Transamniotic Stem Cell Therapy for Experimental Congenital Diaphragmatic Hernia: Structural, Transcriptional, and Cell Kinetics Analyses in the Nitrofen Model.

PURPOSE: We examined select pulmonary effects and donor cell kinetics after transamniotic stem cell therapy (TRASCET) in a model of congenital diaphragmatic hernia (CDH). METHODS: Pregnant dams (n = 58) received nitrofen on gestational day 9.5 (E9) to induce fetal CDH. Fetuses (n = 681) were divided into 4 groups: untreated (n = 99) and 3 groups receiving volume-matched intra-amniotic injections on E17 of either saline (n = 142), luciferase-labeled amniotic fluid-derived mesenchymal stem cells (afMSCs; n = 299), or acellular recombinant luciferase (n = 141). Pulmonary morphometry, quantitative gene expression of pulmonary vascular tone mediators, or screening for labeled afMSCs were performed at term (E22). Statistical comparisons were by Mann-Whitney U-test, nested ANOVA, and Wald test. RESULTS: TRASCET led to significant downregulation of endothelial nitric oxide synthase and endothelin receptor-A expressions compared to both untreated and saline groups (both p < 0.001). TRASCET also led to a significant decrease in arteriole wall thickness compared to the untreated group (p < 0.001) but not the saline group (p = 0.180). Donor afMSCs were identified in the bone marrow and umbilical cord (p = 0.035 and 0.015, respectively, vs. plain luciferase controls). CONCLUSIONS: The effects of TRASCET in experimental CDH appear to be centered on the pulmonary vasculature and to derive from circulating donor cells.

Initial Mechanistic Screening of Transamniotic Stem Cell Therapy in the Rodent Model of Spina Bifida: Host Bone Marrow and Paracrine Activity.

PURPOSE: Transamniotic stem cell therapy (TRASCET) with mesenchymal stem cells (MSCs) can induce spina bifida coverage with neoskin. We initiated a mechanistic analysis of this host response. METHODS: Pregnant dams (n = 28) exposed to retinoic acid to induce fetal spina bifida were divided into an untreated group and 2 groups receiving intra-amniotic injections on gestational day 17 (E17; term = E21-22) of either amniotic fluid-derived MSCs (afMSCs; n = 105) or saline (n = 107). Gene expressions of multiple paracrine and cell clonality markers were quantified at term by RT-qPCR at the defect and fetal bone marrow. Defects were examined histologically for neoskin coverage. Comparisons were by Mann-Whitney U tests and logistic regression. RESULTS: Defect coverage was associated with significant downregulation of both epidermal growth factor (Egf; p = 0.031) and fibroblast growth factor-2 (Fgf-2; p = 0.042) expressions at the defect and with significant downregulation of transforming growth factor-beta-1 (Tgfb-1; p = 0.021) and CD45 (p = 0.028) expressions at the fetal bone marrow. CONCLUSIONS: Coverage of experimental spina bifida is associated with local and bone marrow negative feedback of select paracrine factors, as well as increased relative mesenchymal stem cell activity in the bone marrow. Further analyses informed by these findings may lead to strategies of nonsurgical induction of prenatal coverage of spina bifida.

Thoracoscopic Excision of Mediastinal Bronchogenic Cysts in Children: A Case Series.

Background: Bronchogenic cysts result from a congenital anomalous budding of the tracheobronchial tree. Resection is usually recommended to avoid complications. Mediastinal bronchogenic cysts present a unique challenge due to their proximity to vital structures. The purpose of this study is to review our experience with mediastinal bronchogenic cysts. Methods: A single-institution retrospective review evaluated all mediastinal bronchogenic cyst excisions between January 2012 and November 2022. Patient demographics were assessed, including age at diagnosis, presenting symptoms, imaging workup, and cyst characteristics. Operative approach, complications, and surgical pathology were reported. Results: Five patients were identified. Age at diagnosis ranged from 18 to 27 months. No patient was diagnosed prenatally. All patients had symptoms at the time of diagnosis, including cough, wheezing, and respiratory distress. Three cysts were paratracheal, and two were paraesophageal. Age at surgery ranged from 26 to 30 months. All bronchogenic cysts were successfully resected thoracoscopically. Individual technical challenges included narrowing of the mainstem bronchus preventing lung isolation, significant mediastinal inflammation, the necessity for cyst evacuation to delineate the extent of the cyst, adherence of cyst wall to bronchus or trachea requiring cold dissection, and a stalk of tissue with an intimate connection to the carina that was amputated. No intraoperative or postoperative complication occurred. Surgical pathology was consistent with a bronchogenic cyst in all cases. Median length of hospital stay was two days. Conclusion: Thoracoscopy is a safe and effective procedure for mediastinal bronchogenic cyst excision in children. Certain technical maneuvers are highlighted, which may facilitate resection.

Enhanced recovery after surgery improves clinical outcomes in adolescent bariatric surgery.

BACKGROUND: Enhanced recovery after surgery (ERAS) protocols are evidence-based, multimodal approaches to optimize patient recovery and minimize complications. OBJECTIVES: Our team evaluated clinical outcomes following the implementation of an ERAS protocol for adolescents undergoing metabolic and bariatric surgery. SETTING: Academic hospital, New York, NY, USA. METHODS: We performed a single-institution longitudinal assessment of adolescents who underwent laparoscopic vertical sleeve gastrectomy (VSG) between August 2021 and November 2022. Unpaired t-tests and Fisher's exact test were used to compare means between groups and categorical factors. RESULTS: Forty-three patients were included in the study, 21 who participated in the ERAS protocol and 22 control patients. ERAS cohort was 52% females, with a median age of 17.5 years and a median body mass index (BMI) of 46.3 kg/m2. The non-ERAS cohort was 59% females, with a median age of 16.7 years and a median BMI of 44.0 kg/m2. There were no significant differences between baseline characteristics. Patients in the ERAS group had a shorter time to oral intake (10.7 hours versus 21.5 hours, P < .01), lower morphine milligram equivalents (18.2 versus 97.0, P < .01), and shorter length of stay (1.5 days versus 2.0 days, P = .01). There were no significant differences between return visits to the emergency department (ED) within 30 days (3 versus 2, P = .66) or readmissions (0 versus 1, P = 1.0). CONCLUSIONS: The described ERAS protocol is safe and effective in adolescents undergoing laparoscopic VSG and is associated with shorter time to oral intake, reduced opioid requirements, and shorter hospital lengths of stay with no increase in return ED visits or readmissions.

Pediatric sternal fractures: A single center retrospective review.

PURPOSE: We sought to investigate the diagnosis, management, and outcomes of pediatric sternal fractures. METHODS: We used ICD codes to search our trauma registry and Hound Dog software to search the hospital data warehouse for all cases of radiologically confirmed sternal fracture in patients <21 years from January 1, 1997 to July 1, 2017. We extracted demographics, mechanism of injury, diagnostic modality, associated injuries, and clinical outcomes. RESULTS: We identified 65 children with sternal fractures. 46/65 (71%) were male, with median age 11 years. 34/65 (52%) presented to our emergency department (ED) and the remainder to outpatient clinics. 41/65 (63%) were diagnosed by chest X-ray, 11/65 (17%) by chest CT, 7/65 (11%) by sternal X-ray, and 5/65 (8%) by MRI. Mechanism of injury varied by age. The majority, 50/65 (77%), were isolated injuries and there were no cardiac injuries, aortic injuries or deaths. 18/33 (45%) of those who presented initially to the ED were admitted, and of these 7/18 (39%) had isolated sternal fractures. CONCLUSIONS: In this series, most sternal fractures were isolated with low morbidity. Sternal fracture alone should not prompt aggressive workup for intrathoracic injuries and stable patients with isolated sternal fractures can be safely followed without admission. LEVEL OF EVIDENCE: IV.

Congenital diaphragmatic hernia as a potential target for transamniotic stem cell therapy.

PURPOSE: We sought to determine whether TRASCET could impact congenital diaphragmatic hernia (CDH). METHODS: Twelve pregnant dams received Nitrofen on gestational day 9.5 (E9; term = 22 days) to induce fetal CDH. Fetuses were divided into three groups: untreated (n = 31) and two groups receiving volume-matched intraamniotic injections of either saline (n = 37) or a suspension of 2 × 106 cells/mL of amniotic fluid-derived mesenchymal stem cells (afMSCs; n = 65) on E17. Animals were euthanized at term. Expression of fibroblast growth factor-10 (FGF-10), vascular endothelial growth factor-A (VEGF-A), and surfactant protein-C (SPC) was quantified by qRT-PCR. Statistical analysis was by the Mann-Whitney U test with Bonferroni adjusted criterion (p ≤ 0.01). RESULTS: Among survivors with CDH (n = 27/133), the TRASCET group showed significant downregulation of FGF-10 and VEGF-A gene expressions compared to the untreated (p < 0.001 for both) and saline groups (p = 0.005 and p = 0.004, respectively). SPC expression was higher in the TRASCET group compared to the untreated group (p = 0.01), but not the saline group (p = 0.043). Lung laterality had minimal impact on these comparisons. CONCLUSIONS: Transamniotic stem cell therapy affects select processes of lung development in experimental congenital diaphragmatic hernia. Further scrutiny into this novel therapy as a potential component of the prenatal management of this disease is warranted. LEVEL OF EVIDENCE: N/A (animal and laboratory study).

Donor mesenchymal stem cell kinetics after transamniotic stem cell therapy (TRASCET) in a rodent model of gastroschisis.

PURPOSE: We sought to comprehensively scrutinize donor mesenchymal stem cell kinetics following transamniotic stem cell therapy (TRASCET) in experimental gastroschisis. METHODS: A gastroschisis was surgically created in 102 rat fetuses at gestation day 18 (term = 22 days), immediately followed by volume-matched amniotic injections of either amniotic fluid mesenchymal stem cells (afMSCs) labeled with a luciferase reporter gene (n = 58), or luciferase protein alone (n = 44). Samples from multiple anatomical sites from survivors were screened for luciferase activity via microplate luminometry at term. Statistical analysis included Mann-Whitney U-test, Wald test, and kappa coefficient (p < 0.05). RESULTS: Overall survival was 42% (43/102), with no significant difference between the two groups (p = 0.82). When controlled by acellular luciferase, donor afMSCs were identified selectively in the placenta (p < 0.001) and bowel (p = 0.005), independently of the dams (respectively, p < 0.001 and p = 0.041). Bowel homing was documented exclusively in areas exposed to the amniotic cavity. There was no mutual correlation between placental and bowel homing (kappa = -0.02; p = 0.91). CONCLUSIONS: Amniotic mesenchymal stem cells home to specific sites after TRASCET in the setting of gastroschisis. Placental homing and intestinal homing are central yet seemingly independent constituents of cell trafficking, suggesting that both direct amniotic seeding and hematogenous routing take place. LEVEL OF EVIDENCE: N/A (animal and laboratory study).

A comparison between placental and amniotic mesenchymal stem cells in transamniotic stem cell therapy for experimental gastroschisis.

PURPOSE: We compared placental and amniotic fluid-derived mesenchymal stem cells (pMSCs and afMSCs, respectively) in transamniotic stem cell therapy for experimental gastroschisis. METHODS: Gastroschisis was surgically created in 126 rat fetuses at gestational day 18 (term = 22 days), immediately followed by volume-matched intraamniotic injections of suspensions of afMSCs (n = 32), pMSCs (n = 33), or normal saline (NS) (n = 33). Untreated fetuses served as controls (n = 28). Blinded observers performed computerized measurements of total and segmental (serosa, muscularis, and mucosa) intestinal wall thickness on the herniated bowel at term. Statistical analysis included ANOVA, the Wald test, and Levene's test (p < 0.05). RESULTS: Among survivors, there were statistically significant decreases in segmental and total bowel wall thicknesses in both the afMSC and pMSC groups vs. the untreated (p < 0.001 to 0.003) and saline (p < 0.001 to 0.011) groups. There were significant differences between the afMSC and pMSC groups favoring the former in both therapeutic impact and its variability (p < 0.001 to 0.031). Labeled cells were comparably identified within the intestinal wall in the afMSC and pMSC groups. CONCLUSIONS: Both placental and amniotic mesenchymal stem cells can mitigate bowel damage in experimental gastroschisis as agents of transamniotic stem cell therapy. However, amniotic cells lead to improved and more consistent outcomes. LEVEL OF EVIDENCE: N/A (animal and laboratory study).

Hematogenous Donor Cell Routing Pathway After Transamniotic Stem Cell Therapy.

Donor mesenchymal stem cells (MSCs) have been documented in fetal and maternal circulations after plain intra-amniotic injection, with diverse therapeutic effects. We sought to determine the pathway of this unique cell kinetic route. Rat fetuses (n = 226) were divided into two groups based on the content of intra-amniotic injections performed on gestational day 17 (E17): either a concentrated suspension of luciferase-labeled syngeneic amniotic fluid-derived MSCs (afMSCs; n = 111), or acellular luciferase (n = 115). Samples from placenta, chorion, amnion, amniotic fluid, stomach fluid, peripheral blood, and umbilical cord were procured at five daily time points thereafter until term (E18-22) for luminometry. In addition, 53 sets of fresh gestational membranes (chorion/amnion combined) from nonmanipulated term fetuses were secured to transwell inserts for in vitro analysis of MSC migration using luciferase-labeled afMSCs. Statistical analyses included the Mann-Whitney U-test, Wald test, nonlinear regression modeling, and Fisher's exact test. In vivo, luciferase activity was observed in the amnion, chorion, and placenta of fetuses receiving cells, but not in those receiving acellular luciferase (P < 0.001). There was a consistent nonlinear age-dependent relationship of luciferase activity between the amnion, chorion, and placenta following a parabolic bimodal pattern characterized by significantly higher early preterm (E18) and late-term (E22) activities (P < 0.001), with no differences between E21 and E22 (P = 0.12). In vitro, the presence of cells was documented by luminometry in 21/53 (39.6%) of the assays, in suspension and/or attached to the plastic substrate, and within all screened gestational membrane sets, irrespective of stimuli with collagen coating or fetal bovine serum. We conclude that, after intra-amniotic injection, donor MSCs undergo controlled cell routing, as opposed to passive clearance. Transgestational membrane transport appears to constitute the path for donor cells to reach the placenta, a known gateway to the fetal circulation, significantly expanding the potential applications of transamniotic stem cell therapy.

Postnatal fate of donor mesenchymal stem cells after transamniotic stem cell therapy in a healthy model.

PURPOSE: We sought to examine donor mesenchymal stem cell (MSC) fate after birth following transamniotic stem cell therapy (TRASCET) in a healthy model. METHODS: Lewis rat fetuses (n = 91) were divided into two groups based on the content of volume-matched intraamniotic injections performed on gestational day 17 (term = 21-22 days): either a suspension of amniotic fluid-derived MSCs (afMSCs) labeled with luciferase (n = 38) or acellular luciferase only (n = 53). Infused afMSCs consisted of syngeneic Lewis rat cells phenotyped by flow cytometry. Samples from 14 anatomical sites (heart, lung, brain, liver, spleen, pancreas, bowel, kidney, thyroid, skin, skeletal muscle, thymus, peripheral blood and bone marrow) from survivors were screened for luciferase activity 16 days after birth. Statistical analysis was by logistic regression and the Wald test (p < 0.05). RESULTS: Overall survival was 32% (29/91). When controlled by the acellular luciferase injections, donor afMSCs were not identified at any anatomical site in any neonate as measured by relative light units (all p > 0.05). Donor afMSC viability was confirmed in term placentas. CONCLUSIONS: Donor mesenchymal stem cells are not detectable in the neonate after intraamniotic injection in a normal syngeneic rodent model. This finding suggests that clinical trials of transamniotic stem cell therapy may be amenable to regulatory approval. LEVEL OF EVIDENCE: N/A (animal and laboratory study).

Transamniotic stem cell therapy (TRASCET) in a rabbit model of spina bifida.

PURPOSE: Transamniotic stem cell therapy (TRASCET) with select mesenchymal stem cells (MSCs) has been shown to induce partial or complete skin coverage of spina bifida in rodents. Clinical translation of this emerging therapy hinges on its efficacy in larger animal models. We sought to study TRASCET in a model requiring intra-amniotic injections 60 times larger than those performed in the rat. METHODS: Rabbit fetuses (n = 65) with surgically created spina bifida were divided into three groups. One group (untreated) had no further manipulations. Two groups received volume-matched intra-amniotic injections of either saline or a concentrated suspension of amniotic fluid MSCs (afMSCs) at the time of operation. Infused afMSCs consisted of banked heterologous rabbit afMSCs with mesenchymal identity confirmed by flow cytometry, labeled with green fluorescent protein. Defect coverage at term was blindly categorized only if the presence of a distinctive neoskin was confirmed histologically. Statistical comparisons were by logistic regression and the likelihood ratio test. RESULTS: Among survivors with spina bifida (n = 19), there were statistically significant higher rates of defect coverage (all partial) in the afMSC group when compared with the saline and untreated groups (0-50%; p = 0.022-0.036), with no difference between the saline and untreated groups (p = 1.00). Donor afMSCs were identified locally, though sparsely and not in the neoskin. CONCLUSIONS: Concentrated intra-amniotic injection of amniotic mesenchymal stem cells can induce partial coverage of experimental spina bifida in a leporine model. Transamniotic stem cell therapy may become a feasible strategy in the prenatal management of spina bifida. LEVEL OF EVIDENCE: N/A (animal and laboratory study).

Development and Implementation of a Surgical Quality Improvement Pathway for Pediatric Intussusception Patients.

Children with intussusception can be admitted or discharged from the emergency department (ED) following enema reduction, but little is known about best practices for surgical follow-up and the need for a return to care. METHODS: We developed a standardized clinical assessment and management plan (SCAMP) for ileocolic intussusception to enable the discharge from the ED of successfully reduced patients meeting certain criteria with 2 planned follow-up phone calls by surgical personnel after discharge. Outcomes included incidence of complications in discharged patients, bacteremia, the success of follow-up phone calls, rates of recurrent intussusception, and return to care. RESULTS: Of the 118 patient encounters treated through the SCAMP in 2 pilot studies from February 2013 to December 2017, 76% met discharge criteria, of whom 88% underwent outpatient management. There were no instances of bowel perforation, necrosis, or death in the discharged group. No patients developed bacteremia despite withholding antibiotics for the indication of intussusception. Sixty-two percent and 59% of patients received 24-hour follow-up phone calls, and 28% and 55% of patients received second follow-up phone calls in pilots 1 and 2, respectively. Of those successfully discharged, 74% did not return to care, 19% returned for recurrent intussusception, and 7% returned for unrelated symptoms. Nearly all patients who returned to care did so through the ED and not the clinic. CONCLUSIONS: Implementation of the SCAMP demonstrated that patients meeting certain criteria could be safely discharged from the ED, avoid antibiotics, and safely undergo phone-based follow-up for concerns of recurrent intussusception.

The specification of glycinergic neurons and the role of glycinergic transmission in development.

Glycine's role as an inhibitory neurotransmitter in the adult vertebrate nervous system has been well characterized in a number of different model organisms. However, a full understanding of glycinergic transmission requires a knowledge of how glycinergic synapses emerge and the role of glycinergic signaling during development. Recent literature has provided a detailed picture of the developmental expression of many of the molecular components that comprise the glycinergic phenotype, namely the glycine transporters and the glycine receptor subunits; the transcriptional networks leading to the expression of this important neurotransmitter phenotype are also being elucidated. An equally important focus of research has revealed the critical role of glycinergic signaling in sculpting many different aspects of neural development. This review examines the current literature detailing the expression patterns of the components of the glycinergic phenotype in various vertebrate model organisms over the course of development and the molecular mechanisms governing the expression of the glycinergic phenotype. The review then surveys the recent work on the role of glycinergic signaling in the developing nervous system and concludes with an overview of areas for further research.