Adaptable haemodynamic endothelial cells for organogenesis and tumorigenesis.

Endothelial cells adopt tissue-specific characteristics to instruct organ development and regeneration1,2. This adaptability is lost in cultured adult endothelial cells, which do not vascularize tissues in an organotypic manner. Here, we show that transient reactivation of the embryonic-restricted ETS variant transcription factor 2 (ETV2)3 in mature human endothelial cells cultured in a serum-free three-dimensional matrix composed of a mixture of laminin, entactin and type-IV collagen (LEC matrix) 'resets' these endothelial cells to adaptable, vasculogenic cells, which form perfusable and plastic vascular plexi. Through chromatin remodelling, ETV2 induces tubulogenic pathways, including the activation of RAP1, which promotes the formation of durable lumens4,5. In three-dimensional matrices-which do not have the constraints of bioprinted scaffolds-the 'reset' vascular endothelial cells (R-VECs) self-assemble into stable, multilayered and branching vascular networks within scalable microfluidic chambers, which are capable of transporting human blood. In vivo, R-VECs implanted subcutaneously in mice self-organize into durable pericyte-coated vessels that functionally anastomose to the host circulation and exhibit long-lasting patterning, with no evidence of malformations or angiomas. R-VECs directly interact with cells within three-dimensional co-cultured organoids, removing the need for the restrictive synthetic semipermeable membranes that are required for organ-on-chip systems, therefore providing a physiological platform for vascularization, which we call 'Organ-On-VascularNet'. R-VECs enable perfusion of glucose-responsive insulin-secreting human pancreatic islets, vascularize decellularized rat intestines and arborize healthy or cancerous human colon organoids. Using single-cell RNA sequencing and epigenetic profiling, we demonstrate that R-VECs establish an adaptive vascular niche that differentially adjusts and conforms to organoids and tumoroids in a tissue-specific manner. Our Organ-On-VascularNet model will permit metabolic, immunological and physiochemical studies and screens to decipher the crosstalk between organotypic endothelial cells and parenchymal cells for identification of determinants of endothelial cell heterogeneity, and could lead to advances in therapeutic organ repair and tumour targeting.

Laparo-Endoscopic Rendez-Vous in the Treatment of Cholecysto-Choledocolithiasis in the Pediatric Population.

OBJECTIVES: The incidence of choledocolithiasis is reported to be increasing in children. As for the adult population, several different therapeutic strategies have been described, however it is unclear which of those should be considered the gold standard. There is evidence-based literature in adults that supports a combined "rendez-vous" endoscopic retrograde cholangiopancreatography-laparoscopic cholecystectomy technique. This allows management of the choledocholithiasis during the same anesthetic episode as the cholecystectomy. By contrast, there are just two case reports in children reporting this approach. The aim of this study is to report our experience with this technique in a series of children with choledocholithiasis. METHODS: All patients who underwent the "rendez-vous technique" at our institution between 2009 and 2020 were reviewed and evaluated for outcomes and complications. RESULTS: Eleven children with cholecysto-choledocholithiasis were evaluated: the procedure was successful in 10 whereas in one patient it was aborted due to technical difficulties. All patients resolved their clinical condition without major complications. CONCLUSION: To our knowledge, this is the first consistent series of "rendez-vous technique" in the pediatric population, proving its feasibility and safety.

Whole rat stomach decellularisation using a detergent-enzymatic protocol.

BACKGROUND: Conditions leading to reduced gastric volume are difficult to manage and are associated to poor quality-of-life. Stomach augmentation using a tissue-engineered stomach is a potential solution to restore adequate physiology and food reservoir. Aim of this study was to evaluate the decellularisation of whole rat stomach using a detergent-enzymatic protocol. METHODS: Stomachs harvested from rats were decellularised through luminal and vascular cannulation using 24-h detergent-enzymatic treatment and completely characterized by appropriate staining, DNA and Extracellular matrix -component quantifications. RESULTS: The detergent-enzymatic protocol allows a complete decellularisation of the gastric tissue, with a complete removal of the DNA with two cycles as confirmed by both quantifications and histological analysis. Extracellular matrix components, collagen, fibronectin, laminin and elastin, were optimally preserved by the treatment, while glycosaminoglycans were reduced. CONCLUSION: Gastric tissue can be efficiently decellularised. Scaffolds retained original structure and important components that could enhance integration with other tissues for in vivo transplant. The use of naturally derived material could be potentially considered for the treatment of both congenital and acquired conditions.

Spinal ultrasound in patients with anorectal malformations: is this the end of an era?

PURPOSE: Even if lumbar magnetic resonance imaging (MRI) is considered the gold standard in the diagnosis of occult spinal dysraphism (SD) in patients with anorectal malformations (ARMs), spinal ultrasound (US) performed up to 5 months of life have been largely used as a screening test. The aim of the present study was to evaluate the accuracy in terms of sensibility and specificity of neonatal US to detect occult SD in patients with ARMs. METHODS: Retrospective analysis of all patients treated for ARMs between 1999 and 2013 at our institution who underwent both spinal US (up to 5 months of life) and MRI. Sensibility and specificity have been calculated for US based on MRI results. RESULTS: Of 244 patients treated for ARMs at our institution, 82 (34 females, 48 males) underwent both the imaging studies and have been included in this study. ARMs types were: anal stenosis (7), recto-vestibular fistula (19), recto-perineal fistula (3) and cloaca (5) in female and imperforate anus (7) recto-perineal fistula (14), recto-urethral fistula (22), recto-vesical fistula (5) in males. Forty-seven patients (57, 3 % of total, 18 females, 29 males) had some occult SD (tethered spinal cord, spinal lipoma, syringomyelia) at MRI. Only 7 (14, 8 %) patients of those with spinal anomalies at MRI had pathological US studies. In our population, sensibility and specificity of US for diagnosis of occult SD were, respectively, 14, 8 and 100 %. CONCLUSION: Since it is well known that a screening test must have a high sensibility, our data suggest that spinal ultrasound is not suitable as a screening test for occult spinal dysraphism in patients with ARMs. Furthermore, we strongly advise against the use of US as a screening test for spinal dysraphism to prevent a false sense of security in physician and patients' families.

Comparison of outcomes following radiological reduction of intussusception with or without sedation.

BACKGROUND: Non-operative radiological reduction (NORR) is usually the first line treatment in pediatric ileo-colic intussusception. The aim of our study was to compare outcomes of NORR with or without sedation. METHODS: All patients undergoing to contrast enema NORR for intussusception between 01.01.2015 to 31.12.2020 in two hospitals were included: in one centre patients were sedated (A) while in the other patients were awake (B). Primary outcome was the rate of radiological reduction. Secondary outcomes were length-of-stay, complications and recurrence rate. RESULTS: Seventy-seven and 49 patients were included in group A and B respectively. Successful reduction rate was 72.7% in group A and 61.2% in group B (P>0.05). There were no complications related to the procedure among the 2 groups. Adverse events to sedation were observed in 3 patients. CONCLUSIONS: NORR has similar success rate when performed under sedation or awake, despite the former being graved by additional anesthesiologic risks and thus warrant careful indications.

Role of coagulation tests in the management of acute appendicitis in children.

BACKGROUND: Acute appendicitis (AA) is one of the most common acute surgical conditions in children. Coagulation tests (CoTs) are usually utilized in preoperative assessment to rule out hemorrhagic risks. Our study aimed to evaluate the role of CoTs as predictors for the severity of AA. METHODS: In a retrospective study, we compared the blood tests of two cohorts of pediatric patients with AA (group A and B) evaluated in the Emergency Department of a Pediatric Tertiary care hospital between January 2017 and January 2020. Children in Group A underwent appendectomies while those in Group B were treated with conservative management per hospital protocol. Group A was then subdivided into non-complicated (NCA) and complicated appendicitis (CA), and the CoTs were compared between the two subgroups. RESULTS: Group A consisted of 198 patients and Group B of 150 patients. Blood tests, including CoTs and inflammatory markers, were compared between the 2 groups. We found a statistically significant difference in PT ratio mean value between Group A and B, suggesting that those who underwent appendicectomies had higher PT ratio values. From a pathophysiological point of view, we speculated that the variation of PT ratio in AA might be secondary to a vitamin K absorption deficit due to enteric inflammation. CONCLUSIONS: Our study underlined that a longer PT ratio could be helpful to distinguish CA from NCA. Further investigations may lead to the role of the PT ratio in the choice between conservative and surgical management.

A case of vanishing gastroschisis with short bowel syndrome treated by total parenteral nutrition and intestinal lengthening procedures.

The diagnosis of vanishing gastroschisis is made when in the presence of a full-thickness intrauterine abdominal wall defect the eviscerated loops are incarcerated in the fascial interruption. Four types of vanishing gastroschisis are described (A-D). We report on the case of a newborn with a vanishing gastroschisis-D. Gastroschisis was diagnosed at the 19th week of gestation, confirmed at the 30th, when the herniated loops previously visible to the right of the funiculus were no longer visualized. At the 32nd week, delivery was induced. The neonate weighed 1600 g, and the abdomen was distended, free from skin defects. On surgical exploration, the jejunum was 13 cm in length, with a blind ending. The post-atretic intestine measured 22 cm. A jejunostomy and a colostomy were built. The child received total parenteral nutrition for 13 months due to short bowel syndrome and was then subjected to intestinal lengthening procedure when she was 18 months old. Vanishing gastroschisis is a rare entity with a worse prognosis of the "classic" gastroschisis.

Intussusception and COVID-19 in Infants: Evidence for an Etiopathologic Correlation.

Nonrespiratory conditions related to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections have been largely described. Ileocolic intussusception has been reported in association with SARS-CoV-2 infection in 10 children, raising the possibility of an etiopathologic role for the virus, but none of these cases documented tissue pathology that would have supported SARS-CoV-2 intestinal inflammation. We report 2 cases of intussusception in patients with SARS-CoV-2 infection who were treated at different pediatric tertiary centers in Europe and provide evidence of the presence of the virus in mesenteric and intestinal tissues of the patients.

Endoscopic Treatment of a Severe Vaginal Stenosis Following Battery Insertion in an 11-Year-Old Girl.

Acquired vaginal strictures are rare entities in children. As a result, they are generally difficult to manage and tend to recur despite appropriate initial therapy. This case study reports the staged management of vaginal stenosis following the insertion of a button battery. In this case, an 11-year-old girl experienced at 4 years old a battery insertion in the vaginal canal by her neighbor's son, who was 6-year-old at the time. Two weeks from insertion, the parents noted the foreign body discharge spontaneously. The girl had not complained of any symptoms at the time and had been asymptomatic for many years. In November 2020, she came to the emergency department reporting cramping abdominal pain accompanied by mucopurulent discharge. An abdominal ultrasound showed the presence of hematometrocolpos, and a vaginal stenosis dilation under general anesthesia was performed the following day. After 3 weeks, the stenosis was still present, preventing the passage of Hegar number 4. The girl was subjected to a vaginoscopic stenosis resection utilizing a monopolar hook passed through an operative channel. A Bakri catheter filled with 120 mL of water was left in place. After 10 days, the girl was discharged home with the Bakri inserted. Two weeks after discharge, she was reevaluated in the outpatient setting, where the Bakri was removed with no signs of residual stenosis. Acquired vaginal stenosis could be demanding to treat, particularly with the sole conservative approach. A first-line option can be the Hegar dilation. The endoscopic approach can be a second-line, minimally invasive treatment, but long-term outcomes are difficult to predict.

Testicular torsion during the COVID-19 pandemic: Results of a multicenter study in northern Italy.

INTRODUCTION: The literature reported an increased avoidance of the Emergency Department (ED) during COrona VIrus Disease 19 (COVID-19) pandemic, causing a subsequent increase of morbidity and mortality for acute conditions. Testicular torsion is a surgical emergency, which can lead to the loss of the affected testicle if a delayed treatment occurs. As testicular loss is time-related, outcome was hypothesized to be negatively affected by the pandemic. OBJECTIVE: The aim is to investigate whether presentation, treatment and outcomes of children with testicular torsion were delayed during COVID-19. STUDY DESIGN: Medical records of pediatric patients operated for testicular torsion of six Paediatric Surgical Units in Northern Italy between January 2019 and December 2020 were retrospectively reviewed. Patients were divided as for ones treated during (dC) or before the pandemic (pC). To reflect possible seasonality, related to lockdown restrictions, winter and summer calendar blocks were also analysed. For all cohorts, demographic data, pre-operative evaluation, operative notes and post-operative outcomes were reviewed. Primary outcomes were referral time, time from diagnosis to surgery and ischemic time, while secondary outcomes were orchiectomy and atrophy rates. Statistic was conducted as appropriate. RESULTS: A total of 188 patients with acute testicular torsion were included in the study period, 89 in the pre-COVID-19 (pC) period and 99 during COVID-19 (dC). Time from symptom onset to the access to the Emergency Department (T1) was not different among the two populations (pC: 5,5 h, dC: 6 h, p 0.374), and similarly time from diagnosis to surgery (pC: 2,5 h, dC: 2,5 h, p 0.970) and ischemic time (pC: 8,2 h, dC: 10 h, p 0.655). T1 was <6 h in 46/99 patients (46%) pC and 45/89 patients (51%) dC (p = 0.88, Fisher's exact test). Subgroup analysis accounting for different lockdown measures, confirm the absence of any difference. Orchiectomies rate was 23% (23/99) dC and 21% (19/89) pC (p = 0.861, Fisher's exact test) and rate of post-operative atrophy was 9% dC (7/76) and 14% pC (10/70), p = 0,44, Fisher's exact test. DISCUSSION: Despite worldwide pediatric ED accesses reduction, we reported that neither ischemic time nor the long-term outcomes in children with testicular torsion increased during the COVID-19 pandemic. In the available literature, few studies investigated the topic and are controversial on the results. Similarly to our findings, some studies found that timing and orchiectomy rates were not significantly different during the pandemic, while others reported a correlation to pandemic seasonality. Furthermore, in the recent pediatric literature it has been reported a delayed testicular torsion diagnosis due to shame in informing parents. Strengths of this study are the large numerosity, its multicentric design and a long study period. Its main limitation is being retrospective. CONCLUSIONS: We reported our large cohort from one of the most heavily COVID-19-affected regions, finding that referral, intra-hospital protocols and ischemic time in testicular torsion were not increased during to the pandemic, as well as orchiectomy rate and atrophy.

Lessons learned from pre-clinical testing of xenogeneic decellularized esophagi in a rabbit model.

Decellularization of esophagi from several species for tissue engineering is well described, but successful implantation in animal models of esophageal replacement has been challenging. The purpose of this study was to assess feasibility and applicability of esophageal replacement using decellularized porcine esophageal scaffolds in a new pre-clinical model. Following surgical replacement in rabbits with a vascularizing muscle flap, we observed successful anastomoses of decellularized scaffolds, cues of early neovascularization, and prevention of luminal collapse by the use of biodegradable stents. However, despite the success of the surgical procedure, the long-term survival was limited by the fragility of the animal model. Our results indicate that transplantation of a decellularized porcine scaffold is possible and vascular flaps may be useful to provide a vascular supply, but long-term outcomes require further pre-clinical testing in a different large animal model.

Quality of life and long-term results in patients operated on for Esophageal Atresia.

BACKGROUND: Esophageal atresia (EA) with or without tracheoesophageal fistula (TEF) represents the most severe entity among the foregut malformations, with patients at risk of chronic morbidity. This study aims to investigate on health-related quality of life (QoL) and long-term results in patients with EA. METHODS: 50 patients (30M, 20F, mean age: 14-year-old), among 82 patients operated from January 1995 to December 2005, answered the questionnaire. Two groups where compared: 8-12 years (A), 12-18 years (B). The survey investigated on 5 itmes: dysphagia; GERD; pulmonary affections (asthma, bronchitis, episodes of chronic cough during a year); growth and nutritional status (BMI); QoL scores. A control group of 50 healthy children was used. RESULTS: 42 type III AE (3 long-gap and 1 VACTER association). Median BMI was 18,72 (SD 3,38) (range: 30,52-12,80). In 22 pts BMI < 18,5. 19 pts were positive for dysphagia (EAT-10 score > = 3). A correlation was found between dysphagia and low BMI (Pearson 0,37). Dysphagia and low BMI were more diffuse in the younger population, while decreased in group B. 15 pts treated in their life for GERD (8 with medical therapy; 7 fundoplication). GerdQ test data showed only 4 pts with score > 6. GERD and dysphagia: 11 pts with GERD presented EAT-10 test > 3 (Pearson value confirmed this correlation: 0,59). Respiratory pathologies: 26 pts with positive anamnesis for chronic pulmonary affections (recurrent bronchitis in 21 pts; chronic cough in 15; the association of both in 12; 11 pts with asthma). PedsQL 4.0 median value was 15,4 (SD 10,1), control group value was 15,6 (SD 7,6); difference between the 2 groups was not statistically significant (p-value: 0,11). Correlation test for comparing high value of PedsQL and long-term complication in AE: the strongest association was with the dysphagia (Pearson: 0,55). CONCLUSIONS: Dysphagia resulted the most disabling symptom in group A but swallowing function slightly improved with the growth. Generally, AE seems not heavily influence patients' QoL.

Incidental Ultrasound Diagnosis of Neonatal Intussusception Secondary to Meckel's Diverticulum in a Neurologically Impaired Child.

BACKGROUND Intussusception is the most common cause of intestinal obstruction in children, with a peak incidence usually before the second year of age, while in neonates it is a rare entity. We describe a delayed and incidental diagnosis of neonatal intussusception secondary to Meckel's diverticulum in a neonate with shaken baby syndrome (SBS). This is, to the best of our knowledge, the first reported case of a neonatal intussusception with a Meckel's diverticulum as a lead point in a neurologically impaired child. CASE REPORT A term baby presented at 22 days of age at our Emergency Department in severe conditions due to a suspected SBS. Eight days following hospitalization in the Intensive Care Unit, an isolated episode of rectal bleeding occurred, without any worsening of general conditions or abdominal distension. The ultrasonography showed a "doughnut sign" with high suspicion of ileocecal intussusception. A rectal barium contrast enema was performed but was not resolutive. At exploratory laparotomy an ileocecal intussusception with Meckel's diverticulum acting as a lead point was identified and an intestinal resection was needed due to the ischemic condition of the ileum. The post-operative course was uneventful and the baby recovered well; the residual neurological impairment needed long-term follow-up. CONCLUSIONS Intussusception is a rare entity in neonates and, when severe neurological impairment is present, the diagnosis can be missed because of the compromised condition of the baby and the paucity of gastrointestinal manifestations. In addition, due to the high incidence of lead point in neonatal cases, we recommend reserving non-operative treatment only for selected cases.

Biliary atresia: Potential for a new decade.

Biliary atresia is characterised as an obliterative cholangiopathy of both extra-and intra-hepatic bile ducts. There is marked aetiological heterogeneity with a number of different variants, some syndromic and others perhaps virally-mediated. Current research aims to try and define possible mechanisms and pathogenesis though an actual breakthrough remains elusive. There has been little in the way of surgical advances beyond subtle variations in the Kasai portoenterostomy and laparoscopic equivalents have no declared advantage and have yet to prove equivalence in measures of outcome. The next target has been to maximise potential with better adjuvant therapy, though the evidence base for most currently available therapies such as steroids and ursodeoxycholic acid remains limited. Still high-dose steroid use is widespread, certainly in Europe and the Far East. Clearance of jaundice can be achieved in 50-60% of those subjected to portoenterostomy at <70 days and should be an achievable benchmark. Transplantation is a widely available "rescue" therapy though whether it should be an alternative as a primary procedure is arguable but becoming increasingly heard. The aim of clinical practice remains to get these infants for surgery as early as is possible though this can be difficult to accomplish in practice, and "low-cost" screening projects using stool colour charts have been limited outside of Taiwan and Japan. Centralisation of resources (medical and surgical) is associated with a diminution of time to portoenterostomy but application has been limited by entrenched health delivery models or geographical constraints.

Foreign-body Ingestions in Children During COVID-19 Pandemic in a Pediatric Referral Center.

In February 2020, the COVID-19 pandemic overwhelmed Italy. We retrospectively reviewed all attendances and emergency (A&E) admissions due to foreign-body ingestions (FBIs) to an Italian pediatric referral hospital, from February 24 to April 24, 2020, COVID-19 lockdown and compared them with the same period in the previous 4 years. A total of 101 cases were recorded. Mean age of admission was 4.6 years. Groups did not differ for gender (P = 0.4) or age (P = 0.3). Among FBIs ingestions, 24.0% occurred in children with <2 years of age and 47.5% in children from 2 to 6 years of age. In the 2020 study period, 9 patients were seen for batteries ingestion compared with a median value of one among compared periods. The rates of batteries ingestions increased significantly over the observational period (P < 0.001). We report a dramatic increase in batteries ingestions in children, a potentially fatal event, during the COVID-19 pandemic lockdown.

An In Vitro Whole-Organ Liver Engineering for Testing of Genetic Therapies.

Explosion of gene therapy approaches for treating rare monogenic and common liver disorders created an urgent need for disease models able to replicate human liver cellular environment. Available models lack 3D liver structure or are unable to survive in long-term culture. We aimed to generate and test a 3D culture system that allows long-term maintenance of human liver cell characteristics. The in vitro whole-organ "Bioreactor grown Artificial Liver Model" (BALM) employs a custom-designed bioreactor for long-term 3D culture of human induced pluripotent stem cells-derived hepatocyte-like cells (hiHEPs) in a mouse decellularized liver scaffold. Adeno-associated viral (AAV) and lentiviral (LV) vectors were introduced by intravascular injection. Substantial AAV and LV transgene expression in the BALM-grown hiHEPs was detected. Measurement of secreted proteins in the media allowed non-invasive monitoring of the system. We demonstrated that humanized whole-organ BALM is a valuable tool to generate pre-clinical data for investigational medicinal products.

Decellularised skeletal muscles allow functional muscle regeneration by promoting host cell migration.

Pathological conditions affecting skeletal muscle function may lead to irreversible volumetric muscle loss (VML). Therapeutic approaches involving acellular matrices represent an emerging and promising strategy to promote regeneration of skeletal muscle following injury. Here we investigated the ability of three different decellularised skeletal muscle scaffolds to support muscle regeneration in a xenogeneic immune-competent model of VML, in which the EDL muscle was surgically resected. All implanted acellular matrices, used to replace the resected muscles, were able to generate functional artificial muscles by promoting host myogenic cell migration and differentiation, as well as nervous fibres, vascular networks, and satellite cell (SC) homing. However, acellular tissue mainly composed of extracellular matrix (ECM) allowed better myofibre three-dimensional (3D) organization and the restoration of SC pool, when compared to scaffolds which also preserved muscular cytoskeletal structures. Finally, we showed that fibroblasts are indispensable to promote efficient migration and myogenesis by muscle stem cells across the scaffolds in vitro. This data strongly support the use of xenogeneic acellular muscles as device to treat VML conditions in absence of donor cell implementation, as well as in vitro model for studying cell interplay during myogenesis.

Multi-stage bioengineering of a layered oesophagus with in vitro expanded muscle and epithelial adult progenitors.

A tissue engineered oesophagus could overcome limitations associated with oesophageal substitution. Combining decellularized scaffolds with patient-derived cells shows promise for regeneration of tissue defects. In this proof-of-principle study, a two-stage approach for generation of a bio-artificial oesophageal graft addresses some major challenges in organ engineering, namely: (i) development of multi-strata tubular structures, (ii) appropriate re-population/maturation of constructs before transplantation, (iii) cryopreservation of bio-engineered organs and (iv) in vivo pre-vascularization. The graft comprises decellularized rat oesophagus homogeneously re-populated with mesoangioblasts and fibroblasts for the muscle layer. The oesophageal muscle reaches organised maturation after dynamic culture in a bioreactor and functional integration with neural crest stem cells. Grafts are pre-vascularised in vivo in the omentum prior to mucosa reconstitution with expanded epithelial progenitors. Overall, our optimised two-stage approach produces a fully re-populated, structurally organized and pre-vascularized oesophageal substitute, which could become an alternative to current oesophageal substitutes.

Mouse decellularised liver scaffold improves human embryonic and induced pluripotent stem cells differentiation into hepatocyte-like cells.

Liver transplantation is the definitive treatment of liver failure but donor organ shortage limits its availability. Stem cells are highly expandable and have the potential to differentiate into any specialist cell. Use of patient-derived induced Pluripotent Stem Cells (hiPSCs) has the additional advantage for organ regeneration therapies by removing the need for immunosuppression. We compared hepatocyte differentiation of human embryonic stem cells (hESCs) and hiPSCs in a mouse decellularised liver scaffold (3D) with standard in vitro protocol (2D). Mouse livers were decellularised preserving micro-architecture, blood vessel network and extracellular matrix. hESCs and hiPSCs were primed towards the definitive endoderm. Cells were then seeded either in 3D or 2D cultures and the hepatocyte differentiation was continued. Both hESCs and hiPSCs differentiated more efficiently in 3D than in 2D, with higher and earlier expression of mature hepatocyte marker albumin, lipid and glycogen synthesis associated with a decrease in expression of fetal hepatocyte marker alpha-fetoprotein. Thus we conclude that stem cell hepatocyte differentiation in 3D culture promotes faster cell maturation. This finding suggests that optimised 3D protocols could allow generation of mature liver cells not achieved so far in standard 2D conditions and lead to improvement in cell models of liver disease and regenerative medicine applications.