Florian Obermayr

Expansion and differentiation of neural progenitors derived from the human adult enteric nervous system.

BACKGROUND & AIMS Neural stem and progenitor cells from the enteric nervous system have been proposed for use in cell-based therapies against specific neurogastrointestinal disorders. Recently, enteric neural progenitors were generated from human neonatal and early postnatal (until 5 years after birth) gastrointestinal tract tissues. We investigated the proliferation and differentiation of enteric nervous system progenitors isolated from human adult gastrointestinal tract. METHODS Human enteric spheroids were generated from adult small and large intestine tissues and then expanded and differentiated, depending on the applied cell culture conditions. For implantation studies, spheres were grafted into fetal slice cultures and embryonic aganglionic hindgut explants from mice. Differentiating enteric neural progenitors were characterized by 5-bromo-2-deoxyuridine labeling, in situ hybridization, immunocytochemistry, quantitative real-time polymerase chain reaction, and electrophysiological studies. RESULTS The yield of human neurosphere-like bodies was increased by culture in conditional medium derived from fetal mouse enteric progenitors. We were able to generate proliferating enterospheres from adult human small or large intestine tissues; these enterospheres could be subcultured and maintained for several weeks in vitro. Spheroid-derived cells could be differentiated into a variety of neuronal subtypes and glial cells with characteristics of the enteric nervous system. Experiments involving implantation into organotypic intestinal cultures showed the differentiation capacity of neural progenitors in a 3-dimensional environment. CONCLUSIONS It is feasible to isolate and expand enteric progenitor cells from human adult tissue. These findings offer new strategies for enteric stem cell research and future cell-based therapies.

Development and developmental disorders of the enteric nervous system

The enteric nervous system (ENS) arises from neural crest-derived cells that migrate into and along the gut, leading to the formation of a complex network of neurons and glial cells that regulates motility, secretion and blood flow. This Review summarizes the progress made in the past 5 years in our understanding of ENS development, including the migratory pathways of neural crest-derived cells as they colonize the gut. The importance of interactions between neural crest-derived cells, between signalling pathways and between developmental processes (such as proliferation and migration) in ensuring the correct development of the ENS is also presented. The signalling pathways involved in ENS development that were determined using animal models are also described, as is the evidence for the involvement of the genes encoding these molecules in Hirschsprung disease—the best characterized paediatric enteric neuropathy. Finally, the aetiology and treatment of Hirschsprung disease in the clinic and the potential involvement of defects in ENS development in other paediatric motility disorders are outlined.

Transplanted progenitors generate functional enteric neurons in the postnatal colon

Cell therapy has the potential to treat gastrointestinal motility disorders caused by diseases of the enteric nervous system. Many studies have demonstrated that various stem/progenitor cells can give rise to functional neurons in the embryonic gut; however, it is not yet known whether transplanted neural progenitor cells can migrate, proliferate, and generate functional neurons in the postnatal bowel in vivo. We transplanted neurospheres generated from fetal and postnatal intestinal neural crest-derived cells into the colon of postnatal mice. The neurosphere-derived cells migrated, proliferated, and generated neurons and glial cells that formed ganglion-like clusters within the recipient colon. Graft-derived neurons exhibited morphological, neurochemical, and electrophysiological characteristics similar to those of enteric neurons; they received synaptic inputs; and their neurites projected to muscle layers and the enteric ganglia of the recipient mice. These findings show that transplanted enteric neural progenitor cells can generate functional enteric neurons in the postnatal bowel and advances the notion that cell therapy is a promising strategy for enteric neuropathies.

Assisted reproductive techniques and the risk of anorectal malformations: a German case-control study

BackgroundThe use of assisted reproductive techniques (ART) for treatment of infertility is increasing rapidly worldwide. However, various health effects have been reported including a higher risk of congenital malformations. Therefore, we assessed the risk of anorectal malformations (ARM) after in-vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI).MethodsData of the German Network for Congenital Uro-REctal malformations (CURE-Net) were compared to nationwide data of the German IVF register and the Federal Statistical Office (DESTATIS). Odds ratios (95% confidence intervals) were determined to quantify associations using multivariable logistic regression accounting for potential confounding or interaction by plurality of births.ResultsIn total, 295 ARM patients born between 1997 and 2011 in Germany, who were recruited through participating pediatric surgeries from all over Germany and the German self-help organisation SoMA, were included. Controls were all German live-births (n = 10,069,986) born between 1997 and 2010. Overall, 30 cases (10%) and 129,982 controls (1%) were born after IVF or ICSI, which translates to an odds ratio (95% confidence interval) of 8.7 (5.9–12.6) between ART and ARM in bivariate analyses. Separate analyses showed a significantly increased risk for ARM after IVF (OR, 10.9; 95% CI, 6.2–19.0; P < 0.0001) as well as after ICSI (OR, 7.5; 95% CI, 4.6–12.2; P < 0.0001). Furthermore, separate analyses of patients with isolated ARM, ARM with associated anomalies and those with a VATER/VACTERL association showed strong associations with ART (ORs 4.9, 11.9 and 7.9, respectively). After stratification for plurality of birth, the corresponding odds ratios (95% confidence intervals) were 7.7 (4.6–12.7) for singletons and 4.9 (2.4–10.1) for multiple births.ConclusionsThere is a strongly increased risk for ARM among children born after ART. Elevations of risk were seen after both IVF and ICSI. Further, separate analyses of patients with isolated ARM, ARM with associated anomalies and those with a VATER/VACTERL association showed increased risks in each group. An increased risk of ARM was also seen among both singletons and multiple births.

Perioperative Outcome of Patients with Congenital Diaphragmatic Hernia Undergoing Open Versus Minimally Invasive Surgery

PURPOSE The aim of this study was to evaluate outcome of patients with congenital diaphragmatic hernia (CDH) undergoing open versus minimally invasive surgery. SUBJECTS AND METHODS Patient records of 33 children undergoing surgery for CDH between March 2002 and September 2008 were reviewed. Patient data were compared regarding operating time, intraoperative maximum CO(2) partial pressure (pCO(2 max)) values, postoperative ventilation time, complications, and recurrences. RESULTS Median age at time of operation was 4 days (range, 0-1017 days), and median weight was 3800 g (range, 2000-13,200 g). Laparotomy was performed in 12 children. Seventeen patients underwent thoracoscopic repair, and four children had a laparoscopic approach. Operating time was significantly longer (P=.004) in the minimally invasive group. Median values of pCO(2 max) during operation were not significantly different (P=.25) in the minimally invasive surgery group. The pCO(2 max) values in the postoperative course were significantly lower (P=.013) in the minimally invasive group, whereas median ventilation times postoperatively were significantly longer (P=.024) in the open surgery group. CONCLUSIONS Median values of pCO(2 max) in the postoperative course were significantly lower in the minimally invasive surgery group. In addition, postoperative ventilation time was shorter when children underwent minimally invasive surgery. In conclusion, minimally invasive surgery seems to offer advantages for selected patients with CDH.

Assisted Reproductive Techniques and Risk of Exstrophy-Epispadias Complex: A German Case-Control Study

PURPOSE We assessed the risk of exstrophy-epispadias complex in children conceived by in vitro fertilization or intracytoplasmic sperm injection. MATERIALS AND METHODS Data from the German Network for Congenital Uro-REctal malformations were compared to nationwide data from the German In Vitro Fertilization Register and the German Federal Statistical Office. Odds ratios (95% CI) were determined to quantify associations using logistic regression. RESULTS A total of 123 patients with exstrophy-epispadias complex born in Germany between 1997 and 2011 were recruited through participating departments of pediatric urology and pediatric surgery throughout the country as well as the German self-help organizations Blasenekstrophie/Epispadie e.V. and Kloakenekstrophie. All German live births (10,069,986) between 1997 and 2010 comprised the controls. Overall, 12 subjects (10%) and 129,982 controls (1%) were conceived by in vitro fertilization or intracytoplasmic sperm injection. Conception by assisted reproductive technique was associated with a more than eightfold increased risk of exstrophy-epispadias complex compared to spontaneous conception (OR 8.3, 95% CI 4.6-15.0, p <0.001). Separate analyses showed a significantly increased risk of exstrophy-epispadias complex in children conceived by in vitro fertilization (OR 14.0, 95% CI 6.5-30.0, p <0.0001) or intracytoplasmic sperm injection (OR 5.3, 95% CI 2.2-12.9, p <0.0001). CONCLUSIONS This study provides evidence that assisted reproductive techniques such as in vitro fertilization and intracytoplasmic sperm injection are associated with a markedly increased risk of having a child born with exstrophy-epispadias complex. However, it remains unclear whether this finding may be due to assisted reproduction per se and/or underlying infertility/subfertility etiology or parent characteristics.

Genetic fate-mapping of tyrosine hydroxylase-expressing cells in the enteric nervous system

During development of the enteric nervous system, a subpopulation of enteric neuron precursors transiently expresses catecholaminergic properties. The progeny of these transiently catecholaminergic (TC) cells have not been fully characterized.

Surgical Intervention to Rescue Hirschsprung Disease in a Rat Model.

Background/Aims Rats with a spontaneous null mutation in endothelin receptor type B or Ednrb (sl/sl; spotting lethal) lack enteric neurons in the distal bowel and usually die within the first week after birth. This early postnatal lethality limits their use for examining the potential of cell therapy to treat Hirschsprung disease, and for studies of the influence of EDNRB on the mature CNS and vascular systems. Methods We have developed a surgical intervention to prolong the life of the spotting lethal sl/sl rat, in which we perform a colostomy on postnatal (P) day 4–6 rats to avoid the fatal obstruction caused by the lack of colonic enteric neurons. Results The stomas remained patent and functional and the rats matured normally following surgery. Weight gains were comparable between control and Hirschsprung phenotype (sl/sl) rats, which were followed until 4 weeks after surgery (5 weeks old). We confirmed the absence of enteric neurons in the distal colon of rats whose lives were saved by the surgical intervention. Conclusions This study provides a novel approach for studying EDNRB signalling in multiple organ systems in mature rats, including an animal model to study the efficacy of cell therapy to treat Hirschsprung disease.

Ureteropelvic junction obstruction and calyceal diverticulum in a child with Turner syndrome and horseshoe kidney

Laparoscopic dismembered pyeloplasty for ureteropelvic junction (UPJ) obstruction is considered to be a routine procedure in many pediatric surgical centers. UPJ obstruction is known to be associated with horseshoe kidney and several reports on successful laparoscopic repair in such cases exist. The case of a 9-month-old girl with Turner syndrome is reported. A horseshoe kidney with grade 4 hydronephrosis on the left side was diagnosed by ultrasound during the neonatal period. MAG3 diuretic renography and dynamic magnetic resonance imaging nephrography revealed a differential renal function of 31% and 69% on the left and right side, respectively. No drainage from the left renal pelvis could be demonstrated. Laparoscopy showed a combined UPJ obstruction and a calyceal diverticulum with a narrow infundibulum of the upper pole calices on the left side of the horseshoe kidney. Laparoscopic dismembered pyeloplasty and an additional infundibulopelvic anastomosis was performed. No intraoperative complications occurred. The immediate postoperative course was uneventful. Unobstructed drainage and stable differential renal function on the left side could be demonstrated on MAG3 diuretic renography 6 weeks postoperatively. In conclusion, laparoscopic repair of complex malformations of the upper urinary tract is feasible and leads to good functional outcome in selected cases.

Expression of the Wnt Receptor Frizzled-4 in the Human Enteric Nervous System of Infants

The Wnt signalling pathway plays a crucial role in the development of the nervous system. This signalling cascade is initiated upon binding of the secreted Wnt ligand to a member of the family of frizzled receptors. In the present study, we analysed the presence of frizzled-4 in the enteric nervous system of human infants. Frizzled-4 could be identified by immunohistochemistry in a subpopulation of enteric neuronal and glial cells in the small and large intestine. Detection of frizzled-4 in the tunica muscularis by RT-PCR confirmed this receptors expression on the mRNA level. Interestingly, we observed distinct cell populations that co-expressed frizzled-4 with the intermediate filament protein nestin and the neurotrophin receptor p75NTR, which have been reported to be expressed in neural progenitor cells. Flow cytometry analysis revealed that 60% of p75NTR positive cells of the tunica muscularis were positive for frizzled-4. Additionally, in pathological samples of Hirschsprungs disease, the expression of this Wnt receptor correlated with the number of myenteric ganglion cells and decreased from normoganglionic to aganglionic areas of large intestine. The expression pattern of frizzled-4 indicates that this Wnt receptor could be involved in postnatal development and/or function of the enteric nervous system.