Long-gap esophageal atresia: gastric transposition or esophageal lengthening with delayed primary anastomosis? A systematic review.

PURPOSE: This study aims to evaluate different surgical approaches to long-gap esophageal atresia (LGEA) with or without tracheoesophageal fistula (TEF) is unclear. METHODS: A systematic literature review was done comparing gastric transposition versus esophageal lengthening with delayed primary anastomosis in infants with LGEA+/-TEF. The primary outcome was time to full oral feeds. Secondary outcomes were time to full enteric feeds, need for further surgery, growth, mortality, and postoperative adverse events. RESULTS: No comparative studies were found. However, the literature was re-interrogated for non-comparative studies. Four hundred thirty-eight articles were identified and screened, and 18 met the inclusion criteria. All were case series. Forty-three infants underwent gastric transposition, and 106 had esophageal lengthening with delayed primary anastomosis. One study on gastric transposition reported time to full oral feeds, and one study in each group reported growth. Time to full enteric feeds was reported in one study in each group. 30% of infants had further surgery following gastric transposition, including hiatus hernia repair (5/43, 12%) and esophageal dilation (7/43, 16%). Following esophageal lengthening, 62/106 (58%) had anti-reflux surgery, 58/106 (55%) esophageal dilatation and 11/106 (10%) esophageal stricture resection. Anastomotic complications occurred in 13/43 (30%), gastrointestinal in 16/43 (37%), respiratory in 17/43 (40%), and nerve injury in 2/43 (5%) of the gastric transposition group. In the esophageal lengthening group, anastomotic complications occurred in 68/106 (64%), gastrointestinal in 62/106 (58%), respiratory in 6/106 (6%), and none sustained nerve injury. Each group had one death due to a cause not directly related to the surgical procedure. CONCLUSIONS: This systematic review highlights the morbidity associated with both surgical procedures and the variety in reporting outcomes.

One-year impact of a bowel management program in treating fecal incontinence in patients with anorectal malformations.

BACKGROUND: Many patients with anorectal malformations (ARM) need a bowel management program (BMP) to manage lifelong problems of fecal incontinence or severe constipation. We aimed to evaluate the sustainability of the results in such a program. METHODS: A single-institution retrospective review was performed in children with ARM who attended our BMP (2015-2019). Standardized definitions and validated tools were used to assess fecal continence (Baylor Continence Scale), constipation (Cleveland Constipation Scoring System), urinary symptoms (Vancouver Symptoms Score), and the Pediatric Quality of Life (PedsQL) and health-related quality of life (HRQOL) at the start of BMP and 1-year after completion of the program. RESULTS: 222 patients with ARM at a median age of 6.7 (IQR, 4.9-10.1) years were identified. All (100%) soiled at intake with 149 (67.1%) patients being treated with rectal or antegrade enemas and 73 (32.9%) with oral laxatives. At 1 year 150 (70.4%) were clean, 72.7% were on enemas and 27.3% were on laxatives (p = 0.08). 109 out of 148 (73.6%) patients were clean on enemas. A further 41 out of 66 (62.1%) patients were continent on laxatives with voluntary bowel movements and clean. In the group that was clean, there was improvement in Baylor Continence Scale (25 vs. 13.0, p < 0.000000002), Vancouver (11 vs. 6, p = 0.0110) scores, and clinically relevant improvement in the total PedsQL HRQL (78-85) and the PedsQL HRQL physical function (86-92) and psychosocial domain (77-82). There was no improvement in Cleveland (10 vs. 9, p = 0.31) score. CONCLUSION: An intensive BMP offers significant benefits in the treatment of fecal incontinence in ARM. It appears to also improve urinary incontinence and urinary voiding as well as the patient's quality of life. These changes are sustainable over at least one year.

Case report of a skip segment Hirschsprung's disease: A real phenomenon.

INTRODUCTION AND IMPORTANCE: Hirschsprung's disease is a congenital anomaly that results from an incomplete craniocaudal migration and maturation of intestinal ganglion progenitor cells leading to distal intestinal aganglionosis. Skip segment Hirschsprung's disease is an extremely rare phenomenon. We report a case involving only the small bowel with confirmed colonic ganglionosis. CASE PRESENTATION: A case report of a 14-month-old with a skipped segment involving the distal 50 cm of the small bowel associated with colonic ganglionosis is presented. A current review of the literature is discussed. CLINICAL DISCUSSION: Our patient had persistent obstructive symptoms despite undergoing a technically good, ganglionic pull-through operation at an outside institution. A laparoscopic-assisted pull-through might have documented a small bowel wall diameter discrepancy. CONCLUSION: Although rare, skip segment Hirschsprung's disease is a real phenomenon that paediatric surgeons should be aware of and could involve small and large bowels.

The use of computed tomography versus clinical acumen in diagnosing appendicitis in children: A two-institution international study.

BACKGROUND: Appendicitis in children can be diagnosed utilizing clinical and laboratory findings, with the assistance of ultrasound (US) and/or computed tomography (CT). However, repeated exposure to ionizing radiation increases the lifetime risk of cancer. We compared the work-up of suspected appendicitis between a children's hospital in the United States (USA) and one in Spain to identify differences in imaging use and associated outcomes. METHODS: A two-institution retrospective review was performed for surgical consultations of suspected appendicitis from 2015-2017. We compared imaging use, the utilization of overnight observation, and diagnostic accuracy rates between the two centers. RESULTS: A total of 1,952 children were evaluated. Among the 1,288 in the USA center, 754(58.5%) underwent CT during their evaluation. The most common imaging modality was US only (39.9%), then CT only (39.3%), CT+US (19.3%), and no imaging (i.e. only clinical acumen) (1.6%). In Spain, only 19 (2.9%) of 664 children underwent CT compared to the USA (p < 0.0001). Only clinical acumen was the most common modality employed (48.6%), followed by US only (48.5%), US+CT (2.4%), and CT only (0.5%). In the USA, 16.8% were observed overnight, 2.3% of whom received no imaging. In Spain, 33.4% were observed overnight, 32.4% of whom had no imaging (p < 0.0001). The accuracy rates for diagnosing appendicitis in the USA and Spain centers were 94.7% and 95.1%, respectively. CONCLUSION: Use of clinical acumen and/or US have similar clinical outcomes and similar accuracy rates compared to heavy reliance on CT imaging for diagnosing appendicitis, with associated decrease in radiation exposure. The disparate diagnostic approach of the two centers may reflect that physical examination is a dying art in North America. LEVEL OF EVIDENCE: III.

Single-site retroperitoneoscopy in pediatric metastatic lymphadenopathy.

BACKGROUND: Retroperitoneoscopic surgery (RS) is increasingly used for the diagnosis, staging, and treatment of solid tumors, but rarely in pediatric surgical oncology for retroperitoneal lymph node dissection (RPLND). Herein, we use single-site RS for RPLND in children and compare the perioperative outcomes with those for the transperitoneal laparoscopic approach (TPLA). METHODS: A single institution retrospective chart review was performed for patients undergoing single-site RS and TPLA (January 2018 till June 2019). We compared patient demographics, diagnoses, operative times, complications, postoperative analgesia, and length of hospital stay between both groups. RESULTS: Eight patients (median age of 16.5 years) undergoing single-site RS for RPLND and five patients (median age 17 years) undergoing TPLA RPLND were compared. Groups were comparable in age, median operative duration (232 vs 234 min, p = 0.77), and complications (1 vs 1, p = 0.72). Median postoperative hospital stay and total morphine equivalent doses used postoperatively were significantly lower in the RS group, (0.5 vs 2 days, [p = 0.03] and 0.1 vs 0.4 mg/kg [p = 0.01], respectively). Eight patients underwent ipsilateral modified template RPLND for paratesticular RMS (six single-site RS and two TPLA) and lymph node metastases were found in 50% of these patients. The rest were resections of metastatic lesions for germ cell tumor and neuroblastoma (two single-site RS and three TPLA). CONCLUSIONS: Single-site RS is a safe and feasible technique in carefully selected pediatric surgical oncology patients. RS provides an excellent view of the retroperitoneum, requires less postoperative analgesia, and is associated with faster recovery. LEVEL OF EVIDENCE RATING: IV.

Splenic function is not maintained long-term after partial splenectomy in children with sickle cell disease.

BACKGROUND: Partial splenectomy (PS) may allow preservation of splenic function in cases where splenectomy is indicated for hematologic diseases; however, the long-term outcomes are uncertain. We investigated the long-term outcomes of PS in patients with sickle cell disease (SCD). METHODS: A single-institution retrospective chart review was performed for children with SCD who underwent PS from 1997 to 2017. For comparison, we reviewed outcomes for patients who underwent PS for hereditary spherocytosis (HS). The primary endpoint was viability of the splenic remnant as inferred by the presence of remnant perfusion on ultrasound and/or liver spleen scan. RESULTS: Nine patients with SCD and 26 patients with HS underwent PS at a median age of 11 (IQR, 9-14) and 7.5 (IQR, 6-13) years, respectively. All underwent laparoscopic PS with three (7.9%) conversions to open. Two SCD patients were lost to long-term follow-up. The remaining seven SCD patients had initial postoperative splenic remnant perfusion demonstrated by ultrasonography. By 42 months postoperatively, however, none had a functioning splenic remnant. The median time to loss of splenic remnant was 12.6 (IQR 9.2-28.5) months. In contrast, all HS patients demonstrated robust splenic remnant blood flow with a median follow-up of 46 (IQR 37-82) months. CONCLUSION: No patient with SCD who underwent PS had viable splenic tissue for more than 42 months, likely due to continued autoinfarction typical of patients with this disease. Therefore, we believe that PS to preserve splenic function is not indicated in patients with SCD. LEVEL OF EVIDENCE: III.

Management of pancreatic pseudocysts in pediatric oncology patients.

BACKGROUND: Management of children with pancreatic pseudocysts has historically been adopted from the adult experience where pancreatic pseudocysts greater than 6 cm are unlikely to resolve without intervention. We reviewed the clinical course of pediatric oncology patients with pancreatic pseudocysts. METHODS: A retrospective review of patients treated over a 15-year period was performed. Variables evaluated included cancer type, medications administered, clinical and imaging characteristics of the pancreatic pseudocysts, treatment and outcome. RESULTS: A total of 132 patients with a median age of 13 (IQR, 9-17) years were identified with pancreatitis. Thirty-one (23.5%) patients developed a pancreatic pseudocyst, of which 84% were associated with PEG-asparaginase treatment. The median pseudocyst size was 7.6 (IQR, 4.4-9.9) cm with 59% being greater than 6 cm. Twenty-two (71%) patients with a pancreatic pseudocyst underwent successful conservative management, while only 9 (29%) required procedural intervention including six percutaneous drainage, one of whom recurred and required surgical cyst-enteric drainage. Two other patients had primary surgical cyst-enteric drainage and one patient underwent endoscopic retrograde cholangiopancreatography with stenting. The indication for intervention was worsening pain rather than pseudocyst imaging characteristics, size or serum amylase/lipase. CONCLUSION: Most medication-induced pancreatic pseudocysts in children being treated for cancer, regardless of pseudocyst size, can be managed non-operatively or with transgastric percutaneous drainage. The need for intervention can be safely dictated by patient symptoms. LEVEL OF EVIDENCE: III.

A Quality Improvement Intervention to Decrease Postoperative Opioid Prescriptions in Pediatric Oncology Patients.

PURPOSE: This quality improvement initiative aimed to minimize opioid prescribing after oncologic pediatric surgery. METHODS: Retrospective surgical data collected at a pediatric cancer hospital from July 2016 to June 2018 included hospitalization details, oral morphine equivalents prescribed, unplanned visits/calls because of pain, and parental/patient satisfaction with pain control. The quality improvement initiative promoted opioid prescription at discharge on the basis of prior inpatient requirements and education regarding nonopioid analgesia. Upon commencing this project in July 2018, we collected data prospectively. RESULTS: The retrospective and the prospective cohorts included 271 and 99 patients, respectively. Mean (SD) oral morphine equivalents (mg/kg) prescribed upon discharge was significantly reduced in the prospective (0.75±1.34) versus retrospective cohorts (5.48±6.94, P<0.001). The unplanned visits/calls regarding pain were 23 (retrospective, 8.5%) and 2 (prospective, 2.0%). In total, 44 patients (44.4%) received an opioid prescription at discharge in the prospective cohort, significantly fewer than retrospective cohort (251, 92.6%, P<0.001), and used a mean of 34.3 of 159.8 (21.5%) doses dispensed. Length of stay was comparable (P=0.88) between cohorts. Prospective satisfaction rate was 96.2%, leaving 3 patients (3.8%) not satisfied with their pain control regimen. CONCLUSIONS: Dramatic reduction of opioid prescriptions after oncologic surgery can be achieved without detriment to patient satisfaction or readmissions. LEVEL OF EVIDENCE: Level V.

Acute Chest Syndrome After Splenectomy in Children With Sickle Cell Disease.

BACKGROUND: Individuals with sickle cell disease (SCD) are at high risk of developing life-threatening complications, particularly acute chest syndrome (ACS) postoperatively. The perioperative factors associated with the development of ACS in children with SCD after splenectomy have not been clearly identified. MATERIALS AND METHODS: We retrospectively reviewed medical records of all children who underwent splenectomy at our institution between 1997 and 2017 with the goal of identifying perioperative factors associated with postoperative ACS. Categorical and noncategorical variables were compared using Fisher's exact test and Student's two-tailed t-test, respectively. RESULTS: Sixty-five patients with SCD underwent splenectomy at a median of 4.0 (interquartile range [IQR] 2.0-8.0) years of age. A laparoscopic approach was used for 64 (98.5%) patients. Fifty-six (86.2%) underwent laparoscopic total splenectomy, and eight (12.3%) underwent laparoscopic partial splenectomy, of which two were converted to open. One had an open partial splenectomy (1.5%). Of the 65 patients, 10 (15.4%) developed ACS with a mean time to diagnosis of 49.0 ± 34.5 h. Children who developed ACS had a higher postoperative median pain score of 6.8 (IQR 5.1-9.1) versus 2.7 (IQR 1.6-4.2), P < 0.001, higher median pain score area under the curve 111.5 (IQR 76.9-169.1) versus 47.3 (IQR 30.5-78.3), P = 0.01, and received more total morphine equivalents (median 1.4 [IQR 0.4-2.7] versus 0.5 [IQR 0.3-0.9] mg/kg, respectively; P = 0.003), compared with children who did not develop ACS. CONCLUSIONS: Significant postoperative pain may be an early sign of ACS that could be worsened by opioid use, supporting the investigation of nonopioid pain control options in this patient population.

Pyloric stenosis: an enigma more than a century after the first successful treatment.

Despite hypertrophic pyloric stenosis (HPS) being one of the most frequently treated pediatric surgical conditions, its etiology remains incompletely understood. We review the diagnosis and treatment of this condition with an emphasis on the evolution of surgical techniques that led to laparoscopic pyloromyotomy, the most frequently performed technique for HPS today. In addition, we review key developments in the understanding of HPS etiology and treatment, including the postulated etiology of work-induced hypertrophy of the pylorus, its association with prokinetic macrolide antibiotics, and the emerging role of atropine sulfate as a medical treatment for HPS or a rescue treatment for incomplete myotomy.

Intraislet Pancreatic Ducts Can Give Rise to Insulin-Positive Cells.

A key question in diabetes research is whether new ß-cells can be derived from endogenous, nonendocrine cells. The potential for pancreatic ductal cells to convert into ß-cells is a highly debated issue. To date, it remains unclear what anatomical process would result in duct-derived cells coming to exist within preexisting islets. We used a whole-mount technique to directly visualize the pancreatic ductal network in young wild-type mice, young humans, and wild-type and transgenic mice after partial pancreatectomy. Pancreatic ductal networks, originating from the main ductal tree, were found to reside deep within islets in young mice and humans but not in mature mice or humans. These networks were also not present in normal adult mice after partial pancreatectomy, but TGF-ß receptor mutant mice demonstrated formation of these intraislet duct structures after partial pancreatectomy. Genetic and viral lineage tracings were used to determine whether endocrine cells were derived from pancreatic ducts. Lineage tracing confirmed that pancreatic ductal cells can typically convert into new ß-cells in normal young developing mice as well as in adult TGF-ß signaling mutant mice after partial pancreatectomy. Here the direct visual evidence of ducts growing into islets, along with lineage tracing, not only represents strong evidence for duct cells giving rise to ß-cells in the postnatal pancreas but also importantly implicates TGF-ß signaling in this process.

Pancreatic cell tracing, lineage tagging and targeted genetic manipulations in multiple cell types using pancreatic ductal infusion of adeno-associated viral vectors and/or cell-tagging dyes.

Genetic manipulations, with or without lineage tracing for specific pancreatic cell types, are very powerful tools for studying diabetes, pancreatitis and pancreatic cancer. Nevertheless, the use of Cre/loxP systems to conditionally activate or inactivate the expression of genes in a cell type- and/or temporal-specific manner is not applicable to cell tracing and/or gene manipulations in more than one lineage at a time. Here we report a technique that allows efficient delivery of dyes for cell tagging into the mouse pancreas through the duct system, and that also delivers viruses carrying transgenes or siRNA under a specific promoter. When this technique is applied in genetically modified mice, it enables the investigator to perform either double lineage tracing or cell lineage tracing combined with gene manipulation in a second lineage. The technique requires <40 min.

Barrier function of the coelomic epithelium in the developing pancreas.

Tight spatial regulation of extracellular morphogen signaling within the close confines of a developing embryo is critical for proper organogenesis. Given the complexity of extracellular signaling in developing organs, together with the proximity of adjacent organs that use disparate signaling pathways, we postulated that a physical barrier to signaling may exist between organs in the embryo. Here we describe a previously unrecognized role for the embryonic coelomic epithelium in providing a physical barrier to contain morphogenic signaling in the developing mouse pancreas. This layer of cells appears to function both to contain key factors required for pancreatic epithelial differentiation, and to prevent fusion of adjacent organs during critical developmental windows. During early foregut development, this barrier appears to play a role in preventing splenic anlage-derived activin signaling from inducing intestinalization of the pancreas-specified epithelium.

M2 macrophages promote beta-cell proliferation by up-regulation of SMAD7.

Determination of signaling pathways that regulate beta-cell replication is critical for beta-cell therapy. Here, we show that blocking pancreatic macrophage infiltration after pancreatic duct ligation (PDL) completely inhibits beta-cell proliferation. The TGFß superfamily signaling inhibitor SMAD7 was significantly up-regulated in beta cells after PDL. Beta cells failed to proliferate in response to PDL in beta-cell-specific SMAD7 mutant mice. Forced expression of SMAD7 in beta cells by itself was sufficient to promote beta-cell proliferation in vivo. M2, rather than M1 macrophages, seem to be the inducers of SMAD7-mediated beta-cell proliferation. M2 macrophages not only release TGFß1 to directly induce up-regulation of SMAD7 in beta cells but also release EGF to activate EGF receptor signaling that inhibits TGFß1-activated SMAD2 nuclear translocation, resulting in TGFß signaling inhibition. SMAD7 promotes beta-cell proliferation by increasing CyclinD1 and CyclinD2, and by inducing nuclear exclusion of p27. Our study thus reveals a molecular pathway to potentially increase beta-cell mass through enhanced SMAD7 activity induced by extracellular stimuli.

Pancreatic duct cells as a source of VEGF in mice.

AIMS/HYPOTHESIS: Vascular endothelial growth factor (VEGF) is essential for proper pancreatic development, islet vascularisation and insulin secretion. In the adult pancreas, VEGF is thought to be predominantly secreted by beta cells. Although human duct cells have previously been shown to secrete VEGF at angiogenic levels in culture, an analysis of the kinetics of VEGF synthesis and secretion, as well as elucidation of an in vivo role for this ductal VEGF in affecting islet function and physiology, has been lacking. METHODS: We analysed purified duct cells independently prepared by flow cytometry, surgical isolation or laser-capture microdissection. We infected duct cells in vivo with Vegf (also known as Vegfa) short hairpin RNA (shRNA) in an intrapancreatic ductal infusion system and examined the effect of VEGF knockdown in duct cells in vitro and in vivo. RESULTS: Pancreatic duct cells express high levels of Vegf mRNA. Compared with beta cells, duct cells had a much higher ratio of secreted to intracellular VEGF. As a bioassay, formation of tubular structures by human umbilical vein endothelial cells was essentially undetectable when cultured alone and was substantially increased when co-cultured with pancreatic duct cells but significantly reduced when co-cultured with duct cells pretreated with Vegf shRNA. Compared with islets transplanted alone, improved vascularisation and function was detected in the islets co-transplanted with duct cells but not in islets co-transplanted with duct cells pretreated with Vegf shRNA. CONCLUSIONS/INTERPRETATION: Human islet preparations for transplantation typically contain some contaminating duct cells and our findings suggest that the presence of duct cells in the islet preparation may improve transplantation outcomes.

A smad signaling network regulates islet cell proliferation.

Pancreatic ß-cell loss and dysfunction are critical components of all types of diabetes. Human and rodent ß-cells are able to proliferate, and this proliferation is an important defense against the evolution and progression of diabetes. Transforming growth factor-ß (TGF-ß) signaling has been shown to affect ß-cell development, proliferation, and function, but ß-cell proliferation is thought to be the only source of new ß-cells in the adult. Recently, ß-cell dedifferentiation has been shown to be an important contributory mechanism to ß-cell failure. In this study, we tie together these two pathways by showing that a network of intracellular TGF-ß regulators, smads 7, 2, and 3, control ß-cell proliferation after ß-cell loss, and specifically, smad7 is necessary for that ß-cell proliferation. Importantly, this smad7-mediated proliferation appears to entail passing through a transient, nonpathologic dedifferentiation of ß-cells to a pancreatic polypeptide-fold hormone-positive state. TGF-ß receptor II appears to be a receptor important for controlling the status of the smad network in ß-cells. These studies should help our understanding of properly regulated ß-cell replication.

Specific transduction and labeling of pancreatic ducts by targeted recombinant viral infusion into mouse pancreatic ducts.

Specific labeling of pancreatic ducts has proven to be quite difficult. Such labeling has been highly sought after because of the power it would confer to studies of pancreatic ductal carcinogenesis, as well as studies of the source of new insulin-producing ß-cells. Cre-loxp recombination could, in theory, lineage-tag pancreatic ducts, but results have been conflicting, mainly due to low labeling efficiencies. Here, we achieved a high pancreatic duct labeling efficiency using a recombinant adeno-associated virus (rAAV) with a duct-specific sox9 promoter infused into the mouse common biliary/pancreatic duct. We saw rapid, diffuse duct-specific labeling, with 50 and 89% labeling in the pancreatic tail and head region, respectively. This highly specific labeling of ducts should greatly enhance our ability to study the role of pancreatic ducts in numerous aspects of pancreatic growth, development and function.

α-Cells are dispensable in postnatal morphogenesis and maturation of mouse pancreatic islets.

Glucagon-producing α-cells are the second-most abundant cell type in the islet. Whereas α-cells make up less than 20% of the cells in a mature mouse islet, they occupy a much larger proportion of the pancreatic endocrine cell population during the early postnatal period, the time when morphological and functional maturation occurs to form adult islets. To determine whether α-cells have a role in postnatal islet development, a diphtheria toxin-mediated α-cell ablation mouse model was established. Rapid and persistent depletion of α-cells was achieved by daily injection of the toxin for 2 wk starting at postnatal day 1 (P1). Total pancreatic glucagon content in the α-cell-ablated mice was undetectable at P14 and still less than 0.3% of that of the control mice at 4 mo of age. Histological analyses revealed that formation of spherical islets occurred normally, and the islet size distribution was not changed despite the near-total lack of α-cells. Furthermore, there were no differences in expression of ß-cell maturation marker proteins, including urocortin 3 and glucose transporter 2, in the α-cell-ablated islets at P14. Mice lacking α-cells grew normally and appeared healthy. Both glucose and insulin tolerance tests demonstrated that the α-cell-ablated mice had normal glucose homeostasis. These results indicate that α-cells do not play a critical role in postnatal islet morphogenesis or functional maturation of ß-cells.

Neurogenin3 activation is not sufficient to direct duct-to-beta cell transdifferentiation in the adult pancreas.

It remains controversial whether adult pancreatic ducts harbor facultative beta cell progenitors. Because neurogenin3 (Ngn3) is a key determinant of pancreatic endocrine cell neogenesis during embryogenesis, many studies have also relied upon Ngn3 expression as evidence of beta cell neogenesis in adults. Recently, however, Ngn3 as a marker of adult beta cell neogenesis has been called into question by reports of Ngn3 expression in fully-developed beta cells. Nevertheless, direct evidence as to whether Ngn3 activation in adult pancreatic duct cells may lead to duct-to-beta cell transdifferentiation is lacking. Here we studied two models of Ngn3 activation in adult pancreatic duct cells (low-dose alloxan treatment and pancreatic duct ligation) and lineage-traced Ngn3-activated duct cells by labeling them through intraductal infusion with a cell-tagging dye, CFDA-SE No dye-labeled beta cells were found during the follow-up in either model, suggesting that activation of Ngn3 in duct cells is not sufficient to direct their transdifferentiation into beta cells. Therefore, Ngn3 activation in duct cells is not a signature for adult beta cell neogenesis.

No evidence for ß cell neogenesis in murine adult pancreas.

Whether facultative ß cell progenitors exist in the adult pancreas is a major unsolved question. To date, lineage-tracing studies have provided conflicting results. To track ß cell neogenesis in vivo, we generated transgenic mice that transiently coexpress mTomato and GFP in a time-sensitive, nonconditional Cre-mediated manner, so that insulin-producing cells express GFP under control of the insulin promoter, while all other cells express mTomato (INSCremTmG mice). Newly differentiated ß cells were detected by flow cytometry and fluorescence microscopy, taking advantage of their transient coexpression of GFP and mTomato fluorescent proteins. We found that ß cell neogenesis predominantly occurs during embryogenesis, decreases dramatically shortly after birth, and is completely absent in adults across various models of ß cell loss, ß cell growth and regeneration, and inflammation. Moreover, we demonstrated upregulation of neurogenin 3 (NGN3) in both proliferating ducts and preexisting ß cells in the ligated pancreatic tail after pancreatic ductal ligation. These results are consistent with some recent reports, but argue against the widely held belief that NGN3 marks cells undergoing endocrine neogenesis in the pancreas. Our data suggest that ß cell neogenesis in the adult pancreas occurs rarely, if ever, under either normal or pathological conditions.