(Re)generating human beta cells : status, pitfalls, and perspectives

Diabetes mellitus results from disturbed glucose homeostasis due to an absolute (type 1) or relative (type 2) deficiency of insulin, a peptide hormone almost exclusively produced by the beta cells of the endocrine pancreas in a tightly regulated manner. Current therapy only delays disease progression through insulin injection and/or oral medications that increase insulin secretion or sensitivity, decrease hepatic glucose production, or promote glucosuria. These drugs have turned diabetes into a chronic disease as they do not solve the underlying beta cell defects or entirely prevent the long-term complications of hyperglycemia. Beta cell replacement through islet transplantation is a more physiological therapeutic alternative but is severely hampered by donor shortage and immune rejection. A curative strategy should combine newer approaches to immunomodulation with beta cell replacement. Success of this approach depends on the development of practical methods for generating beta cells, either in vitro or in situ through beta cell replication or beta cell differentiation. This review provides an overview of human beta cell generation

Semi-automated digital measurement as the method of choice for beta cell mass analysis

Pancreas injury by partial duct ligation (PDL) activates beta cell differentiation and proliferation in adult mouse pancreas but remains controversial regarding the anticipated increase in beta cell volume. Several reports unable to show beta cell volume augmentation in PDL pancreas used automated digital image analysis software. We hypothesized that fully automatic beta cell morphometry without manual micrograph artifact remediation introduces bias and therefore might be responsible for reported discrepancies and controversy. However, our present results prove that standard digital image processing with automatic thresholding is sufficiently robust albeit less sensitive and less adequate to demonstrate a significant increase in beta cell volume in PDL versus Sham-operated pancreas. We therefore conclude that other confounding factors such as quality of surgery, selection of samples based on relative abundance of the transcription factor Neurogenin 3 (Ngn3) and tissue processing give rise to inter-laboratory inconsistencies in beta cell volume quantification in PDL pancreas

A rare case of hypoglycemia in a patient with elevated right hemidiaphragm.

A 57-year-old woman was admitted to the emergency department, presenting with episodes of altered consciousness and behaviour which, upon further examination, were linked to periods of recurrent hypoglycaemia. Imaging revealed a large mass in the right thoracic cavity while blood analysis demonstrated diminished C-peptide, (pro-)insulin, insulin-like growth factor 1 (IGF-I) and IGF binding protein 3 levels. Based on these findings, an IGF-II secreting tumour was suspected. Before the excision of the tumour, euglycaemia could only be achieved by means of intravenous glucose administration and the use of oral corticosteroids. Anatomopathologically the diagnosis of a solitary fibrous tumour (SFT) was confirmed. Immunoblot analysis on the serum revealed elevated 'big'-IGF-II levels, confirming our initial diagnosis of Doege-Potter syndrome in SFT

A rare case of hypoglycemia in a patient with elevated right hemidiaphragm.

A 57-year-old woman was admitted to the emergency department, presenting with episodes of altered consciousness and behaviour which, upon further examination, were linked to periods of recurrent hypoglycaemia. Imaging revealed a large mass in the right thoracic cavity while blood analysis demonstrated diminished C-peptide, (pro-)insulin, insulin-like growth factor 1 (IGF-I) and IGF binding protein 3 levels. Based on these findings, an IGF-II secreting tumour was suspected. Before the excision of the tumour, euglycaemia could only be achieved by means of intravenous glucose administration and the use of oral corticosteroids. Anatomopathologically the diagnosis of a solitary fibrous tumour (SFT) was confirmed. Immunoblot analysis on the serum revealed elevated 'big'-IGF-II levels, confirming our initial diagnosis of Doege-Potter syndrome in SFT

Insulitis and lymphoid structures in the islets of Langerhans of a 66-year-old patient with long-standing type 1 diabetes

Insulitis is a characteristic inflammatory lesion consisting of immune cell infiltrates around and within the pancreatic islets of patients with recent-onset type 1 diabetes (T1D). The infiltration is typically mild, both in terms of the number of infiltrating cells and the number of islets affected. Here, we present an unusual histopathological case study of a 66-year-old female patient with long-standing T1D, insulitis, and islet-associated lymphoid tissue. Most islets in the head of the pancreas of this patient were insulin-deficient, whereas the islets in the tail appeared normal. Insulitis was present in 0.84% of the insulin-containing islets and three islets had large lymphocytic infiltrates resembling tertiary lymphoid structures (TLS). Of note, this is the first description of potential TLS in the endocrine pancreas of a patient with T1D. Their association with a marked residual beta cell mass is of interest and may hint at new insights into disease progression and regulation of autoimmunity.status: publishe

Towards a Functional Cure for Diabetes Using Stem Cell-Derived Beta Cells: Are We There Yet?

Diabetes mellitus is a pandemic metabolic disorder that results from either the autoimmune destruction or the dysfunction of insulin-producing pancreatic beta cells. A promising cure is beta cell replacement through the transplantation of islets of Langerhans. However, donor shortage hinders the widespread implementation of this therapy. Human pluripotent stem cells, including embryonic stem cells and induced pluripotent stem cells, represent an attractive alternative beta cell source for transplantation. Although major advances over the past two decades have led to the generation of stem cell-derived beta-like cells that share many features with genuine beta cells, producing fully mature beta cells remains challenging. Here, we review the current status of beta cell differentiation protocols and highlight specific challenges that are associated with producing mature beta cells. We address the challenges and opportunities that are offered by monogenic forms of diabetes. Finally, we discuss the remaining hurdles for clinical application of stem cell-derived beta cells and the status of ongoing clinical trials.SCOPUS: re.jinfo:eu-repo/semantics/publishe

Reversal of hyperglycemia in diabetic mice by a marginal islet mass together with human blood outgrowth endothelial cells is independent of the delivery technique and blood clot-induced processes

We recently reported that human blood outgrowth endothelial cells (BOEC) are supportive to reverse hyperglycemia in marginal islet mass-transplanted diabetic mice. In this report, we investigated whether the observed effect was evoked by islet packing in a blood clot prior to transplantation or could be mimicked by another method of islet/cell delivery. A marginal islet mass with or without BOEC was grafted underneath the kidney capsule of diabetic recipient mice via a (blood clot-independent) tubing system and compared with previous islet packing in a blood clot. The effect on metabolic outcome of both delivery techniques as well as the additive effect of BOEC was subsequently evaluated. Marginal islet mass transplantation via a tubing system required more islets per recipient than via a blood clot. Using the tubing method, transplantation of a marginal islet mass combined with 5x10 (5) BOEC resulted in reversal of hyperglycemia, improved glucose tolerance and increased kidney insulin content. The present study provides evidence that (1) previous packing in a blood clot results in more effective islet delivery compared with tubing; (2) BOEC exert a beneficial effect on marginal islet transplantation, independent of grafting technique and potential blood clot-induced processes. These data further support the use of BOEC in (pre-) clinical studies that aim to improve current islet transplantation protocols.status: publishe

Vegf-A mRNA transfection as a novel approach to improve mouse and human islet graft revascularisation

AIMS/HYPOTHESIS: The initial avascular period following islet transplantation seriously compromises graft function and survival. Enhancing graft revascularisation to improve engraftment has been attempted through virus-based delivery of angiogenic triggers, but risks associated with viral vectors have hampered clinical translation. In vitro transcribed mRNA transfection circumvents these risks and may be used for improving islet engraftment. METHODS: Mouse and human pancreatic islet cells were transfected with mRNA encoding the angiogenic growth factor vascular endothelial growth factor A (VEGF-A) before transplantation under the kidney capsule in mice. RESULTS: At day 7 post transplantation, revascularisation of grafts transfected with Vegf-A (also known as Vegfa) mRNA was significantly higher compared with non-transfected or Gfp mRNA-transfected controls in mouse islet grafts (2.11- and 1.87-fold, respectively) (vessel area/graft area, mean ± SEM: 0.118 ± 0.01 [n = 3] in Vegf-A mRNA transfected group (VEGF) vs 0.056 ± 0.01 [n = 3] in no RNA [p < 0.05] vs 0.063 ± 0.02 [n = 4] in Gfp mRNA transfected group (GFP) [p < 0.05]); EndoC-bH3 grafts (2.85- and 2.48-fold. respectively) (0.085 ± 0.02 [n = 4] in VEGF vs 0.030 ± 0.004 [n = 4] in no RNA [p < 0.05] vs 0.034 ± 0.01 [n = 5] in GFP [p < 0.05]); and human islet grafts (3.17- and 3.80-fold, respectively) (0.048 ± 0.013 [n = 3] in VEGF vs 0.015 ± 0.0051 [n = 4] in no RNA [p < 0.01] vs 0.013 ± 0.0046 [n = 4] in GFP [p < 0.01]). At day 30 post transplantation, human islet grafts maintained a vascularisation benefit (1.70- and 1.82-fold, respectively) (0.049 ± 0.0042 [n = 8] in VEGF vs 0.029 ± 0.0052 [n = 5] in no RNA [p < 0.05] vs 0.027 ± 0.0056 [n = 4] in GFP [p < 0.05]) and a higher beta cell volume (1.64- and 2.26-fold, respectively) (0.0292 ± 0.0032 μl [n = 7] in VEGF vs 0.0178 ± 0.0021 μl [n = 5] in no RNA [p < 0.01] vs 0.0129 ± 0.0012 μl [n = 4] in GFP [p < 0.001]). CONCLUSIONS/INTERPRETATION: Vegf-A mRNA transfection before transplantation provides a promising and safe strategy to improve engraftment of islets and other cell-based implants

Vegf-A mRNA transfection as a novel approach to improve mouse and human islet graft revascularisation

AIMS/HYPOTHESIS: The initial avascular period following islet transplantation seriously compromises graft function and survival. Enhancing graft revascularisation to improve engraftment has been attempted through virus-based delivery of angiogenic triggers, but risks associated with viral vectors have hampered clinical translation. In vitro transcribed mRNA transfection circumvents these risks and may be used for improving islet engraftment. METHODS: Mouse and human pancreatic islet cells were transfected with mRNA encoding the angiogenic growth factor vascular endothelial growth factor A (VEGF-A) before transplantation under the kidney capsule in mice. RESULTS: At day 7 post transplantation, revascularisation of grafts transfected with Vegf-A (also known as Vegfa) mRNA was significantly higher compared with non-transfected or Gfp mRNA-transfected controls in mouse islet grafts (2.11- and 1.87-fold, respectively) (vessel area/graft area, mean ± SEM: 0.118 ± 0.01 [n = 3] in Vegf-A mRNA transfected group (VEGF) vs 0.056 ± 0.01 [n = 3] in no RNA [p < 0.05] vs 0.063 ± 0.02 [n = 4] in Gfp mRNA transfected group (GFP) [p < 0.05]); EndoC-bH3 grafts (2.85- and 2.48-fold. respectively) (0.085 ± 0.02 [n = 4] in VEGF vs 0.030 ± 0.004 [n = 4] in no RNA [p < 0.05] vs 0.034 ± 0.01 [n = 5] in GFP [p < 0.05]); and human islet grafts (3.17- and 3.80-fold, respectively) (0.048 ± 0.013 [n = 3] in VEGF vs 0.015 ± 0.0051 [n = 4] in no RNA [p < 0.01] vs 0.013 ± 0.0046 [n = 4] in GFP [p < 0.01]). At day 30 post transplantation, human islet grafts maintained a vascularisation benefit (1.70- and 1.82-fold, respectively) (0.049 ± 0.0042 [n = 8] in VEGF vs 0.029 ± 0.0052 [n = 5] in no RNA [p < 0.05] vs 0.027 ± 0.0056 [n = 4] in GFP [p < 0.05]) and a higher beta cell volume (1.64- and 2.26-fold, respectively) (0.0292 ± 0.0032 μl [n = 7] in VEGF vs 0.0178 ± 0.0021 μl [n = 5] in no RNA [p < 0.01] vs 0.0129 ± 0.0012 μl [n = 4] in GFP [p < 0.001]). CONCLUSIONS/INTERPRETATION: Vegf-A mRNA transfection before transplantation provides a promising and safe strategy to improve engraftment of islets and other cell-based implants.status: publishe

Conditional islet hypovascularisation does not preclude beta cell expansion during pregnancy in mice

Endothelial-endocrine cell interactions and vascular endothelial growth factor (VEGF)-A signalling are deemed essential for maternal islet vascularisation, glucose control and beta cell expansion during mouse pregnancy. The aim of this study was to assess whether pregnancy-associated beta cell expansion was affected under conditions of islet hypovascularisation. Soluble fms-like tyrosine kinase 1 (sFLT1), a VEGF-A decoy receptor, was conditionally overexpressed in maternal mouse beta cells from 1.5 to 14.5 days post coitum. Islet vascularisation, glycaemic control, beta cell proliferation, individual beta cell size and total beta cell volume were assessed in both pregnant mice and non-pregnant littermates. Conditional overexpression of sFLT1 in beta cells resulted in islet hypovascularisation and glucose intolerance in both pregnant and non-pregnant mice. In contrast to non-pregnant littermates, glucose intolerance in pregnant mice was transient. sFLT1 overexpression did not affect pregnancy-associated changes in beta cell proliferation, individual beta cell size or total beta cell volume. Reduced intra-islet VEGF-A signalling results in maternal islet hypovascularisation and impaired glycaemic control but does not preclude beta cell expansion during mouse pregnancy