Luc Baeyens

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.

Notch Signaling as Gatekeeper of Rat Acinar-to-β-Cell Conversion in Vitro

BACKGROUND & AIMS Exocrine acinar cells in the pancreas are highly differentiated cells that retain a remarkable degree of plasticity. After isolation and an initial phase of dedifferentiation in vitro, rodent acinar cells can convert to endocrine beta-cells when cultured in the presence of appropriate factors. The mechanisms regulating this phenotypic conversion are largely unknown. METHODS Using rat acinar cell cultures, we studied the role of Notch signaling in a model of acinar-to-beta-cell conversion. RESULTS We report a novel lectin-based cell labeling method to demonstrate the acinar origin of newly formed insulin-expressing beta-cells. This method allows for specific tracing of the acinar cells. We demonstrate that growth factor-induced conversion of adult acinar cells to beta-cells is negatively regulated by Notch1 signaling. Activated Notch1 signaling prevents the reexpression of the proendocrine transcription factor Neurogenin-3, the key regulator of endocrine development in the embryonic pancreas. Interfering with Notch1 signaling allows modulating the acinar cell susceptibility to the differentiation-inducing factors. Its inhibition significantly improves beta-cell neoformation with approximately 30% of acinar cells that convert to beta-cells. The newly formed beta-cells mature when transplanted ectopically and are capable of restoring normal blood glycemia in diabetic recipients. CONCLUSIONS We report for the first time an efficient way to reprogram one third of the acinar cells to beta-cells by adult cell type conversion. This could find application in cell replacement therapy of type 1 diabetes, provided that it can be translated from rodent to human models.

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.

Ngn3 expression during postnatal in vitro beta cell neogenesis induced by the JAK/STAT pathway.

The basic helix–loop–helix protein Neurogenin3 specifies precursor cells of the endocrine pancreas during embryonic development, and is thought to be absent postnatally. We have studied Ngn3 expression during in vitro generation of beta-cells from adult rat exocrine pancreas tissue treated with epidermal growth factor and leukaemia inhibitory factor. This treatment induced a transient expression of both Ngn3 and its upstream activator hepatocyte nuclear factor 6. Inhibition of EGF and LIF signalling by pharmacological antagonists of the JAK2/STAT3 pathway, or knockdown of Ngn3 by RNA interference prevented the generation of new insulin-positive cells. This study demonstrates that in vitro growth factor stimulation can induce recapitulation of an embryonic endocrine differentiation pathway in adult dedifferentiated exocrine cells. This could prove to be important for understanding the mechanism of beta-cell regeneration and for therapeutic ex vivo neogenesis of beta cells.

Evaluation of the radiation dose in micro-CT with optimization of the scan protocol

INTRODUCTION Micro-CT provides non-invasive anatomic evaluation of small animals. Serial micro-CT measurements are, however, hampered by the severity of ionizing radiation doses cumulating over the total period of follow-up. The dose levels may be sufficient to influence experimental outcomes such as animal survival or tumor growth. AIM This study was designed to evaluate the radiation dose of micro-CT and to optimize the scanning protocol for longitudinal micro-CT scans. METHODS AND MATERIALS Normal C57Bl/6 mice were euthanized. Radiation exposure was measured using individually calibrated lithium fluoride thermoluminescent dosimeters (TLDs). Thirteen TLDs were placed in the mice at the thyroid, lungs, liver, stomach, colon, bladder and near the spleen. Micro-CT (SkyScan 1178) was performed using two digital X-ray cameras which scanned over 180 degrees at a resolution of 83 microm, a rotation step of 1.08 degrees , 50 kV, 615 microA and 121 s image acquisition time. The TLDs were removed after each scan. CTDI(100) was measured with a 100 mm ionization chamber, centrally positioned in a 2.7 cm diameter water phantom, and rotation steps were increased to reduce both scan time and radiation dose. RESULTS Internal TLD analysis demonstrated median organ dose of 5.5 +/- 0.6 mGy per mA s, confirmed by CTDI(100) with result of 6.6 mGy per mA s. A rotation step of 2.16 resulted in qualitatively accurate images. At a resolution of 83 microm the scan time is reduced to 63 s with an estimated dose of 2.9 mGy per mA s. At 166 microm resolution, the scan time is limited to 27 s, with a concordant dose of 1.2 mGy per mA s. CONCLUSIONS The radiation dose of a standard micro-CT scan is relatively high and could influence the experimental outcome. We believe that the presented adaptation of the scan protocol allows for accurate imaging without the risk of interfering with the experimental outcome of the study.

Can β‐cells be derived from exocrine pancreas?

A major goal of research aiming at improving islet cell replacement therapy is to find the most suitable progenitor cell type from which functional β‐cells can be generated in large numbers. Many possibilities have been raised, including β‐cells themselves, embryonic or adult stem cells and reprogramming of other cell types. Some of these progenitor types may be active or reside in a dormant state in adults in vivo, while others can be rather considered to be products of tissue engineering in vitro. Starting from the available pancreas organs from cadaveric donors, an attractive possibility is to reprogram acinar exocrine cells into β‐cells. Indeed, acinar cells isolated from adult rats display a pronounced plasticity in culture. After an initial step of dedifferentiation, they can be redirected to the β‐cell phenotype by adding agonists of the JAK2/STAT3 signalling pathway to the medium (epidermal growth factor and leukaemia inhibitory factor). The acinar cells that undergo exocrine‐to‐endocrine transdifferentiation first need to re‐express neurogenin‐3 and then need to escape inhibition by Notch signalling. The insulin‐expressing cells that are generated in this way are glucose‐regulated and can normalize glycaemia after transplantation into diabetic immunocompromised mice. It will now be important to translate these findings to human cells.

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.

Bicyclist's vulva: observational study

# Bicyclists vulva: observational study {#article-title-2} Many chronic injuries related to athletic bicycling are now recognised: cyclists nipples,1 neuropathic syndromes,2 and skin problems caused by the saddle. We have seen a new clinical problem in female high level cycling competitors: bicyclists vulva (figure). ![][1] Bicyclists vulva Six women, aged 21–38 years, had a unilateral chronic swelling of the labium majus after a few years of intensive bicycling (an average of 462.5 km per week). All six had typical unilateral lymphoedema (five on the right side, one on the left) which was more severe after more intense and longer training. The position of the bicycle saddle, the type of shorts worn, and the womens perineal hygiene were optimum. There was no family history of lymphoedema in any of the women, nor any common factor that might explain the lymphoedema. All six women regularly had inflammatory skin problems related to the saddle and five had scars and perineal lesions such as chafing, perineal folliculitis, and nodules. Further clinical and ultrasound examination showed no pelvic anomaly. Three of the cyclists (aged 27, 21, and 21 years) underwent three phase lymphoscintigraphy of their legs.3 We found similar lymphatic anomalies in all three. One had bilateral intra-abdominal abnormalities at the level of the iliac nodes and functional insufficiency of the superficial lymphatic system on the left side, the same side as the oedematous labium majus. In the other two, who presented … [1]: /embed/graphic-1.gif

Expression and function of leukaemia inhibitory factor and its receptor in normal and regenerating rat pancreas

Aims/hypothesisIt was recently reported that culturing adult exocrine cells in the presence of epidermal growth factor and leukaemia inhibitory factor (LIF) resulted in their transdifferentiation into endocrine beta cells. The aim of this study was to examine the expression and function of LIF in the pancreas.Materials and methodsWe studied the expression of LIF and its receptor components, LIF-receptor-β and gp130, by immunohistochemistry, western blotting and RT-PCR in normal rat pancreas, pancreas with duct ligation-induced islet neogenesis, and in pancreatic cell cultures. Isolated duct fragments were cultured in the presence of LIF and a janus kinase 2 (JAK2) inhibitor.ResultsLIF was detected by immunohistochemistry, western blot and RT-PCR in the ducts of the normal pancreas. Both LIF-receptor-β and gp130 were detected by RT-PCR in the pancreas. Immunostaining revealed gp130 exclusively in the ducts and centro-acinar cells. After duct ligation-induced tissue injury, upregulation of LIF and its receptor occurred in rat pancreas. Metaplastic exocrine cells also started to express LIF and this was increased after alloxan treatment. Signalling via LIF-receptor-β/gp130 involves the JAK/signal transducer and activator of transcription (STAT) pathway. LIF induced increased activation of STAT3 in pancreatic cells. In isolated duct fragments, addition of LIF resulted in a significant increase in duct cell proliferation, while a specific inhibitor of the JAK/STAT signalling pathway inhibited proliferation.Conclusion/interpretationOur observations show that LIF and its receptor are expressed in cells from pancreatic ducts. The cytokine plays a role in pancreatic physiology, controls duct cell proliferation and is involved in repair processes following pancreatic injury.

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 …