Novel time-resolved reporter mouse reveals spatial and transcriptional heterogeneity during alpha cell differentiation.

AIMS/HYPOTHESIS: Glucagon-expressing pancreatic alpha cells have attracted much attention for their plasticity to transdifferentiate into insulin-producing beta cells; however, it remains unclear precisely when, and from where, alpha cells emerge and what regulates alpha cell fate. We therefore explored the spatial and transcriptional heterogeneity of alpha cell differentiation using a novel time-resolved reporter system. METHODS: We established the mouse model, 'Gcg-Timer', in which newly generated alpha cells can be distinguished from more-differentiated cells by their fluorescence. Fluorescence imaging and transcriptome analysis were performed with Gcg-Timer mice during the embryonic and postnatal stages. RESULTS: Fluorescence imaging and flow cytometry demonstrated that green fluorescence-dominant cells were present in Gcg-Timer mice at the embryonic and neonatal stages but not after 1 week of age, suggesting that alpha cell neogenesis occurs during embryogenesis and early neonatal stages under physiological conditions. Transcriptome analysis of Gcg-Timer embryos revealed that the mRNAs related to angiogenesis were enriched in newly generated alpha cells. Histological analysis revealed that some alpha cells arise close to the pancreatic ducts, whereas the others arise away from the ducts and adjacent to the blood vessels. Notably, when the glucagon signal was suppressed by genetic ablation or by chemicals, such as neutralising glucagon antibody, green-dominant cells emerged again in adult mice. CONCLUSIONS/INTERPRETATION: Novel time-resolved analysis with Gcg-Timer reporter mice uncovered spatiotemporal features of alpha cell neogenesis that will enhance our understanding of cellular identity and plasticity within the islets. DATA AVAILABILITY: Raw and processed RNA sequencing data for this study has been deposited in the Gene Expression Omnibus under accession number GSE229090.

Targeting ß-Cell Plasticity: A Promising Approach for Diabetes Treatment.

The ß-cells within the pancreas play a pivotal role in insulin production and secretion, responding to fluctuations in blood glucose levels. However, factors like obesity, dietary habits, and prolonged insulin resistance can compromise ß-cell function, contributing to the development of Type 2 Diabetes (T2D). A critical aspect of this dysfunction involves ß-cell dedifferentiation and transdifferentiation, wherein these cells lose their specialized characteristics and adopt different identities, notably transitioning towards progenitor or other pancreatic cell types like α-cells. This process significantly contributes to ß-cell malfunction and the progression of T2D, often surpassing the impact of outright ß-cell loss. Alterations in the expressions of specific genes and transcription factors unique to ß-cells, along with epigenetic modifications and environmental factors such as inflammation, oxidative stress, and mitochondrial dysfunction, underpin the occurrence of ß-cell dedifferentiation and the onset of T2D. Recent research underscores the potential therapeutic value for targeting ß-cell dedifferentiation to manage T2D effectively. In this review, we aim to dissect the intricate mechanisms governing ß-cell dedifferentiation and explore the therapeutic avenues stemming from these insights.

Transcriptomic and immunophenotypic profiling reveals molecular and immunological hallmarks of colorectal cancer tumourigenesis.

OBJECTIVE: Biological insights into the stepwise development and progression of colorectal cancer (CRC) are imperative to develop tailored approaches for early detection and optimal clinical management of this disease. Here, we aimed to dissect the transcriptional and immunologic alterations that accompany malignant transformation in CRC and to identify clinically relevant biomarkers through spatial profiling of pT1 CRC samples. DESIGN: We employed digital spatial profiling (GeoMx) on eight pT1 CRCs to study gene expression in the epithelial and stromal segments across regions of distinct histology, including normal mucosa, low-grade and high-grade dysplasia and cancer. Consecutive histology sections were profiled by imaging mass cytometry to reveal immune contextures. Finally, publicly available single-cell RNA-sequencing data was analysed to determine the cellular origin of relevant transcripts. RESULTS: Comparison of gene expression between regions within pT1 CRC samples identified differentially expressed genes in the epithelium (n=1394 genes) and the stromal segments (n=1145 genes) across distinct histologies. Pathway analysis identified an early onset of inflammatory responses during malignant transformation, typified by upregulation of gene signatures such as innate immune sensing. We detected increased infiltration of myeloid cells and a shift in macrophage populations from pro-inflammatory HLA-DR+CD204- macrophages to HLA-DR-CD204+ immune-suppressive subsets from normal tissue through dysplasia to cancer, accompanied by the upregulation of the CD47/SIRPα 'don't eat me signal'. CONCLUSION: Spatial profiling revealed the molecular and immunological landscape of CRC tumourigenesis at early disease stage. We identified biomarkers with strong association with disease progression as well as targetable immune processes that are exploitable in a clinical setting.

Spatial and transcriptional heterogeneity of pancreatic beta cell neogenesis revealed by a time-resolved reporter system.

AIMS/HYPOTHESIS: While pancreatic beta cells have been shown to originate from endocrine progenitors in ductal regions, it remains unclear precisely where beta cells emerge from and which transcripts define newborn beta cells. We therefore investigated characteristics of newborn beta cells extracted by a time-resolved reporter system. METHODS: We established a mouse model, 'Ins1-GFP; Timer', which provides spatial information during beta cell neogenesis with high temporal resolution. Single-cell RNA-sequencing (scRNA-seq) was performed on mouse beta cells sorted by fluorescent reporter to uncover transcriptomic profiles of newborn beta cells. scRNA-seq of human embryonic stem cell (hESC)-derived beta-like cells was also performed to compare newborn beta cell features between mouse and human. RESULTS: Fluorescence imaging of Ins1-GFP; Timer mouse pancreas successfully dissected newly generated beta cells as green fluorescence-dominant cells. This reporter system revealed that, as expected, some newborn beta cells arise close to the ducts (ßduct); unexpectedly, the others arise away from the ducts and adjacent to blood vessels (ßvessel). Single-cell transcriptomic analyses demonstrated five distinct populations among newborn beta cells, confirming spatial heterogeneity of beta cell neogenesis such as high probability of glucagon-positive ßduct, musculoaponeurotic fibrosarcoma oncogene family B (MafB)-positive ßduct and musculoaponeurotic fibrosarcoma oncogene family A (MafA)-positive ßvessel cells. Comparative analysis with scRNA-seq data of mouse newborn beta cells and hESC-derived beta-like cells uncovered transcriptional similarity between mouse and human beta cell neogenesis including microsomal glutathione S-transferase 1 (MGST1)- and synaptotagmin 13 (SYT13)-highly-expressing state. CONCLUSIONS/INTERPRETATION: The combination of time-resolved histological imaging with single-cell transcriptional mapping demonstrated novel features of spatial and transcriptional heterogeneity in beta cell neogenesis, which will lead to a better understanding of beta cell differentiation for future cell therapy. DATA AVAILABILITY: Raw and processed single-cell RNA-sequencing data for this study has been deposited in the Gene Expression Omnibus under accession number GSE155742.

Primary TSC2-/meth Cells Induce Follicular Neogenesis in an Innovative TSC Mouse Model.

Cutaneous lesions are one of the hallmarks of tuberous sclerosis complex (TSC), a genetic disease in which mTOR is hyperactivated due to the lack of hamartin or tuberin. To date, novel pharmacological treatments for TSC cutaneous lesions that are benign but still have an impact on a patient's life are needed, because neither surgery nor rapamycin administration prevents their recurrence. Here, we demonstrated that primary TSC2-/meth cells that do not express tuberin for an epigenetic event caused cutaneous lesions and follicular neogenesis when they were subcutaneously injected in nude mice. Tuberin-null cells localized in the hair bulbs and alongside mature hairs, where high phosphorylation of S6 and Erk indicated mTOR hyperactivation. Interestingly, 5-azacytidine treatment reduced hair follicles, indicating that chromatin remodeling agents might be effective on TSC lesions in which cells lack tuberin for an epigenetic event. Moreover, we demonstrated that the primary TSC2-/meth cells had metastatic capability: when subcutaneously injected, they reached the bloodstream and lymphatics and invaded the lungs, causing the enlargement of the alveolar walls. The capability of TSC2-/meth cells to survive and migrate in vivo makes our mouse model ideal to follow the progression of the disease and test potential pharmacological treatments in a time-dependent manner.

Local islet remodelling associated with duct lesion-islet complex in adult human pancreas.

AIMS/HYPOTHESIS: Islets are thought to be stably present in the adult human pancreas to maintain glucose homeostasis. However, identification of the pancreatic intraepithelial neoplasia (PanIN)-islet complex in mice and the presence of PanIN lesions in adult humans suggest that similar remodelling of islet structure and environment may occur in the human pancreas. To identify islet remodelling in a clinically related setting, we examine human donor pancreases with 3D histology to detect and characterise the human PanIN-islet complex. METHODS: Cadaveric donor pancreases (26-65 years old, n = 10) were fixed and sectioned (350 µm) for tissue labelling, clearing and microscopy to detect local islet remodelling for 3D analysis of the microenvironment. The remodelled microenvironment was subsequently examined via microtome-based histology for clinical assessment. RESULTS: In nine pancreases, we identified the unique peri-lobular islet aggregation associated with the PanIN lesion (16 lesion-islet complexes detected; size: 3.18 ± 1.34 mm). Important features of the lesion-islet microenvironment include: (1) formation of intra-islet ducts, (2) acinar atrophy, (3) adipocyte association, (4) inflammation (CD45+), (5) stromal accumulation (α-SMA+), (6) increase in Ki-67 proliferation index but absence of Ki-67+ alpha/beta cells and (7) in-depth and continuous duct-islet cell contacts, forming a cluster. The duct-islet cell cluster and intra-islet ducts suggest likely islet cell neogenesis but not replication. CONCLUSIONS/INTERPRETATION: We identify local islet remodelling associated with PanIN-islet complex in the adult human pancreas. The tissue remodelling and the evidence of inflammation and stromal accumulation suggest that the PanIN-islet complex is derived from tissue repair after a local injury.

Protein malnutrition early in life increased apoptosis but did not alter the ß-cell mass during gestation.

We evaluated whether early-life protein restriction alters structural parameters that affect ß-cell mass on the 15th day and 20th day of gestation in control pregnant (CP), control non-pregnant (CNP), low-protein pregnant (LPP) and low-protein non-pregnant (LPNP) rats from the fetal to the adult life stage as well as in protein-restricted rats that recovered after weaning (recovered pregnant (RP) and recovered non-pregnant). On the 15th day of gestation, the CNP group had a higher proportion of smaller islets, whereas the CP group exhibited a higher proportion of islets larger than the median. The ß-cell mass was lower in the low-protein group than that in the recovered and control groups. Gestation increased the ß-cell mass, ß-cell proliferation frequency and neogenesis frequency independently of the nutritional status. The apoptosis frequency was increased in the recovered groups compared with that in the other groups. On the 20th day of gestation, a higher proportion of islets smaller than the median was observed in the non-pregnant groups, whereas a higher proportion of islets larger than the median was observed in the RP, LPP and CP groups. ß-Cell mass was lower in the low-protein group than that in the recovered and control groups, regardless of the physiological status. The ß-cell proliferation frequency was lower, whereas the apoptosis rate was higher in recovered rats compared with those in the low-protein and control rats. Thus, protein malnutrition early in life did not alter the mass of ß-cells, especially in the first two-thirds of gestation, despite the increase in apoptosis.

CXCL13 Signaling in the Tumor Microenvironment.

Chemokines have emerged as important players in tumorigenic process. An extensive body of literature generated over the last two or three decades strongly implicate abnormally activated or functionally disrupted chemokine signaling in liaising most-if not all-hallmark processes of cancer. It is well-known that chemokine signaling networks within the tumor microenvironment are highly versatile and context-dependent: exert both pro-tumoral and antitumoral activities. The C-X-C motif chemokine ligand 13 (CXCL13), and its cognate receptor CXCR5, represents an emerging example of chemokine signaling axes, which express the ability to modulate tumor growth and progression in either way. Collateral evidence indicate that CXCL13-CXCR5 axis may directly modulate tumor growth by inducing proliferation of cancer cells, as well as promoting invasive phenotypes and preventing their apoptosis. In addition, CXCL13-CXCR5 axis may also indirectly modulate tumor growth by regulating noncancerous cells, particularly the immune cells, within the tumor microenvironment. Here, we review the role of CXCL13, together with CXCR5, in the human tumor microenvironment. We first elaborate their patterns of expression, regulation, and biological functions in normal physiology. We then consider how their aberrant activity, as a result of differential overexpression or co-expression, may directly or indirectly modulate the growth of tumors through effects on both cancerous and noncancerous cells.

ß-cell replenishment: Possible curative approaches for diabetes mellitus.

AIMS: Diabetes mellitus (DM) is a disorder of heterogeneous etiology marked by persistent hyperglycemia. Exogenous insulin is the only treatment for type 1 diabetes (T1D). Islet transplantation is a potential long cure for T1D but is disapproved due to the possibility of immune rejection in the later stage. The approaches used for treating type 2 diabetes (T2D) include diet restrictions, weight management and pharmacological interventions. These procedures have not been able to boost the quality of life for diabetic patients owing to the complexity of the disorder. DATA SYNTHESIS: Hence, research has embarked on permanent ways of managing, or even curing the disease. One of the possible approaches to restore the pancreas with new glucose-responsive ß-cells is by their regeneration. Regeneration of ß-cells include islet neogenesis, dedifferentiation, and trans-differentiation of the already differentiated cells. CONCLUSIONS: This review briefly describes the islet development, functions of ß-cells, mechanism and factors involved in ß-cell death. It further elaborates on the potential of the existing and possible therapeutic modalities involved in the in-vivo replenishment of ß-cells with a focus on exercise, diet, hormones, small molecules, and phytochemicals.

Self-organization process in newborn skin organoid formation inspires strategy to restore hair regeneration of adult cells.

Organoids made from dissociated progenitor cells undergo tissue-like organization. This in vitro self-organization process is not identical to embryonic organ formation, but it achieves a similar phenotype in vivo. This implies genetic codes do not specify morphology directly; instead, complex tissue architectures may be achieved through several intermediate layers of cross talk between genetic information and biophysical processes. Here we use newborn and adult skin organoids for analyses. Dissociated cells from newborn mouse skin form hair primordia-bearing organoids that grow hairs robustly in vivo after transplantation to nude mice. Detailed time-lapse imaging of 3D cultures revealed unexpected morphological transitions between six distinct phases: dissociated cells, cell aggregates, polarized cysts, cyst coalescence, planar skin, and hair-bearing skin. Transcriptome profiling reveals the sequential expression of adhesion molecules, growth factors, Wnts, and matrix metalloproteinases (MMPs). Functional perturbations at different times discern their roles in regulating the switch from one phase to another. In contrast, adult cells form small aggregates, but then development stalls in vitro. Comparative transcriptome analyses suggest suppressing epidermal differentiation in adult cells is critical. These results inspire a strategy that can restore morphological transitions and rescue the hair-forming ability of adult organoids: (i) continuous PKC inhibition and (ii) timely supply of growth factors (IGF, VEGF), Wnts, and MMPs. This comprehensive study demonstrates that alternating molecular events and physical processes are in action during organoid morphogenesis and that the self-organizing processes can be restored via environmental reprogramming. This tissue-level phase transition could drive self-organization behavior in organoid morphogenies beyond the skin.

Modified IVAN Technique: Long-Term Follow-Up of 20 Cases Over 2 to 11 Years.

When natural teeth fail, frequently there is a loss of hard and soft tissue. This may complicate subsequent dental implant placement by creating insufficient bone to house the implant. This also occurs when the tooth has been missing for an extended period, especially in the premaxilla, where the bone is less dense and often lacks sufficient volume of facial bone. Site reconstruction to accommodate implant placement often requires both hard and soft tissue augmentation. The modified interpositional vascularized augmentation neogenesis (mIVAN) technique achieves the desired treatment goals in both delayed and immediate placement scenarios. The technique will be discussed as well as the long-term follow-up on 20 cases.

Activin A-induced signalling controls hair follicle neogenesis.

Acquisition of potent human dermal papilla (DP) cells that can induce hair follicle neogenesis is an overarching concern, and various approaches have been tried. In an attempt to solve the problem, we previously introduced the three-dimensional (3D) culture of human DP cells and observed de novo formation of hair follicles when conducting a patch hair reconstitution assay using 3D cultured DP spheres with mouse epidermal cells. In this study, we have subsequently focused our attention on activin A, one of the notably upregulated proteins in DP spheres compared with 2D cultured DP cells. We then adopted a small interfering RNA-mediated gene knock-down approach and hair reconstitution assay to investigate the role of activin A. We observed that human DP spheres with activin A knock-down are severely impaired in hair follicle neogenesis when combined with mouse epidermal cells. In addition, activin receptor 2B (ActvR2B) knock-down mouse epidermal cells showed severe impairment of hair follicle neogenesis when combined with human DP spheres. Moreover, recombinant activin A treatment of mouse epidermal cells increased the expression of downstream genes of the activin pathway. Taken together, our data strongly suggest that activin A-induced signalling plays a critical role in hair follicle neogenesis, which has not been previously reported.

Ectopic lymphoid follicles: inducible centres for generating antigen-specific immune responses within tissues.

Lymphoid neogenesis is traditionally viewed as a pre-programmed process that promotes the formation of lymphoid organs during development. Here, the spatial organization of T and B cells in lymph nodes and spleen into discrete structures regulates antigen-specific responses and adaptive immunity following immune challenge. However, lymphoid neogenesis is also triggered by chronic or persistent inflammation. Here, ectopic (or tertiary) lymphoid organs frequently develop in inflamed tissues as a response to infection, auto-immunity, transplantation, cancer or environmental irritants. Although these structures affect local immune responses, the contribution of these lymphoid aggregates to the underlining pathology are highly context dependent and can elicit either protective or deleterious outcomes. Here we review the cellular and molecular mechanisms responsible for ectopic lymphoid neogenesis and consider the relevance of these structures in human disease.

Life and death of ß cells in Type 1 diabetes: A comprehensive review.

Type 1 diabetes (T1D) is an autoimmune disorder characterized by the destruction of insulin-producing pancreatic ß cells. Immune modulators have achieved some success in modifying the course of disease progression in T1D. However, there are parallel declines in C-peptide levels in treated and control groups after initial responses. In this review, we discuss mechanisms of ß cell death in T1D that involve necrosis and apoptosis. New technologies are being developed to enable visualization of insulitis and ß cell mass involving positron emission transmission that identifies ß cell ligands and magnetic resonance imaging that can identify vascular leakage. Molecular signatures that identify ß cell derived insulin DNA that is released from dying cells have been described and applied to clinical settings. We also consider changes in ß cells that occur during disease progression including the induction of DNA methyltransferases that may affect the function and differentiation of ß cells. Our findings from newer data suggest that the model of chronic long standing ß cell killing should be reconsidered. These studies indicate that the pathophysiology is accelerated in the peridiagnosis period and manifest by increased rates of ß cell killing and insulin secretory impairments over a shorter period than previously thought. Finally, we consider cellular explanations to account for the ongoing loss of insulin production despite continued immune therapy that may identify potential targets for treatment. The progressive decline in ß cell function raises the question as to whether ß cell failure that is independent of immune attack may be involved.

Characterisation of Tertiary Lymphoid Organs in Explanted Rejected Donor Kidneys.

PURPOSE: Tertiary lymphoid organs (TLOs) have been described within organ allografts, but whether they promote destructive or beneficial alloimmune responses remains controversial. This study aimed to characterize TLO distribution in human chronically rejected renal allografts and to explore their functions. METHODS: A total of 29 explanted chronically rejected and 12 acutely rejected renal allografts were analyzed by immunohistochemistry. The distribution of TLOs, T cells, follicular dendritic cells, B cells, and follicular regulatory T (Tfr) cells, as well as Ki67, peripheral lymph node addressin (PNAd), podoplanin, AID, IL-17, IL-21, IL-10, and C4d expression were detected by immunohistochemistry. Correlations between lymphoid neogenesis and the expression of IL-17, IL-21, C4d, podoplanin, IL-10, and Foxp3 were evaluated. In addition, the duration of graft function was compared between allografts that harbored or lacked TLOs. RESULTS: TLOs were detected in 27.6% of chronically rejected renal grafts, but they rarely had germinal centers. Lymphoid neogenesis negatively correlated with CXCR5 expression, and almost completely correlated with IL-17 expression. Those grafts that harbored a TLO functioned for an average of 5.98 years and those without a TLO lasted only about half as long with an average of 2.91 years. However, in grafts that harbored a TLO, Foxp3(+) cells were comparitively less than those without a TLO. Foxp3(+)CXCR5(+) Tfr cells and IL-10(+) cells were rare in grafts, irrespective of the presence of a TLO. CONCLUSION: TLOs in chronically rejected kidney allografts may be an epiphenomenon of the inflammatory process that is related to graft duration.

The pathological features of ectopic lymphoid neogenesis in idiopathic dacryoadenitis.

BACKGROUND: Lymphoid neogenesis has been reported in various diseases but not in idiopathic dacryoadenitis. The aim of this paper is to discuss the pathological features of lymphoid neogenesis in idiopathic dacryoadenitis. METHODS: 20 cases of idiopathic dacryoadenitis were collected retrospectively. Lymphoid neogenesis was graded by lymphocytic aggregates and germinal center-like structure formation. T and B cell compartmentalization, follicular dendritic cells and the expression of CXCL13 and CCL21 were analyzed. RESULTS: Grade 1 lymphoid neogenesis was observed in 10 of 20 cases (50 %), grade 2 in 18 of 20 cases (90 %) and grade 3 in 14 of 20 (70 %). The existence of T and B cell compartmentalization and follicular dendritic cells increased in parallel to the grade of lymphoid neogenesis. The expression of CXCL13 significantly increased in the higher grade of lymphoid neogenesis, but no correlation was found between CCL21 and grades of lymphoid neogenesis. CONCLUSIONS: Ectopic lymphoid neogenesis participates in the pathogenesis of idiopathic dacryoadenitis and appears as a dynamic process.

Division of labour: how does folate metabolism partition between one-carbon metabolism and amino acid oxidation?

One-carbon metabolism is usually represented as having three canonical functions: purine synthesis, thymidylate synthesis and methylation reactions. There is however a fourth major function: the metabolism of some amino acids (serine, glycine, tryptophan and histidine), as well as choline. These substrates can provide cells with more one-carbon groups than they need for these three canonical functions. Therefore, there must be mechanisms for the disposal of these one-carbon groups (when in excess) which maintain the complement of these groups required for the canonical functions. The key enzyme for these mechanisms is 10-formyl-THF (tetrahydrofolate) dehydrogenase (both mitochondrial and cytoplasmic isoforms) which oxidizes the formyl group to CO2 with the attendant reduction of NADP(+) to NADPH and release of THF. In addition to oxidizing the excess of these compounds, this process can reduce substantial quantities of NADP(+) to NADPH.

Self-assembling peptide hydrogel scaffolds support stem cell-based hair follicle regeneration.

Recent studies show that designer peptide nanofibers can mimic properties of extracellular matrix molecules, promising great potential as scaffold materials for tissue engineering. However, their ability in supporting organogenesis has not been studied. Here we examined the effect of self-assembling peptide hydrogels in supporting skin derived precursors (SKPs) in hair follicle neogenesis. We found that hydrogels formed by RADA16, PRG which contains RGD, and particularly the combination of RADA16 and PRG (RADA-PRG) enhanced SKP proliferation. Notably, the RADA-PRG hydrogel, which exhibited advantages of RADA16 in adequate nanofiber formation and PRG in providing the integrin binding sequence, exhibited superior effects in enhancing SKP survival, expression of hair induction signature genes such as Akp2 and Bmp6, and more importantly de novo hair genesis in mice. Thus our results suggest that RADA-PRG may serve as a novel scaffold material for stem cell transplantation and tissue engineering.

[Stem-cell therapy, ß-cell- and islet cell-neogenesis: possible treatments of type 1 diabetes in the future?]. / Ossejtterápia, ß-sejt- és Langerhans-sziget-neogenezis: a jövo lehetséges terápiái 1-es típusú diabetesben?

In type 1 diabetic patients perfect normoglycaemia can only be achieved by successful transplantation of the pancreas or Langerhans' islets. Surgical transplantation of the whole pancreas is an invasive operation exerting great burden on the patients. Transplantation of the islets of Langerhans does not burden the patients but the survival of the islet grafts is limited. Both interventions are hampered by the lack of donor organs. However, much of these difficulties could be overcome by the use of "artificial ß-cells" which ought to have an ultrastructure identical with that of natural ß-cells and produce and secrete insulin in a glucose dependent manner. At present three such methods are at our disposal: transformation of the ductal cells of the exocrine pancreas into ß-cells, development of ß-cells from stem-cells, and neogenesis of Langerhans' islets induced by viral delivery of transcription factors. The author summarises the experience and experimental results obtained with the use of the three methods.

Activation of GLP-1 and gastrin signalling induces in vivo reprogramming of pancreatic exocrine cells into beta cells in mice.

AIMS/HYPOTHESIS: Lineage conversion of non-beta cells into insulin-producing cells has been proposed as a therapy for the cure of diabetes. Glucagon-like peptide-1 (GLP-1) and its derivatives can induce beta cell neogenesis in vitro and beta cell mass expansion in vivo, but GLP-1 signalling has not been shown to regulate cell fate decisions in vivo. We therefore tested the impact of GLP-1 receptor (GLP1R) expression on beta cell differentiation in vivo. METHODS: Mice overexpressing GLP1R in pancreatic exocrine cells were generated by Cre-mediated recombination in sex-determining region Y-box 9 (SOX9)-expressing cells and then treated with exendin-4 and/or gastrin. Histological analysis was performed to detect cellular reprogramming from the exocrine lineage into insulin-producing cells. RESULTS: Whereas no newly generated beta cells were detected in the mice treated with exendin-4 alone, treatment with gastrin only induced the conversion of exocrine cells into insulin-producing cells. Furthermore, the overexpression of GLP1R, together with gastrin and exendin-4, synergistically promoted beta cell neogenesis accompanied by the formation of islet-like clusters. These newly generated beta cells expressed beta cell specific transcription factors, such as pancreatic and duodenal homeobox 1 (PDX1), NK6 homeobox 1 (NKX6.1) and musculoaponeurotic fibrosarcoma oncogene family A (MafA). These mice showed no histological evidence of pancreatitis or pancreatic dysplasia in their acini and had normal plasma amylase levels. CONCLUSIONS/INTERPRETATION: Activation of GLP-1 and gastrin signalling induces beta cell neogenesis in the exocrine lineage without any deleterious pancreatic changes, which may lead to a potential therapy to cure diabetes by generating surrogate beta cells.