Publications by authors named "Luc Bouwens"

64 Publications

Transcutaneous Vagal Nerve Stimulation Alone or in Combination With Radiotherapy Stimulates Lung Tumor Infiltrating Lymphocytes But Fails to Suppress Tumor Growth.

Front Immunol 2021 1;12:772555. Epub 2021 Dec 1.

Laboratory for Molecular and Cellular Therapy, Department of Biomedical Sciences, Vrije Universiteit Brussel, Brussels, Belgium.

The combination of radiotherapy (RT) with immunotherapy represents a promising treatment modality for non-small cell lung cancer (NSCLC) patients. As only a minority of patients shows a persistent response today, a spacious optimization window remains to be explored. Previously we showed that fractionated RT can induce a local immunosuppressive profile. Based on the evolving concept of an immunomodulatory role for vagal nerve stimulation (VNS), we tested its therapeutic and immunological effects alone and in combination with fractionated RT in a preclinical-translational study. Lewis lung carcinoma-bearing C57Bl/6 mice were treated with VNS, fractionated RT or the combination while a patient cohort with locally advanced NSCLC receiving concurrent radiochemotherapy (ccRTCT) was enrolled in a clinical trial to receive either sham or effective VNS daily during their 6 weeks of ccRTCT treatment. Preclinically, VNS alone or with RT showed no therapeutic effect yet VNS alone significantly enhanced the activation profile of intratumoral CD8 T cells by upregulating their IFN-γ and CD137 expression. In the periphery, VNS reduced the RT-mediated rise of splenic, but not blood-derived, regulatory T cells (Treg) and monocytes. In accordance, the serological levels of protumoral CXCL5 next to two Treg-attracting chemokines CCL1 and CCL22 were reduced upon VNS monotherapy. In line with our preclinical findings on the lack of immunological changes in blood circulating immune cells upon VNS, immune monitoring of the peripheral blood of VNS treated NSCLC patients (n=7) did not show any significant changes compared to ccRTCT alone. As our preclinical data do suggest that VNS intensifies the stimulatory profile of the tumor infiltrated CD8 T cells, this favors further research into non-invasive VNS to optimize current response rates to RT-immunotherapy in lung cancer patients.
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http://dx.doi.org/10.3389/fimmu.2021.772555DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8671299PMC
December 2021

Discovery and 3D imaging of a novel ΔNp63-expressing basal cell type in human pancreatic ducts with implications in disease.

Gut 2021 Jul 30. Epub 2021 Jul 30.

Laboratory of Medical and Molecular Oncology, Vrije Universiteit Brussel, Brussel, Belgium

Objective: The aggressive basal-like molecular subtype of pancreatic ductal adenocarcinoma (PDAC) harbours a ΔNp63 (p40) gene expression signature reminiscent of a basal cell type. Distinct from other epithelia with basal tumours, ΔNp63 basal cells reportedly do not exist in the normal pancreas.

Design: We evaluated ΔNp63 expression in human pancreas, chronic pancreatitis (CP) and PDAC. We further studied in depth the non-cancerous tissue and developed a three-dimensional (3D) imaging protocol (FLIP-IT, Fluorescence Light sheet microscopic Imaging of Paraffin-embedded or Intact Tissue) to study formalin-fixed paraffin-embedded samples at single cell resolution. Pertinent mouse models and HPDE cells were analysed.

Results: In normal human pancreas, rare ΔNp63 cells exist in ducts while their prevalence increases in CP and in a subset of PDAC. In non-cancer tissue, ΔNp63 cells are atypical KRT19 duct cells that overall lack SOX9 expression while they do express canonical basal markers and pertain to a niche of cells expressing gastrointestinal stem cell markers. 3D views show that the basal cells anchor on the basal membrane of normal medium to large ducts while in CP they exist in multilayer dome-like structures. In mice, ΔNp63 is not found in adult pancreas nor in selected models of CP or PDAC, but it is induced in organoids from larger Sox9 ducts. In HPDE, ΔNp63 supports a basal cell phenotype at the expense of a classical duct cell differentiation programme.

Conclusion: In larger human pancreatic ducts, basal cells exist. ΔNp63 suppresses duct cell identity. These cells may play an important role in pancreatic disease, including PDAC ontogeny, but are not present in mouse models.
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http://dx.doi.org/10.1136/gutjnl-2020-322874DOI Listing
July 2021

Fractionated Radiation Severely Reduces the Number of CD8+ T Cells and Mature Antigen Presenting Cells Within Lung Tumors.

Int J Radiat Oncol Biol Phys 2021 09 15;111(1):272-283. Epub 2021 Apr 15.

Laboratory of Molecular and Cellular Therapy, Department of Biomedical Sciences, Vrije Universiteit Brussel, Brussels, Belgium. Electronic address:

Purpose: The combination of standard-of-care radiation therapy (RT) with immunotherapy is moving to the mainstream of non-small cell lung cancer treatment. Multiple preclinical studies reported on the CD8 T cell stimulating properties of RT, resulting in abscopal therapeutic effects. A literature search demonstrates that most preclinical lung cancer studies applied subcutaneous lung tumor models. Hence, in-depth immunologic evaluation of clinically relevant RT in orthotopic lung cancer models is lacking.

Methods And Materials: We studied the therapeutic and immunologic effects of low-dose fractionated RT on lungs from C57BL/6 mice, challenged 2 weeks before with firefly luciferase expressing Lewis lung carcinoma cells via the tail vein. Low-dose fractionation was represented by 4 consecutive daily fractions of image guided RT at 3.2 Gy.

Results: We showed reduced lung tumor growth upon irradiation using in vivo bioluminescence imaging and immunohistochemistry. Moreover, significant immunologic RT-induced changes were observed in irradiated lungs and in the periphery (spleen and blood). First, a significant decrease in the number of CD8 T cells and trends toward more CD4 and regulatory T cells were seen after RT in all evaluated tissues. Notably, only in the periphery did the remaining CD8 T cells show a more activated phenotype. In addition, a significant expansion of neutrophils and monocytes was observed upon RT locally and systemically. Locally, RT increased the influx of tumor-associated macrophages and conventional type 2 dendritic cells, whereas the alveolar macrophages and conventional type 1 DCs dramatically decreased. Functionally, these antigen-presenting cells severely reduced their CD86 expression, suggesting a reduced capacity to induce potent immunity.

Conclusions: Our results imply that low-dose fractionated RT of tumor-bearing lung tissue shifts the immune cell balance toward an immature myeloid cell dominating profile. These data argue for myeloid cell repolarizing strategies to enhance the abscopal effects in patients with non-small cell lung cancer treated with fractionated RT.
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http://dx.doi.org/10.1016/j.ijrobp.2021.04.009DOI Listing
September 2021

Single-cell profiling of myeloid cells in glioblastoma across species and disease stage reveals macrophage competition and specialization.

Nat Neurosci 2021 04 29;24(4):595-610. Epub 2021 Mar 29.

Myeloid Cell Immunology Lab, VIB Center for Inflammation Research, Brussels, Belgium.

Glioblastomas are aggressive primary brain cancers that recur as therapy-resistant tumors. Myeloid cells control glioblastoma malignancy, but their dynamics during disease progression remain poorly understood. Here, we employed single-cell RNA sequencing and CITE-seq to map the glioblastoma immune landscape in mouse tumors and in patients with newly diagnosed disease or recurrence. This revealed a large and diverse myeloid compartment, with dendritic cell and macrophage populations that were conserved across species and dynamic across disease stages. Tumor-associated macrophages (TAMs) consisted of microglia- or monocyte-derived populations, with both exhibiting additional heterogeneity, including subsets with conserved lipid and hypoxic signatures. Microglia- and monocyte-derived TAMs were self-renewing populations that competed for space and could be depleted via CSF1R blockade. Microglia-derived TAMs were predominant in newly diagnosed tumors, but were outnumbered by monocyte-derived TAMs following recurrence, especially in hypoxic tumor environments. Our results unravel the glioblastoma myeloid landscape and provide a framework for future therapeutic interventions.
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http://dx.doi.org/10.1038/s41593-020-00789-yDOI Listing
April 2021

MECOM permits pancreatic acinar cell dedifferentiation avoiding cell death under stress conditions.

Cell Death Differ 2021 09 24;28(9):2601-2615. Epub 2021 Mar 24.

Laboratory of Medical and Molecular Oncology, Oncology Research Center, Vrije Universiteit Brussel, Laarbeeklaan 103, Jette, 1090, België.

Maintenance of the pancreatic acinar cell phenotype suppresses tumor formation. Hence, repetitive acute or chronic pancreatitis, stress conditions in which the acinar cells dedifferentiate, predispose for cancer formation in the pancreas. Dedifferentiated acinar cells acquire a large panel of duct cell-specific markers. However, it remains unclear to what extent dedifferentiated acini differ from native duct cells and which genes are uniquely regulating acinar cell dedifferentiation. Moreover, most studies have been performed on mice since the availability of human cells is scarce. Here, we applied a non-genetic lineage tracing method of human pancreatic exocrine acinar and duct cells that allowed cell-type-specific gene expression profiling by RNA sequencing. Subsequent to this discovery analysis, one transcription factor that was unique for dedifferentiated acinar cells was functionally characterized. RNA sequencing analysis showed that human dedifferentiated acinar cells expressed genes in "Pathways of cancer" with a prominence of MECOM (EVI-1), a transcription factor that was not expressed by duct cells. During mouse embryonic development, pre-acinar cells also transiently expressed MECOM and in the adult mouse pancreas, MECOM was re-expressed when mice were subjected to acute and chronic pancreatitis, conditions in which acinar cells dedifferentiate. In human cells and in mice, MECOM expression correlated with and was directly regulated by SOX9. Mouse acinar cells that, by genetic manipulation, lose the ability to upregulate MECOM showed impaired cell adhesion, more prominent acinar cell death, and suppressed acinar cell dedifferentiation by limited ERK signaling. In conclusion, we transcriptionally profiled the two major human pancreatic exocrine cell types, acinar and duct cells, during experimental stress conditions. We provide insights that in dedifferentiated acinar cells, cancer pathways are upregulated in which MECOM is a critical regulator that suppresses acinar cell death by permitting cellular dedifferentiation.
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http://dx.doi.org/10.1038/s41418-021-00771-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8408219PMC
September 2021

Dynamic Regulation of Expression of KRAS and Its Effectors Determines the Ability to Initiate Tumorigenesis in Pancreatic Acinar Cells.

Cancer Res 2021 05 18;81(10):2679-2689. Epub 2021 Feb 18.

Université catholique de Louvain, de Duve Institute, Brussels, Belgium.

Pancreatic acinar cells are a cell type of origin for pancreatic cancer that become progressively less sensitive to tumorigenesis induced by oncogenic Kras mutations after birth. This sensitivity is increased when Kras mutations are combined with pancreatitis. Molecular mechanisms underlying these observations are still largely unknown. To identify these mechanisms, we generated the first CRISPR-edited mouse models that enable detection of wild-type and mutant KRAS proteins . Analysis of these mouse models revealed that more than 75% of adult acinar cells are devoid of detectable KRAS protein. In the 25% of acinar cells expressing KRAS protein, transcriptomic analysis highlighted a slight upregulation of the RAS and MAPK pathways. However, at the protein level, only marginal pancreatic expression of essential KRAS effectors, including C-RAF, was observed. The expression of KRAS and its effectors gradually decreased after birth. The low sensitivity of adult acinar cells to Kras mutations resulted from low expression of KRAS and its effectors and the subsequent lack of activation of RAS/MAPK pathways. Pancreatitis triggered expression of KRAS and its effectors as well as subsequent activation of downstream signaling; this induction required the activity of EGFR. Finally, expression of C-RAF in adult pancreas was required for pancreatic tumorigenesis. In conclusion, our study reveals that control of the expression of KRAS and its effectors regulates the sensitivity of acinar cells to transformation by oncogenic Kras mutations. SIGNIFICANCE: This study generates new mouse models to study regulation of KRAS during pancreatic tumorigenesis and highlights a novel mechanism through which pancreatitis sensitizes acinar cells to Kras mutations.
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http://dx.doi.org/10.1158/0008-5472.CAN-20-2976DOI Listing
May 2021

Aspalathin Protects Insulin-Producing β Cells against Glucotoxicity and Oxidative Stress-Induced Cell Death.

Mol Nutr Food Res 2020 04 12;64(8):e1901009. Epub 2020 Feb 12.

Cell Differentiation Lab, Vrije Universiteit Brussel, Laarbeeklaan 103, 1090 Jette, Brussels, Belgium.

Scope: Aspalathin, the main polyphenolic phytochemical of rooibos (Aspalathus linearis), has been attributed with health promoting properties, including a glucose lowering effect that can prove interesting for application as nutraceutical or therapeutic in (pre-)diabetics. Preservation of β cell mass in the pancreas is considered a key issue for diabetes prevention or treatment, therefore the aim is to investigate whether aspalathin also has β cell cytoprotective potential.

Methods And Results: Rat pancreatic islets and the β cell line Insulinoma 1E (INS1E) are studied in vitro after exposure to various cytotoxic agents, namely streptozotocin (STZ), hydrogen peroxide, or chronic high glucose. The effect of aspalathin on cell survival and apoptosis is studied. Expression of relevant cytoprotective genes is analyzed by qRT-PCR and proteins by Western blot. Aspalathin is found to protect β cells against cytotoxicity and apoptosis. This is associated with increased translocation of nuclear factor erythroid 2-related factor 2 (NRF2) and expression of its antioxidant target genes heme oxygenase 1 (Hmox1), NAD(P)H quinone dehydrogenase 1 (Nqo-1), and superoxide dismutase 1 (Sod1).

Conclusion: It is proposed that aspalathin protects β cells against glucotoxicity and oxidative stress by increasing the expression of NRF2-regulated antioxidant enzymes. This indicates that aspalathin is an interesting β cell cytoprotectant.
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http://dx.doi.org/10.1002/mnfr.201901009DOI Listing
April 2020

Measuring the Pancreatic β Cell Mass in Vivo with Exendin SPECT during Hyperglycemia and Severe Insulitis.

Mol Pharm 2019 09 8;16(9):4024-4030. Epub 2019 Aug 8.

Department of Radiology and Nuclear Medicine , Radboud university medical center , PO Box 9101, 6500 HB Nijmegen , The Netherlands.

Objective: Targeting the glucagon-like peptide-1 receptor with radiolabeled exendin is a very promising method to noninvasively determine the β cell mass in the pancreas, which is needed to unravel the pathophysiology of type 1 and type 2 diabetes. The present study aimed to explore the effects of both hyperglycemia and insulitis on the uptake of exendin in a spontaneous type 1 diabetes mouse model, nonobese diabetic (NOD) mice.

Methods: NOD mice ( = 75, 7-21 weeks old) were injected intravenously with [In]In-DTPA-exendin-3, and single-photon emission computed tomography (SPECT) images were acquired 1 h pi. The pancreatic accumulation of [In]In-DTPA-exendin-3 was quantified in vivo using SPECT and by ex vivo counting and correlated to the β cell mass (BCM). The influence of insulitis and hyperglycemia on the exendin uptake was assessed.

Results: The pancreas could be visualized longitudinally using SPECT. A linear correlation was found between the BCM (%) and pancreatic uptake (%ID/g) as measured by ex vivo counting (Pearson = 0.64, < 0.001), which was not affected by either insulitis (Pearson = 0.66, = 0.83) or hyperglycemia (Pearson = 0.57, = 0.51). Biodistribution and ex vivo autoradiography revealed remaining [In]In-DTPA-exendin-3 uptake in the pancreas despite total ablation of BCM.

Conclusions: Despite hyperglycemia and severe insulitis, we have found a good correlation between BCM and pancreatic exendin uptake, even in a suboptimal model with relatively high background activity.
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http://dx.doi.org/10.1021/acs.molpharmaceut.9b00728DOI Listing
September 2019

Adult human pancreatic acinar cells dedifferentiate into an embryonic progenitor-like state in 3D suspension culture.

Sci Rep 2019 03 11;9(1):4040. Epub 2019 Mar 11.

Cell Differentiation Laboratory, Vrije Universiteit Brussel, 1090, Brussels, Belgium.

Human pancreatic exocrine cells were cultured in 3D suspension and formed pancreatospheres composed of acinar-derived and duct-like cells. We investigated, up to 6 days, the fate of human pancreatic acinar cells using fluorescein-conjugated Ulex Europaeus Agglutinin 1 lectin, a previously published acinar-specific non-genetic lineage tracing strategy. At day 4, fluorescence-activated cell sort for the intracellularly incorporated FITC-conjugated UEA1 lectin and the duct-specific CA19.9 surface marker, distinguished acinar-derived cells (UEA1CA19.9) from duct-like cells (UEA1CA19.9) and acinar-to-duct-like transdifferentiated cells (UEA1CA19.9). mRNA expression analysis of the acinar-derived (UEA1CA19.9) and duct-like (UEA1CA19.9) cell fractions with concomitant immunocytochemical analysis of the pancreatospheres revealed acquisition of an embryonic signature in the UEA1CA19.9 acinar-derived cells characterized by de novo expression of SOX9 and CD142, robust expression of PDX1 and surface expression of GP2. The colocalisation of CD142, a multipotent pancreatic progenitor surface marker, PDX1, SOX9 and GP2 is reminiscent of a cellular state present during human embryonic development. Addition of TGF-beta signalling inhibitor Alk5iII, induced a 28-fold increased KI67-labeling in pancreatospheres, more pronounced in the CD142GP2 acinar-derived cells. These findings with human cells underscore the remarkable plasticity of pancreatic exocrine acinar cells, previously described in rodents, and could find applications in the field of regenerative medicine.
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http://dx.doi.org/10.1038/s41598-019-40481-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6411888PMC
March 2019

A nanobody-based tracer targeting DPP6 for non-invasive imaging of human pancreatic endocrine cells.

Sci Rep 2017 11 9;7(1):15130. Epub 2017 Nov 9.

In vivo Cellular and Molecular Imaging Laboratory (ICMI), Vrije Universiteit Brussel (VUB), Brussels, Belgium.

There are presently no reliable ways to quantify endocrine cell mass (ECM) in vivo, which prevents an accurate understanding of the progressive beta cell loss in diabetes or following islet transplantation. To address this unmet need, we coupled RNA sequencing of human pancreatic islets to a systems biology approach to identify new biomarkers of the endocrine pancreas. Dipeptidyl-Peptidase 6 (DPP6) was identified as a target whose mRNA expression is at least 25-fold higher in human pancreatic islets as compared to surrounding tissues and is not changed by proinflammatory cytokines. At the protein level, DPP6 localizes only in beta and alpha cells within the pancreas. We next generated a high-affinity camelid single-domain antibody (nanobody) targeting human DPP6. The nanobody was radiolabelled and in vivo SPECT/CT imaging and biodistribution studies were performed in immunodeficient mice that were either transplanted with DPP6-expressing Kelly neuroblastoma cells or insulin-producing human EndoC-βH1 cells. The human DPP6-expressing cells were clearly visualized in both models. In conclusion, we have identified a novel beta and alpha cell biomarker and developed a tracer for in vivo imaging of human insulin secreting cells. This provides a useful tool to non-invasively follow up intramuscularly implanted insulin secreting cells.
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http://dx.doi.org/10.1038/s41598-017-15417-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5680294PMC
November 2017

Circulating microRNA-375 as biomarker of pancreatic beta cell death and protection of beta cell mass by cytoprotective compounds.

PLoS One 2017 17;12(10):e0186480. Epub 2017 Oct 17.

Cell Differentiation Lab, Vrije Universiteit Brussel (VUB), Brussels, Belgium.

Objective: Previous studies demonstrated that circulating microRNA-375 (miR-375) is a suitable plasma biomarker for real-time detection of beta cell death. The present study evaluated the use of this biomarker to assess the beta cytoprotective effect of phenylpropenoic acid glucoside (PPAG), which was previously demonstrated to protect beta cells against various types of injury, and of exendin-4, which is an established antidiabetic drug.

Methods: PPAG or exendin-4 were administered in mice treated with streptozotocin (STZ) to acutely induce beta cell death. Beta cell mass and apoptotic death were measured in pancreatic tissue sections. Circulating miR-375 was measured in blood plasma by RT-qPCR. The release of miR-375 was also measured in vitro by MIN-6 beta cells.

Results: Administration of STZ resulted in measurable circulating levels of miR-375, a decrease in beta cell mass and increase in frequency of apoptotic beta cells. In vitro, there was a good correlation between miR-375 release and the extent of beta cell death. Treatment of mice with PPAG or exendin-4 significantly attenuated STZ-induced loss of beta cell mass and beta cell apoptosis, and normalized the blood level of miR-375.

Conclusions: These findings show the potential use of serological miR-375 measurements to evaluate the beta cytoprotective effect of (potential) antidiabetic drugs in vivo.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0186480PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5645134PMC
October 2017

Acinar cells in the neonatal pancreas grow by self-duplication and not by neogenesis from duct cells.

Sci Rep 2017 10 3;7(1):12643. Epub 2017 Oct 3.

Cell Differentiation Lab, Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel, Brussels, Belgium.

Pancreatic acinar cells secrete digestive enzymes necessary for nutrient digestion in the intestine. They are considered the initiating cell type of pancreatic cancer and are endowed with differentiation plasticity that has been harnessed to regenerate endocrine beta cells. However, there is still uncertainty about the mechanisms of acinar cell formation during the dynamic period of early postnatal development. To unravel cellular contributions in the exocrine acinar development we studied two reporter mouse strains to trace the fate of acinar and duct cells during the first 4 weeks of life. In the acinar reporter mice, the labelling index of acinar cells remained unchanged during the neonatal pancreas growth period, evidencing that acinar cells are formed by self-duplication. In line with this, duct cell tracing did not show significant increase in acinar cell labelling, excluding duct-to-acinar cell contribution during neonatal development. Immunohistochemical analysis confirms massive levels of acinar cell proliferation in this early period of life. Further, also increase in acinar cell size contributes to the growth of pancreatic mass.We conclude that the growth of acinar cells during physiological neonatal pancreas development is by self-duplication (and hypertrophy) rather than neogenesis from progenitor cells as was suggested before.
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http://dx.doi.org/10.1038/s41598-017-12721-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5626771PMC
October 2017

Co-localization of acinar markers and insulin in pancreatic cells of subjects with type 2 diabetes.

PLoS One 2017 15;12(6):e0179398. Epub 2017 Jun 15.

Department of Clinical and Experimental Medicine, Islet Cell Laboratory, University of Pisa, Pisa, Italy.

To search for clues suggesting that beta cells may generate by transdifferentiation in humans, we assessed the presence of cells double positive for exocrine (amylase, carboxypeptidase A) and endocrine (insulin) markers in the pancreas of non-diabetic individuals (ND) and patients with type 2 diabetes (T2D). Samples from twelve ND and twelve matched T2D multiorgan donors were studied by electron microscopy, including amylase and insulin immunogold labeling; carboxypeptidase A immunofluorescence light microscopy assessment was also performed. In the pancreas from four T2D donors, cells containing both zymogen-like and insulin-like granules were observed, scattered in the exocrine compartment. Nature of granules was confirmed by immunogold labeling for amylase and insulin. Double positive cells ranged from 0.82 to 1.74 per mm2, corresponding to 0.26±0.045% of the counted exocrine cells. Intriguingly, cells of the innate immune systems (mast cells and/or macrophages) were adjacent to 33.3±13.6% of these hybrid cells. No cells showing co-localization of amylase and insulin were found in ND samples by electron microscopy. Similarly, cells containing both carboxypeptidase A and insulin were more frequently observed in the diabetic pancreata. These results demonstrate more abundant presence of cells containing both acinar markers and insulin in the pancreas of T2D subjects, which suggests possible conversion from one cellular type to the other and specific association with the diseased condition.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0179398PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5472296PMC
September 2017

Phenylpropenoic Acid Glucoside from Rooibos Protects Pancreatic Beta Cells against Cell Death Induced by Acute Injury.

PLoS One 2016 14;11(6):e0157604. Epub 2016 Jun 14.

Cell Differentiation Lab, Diabetes Research Center, Vrije Universiteit Brussel (VUB), Brussels, Belgium.

Objective: Previous studies demonstrated that a phenylpropenoic acid glucoside (PPAG) from rooibos (Aspalathus linearis) extract had anti-hyperglycemic activity and significant protective effects on the pancreatic beta cell mass in a chronic diet-induced diabetes model. The present study evaluated the cytoprotective effect of the phytochemical on beta cells exposed to acute cell stress.

Methods: Synthetically prepared PPAG was administered orally in mice treated with a single dose of streptozotocin to acutely induce beta cell death and hyperglycemia. Its effect was assessed on beta cell mass, proliferation and apoptotic cell death. Its cytoprotective effect was also studied in vitro on INS-1E beta cells and on human pancreatic islet cells.

Results: Treatment with the phytochemical PPAG protected beta cells during the first days after the insult against apoptotic cell death, as evidenced by TUNEL staining, and prevented loss of expression of anti-apoptotic protein BCL2 in vivo. In vitro, PPAG protected INS-1E beta cells from streptozotocin-induced apoptosis and necrosis in a BCL2-dependent and independent way, respectively, depending on glucose concentration. PPAG also protected human pancreatic islet cells against the cytotoxic action of the fatty acid palmitate.

Conclusions: These findings show the potential use of PPAG as phytomedicine which protects the beta cell mass exposed to acute diabetogenic stress.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0157604PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4907458PMC
July 2017

Acinar phenotype is preserved in human exocrine pancreas cells cultured at low temperature: implications for lineage-tracing of β-cell neogenesis.

Biosci Rep 2016 06 6;36(3). Epub 2016 May 6.

Cell Differentiation Unit, Diabetes Research Center, Vrije Universiteit Brussel (VUB), Brussels, BE 1090, Belgium

The regenerative medicine field is expanding with great successes in laboratory and preclinical settings. Pancreatic acinar cells in diabetic mice were recently converted into β-cells by treatment with ciliary neurotrophic factor (CNTF) and epidermal growth factor (EGF). This suggests that human acinar cells might become a cornerstone for diabetes cell therapy in the future, if they can also be converted into glucose-responsive insulin-producing cells. Presently, studying pancreatic acinar cell biology in vitro is limited by their high plasticity, as they rapidly lose their phenotype and spontaneously transdifferentiate to a duct-like phenotype in culture. We questioned whether human pancreatic acinar cell phenotype could be preserved in vitro by physico-chemical manipulations and whether this could be valuable in the study of β-cell neogenesis. We found that culture at low temperature (4°C) resulted in the maintenance of morphological and molecular acinar cell characteristics. Specifically, chilled acinar cells did not form the spherical clusters observed in controls (culture at 37°C), and they maintained high levels of acinar-specific transcripts and proteins. Five-day chilled acinar cells still transdifferentiated into duct-like cells upon transfer to 37°C. Moreover, adenoviral-mediated gene transfer evidenced an active Amylase promoter in the 7-day chilled acinar cells, and transduction performed in chilled conditions improved acinar cell labelling. Together, our findings indicate the maintenance of human pancreatic acinar cell phenotype at low temperature and the possibility to efficiently label acinar cells, which opens new perspectives for the study of human acinar-to-β-cell transdifferentiation.
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http://dx.doi.org/10.1042/BSR20150259DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4859086PMC
June 2016

Beta Cell Mass Restoration in Alloxan-Diabetic Mice Treated with EGF and Gastrin.

PLoS One 2015 9;10(10):e0140148. Epub 2015 Oct 9.

Cell Differentiation Lab, Vrije Universiteit Brussel (Brussels Free University), Brussels, Belgium.

One week of treatment with EGF and gastrin (EGF/G) was shown to restore normoglycemia and to induce islet regeneration in mice treated with the diabetogenic agent alloxan. The mechanisms underlying this regeneration are not fully understood. We performed genetic lineage tracing experiments to evaluate the contribution of beta cell neogenesis in this model. One day after alloxan administration, mice received EGF/G treatment for one week. The treatment could not prevent the initial alloxan-induced beta cell mass destruction, however it did reverse glycemia to control levels within one day, suggesting improved peripheral glucose uptake. In vitro experiments with C2C12 cell line showed that EGF could stimulate glucose uptake with an efficacy comparable to that of insulin. Subsequently, EGF/G treatment stimulated a 3-fold increase in beta cell mass, which was partially driven by neogenesis and beta cell proliferation as assessed by beta cell lineage tracing and BrdU-labeling experiments, respectively. Acinar cell lineage tracing failed to show an important contribution of acinar cells to the newly formed beta cells. No appearance of transitional cells co-expressing insulin and glucagon, a hallmark for alpha-to-beta cell conversion, was found, suggesting that alpha cells did not significantly contribute to the regeneration. An important fraction of the beta cells significantly lost insulin positivity after alloxan administration, which was restored to normal after one week of EGF/G treatment. Alloxan-only mice showed more pronounced beta cell neogenesis and proliferation, even though beta cell mass remained significantly depleted, suggesting ongoing beta cell death in that group. After one week, macrophage infiltration was significantly reduced in EGF/G-treated group compared to the alloxan-only group. Our results suggest that EGF/G-induced beta cell regeneration in alloxan-diabetic mice is driven by beta cell neogenesis, proliferation and recovery of insulin. The glucose-lowering effect of the treatment might play an important role in the regeneration process.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0140148PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4599944PMC
June 2016

Regulating the beta cell mass as a strategy for type-2 diabetes treatment.

Curr Drug Targets 2015 ;16(5):516-24

VUB Cell Differentiation Lab, Laarbeeklaan 103, B-1090 Brussels, Belgium.

The incidence of type 2 diabetes (T2D) increases dramatically worldwide and has created an enormous health care burden. Obesity, dyslipidemia and insulin resistance are major risk factors for the development of T2D, but the major factor leading to the disease is failure of the insulin-producing beta cell mass to compensate for increasing insulin demands of the body. Progression of the disease further diminishes the beta cell mass as a result of lipotoxicity and glucotoxicity for which beta cells are particularly sensitive. Hence, treatment aiming to prevent beta cell loss or increase the number of beta cells could inhibit diabetes progression or lead to restoration of normal metabolism. Whereas current and new antidiabetic drugs are mainly targeting insulin secretion and action or glucose uptake, newer interventions must be found that prevent beta cell loss or increase beta cell number. The targets for this are beta cell proliferation, neogenesis and survival. This review examines major evidence from animal experiments suggesting that it is feasible to regulate the beta cell mass by bioactive compounds like growth factors, cytokines, hormones, phytochemicals and small molecules. Often the mode of action remains unclear due to inadequate methods to assess the effects of the compounds on the beta cell dynamics. Furthermore, a major challenge is to identify compounds with sufficient specificity in order to avoid unwanted effects on other cell types. Provided such safety issues can be solved, this may provide a curative approach for diabetes treatment.
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http://dx.doi.org/10.2174/1389450116666150204113928DOI Listing
January 2016

Diabetes: β cells at last.

Nat Rev Endocrinol 2015 Jan 11;11(1):5-6. Epub 2014 Nov 11.

Cell Differentiation Unit, Diabetes Research Center, Vrije Universiteit Brussel (VUB), Laarbeeklaan 103, 1090 Brussels, Belgium.

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http://dx.doi.org/10.1038/nrendo.2014.200DOI Listing
January 2015

A standardized method for in vivo mouse pancreas imaging and semiquantitative β cell mass measurement by dual isotope SPECT.

Mol Imaging Biol 2015 Feb;17(1):58-66

Cell Differentiation Unit, Vrije Universiteit Brussel, Laarbeeklaan 103, 1090, Brussels, Belgium,

Purpose: In order to evaluate future β cell tracers in vivo, we aimed to develop a standardized in vivo method allowing semiquantitative measurement of a prospective β cell tracer within the pancreas.

Procedures: 2-[(123)I]Iodo-L-phenylalanine ([(123)I]IPA) and [Lys(40)([(111)In]DTPA)]exendin-3 ([(111)In]Ex3) pancreatic uptake and biodistribution were evaluated using SPECT, autoradiography, and an ex vivo biodistribution study in a controlled unilaterally nephrectomized mouse β cell depletion model. Semiquantitative measurement of the imaging results was performed using [(123)I]IPA to delineate the pancreas and [(111)In]Ex3 as a β cell tracer.

Results: The uptake of [(123)I]IPA was highest in the pancreas. Aside from the kidneys, the uptake of [(111)In]Ex3 was highest in the pancreas and lungs. Autoradiography showed only uptake of [(111)In]Ex3 in insulin-expressing cells. Semiquantitative measurement of [(111)In]Ex3 in the SPECT images based on the delineation of the pancreas with [(123)I]IPA showed a high correlation with the [(111)In]Ex3 uptake data of the pancreas obtained by dissection. A strong positive correlation was observed between the relative insulin positive area and the pancreas-to-blood ratios of [(111)In]Ex3 uptake as determined by counting with a gamma counter and the semiquantitative analysis of the SPECT images.

Conclusions: [(123)I]IPA is a promising tracer to delineate pancreatic tissue on SPECT images. It shows a high uptake in the pancreas as compared to other abdominal tissues. This study also demonstrates the feasibility and accuracy to measure the β cell mass in vivo in an animal model of diabetes.
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http://dx.doi.org/10.1007/s11307-014-0771-yDOI Listing
February 2015

Phenylpropenoic acid glucoside augments pancreatic beta cell mass in high-fat diet-fed mice and protects beta cells from ER stress-induced apoptosis.

Mol Nutr Food Res 2014 Oct 4;58(10):1980-90. Epub 2014 Aug 4.

Cell Differentiation Lab, Vrije Universiteit Brussel, Brussels, Belgium.

Scope: A major goal of diabetes therapy is to identify novel drugs that preserve or expand pancreatic beta cell mass. Here, we examined the effect of a phenylpropenoic acid glucoside (PPAG) on the beta cell mass, and via which mechanism this effect is established.

Methods And Results: Mice were fed a high-fat and fructose-containing diet to induce obesity and hyperglycemia. PPAG treatment protected obese mice from diet-induced hyperglycemia and resulted in a tripling of beta cell mass. The effect of the phytochemical on beta cell mass was neither due to increased proliferation, as determined by Ki67 immunostaining, nor to neogenesis, which was assessed by genetic lineage tracing. TUNEL staining revealed suppressed apoptosis in PPAG-treated obese mice. In vitro, PPAG protected beta cells from palmitate-induced apoptosis. It protected beta cells against ER stress by increasing expression of antiapoptotic B-cell lymphoma 2 (BCL2) protein without affecting proapoptotic signals.

Conclusions: We identified an antidiabetic phytochemical that protects pancreatic beta cells from ER stress and apoptosis induced by high-fat diet/lipotoxicity. At the tissue level, this led to a tripling of beta cell mass. At the molecular level, the protective effect of the phytochemical was mediated by increasing BCL2 expression in beta cells.
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http://dx.doi.org/10.1002/mnfr.201400211DOI Listing
October 2014

Conversion of human pancreatic acinar cells toward a ductal-mesenchymal phenotype and the role of transforming growth factor β and activin signaling.

Pancreas 2014 Oct;43(7):1083-92

From the *Cell Differentiation Laboratory, and †Cell Therapy Laboratory, Diabetes Research Center, Vrije Universiteit Brussel, Brussels, Belgium.

Objective: Epithelial-mesenchymal transition may interfere with the differentiation of cultured pancreatic acinar cells toward endocrine cells. Therefore, it will be important to investigate into detail the reprogramming of human pancreatic acinar cells toward a mesenchymal phenotype: the association with acinoductal transdifferentiation, the influence of cell adhesion, and the regulation behind this process.

Methods: Human exocrine cells, isolated from donor pancreata, were cultured in suspension or as monolayers. Non-genetic lineage tracing, using labeled ulex europaeus agglutinin 1 lectin, was performed, and the role of the transforming growth factor (TGF-β) superfamily was investigated.

Results: After 7 days in monolayer culture, the human acinar cells coexpressed the mesenchymal marker vimentin and the ductal marker Sox9. However, when the human exocrine cells were cultured in suspension, epithelial-mesenchymal transition was not observed. The spontaneous transition of the human acinar cells toward a ductal and mesenchymal phenotype was decreased by inhibition of the TGF-β and activin signaling pathways.

Conclusions: The human acinar cells spontaneously undergo TGF-β- regulated reprogramming in the monolayer culture. These observations are helpful to develop culture methods for the in vitro reprogramming of pancreatic exocrine to endocrine cells. They are also of potential interest for studies on exocrine acinar cells in the development of pancreatic cancer.
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http://dx.doi.org/10.1097/MPA.0000000000000154DOI Listing
October 2014

Specific targeting of atherosclerotic plaques in ApoE(-/-) mice using a new Camelid sdAb binding the vulnerable plaque marker LOX-1.

Mol Imaging Biol 2014 Oct;16(5):690-8

Laboratory of Cellular and Molecular Immunology (CMIM), Vrije Universiteit Brussel (VUB), Pleinlaan 2, Brussels, 1050, Belgium,

Purpose: Molecular imaging has the potential to provide quantitative information about specific biological aspects of developing atherosclerotic lesions. This requires the generation of reliable, highly specific plaque tracers. This study reports a new camelid single-domain antibody fragment (sdAb) targeting the Lectin-like oxidized low-density lipoprotein receptor (LOX-1), a biomarker for the detection and molecular phenotyping of vulnerable atherosclerotic plaques.

Procedures: A camelid sdAb was generated and selected for high affinity binding to LOX-1. Ex vivo biodistribution and in vivo single photon emission computed tomography (SPECT)/computed tomography (CT) imaging studies were performed in wild-type mice and in fat-fed atherosclerotic apolipoprotein E-deficient mice with (99m)Tc-labeled sdAbs. Gamma-counting and autoradiography analyses were performed on dissected aorta segments with different degrees of plaque burden. The specificity of the LOX-1-targeting sdAb was evaluated by blocking with unlabeled sdAb or by comparison with a nontargeting (99m)Tc-labeled control sdAb.

Results: We generated a sdAb binding LOX-1 with a KD of 280 pM ± 62 pM affinity. After (99m)Tc-labeling, the tracer had radiochemical purity higher then 99 % and retained specificity in in vitro binding studies. Tracer blood clearance was fast with concomitant high kidney retention. At 3 h after injection, uptake in tissues other than plaques was low and not different than background, suggesting a restricted expression pattern of LOX-1. Conversely, uptake in aortic segments increased with plaque content and was due to specific LOX-1 binding. In vivo SPECT/CT imaging 160 min after injection in atherosclerotic mice confirmed specific targeting of LOX-1-expressing aortic plaques.

Conclusions: The LOX-sdAb specifically targets LOX-1-expressing atherosclerotic plaques within hours after injection. The possibility to image LOX-1 rapidly after administration combined with the favourable biodistribution of a sdAb are beneficial for molecular phenotyping of atherosclerotic plaques and the generation of a future prognostic tracer.
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http://dx.doi.org/10.1007/s11307-014-0731-6DOI Listing
October 2014

Molecular imaging with macrophage CRIg-targeting nanobodies for early and preclinical diagnosis in a mouse model of rheumatoid arthritis.

J Nucl Med 2014 May 31;55(5):824-9. Epub 2014 Mar 31.

Laboratory of Cellular and Molecular Immunology, Vrije Universiteit Brussel, Brussels, Belgium.

Unlabelled: An accurate and noninvasive tracer able to detect molecular events underlying the development of rheumatoid arthritis (RA) would be useful for RA diagnosis and drug efficacy assessment. A complement receptor of the Ig superfamily (CRIg) is expressed on synovial macrophages of RA patients, making it an interesting target for molecular imaging of RA. We aim to develop a radiotracer for the visualization of CRIg in a mouse model for RA using radiolabeled single-domain variable antibody VHH fragments (Nanobodies).

Methods: Quantitative polymerase chain reaction was used to locate CRIg expression in mice with collagen-induced arthritis (CIA). A Nanobody, NbV4m119, was generated to specifically target CRIg. Flow cytometry, phosphorimaging, and confocal microscopy were used to confirm NbVm119 binding to CRIg-positive cells. SPECT (SPECT/CT) was used to image arthritic lesions in the inflamed paws of 29 mice using (99m)Tc-NbV4m119 Nanobody.

Results: CRIg is constitutively expressed in the liver and was found to be upregulated in synovial tissues of CIA mice. SPECT/CT imaging revealed that (99m)Tc-NbV4m119 specifically targeted CRIg-positive liver macrophages in naïve wild-type but not in CRIg(-/-) (CRIg knockout) mice. In CIA mice, (99m)Tc-NbV4m119 accumulation in arthritic lesions increased according to the severity of the inflammation. In the knees of mice with CIA, (99m)Tc-NbV4m119 was found to accumulate even before the onset of macroscopic clinical symptoms.

Conclusion: SPECT/CT imaging with (99m)Tc-NbV4m119 visualizes joint inflammation in CIA. Furthermore, imaging could predict which mice will develop clinical symptoms during CIA. Consequently, imaging of joint inflammation with CRIg-specific Nanobodies offers perspectives for clinical applications in RA patients.
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http://dx.doi.org/10.2967/jnumed.113.130617DOI Listing
May 2014

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

Nat Biotechnol 2014 Jan 17;32(1):76-83. Epub 2013 Nov 17.

Diabetes Research Center, Vrije Universiteit Brussel, Brussels, Belgium.

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.
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http://dx.doi.org/10.1038/nbt.2747DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4096987PMC
January 2014

Efficient definitive endoderm induction from mouse embryonic stem cell adherent cultures: a rapid screening model for differentiation studies.

Stem Cell Res 2014 Jan 23;12(1):166-77. Epub 2013 Oct 23.

Cell Differentiation Unit, Diabetes Research Center, Vrije Universiteit Brussel, Belgium.

Definitive endoderm (DE) differentiation from mouse embryonic stem cell (mESC) monolayer cultures has been limited by poor cell survival or low efficiency. Recently, a combination of TGFβ and Wnt activation with BMP inhibition improved DE induction in embryoid bodies cultured in suspension. Based on these observations we developed a protocol to efficiently induce DE cells in monolayer cultures of mESCs. We obtained a good cell yield with 54.92% DE induction as shown by Foxa2, Sox17, Cxcr4 and E-Cadherin expression. These DE-cells could be further differentiated into posterior foregut and pancreatic phenotypes using a culture protocol initially developed for human embryonic stem cell (hESC) differentiation. In addition, this mESC-derived DE gave rise to hepatocyte-like cells after exposure to BMP and FGF ligands. Our data therefore indicate a substantial improvement of monolayer DE induction from mESCs and support the concept that differentiation conditions for mESC-derived DE are similar to those for hESCs. As mESCs are easier to maintain and manipulate in culture compared to hESCs, and considering the shorter duration of embryonic development in the mouse, this method of efficient DE induction on monolayer will promote the development of new differentiation protocols to obtain DE-derivatives, like pancreatic beta-cells, for future use in cell replacement therapies.
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http://dx.doi.org/10.1016/j.scr.2013.10.004DOI Listing
January 2014

[Differentiation of pluripotent stem cells into pancreatic lineages].

Med Sci (Paris) 2013 Aug-Sep;29(8-9):736-43. Epub 2013 Sep 5.

Unité de différenciation cellulaire, Centre de recherche sur le diabète, Vrije Universiteit Brussel, Bruxelles, Belgique.

Diabetes mellitus is the leading metabolic disease and represents a major public health concern worldwide. Whereas the transplantation of pancreas donor-derived islets significantly improves the quality of life of diabetic patients who become insulin independent for few years, it can unfortunately be provided only to few patients in an advanced stage of the disease. This situation is related to the severe shortage in pancreas donors and has prompted the hunt for alternative sources of islet cells. Beside many other strategies aiming at producing new beta cells in vitro or in vivo, a particular focus has been on the plupiropent stem cells because of their abundant availability and their extreme plasticity. Progress in understanding small vertebrates embryonic development has tremendously contributed to the design of differentiation strategies applied to pluripotent stem cells. Nowadays, definitive endoderm and pancreatic progenitors can be efficiently induced from human embryonic stem cells and from human induced pluripotent stem cells. Although we are still lacking the knowledge required for deriving functional beta cells in vitro, transplantation experiments have demonstrated that stem cell-derived pancreas progenitors further generate this phenotype in vivo. All these findings gathered during the last decade witness the closer clinical application of pluripotent stem cell progenies in diabetes cell therapy.
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http://dx.doi.org/10.1051/medsci/2013298012DOI Listing
November 2013

The use of stem cells for pancreatic regeneration in diabetes mellitus.

Nat Rev Endocrinol 2013 Oct 23;9(10):598-606. Epub 2013 Jul 23.

Cell Differentiation Unit, Vrije Universiteit Brussel, Laarbeeklaan 103, Brussels B-1090, Belgium.

The endocrine pancreas represents an interesting arena for regenerative medicine and cell therapeutics. One of the major pancreatic diseases, diabetes mellitus is a metabolic disorder caused by having an insufficient number of insulin-producing β cells. Replenishment of β cells by cell transplantation can restore normal metabolic control. The shortage in donor pancreata has meant that the demand for transplantable β cells has outstripped the supply, which could be met by using alternative sources of stem cells. This situation has opened up new areas of research, such as cellular reprogramming and in vivo β-cell regeneration. Pluripotent stem cells seem to be the best option for clinical applications of β-cell regeneration in the near future, as these cells have been demonstrated to represent an unlimited source of functional β cells. Although compelling evidence shows that the adult pancreas retains regenerative capacity, it remains unclear whether this organ contains stem cells. Alternatively, specialized cell types within or outside the pancreas retain plasticity in proliferation and differentiation. Cellular reprogramming or transdifferentiation of exocrine cells or other types of endocrine cells in the pancreas could provide a long-term solution.
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http://dx.doi.org/10.1038/nrendo.2013.145DOI Listing
October 2013

Signaling pathways during maintenance and definitive endoderm differentiation of embryonic stem cells.

Int J Dev Biol 2013 ;57(1):1-12

Cell Differentiation Unit, Diabetes Research Center, Vrije Universiteit Brussel, Belgium.

Embryonic stem cells (ESCs) have the potential to be used as unlimited resources for tissue replacement therapy, thereby compensating for organ donor shortage. To reach this goal, the molecular principles governing early differentiation events in the developing embryo need to be addressed, understood and properly implemented in vitro. Studies carried out in several vertebrate models have established that Nodal/Activin A, BMP, WNT and FGF signaling pathways regulate early embryo development and that these pathways are similarly used during germ layer formation by cultured ESCs. However, differences have also been identified in the way these pathways function or interact in mouse vs. human ESCs, making it sometimes difficult to extrapolate findings from one system to the other. In this review, we discuss and compare the role of the relevant signaling pathways and their crosstalk during undifferentiated growth and during the endoderm differentiation of mouse and human ESCs.
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http://dx.doi.org/10.1387/ijdb.120115lsDOI Listing
November 2013

Role of BMP signaling in pancreatic progenitor differentiation from human embryonic stem cells.

Stem Cell Rev Rep 2013 Oct;9(5):569-77

Cell Differentiation Unit, Diabetes Research Center, Vrije Universiteit Brussel (VUB), Laarbeeklaan 103, 1090, Brussels, Belgium.

Transplantation of pancreatic progenitors derived from human embryonic stem cells (hESCs) is a promising way to treat diabetes. Strategies to obtain the required cell mass would rely on the up scaling of current differentiation protocols, or the proliferation of committed progenitors. We aimed at finding conditions that maintain a proliferating pancreatic progenitor pool and we assessed the role of BMP4 signaling in this process. hESCs were differentiated into PDX1 positive pancreatic progenitor stage following our established protocol with few modifications, and then the progenitor cells were passaged in a defined proliferation medium (PM). During passage, the effect of BMP4 signaling on the differentiation and proliferation of pancreatic progenitors was examined by RT-PCR and immunofluorescence analysis. We found that PDX1 positive pancreatic progenitors proliferated and gained NKX6.1 expression in the PM, whereas they failed to express NKX6.1 if BMP signaling was inhibited with Noggin. In this latter condition, part of the progenitors rather generated pro-endocrine cells denoted by NGN3 and synaptophysin expression. On the contrary, addition of BMP4 to the PM promoted the early derivation of PDX1 and NKX6.1 coexpressing pancreatic progenitors. Our findings are in line with mouse pancreas development, and indicate that BMP4 signaling is required for the derivation and maintenance of hESC-derived PDX1+NKX6.1+ pancreatic progenitors. These results are instructive for guiding the development of an efficient pancreas differentiation protocol in view of diabetes cell replacement therapy.
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http://dx.doi.org/10.1007/s12015-013-9435-6DOI Listing
October 2013
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