Takumi Fujimura

Acoustic radiation force impulse imaging for assessing graft fibrosis after pediatric living donor liver transplantation: a pilot study.

Graft fibrosis is a common finding during protocol biopsy examinations after pediatric liver transplantation. We evaluated the clinical utility of liver stiffness measurements by acoustic radiation force impulse (ARFI) imaging, a novel ultrasound‐based elastography method, for assessing graft fibrosis after pediatric living donor liver transplantation (LDLT). We performed 73 liver stiffness measurements by ARFI imaging in 65 pediatric LDLT recipients through the upper midline of the abdomen (midline value) and the right intercostal space (intercostal value) around the time of protocol biopsy examinations. Fifty‐nine of these liver stiffness measurements could be compared with histopathological findings. Graft fibrosis was assessed according to the degrees of portal and pericellular fibrosis. Significant fibrosis, which was defined as F2 or worse portal fibrosis and/or moderate or worse pericellular fibrosis, was observed in 14 examinations, which had significantly higher midline (P = 0.005) and intercostal values (P < 0.001) than the others. Liver stiffness measurements by ARFI imaging significantly increased with increases in the portal and pericellular fibrosis grades. For the diagnosis of significant fibrosis, the areas under the receiver operating characteristic curve (AUROCs) were 0.760 (P = 0.005) and 0.849 (P < 0.001) for the midline and intercostal values, respectively. The optimal cutoff values were 1.30 and 1.39 m/second for midline and intercostal values, respectively. Slight but significant elevations were noted in the results of biochemical liver tests: serum levels of γ‐glutamyltransferase showed the highest AUROC (0.809, P = 0.001) with an optimal cutoff value of 20 IU/L. In conclusion, liver stiffness measurements by ARFI imaging had good accuracy for diagnosing graft fibrosis after pediatric LDLT. The pericellular pattern of fibrosis was frequently observed after pediatric LDLT, and moderate pericellular fibrosis was detectable by ARFI imaging. Liver Transpl 19:1202–1213, 2013.

Diagnosing native liver fibrosis and esophageal varices using liver and spleen stiffness measurements in biliary atresia: a pilot study

BackgroundBiliary atresia commonly leads to liver fibrosis and cirrhotic complications, including esophageal varices.ObjectiveTo evaluate liver and spleen stiffness measurements using acoustic radiation force impulse (ARFI) imaging for diagnosing grade of liver fibrosis and predicting the presence of esophageal varices in patients treated for biliary atresia.Materials and methodsARFI imaging of the spleen and native liver was performed in 28 patients with biliary atresia. We studied the relation between ARFI imaging values and liver histology findings (n=22), upper gastrointestinal endoscopy findings (n=16) and several noninvasive test results. Diagnostic accuracy was assessed using receiver operating characteristic curve analyses.ResultsLiver stiffness measurements exhibited a significant difference among the different grades of liver fibrosis (P=0.009), and showed higher values in patients with high-risk esophageal varices than in the other patients (P=0.04). The areas under the receiver operating characteristic curves of liver stiffness measurements for liver fibrosis grades  ≥ F2, ≥F3 and = F4 were 0.83, 0.93 and 0.94, respectively. Patients with high-risk esophageal varices were preferentially diagnosed by the combined liver and spleen stiffness measurements (area under the curve, 0.92).ConclusionLiver and spleen stiffness measurements using ARFI imaging are potential noninvasive markers for liver fibrosis and esophageal varices in patients treated for biliary atresia.

Applications of Mesenchymal Stem Cells and Neural Crest Cells in Craniofacial Skeletal Research

Craniofacial skeletal tissues are composed of tooth and bone, together with nerves and blood vessels. This composite material is mainly derived from neural crest cells (NCCs). The neural crest is transient embryonic tissue present during neural tube formation whose cells have high potential for migration and differentiation. Thus, NCCs are promising candidates for craniofacial tissue regeneration; however, the clinical application of NCCs is hindered by their limited accessibility. In contrast, mesenchymal stem cells (MSCs) are easily accessible in adults, have similar potential for self-renewal, and can differentiate into skeletal tissues, including bones and cartilage. Therefore, MSCs may represent good sources of stem cells for clinical use. MSCs are classically identified under adherent culture conditions, leading to contamination with other cell lineages. Previous studies have identified mouse- and human-specific MSC subsets using cell surface markers. Additionally, some studies have shown that a subset of MSCs is closely related to neural crest derivatives and endothelial cells. These MSCs may be promising candidates for regeneration of craniofacial tissues from the perspective of developmental fate. Here, we review the fundamental biology of MSCs in craniofacial research.

LNGFR+THY-1+ human pluripotent stem cell-derived neural crest-like cells have the potential to develop into mesenchymal stem cells

Mesenchymal stem cells (MSCs) are defined as non-hematopoietic, plastic-adherent, self-renewing cells that are capable of tri-lineage differentiation into bone, cartilage or fat in vitro. Thus, MSCs are promising candidates for cell-based medicine. However, classifications of MSCs have been defined retrospectively; moreover, this conventional criterion may be inaccurate due to contamination with other hematopoietic lineage cells. Human MSCs can be enriched by selection for LNGFR and THY-1, and this population may be analogous to murine PDGFRα+Sca-1+ cells, which are developmentally derived from neural crest cells (NCCs). Murine NCCs were labeled by fluorescence, which provided definitive proof of neural crest lineage, however, technical considerations prevent the use of a similar approach to determine the origin of human LNGFR+THY-1+ MSCs. To further clarify the origin of human MSCs, human embryonic stem cells (ESCs) and human induced pluripotent stem cells (iPSCs) were used in this study. Under culture conditions required for the induction of neural crest cells, human ESCs and iPSCs-derived cells highly expressed LNGFR and THY-1. These LNGFR+THY-1+ neural crest-like cells, designated as LT-NCLCs, showed a strong potential to differentiate into both mesenchymal and neural crest lineages. LT-NCLCs proliferated to form colonies and actively migrated in response to serum concentration. Furthermore, we transplanted LT-NCLCs into chick embryos, and traced their potential for survival, migration and differentiation in the host environment. These results suggest that LNGFR+THY-1+ cells identified following NCLC induction from ESCs/iPSCs shared similar potentials with multipotent MSCs.

Fluorescence Visualization of the Enteric Nervous Network in a Chemically Induced Aganglionosis Model.

Gastrointestinal motility disorders, severe variants in particular, remain a therapeutic challenge in pediatric surgery. Absence of enteric ganglion cells that originate from neural crest cells is a major cause of dysmotility. However, the limitations of currently available animal models of dysmotility continue to impede the development of new therapeutics. Indeed, the short lifespan and/or poor penetrance of existing genetic models of dysmotility prohibit the functional evaluation of promising approaches, such as stem cell replacement strategy. Here, we induced an aganglionosis model using topical benzalkonium chloride in a P0-Cre/GFP transgenic mouse in which the neural crest lineage is labeled by green fluorescence. Pathological abnormalities and functional changes in the gastrointestinal tract were evaluated 2–8 weeks after chemical injury. Laparotomy combined with fluorescence microscopy allowed direct visualization of the enteric neural network in vivo. Immunohistochemical evaluation further confirmed the irreversible disappearance of ganglion cells, glial cells, and interstitial cell of Cajal. Remaining stool weight and bead expulsion time in particular supported the pathophysiological relevance of this chemically-induced model of aganglionosis. Interestingly, we show that chemical ablation of enteric ganglion cells is associated with a long lifespan. By combining genetic labeling of neural crest derivatives and chemical ablation of enteric ganglion cells, we developed a newly customized model of aganglionosis. Our results indicate that this aganglionosis model exhibits decreased gastrointestinal motility and shows sufficient survival for functional evaluation. This model may prove useful for the development of future therapies against motility disorders.

Metastasectomy of Hepatoblastoma Utilizing a Novel Overlay Fluorescence Imaging System

BACKGROUND The curability of hepatoblastoma (HB) largely depends on the achievement of radical surgical resection, even for metastatic tumors. However, the extension of the metastatic tumor when viewed through an endoscope with the conventional white-light mode is often unclear. Advancements in imaging technology utilizing indocyanine green (ICG) have facilitated precise resection of metastatic HBs, owing to the longer retention of ICG in such lesions than in other normal tissues. CASE We utilized an endoscope loaded with the PINPOINT system (NOVADAQ Technologies, Inc., Ontario, Canada), which allows for real-time overlay visualization with the same focal range between the white-light mode and near-infrared mode. Metastatic HBs that have taken up ICG are visualized as an area of green color superimposed on a high-definition white-light image. A 19-year-old female who underwent liver transplantation for an unresectable HB 2 years earlier was noted to have metastases on the diaphragm and the pleura. Preoperative magnetic resonance imaging showed metastatic HBs on the right pleura extending from the ribs and the diaphragm. The margin of the metastatic tumor was more sharply demarcated by the PINPOINT system than that detected in the normal white-light mode. The tumor was successfully resected en bloc with real-time guidance utilizing the overlay image. The alphafetoprotein levels were normalized and have remained within normal limits in the 12 months since the operation. CONCLUSION Novel overlay imaging technology with ICG makes it possible to achieve real-time precise resection of metastatic HBs.

PS5.87LNGFR (+) THY-1 (+) multipotent stem cells derived from human induced pluripotent stem cells

Sox2 is known to play a major role in maintaining neural stem cells in a multipotent state, functioning both as transcriptional activator and repressor. We are interested in understanding how Sox2 can switch between these two functions and, in particular, we want to determine whether this activity change is linked to SUMOylation. We have compared the transcriptional activation ability of wildtype (wt) Sox2 to the activity of a mutant Sox2 construct which should not be SUMOylated (K247R) and to Sox2-SUMO1/2/3 fusion constructs. The activity of wt Sox2 resulted comparable to the one of the K247R, while all the fusion constructs showed reduced activation ability. These results suggest that SUMOylation of Sox2 could cause a loss of its transcriptional activator activity or, potentially, that it causes a switch from activator to repressor function. However, does Sox2 SUMOylation affect Sox2 transcriptional activity in hNSC? In order to answer this question, we performed RNA sequencing on hNSC transfected with wt Sox2, K247R or K247R-SUMO2 fusion construct. We are now analysing the results of this experiment and comparing them with publicly available data from published literature. Only a very small amount of the total Sox2 present within a single cell is expected to be SUMOylated. For this reason, showing that Sox2 is indeed SUMOylated in vivo is a very challenging task. In order achieve this, we are testing different experimental approaches, including subcellular fractionation, protein pull-down followed by western-blot and mass spectrometry. It is known that Sox2 regulates the multipotency of NSC and that cells expressing high levels of Sox2 progress slowly through cell cycle. In order to determine whether SUMOylation plays a role in this regulation, we are performing cell cycle analysis and proliferation assays on hNSC transiently transfected with either Sox2, K247R or K247R-SUMO2.

Technical feasibility of visualizing myenteric plexus using confocal laser endomicroscopy

In preceding studies, we identified that the myenteric plexus (MP) could be visualized with confocal laser endomicroscopy (CLE) by applying neural fluorescent probes lacking clinical safety profiling data from the submucosal side. In this study, we evaluated the technical feasibility of MP visualization using probe‐based CLE (pCLE) from the serosal side with cresyl violet (CV), which has been used clinically for chromoendoscopy.

Dialysis membrane-enforced microelectrode array measurement of diverse gut electrical activity

A variety of electrical activities occur depending on the functional state in each section of the gut, but the application of microelectrode array (MEA) is rather limited. We thus developed a dialysis membranes-enforced technique to investigate diverse and complex spatio-temporal electrical activity in the gut. Muscle sheets isolated from the gastrointestinal (GI) tract of mice along with a piece of dialysis membrane were woven over and under the strings to fix them to the anchor rig, and mounted on an 8×8 MEA (inter-electrode distance=150µm). Small molecules (molecular weight <12,000) were exchanged through the membrane, maintaining a physiological environment. Low impedance MEA was used to measure electrical signals in a wide frequency range. We demonstrated the following examples: 1) pacemaker activity-like potentials accompanied by bursting spike-like potentials in the ileum; 2) electrotonic potentials reflecting local neurotransmission in the ileum; 3) myoelectric complex-like potentials consisting of slow and rapid oscillations accompanied by spike potentials in the colon. Despite their limited spatial resolution, these recordings detected transient electric activities that optical probes followed with difficulty. In Addition, propagation of pacemaker-like potential was visualized in the stomach and ileum. These results indicate that the dialysis membrane-enforced technique largely extends the application of MEA, probably due to stabilisation of the access resistance between each sensing electrode and a reference electrode and improvement of electric separation between sensing electrodes. We anticipate that this technique will be utilized to characterise spatio-temporal electrical activities in the gut in health and disease.

Application of nuclear medicine to achieve less invasive surgery for malignant solid tumors in children

The use of nuclear medicine for the management of malignant tumor, such as radioguided surgery and sentinel lymph node biopsy (SLNB), has been widely accepted in the adult practice. However, there are very few studies to apply those techniques for pediatric diseases. The aim of this study was to investigate the feasibility of application of nuclear medicine in surgery for neuroblastoma (NB) or rhabdomyosarcoma (RMS) in children.