Gunter Leuckx

Transient cytokine treatment induces acinar cell reprogramming and regenerates functional beta cell mass in diabetic mice

Reprogramming of pancreatic exocrine cells into cells resembling beta cells may provide a strategy for treating diabetes. Here we show that transient administration of epidermal growth factor and ciliary neurotrophic factor to adult mice with chronic hyperglycemia efficiently stimulates the conversion of terminally differentiated acinar cells to beta-like cells. Newly generated beta-like cells are epigenetically reprogrammed, functional and glucose responsive, and they reinstate normal glycemic control for up to 248 d. The regenerative process depends on Stat3 signaling and requires a threshold number of Neurogenin 3 (Ngn3)-expressing acinar cells. In contrast to previous work demonstrating in vivo conversion of acinar cells to beta-like cells by viral delivery of exogenous transcription factors, our approach achieves acinar-to-beta-cell reprogramming through transient cytokine exposure rather than genetic modification.

Adult Duct-Lining Cells Can Reprogram into β-like Cells Able to Counter Repeated Cycles of Toxin-Induced Diabetes

It was recently demonstrated that embryonic glucagon-producing cells in the pancreas can regenerate and convert into insulin-producing β-like cells through the constitutive/ectopic expression of the Pax4 gene. However, whether α cells in adult mice display the same plasticity is unknown. Similarly, the mechanisms underlying such reprogramming remain unclear. We now demonstrate that the misexpression of Pax4 in glucagon(+) cells age-independently induces their conversion into β-like cells and their glucagon shortage-mediated replacement, resulting in islet hypertrophy and in an unexpected islet neogenesis. Combining several lineage-tracing approaches, we show that, upon Pax4-mediated α-to-β-like cell conversion, pancreatic duct-lining precursor cells are continuously mobilized, re-express the developmental gene Ngn3, and successively adopt a glucagon(+) and a β-like cell identity through a mechanism involving the reawakening of the epithelial-to-mesenchymal transition. Importantly, these processes can repeatedly regenerate the whole β cell mass and thereby reverse several rounds of toxin-induced diabetes, providing perspectives to design therapeutic regenerative strategies.

The Inactivation of Arx in Pancreatic α-Cells Triggers Their Neogenesis and Conversion into Functional β-Like Cells

Recently, it was demonstrated that pancreatic new-born glucagon-producing cells can regenerate and convert into insulin-producing β-like cells through the ectopic expression of a single gene, Pax4. Here, combining conditional loss-of-function and lineage tracing approaches, we show that the selective inhibition of the Arx gene in α-cells is sufficient to promote the conversion of adult α-cells into β-like cells at any age. Interestingly, this conversion induces the continuous mobilization of duct-lining precursor cells to adopt an endocrine cell fate, the glucagon+ cells thereby generated being subsequently converted into β-like cells upon Arx inhibition. Of interest, through the generation and analysis of Arx and Pax4 conditional double-mutants, we provide evidence that Pax4 is dispensable for these regeneration processes, indicating that Arx represents the main trigger of α-cell-mediated β-like cell neogenesis. Importantly, the loss of Arx in α-cells is sufficient to regenerate a functional β-cell mass and thereby reverse diabetes following toxin-induced β-cell depletion. Our data therefore suggest that strategies aiming at inhibiting the expression of Arx, or its molecular targets/co-factors, may pave new avenues for the treatment of diabetes.

Neurogenin 3+ cells contribute to β-cell neogenesis and proliferation in injured adult mouse pancreas

We previously showed that injury by partial duct ligation (PDL) in adult mouse pancreas activates Neurogenin 3 (Ngn3)+ progenitor cells that can differentiate to β cells ex vivo. Here we evaluate the role of Ngn3+ cells in β cell expansion in situ. PDL not only induced doubling of the β cell volume but also increased the total number of islets. β cells proliferated without extended delay (the so-called ‘refractory’ period), their proliferation potential was highest in small islets, and 86% of the β cell expansion was attributable to proliferation of pre-existing β cells. At sufficiently high Ngn3 expression level, upto 14% of all β cells and 40% of small islet β cells derived from non-β cells. Moreover, β cell proliferation was blunted by a selective ablation of Ngn3+ cells but not by conditional knockout of Ngn3 in pre-existing β cells supporting a key role for Ngn3+ insulin− cells in β cell proliferation and expansion. We conclude that Ngn3+ cell-dependent proliferation of pre-existing and newly-formed β cells as well as reprogramming of non-β cells contribute to in vivo β cell expansion in the injured pancreas of adult mice.

Reprogramming of human pancreatic exocrine cells to β -like cells

Rodent acinar cells exhibit a remarkable plasticity as they can transdifferentiate to duct-, hepatocyte- and islet β-like cells. We evaluated whether exocrine cells from adult human pancreas can similarly respond to proendocrine stimuli. Exocrine cells from adult human pancreas were transduced directly with lentiviruses expressing activated MAPK (mitogen-activated protein kinase) and STAT3 (signal transducer and activator of transcription 3) and cultured as monolayers or as 3D structures. Expression of STAT3 and MAPK in human exocrine cells activated expression of the proendocrine factor neurogenin 3 in 50% to 80% of transduced exocrine cells. However, the number of insulin-positive cells increased only in the exocrine cells grown initially in suspension before 3D culture. Lineage tracing identified human acinar cells as the source of Ngn3- and insulin-expressing cells. Long-term engraftment into immunocompromised mice increased the efficiency of reprogramming to insulin-positive cells. Our data demonstrate that exocrine cells from human pancreas can be reprogrammed to transplantable insulin-producing cells that acquire functionality. Given the large number of exocrine cells in a donor pancreas, this approach presents a novel strategy to expand cell therapy in type 1 diabetes.

Estrogen Receptor α Regulates β-Cell Formation During Pancreas Development and Following Injury

Identifying pathways for β-cell generation is essential for cell therapy in diabetes. We investigated the potential of 17β-estradiol (E2) and estrogen receptor (ER) signaling for stimulating β-cell generation during embryonic development and in the severely injured adult pancreas. E2 concentration, ER activity, and number of ERα transcripts were enhanced in the pancreas injured by partial duct ligation (PDL) along with nuclear localization of ERα in β-cells. PDL-induced proliferation of β-cells depended on aromatase activity. The activation of Neurogenin3 (Ngn3) gene expression and β-cell growth in PDL pancreas were impaired when ERα was turned off chemically or genetically (ERα−/−), whereas in situ delivery of E2 promoted β-cell formation. In the embryonic pancreas, β-cell replication, number of Ngn3+ progenitor cells, and expression of key transcription factors of the endocrine lineage were decreased by ERα inactivation. The current study reveals that E2 and ERα signaling can drive β-cell replication and formation in mouse pancreas.

Partial duct ligation: β-cell proliferation and beyond.

Experimentally induced injury is an established strategy for studying mechanisms of tissue remodeling with the final goal of developing new regenerative therapies. Under normal physiological conditions, proliferation and differentiation of progenitor cells, including even canonical stem cell−like activity, can be stimulated in tissues, such as brain and liver, that have a low cellular turnover rate (1,2). The presence of stem/progenitor cells in the pancreas could be relevant to normal homeostatic maintenance of various cell types in this organ, such as endocrine hormone−expressing cells, enzyme-secreting acinar cells, and the less secretory exocrine duct cells. Further, pancreatic stem/progenitor cells may be a possible source for replenishing cells destroyed by autoimmune disease or other stressors. We speculate that proliferative progenitors might be isolated, expanded, and differentiated in vitro to alleviate the donor scarcity in human islet transplantation and may therefore be developed as a therapy for diabetes. However, the existence and exact location of adult stem- or progenitor-like cells that can give rise to functional β-cells is highly controversial. This Perspective focuses on findings from a severe insult model (partial duct ligation [PDL]) with a long history (3). PDL received renewed attention when a 2008 study combined it with genetic reporter strategies now possible in mice to try to identify and isolate cells acting as β-cell progenitors (4). In vivo β-cell neogenesis under PDL was recently substantiated (5,6). Because the outcomes from this technique appear to vary across laboratories, we summarize and discuss some of the reported discrepancies to help identify current limitations and pitfalls of this model as well as opportunities for forward progress. The mechanisms leading to replacement of the endogenous β-cell pool have been studied under several cell ablation paradigms to test for the existence and type of cells in the adult mouse pancreas that are …

Differentiating neural crest stem cells induce proliferation of cultured rodent islet beta cells

Aims/hypothesisEfficient stimulation of cycling activity in cultured beta cells would allow the design of new strategies for cell therapy in diabetes. Neural crest stem cells (NCSCs) play a role in beta cell development and maturation and increase the beta cell number in co-transplants. The mechanism behind NCSC-induced beta cell proliferation and the functional capacity of the new beta cells is not known.MethodsWe developed a new in vitro co-culture system that enables the dissection of the elements that control the cellular interactions that lead to NCSC-dependent increase in islet beta cells.ResultsMouse NCSCs were cultured in vitro, first in medium that stimulated their proliferation, then under conditions that supported their differentiation. When mouse islet cells were cultured together with the NCSCs, more than 35% of the beta cells showed cycle activity. This labelling index is more than tenfold higher than control islets cultured without NCSCs. Beta cells that proliferated under these culture conditions were fully glucose responsive in terms of insulin secretion. NCSCs also induced beta cell proliferation in islets isolated from 1-year-old mice, but not in dissociated islet cells isolated from human donor pancreas tissue. To stimulate beta cell proliferation, NCSCs need to be in intimate contact with the beta cells.Conclusions/interpretationCulture of islet cells in contact with NCSCs induces highly efficient beta cell proliferation. The reported culture system is an excellent platform for further dissection of the minimal set of factors needed to drive this process and explore its potential for translation to diabetes therapy.

Ectopic Expression of E2F1 Stimulates β-Cell Proliferation and Function

OBJECTIVE Generating functional β-cells by inducing their proliferation may provide new perspectives for cell therapy in diabetes. Transcription factor E2F1 controls G1- to S-phase transition during the cycling of many cell types and is required for pancreatic β-cell growth and function. However, the consequences of overexpression of E2F1 in β-cells are unknown. RESEARCH DESIGN AND METHODS The effects of E2F1 overexpression on β-cell proliferation and function were analyzed in isolated rat β-cells and in transgenic mice. RESULTS Adenovirus AdE2F1-mediated overexpression of E2F1 increased the proliferation of isolated primary rat β-cells 20-fold but also enhanced β-cell death. Coinfection with adenovirus AdAkt expressing a constitutively active form of Akt (protein kinase B) suppressed β-cell death to control levels. At 48 h after infection, the total β-cell number and insulin content were, respectively, 46 and 79% higher in AdE2F1+AdAkt-infected cultures compared with untreated. Conditional overexpression of E2F1 in mice resulted in a twofold increase of β-cell proliferation and a 70% increase of pancreatic insulin content, but did not increase β-cell mass. Glucose-challenged insulin release was increased, and the mice showed protection against toxin-induced diabetes. CONCLUSIONS Overexpression of E2F1, either in vitro or in vivo, can stimulate β-cell proliferation activity. In vivo E2F1 expression significantly increases the insulin content and function of adult β-cells, making it a strategic target for therapeutic manipulation of β-cell function.

Human blood outgrowth endothelial cells improve islet survival and function when co-transplanted in a mouse model of diabetes.

Aims/hypothesisAs current islet-transplantation protocols suffer from significant graft loss and dysfunction, strategies to sustain the long-term benefits of this therapy are required. Rapid and adequate oxygen and nutrient delivery by blood vessels improves islet engraftment and function. The present report evaluated a potentially beneficial effect of adult human blood outgrowth endothelial cells (BOEC) on islet graft vascularisation and function.MethodsHuman BOEC, 5 × 105, were co-transplanted with a rat marginal-islet graft under the kidney capsule of hyperglycaemic NOD severe combined immunodeficiency (SCID) mice, and the effect on metabolic outcome was evaluated.ResultsAlthough vessel density remained unaffected, co-transplantation of islets with BOEC resulted in a significant and specific improvement of glycaemia and increased plasma C-peptide. Moreover, in contrast to control mice, BOEC recipients displayed reduced beta cell death and increases in body weight, beta cell proliferation and graft-vessel and beta cell volume. In vivo cell tracing demonstrated that BOEC remain at the site of transplantation and do not expand. The potential clinical applicability was underscored by the observed metabolic benefit of co-transplanting islets with BOEC derived from a type 1 diabetes patient.Conclusions/interpretationThe present data support the use of autologous BOEC in translational studies that aim to improve current islet-transplantation protocols for the treatment of brittle type 1 diabetes.