β1-integrins. This may bear impact on attempts to influence angiogenesis in a fibrinous environment.

In between the period of transplantation and revascularization, pancreatic islets are exposed to low-oxygen and low-nutrient conditions. In the present study we mimicked those conditions in vitro to study the involvement of different cell death processes, release of danger-associated molecular patterns (DAMP) and associated in vitro immune activation. Under low-oxygen and low-nutrient conditions, apoptosis, autophagy and necroptosis occur in human islets. Necroptosis is responsible for DAMP-release such as dsDNA, uric acid and HMGB1. The sensors of the innate immune system able to recognize these DAMPs are mainly TLR, NOD receptors and C-type lectins. By using cell-lines with a non-functional adaptor molecule MyD88, we were able to show that the islet-derived DAMPs signal mainly via TLR. Immunoisolation in immunoprotective membranes reduced DAMP release and immune activation via retention of the relative large DAMPs in the capsules. Another effective strategy was suppressing necroptosis using the inhibitor nec-1. Although the effect on cell-survival was minor, nec-1 was able to reduce the release of HMGB1 and its associated immune activation. Our data demonstrate that in the immediate post-transplant period islets release DAMPs that in vitro enhance responses of innate immune cells. DAMP release can be reduced in vitro by immunoisolation or intervention with nec-1.

Loss of pancreatic islet beta-cell mass and beta-cell dysfunction are central in the development of type 2 diabetes (T2DM). We recently showed that mature human insulin-containing beta-cells can convert into glucagon-containing alpha-cells ex vivo. This loss of beta-cell identity was characterized by the presence of beta-cell transcription factors (Nkx6.1, Pdx1) in glucagon(+) cells. Here, we investigated whether the loss of beta-cell identity also occurs in vivo, and whether it is related to the presence of (pre)diabetes in humans and nonhuman primates. We observed an eight times increased frequency of insulin(+) cells coexpressing glucagon in donors with diabetes. Up to 5% of the cells that were Nkx6.1(+) but insulin(-) coexpressed glucagon, which represents a five times increased frequency compared with the control group. This increase in bihormonal and Nkx6.1(+)glucagon(+)insulin(-) cells was also found in islets of diabetic macaques. The higher proportion of bihormonal cells and Nkx6.1(+)glucagon(+)insulin(-) cells in macaques and humans with diabetes was correlated with the presence and extent of islet amyloidosis. These data indicate that the loss of beta-cell identity occurs in T2DM and could contribute to the decrease of functional beta-cell mass. Maintenance of beta-cell identity is a potential novel strategy to preserve beta-cell function in diabetes.

Clinical islet transplantation is a promising treatment for patients with type 1 diabetes. However, pancreatic islets vary in size and shape affecting their survival and function after transplantation because of mass transport limitations. To reduce diffusion restrictions and improve islet cell survival, the generation of islets with optimal dimensions by dispersion followed by reassembly of islet cells, can help limit the length of diffusion pathways. This study describes a microwell platform that supports the controlled and reproducible production of three-dimensional pancreatic cell clusters of human donor islets. We observed that primary human islet cell aggregates with a diameter of 100–150 μm consisting of about 1000 cells best resembled intact pancreatic islets as they showed low apoptotic cell death (

In clinical islet transplantation, allogeneic islets of Langerhans are transplanted into the portal vein of patients with type 1 diabetes, enabling the restoration of normoglycemia. After intra-hepatic transplantation several factors are involved in the decay in islet mass and function mainly caused by an immediate blood mediated inflammatory response, lack of vascularization, and allo- and autoimmunity. Bioengineered scaffolds can potentially provide an alternative extra-hepatic transplantation site for islets by improving nutrient diffusion and blood supply to the scaffold. This would ultimately result in enhanced islet viability and functionality compared to conventional intra portal transplantation. In this regard, the biomaterial choice, the three-dimensional (3D) shape and scaffold porosity are key parameters for an optimal construct design and, ultimately, transplantation outcome. We used 3D bioplotting for the fabrication of a 3D alginate-based porous scaffold as an extra-hepatic islet delivery system. In 3D-plotted alginate scaffolds the surface to volume ratio, and thus oxygen and nutrient transport, is increased compared to conventional bulk hydrogels. Several alginate mixtures have been tested for INS1E ?-cell viability. Alginate/gelatin mixtures resulted in high plotting performances, and satisfactory handling properties. INS1E ?-cells, human and mouse islets were successfully embedded in 3D-plotted constructs without affecting their morphology and viability, while preventing their aggregation. 3D plotted scaffolds could help in creating an alternative extra-hepatic transplantation site. In contrast to microcapsule embedding, in 3D plotted scaffold islets are confined in one location and blood vessels can grow into the pores of the construct, in closer contact to the embedded tissue. Once revascularization has occurred, the functionality is fully restored upon degradation of the scaffold.

SummaryIn diabetes mellitus an increased risk exists for vascular complications. A role for advanced glycation endproducts (AGEs) in the acceleration of vascular disease has been suggested. Nε(carboxymethyl)lysine (CML)- and methylglyoxal (MGO)-modified proteins have been identified as majorAGEs. The interaction of these AGEs with the human endothelial cells and macrophages was studied. Changes in adhesion molecule expression, i. e. vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1) and E-selectin were determined by cell-bound Elisa on human endothelial cells after incubation with CML-modified albumin and MGO-modified albumin. The presence of the full-length receptor of AGEs (RAGE) and splice variants of RAGE was determined by specific RT-PCR. In addition, binding studies were performed with CML- and MGOmodified albumin to endothelial cells and P388D1 macrophages. We demonstrated that CML-albumin or MGO-albumin did not induce activation of endothelial cells as measured by the expression of adhesion molecules, while, under the same conditions, TNF-α did. No specific binding of CML-albumin and MGO-albumin on these cells was found. In contrast to endothelial cells, a specific binding of MGO-albumin to P388D1 macrophages was demonstrated, which could be competed by ligands of scavenger receptors. In human umbilical vein and microvascular endothelial cells we found the N-truncated and C-truncated splice variants of RAGE.In conclusion, under our experimental conditions no CML- or MGO-albumin-induced increase in adhesion molecule expression was found on endothelial cells. In agreement with this, no binding of these AGEs was found to endothelial cells. The existence of splice variants of RAGE in endothelial cells might explain the lack of interaction of extracellular AGEs with these cells.

The endothelium plays a central role in the logistics of the immune system by allowing the selective transmigration of leukocytes, as well as the maintenance of the circulation and coagulation homeostasis. Evidence is increasing that the carbohydrate composition of the endothelial cell surface is critical for the cells to exert their physiological function. The major aim of this study is to unravel the mechanisms underlying the expression of carbohydrate structures by endothelial cells, which are involved in leukocyte adhesion and migration. Using quantitative real-time PCR, the expression profile of a selected group of 74 glycosylation-related genes has been determined in human umbilical vein endothelial cells (HUVEC) and human foreskin microvascular endothelial cells (FMVEC) under control and TNFα-induced conditions. The set of genes comprised 59 glycosyltransferases, 6 mannosidases and 9 sulfotransferases. In parallel, the overall cell surface glycan profile has been assessed by the use of glycan-specific lectins and monoclonal antibodies. The results demonstrate that HUVEC and FMVEC differ substantially in the expression of glycosylation-related genes and, accordingly, also in the presence of different glycan epitopes on the cell membrane. Induction of an inflamed phenotype of the cells by treatment with TNFα differentially modulates a set of these genes in HUVEC and FMVEC resulting in a change in the cell membrane associated glycans that are of importance in inflammation-related endothelial cell-surface processes. © 2005 Wiley-Liss, Inc.

Angiogenesis is the formation of new microvessels from existing vasculature. It is a crucial component of normal embryogenic development and of some physiological processes in adulthood. Pathological conditions such as tumor growth and metastasis, which involve tissue remodeling and inflammation, are usually associated with vascular leakage and subsequent angiogenesis. Tumor angiogenesis often augments tumor survival, progression, and metastasis, thus enhancing the malignant characteristics of the disease. Proteolytic degradation of the extracellular matrix that surrounds both the capillary sprouts and the migrating tumor cells is an essential part of tumor angiogenesis and tumor growth. In particular, proteases of the fibrinolytic system and the matrix metalloproteinase family play a role in these processes. In addition to proangiogenic effects, proteases also can negatively regulate angiogenesis. Protein fragments that result from proteolytic degradation of extracellular matrix components and other proteins can exhibit potent antiangiogenic properties. A thorough understanding of tumor-associated proteolytic processes is required to identify specific targets that are suitable for protease-based tumor therapy.

Activin A alters the characteristics of human arterial smooth muscle cells (SMCs) toward a contractile, quiescent phenotype. We hypothesize that activin A may prevent SMC-rich neointimal hyperplasia. Here, we study the effect of adenovirus-mediated expression of activin A on neointima formation in vitro and in vivo. Human saphenous vein organ cultures, in which a neointima is formed spontaneously, were infected either with activin A- or lacZ-adenovirus. Activin A-overexpression reduces neointima formation by 78%, whereas no significant reduction was observed after control infection. In addition, the effect of activin A on neointima formation was assessed in vivo in mice with cuffed femoral arteries. In activin A adenovirus-infected mice (IV injection), neointimal hyperplasia is reduced by 77% compared with the SMC-rich neointima in mock-infected or in noninfected mice. Cultured human saphenous vein SMCs and murine aorta SMCs were incubated with activin A and an increased expression of SM22α and SM α-actin mRNA, and SM α-actin protein was demonstrated. Laser-capture microdissection on sections of cuffed murine arteries and subsequent real-time RT-PCR established in vivo induction of SM α-actin mRNA in the media of activin A–treated mice. In summary, activin A inhibits neointima formation in vitro and in vivo by preventing SMC dedifferentiation.