Hepatocyte differentiation from embryonic stem cells and umbilical cord blood cells

With the development of regeneration medicine, many researchers have attempted hepatic differentiation from nonhepatic-origin cell sources. The differentiation of embryonic stem (ES) cells into hepatocyte-like cells has been reported in several papers. Mouse ES cells have shown a potential to develop into hepatocyte-like cells in vitro on the basis of hepatic gene expression after adding several growth factors. We transplanted cultured embryoid body (EB) cells (male) into female mice. A liver specimen of the recipient was examined by immunohistochemical staining for albumin and fluorescence in situ hybridization for the Y chromosome after transplantation. Both Y chromosome- and albumin-positive cells were recognized in the recipient female liver, and were considered to be hepatocyte-like cells derived from ES cells containing the Y chromosome. Many groups, including ourselves, have studied hepatocyte-like cell differentiation from umbilical cord blood cells (UBCs). We cultured nucleated cells isolated from UBCs. Using immunostaining, ALB-positive and CK-19-positive cells were recognized in the culture. Dual staining of ALB and CK-19 demonstrated that ALB was coexpresed with CK-19, suggesting the existence of hepatic progenitors. In this review, we consider recent studies of the differentiation of hepatocytes from nonhepatic origins, especially ES cells and umbilical cord blood.     

Prediction of iopromide reduction rates during haemodialysis using an in vitro dialysis system.

BACKGROUND: In clinical studies, it has been difficult to evaluate the influence of haemodialysis (HD) parameters on HD clearance (CL(HD)) and reduction rate (RR) of non-ionic contrast medium during HD sessions. We therefore predicted clinical values of CL(HD) and RR of iopromide, a non-ionic contrast medium, from findings obtained from in vitro experiments, and confirmed that these predictive values were comparable with the actual values in clinical cases. METHODS: We developed a correlation equation for predicting CL(HD) on the basis of in vitro HD experiments by varying blood flow rates between 100 and 200 ml/min with a cuprammonium rayon dialyser (AM-SD-10H). Total body clearance of iopromide (CL(PT)) was estimated by the Cockroft-Gault equation. The volume of distribution (V(d)) was obtained from the reported value. By using the HD and three pharmacokinetic parameters (CL(HD), CL(PT) and V(d)), we predicted CL(HD) and RR for seven patients undergoing HD after the administration of iopromide. RESULTS: In the in vitro study, the mean values (+/-SD) of iopromide clearance at blood flow rates of 100, 150 and 200 ml/min were 45.35 (2.54), 53.88 (6.46) and 57.61 (4.72) ml/min, respectively. There were highly significant correlations between clearance and blood flow rate (r = 0.975). Although the predicted CL(HD) showed a tendency towards underestimation, a good correlation was found. Predicted RR values were similar to observed values except for one case. CONCLUSION: The in vitro model used in the present study provides pertinent information about CL(HD) and is helpful for predicting RR during HD in individual patients undergoing HD.     

Teratoma formation and hepatocyte differentiation in mouse liver transplanted with mouse embryonic stem cell-derived embryoid bodies

We previously reported that mouse embryonic stem (ES) cells are capable of differentiating into hepatocytes in cultured embryoid bodies (EBs) and that hepatocytes generate in the recipient liver injected with cultured day-9 EB cells via spleen without the formation of a teratoma. Because ES cells frequently form teratomas in recipient mice, we investigated incidence of teratoma formation when day-9 EBs derived from ES cells were transplanted directly into the subcapsule of mouse liver. In contrast to injection of day-9 EB cells through the portal vein via the spleen, direct subcapsular injection of cultured day-9 EB cells into liver, and even of cultured day-15 EBs, resulted in an high incidence of teratoma in the liver. In teratomas of livers injected directly with day-15 EBs, hepatocytes were detected singly and in clusters. These results imply that undifferentiated cells capable of developing into teratomas exist in cultured EBs, and even in cultured day-15 EBs containing differentiated hepatocytes.     

In Vivo Molecular Imaging Characterizes Pulmonary Gene Expression During Experimental Lung Transplantation

Experimental gene therapy is a promising strategy to prevent ischemia-reperfusion (I/R) injury and allograft rejection after lung transplantation, and methods will eventually be needed to characterize pulmonary transgene expression in vivo in humans. Therefore, we studied positron emission tomography (PET) as a means of performing in vivo molecular imaging in rodent models of lung transplantation. Rats were transfected endotracheally with adenovirus encoding a fusion gene of a mutant Herpes simplex virus-1 thymidine kinase and the green fluorescent protein gene (the former serving as an imaging reporter gene). Twenty-four hours after transfection, lungs were transplanted in groups representing normal transplantation, I/R injury and acute allograft rejection. Imaging was obtained either 24 h after transplantation to study reperfusion injury or 4 days after transplantation to study graft rejection. After imaging, lungs were excised and analyzed for thymidine kinase activity. Imaging detected transgene expression in transplanted lungs even in the presence of acute rejection or I/R injury. The PET imaging signal correlated with in vitro lung tissue assays of thymidine kinase activity (r(2) = 0.534). Thus, noninvasive molecular imaging with PET is a feasible, sensitive and quantitative method for characterizing pulmonary transgene expression in experimental lung transplantation.     

Bone Mineral Density Measurement at Both Spine and Hip for Diagnosing Osteoporosis in Japanese Patients

In Japan, spinal dual-energy X-ray absorptiometry (DXA) has been commonly performed for diagnosing osteoporosis but scanning the proximal femur is not done widely. The latest Japanese guidelines for prevention and treatment of osteoporosis, revised in 2006, recommend bone mineral density (BMD) measurement at both spine and hip for diagnosing osteoporosis, although there have been no reports that proved the necessity of those measurements. One thousand forty-one women and 485 men with clinical suspicion of osteoporosis were enrolled in this study, and DXA was performed at both spine and hip. The proportions of the patients who had inconsistency between diagnosis of osteoporosis from spinal DXA and that of hip were estimated. As a result, 22% of women and 15% of men had an inconsistency with the diagnosis of osteoporosis using DXA at each measurement site. There was inconsistency in diagnosing osteoporosis using DXA at the spine and proximal femur measurement sites. Because spine and femoral DXA measurements complement each other in the diagnosis of osteoporosis, BMD measurement at both spine and hip should be performed for all Japanese patients who are suspected osteoporosis, regardless of age and sex.     

Lack of correlation between results of ABO-incompatible living kidney transplantation and anti-ABO blood type antibody titers under our current immunosuppression

In this study, we examined the impact of preoperative anti-A/B antibody titers on the results of ABO-incompatible living kidney transplantation (LKT). In all, 167 recipients underwent ABO-incompatible LKT at our institution between 1989 and 2002. These patients were subdivided into those transplanted under cyclosporine with azathioprine or mizoribine (Group 1, n=78) and those transplanted under tacrolimus or mycophenolate mofetil (Group 2, n=89). Overall patient survival at 5 and 10 years was 93.8% and 88.0%, respectively. Overall graft survival at 5 and 10 years was 76.9% and 55.9%, respectively. Graft survival in the patients with anti-A/B IgG titers over 1:128 was significantly lower in group 1, whereas no significant correlation between the anti-A/B IgG titers and graft survival was found in group 2. In conclusion, no correlation between anti-A/B antibody titers and the results of ABO-incompatible LKT was seen after tacrolimus or mycophenolate mofetil application.     

Peritubular capillaritis in early renal allograft is associated with the development of chronic rejection and chronic allograft nephropathy

Abstract:  Peritubular capillaritis (PTCitis) has been recognized as one form of acute/active allograft rejection, and its relation to humoral immunity has been suggested. However, its mechanisms remain to be fully clarified, and there are no criteria for evaluating the extent of PTCitis in a biopsied allograft. In this study, we first evaluated the extent of PTCitis in early allografts in patients presenting with acute cellular rejection (ACR) and antibody-mediated rejection (AbAR). We also included patients who showed no evidence of ACR and/or AbAR. Next, we investigated whether or not PTCitis persisted and if peritubular capillary basement membrane (PTCBM) thickening was present in their follow-up biopsy specimens. We adopted the scoring system of PTCitis, which was presented at the Seventh Banff Conference on Allograft Pathology in 2003. In total, 53 patients were included in this study. At first biopsy, 17 showed ACR, eight showed AbAR, 16 showed mild PTCitis only, and 14 were without significant pathologic changes. The PTC score was the highest in the AbAR group, and in some patients the score gradually increased during the follow-up period. Similar changes were also observed in the group with mild PTCitis only. In late allografts, half of the patients with AbAR developed chronic rejection (CR), and the PTCBM score was the highest in that group. Surprisingly, CR was present in more than 30% of patients without ACR and/or AbAR but mild PTCitis only. In the control group, only a few showed CR and/or chronic allograft nephropathy (CAN). In conclusion, it became clear that we should carefully monitor for mild PTCitis in early allografts. In addition, our data also proved the usefulness of the PTC score and PTCBM score.     

Rectovaginal fistulography: a technique for the identification of recurrent elusive fistulas

Introduction and hypothesis  The purpose of this study is to review our experience with a technique for diagnosing small rectovaginal fistulas that occasionally permit passage of air or mucus. Methods  During an in-office visit suspicious areas of the vagina were probed with a cone-tip catheter and injected with a contrast dye to visualize the suspected fistula tract communicating to the rectum under fluoroscopic guidance. The fistulous tracts were further isolated using a flexi-tip glide wire. Results  Five out of nine patients were found to have fistulas not diagnosed by other means. Three patients had recurrent rectovaginal fistula after a vaginal delivery, one patient was identified with a high rectovaginal fistula due to diverticular disease, and one patient had a rectovaginal fistula due to prior hemorrhoidectomy. One patient had a negative test, and the fistula that was diagnosed intraoperatively was due to underlying Crohn’s disease. Conclusion  Direct fistulography is a useful technique to visualize otherwise elusive symptomatic rectovaginal fistula tracts.     

Nerve growth factor combined with vascular endothelial growth factor enhances regeneration of bladder acellular matrix graft in spinal cord injury-induced neurogenic rat bladder

OBJECTIVE

To determine the combined effects of nerve growth factor (NGF) and vascular endothelial growth factor (VEGF) on regeneration of the bladder acellular matrix graft (BAMG) in spinal cord injury (SCI)‐mediated neurogenic bladder in rats.

MATERIALS AND METHODS

In all, 40 female Sprague‐Dawley rats were used. At 8 weeks after spinalization surgery (neurogenic bladder), they were divided into five groups consisting of untreated controls and those whose bladders were injected with either no growth factor, NGF (2 µg/rat), VEGF (2 µg/rat) or both at partial BAMG replacement surgery. After 8 weeks, bladder function was assessed by urodynamic studies and the bladders were harvested for histological examination. Smooth muscle induction, collagen and nerve fibre regeneration were assessed immunohistochemically using antibodies to smooth muscle actin (α‐actin), Masson’s trichrome and protein gene product 9.5, respectively.

RESULTS

Bladder capacity and compliance were significantly increased in all BAMG groups 8 weeks after surgery compared with that before bladder replacement surgery. Bladder capacity and compliance were much higher in the VEGF and NGF combined group than in the control, or NGF and VEGF alone groups. There was no significant difference in the residual volume ratio among all groups.

CONCLUSIONS

This is the first report showing that NGF has a significant synergistic effect on the development, differentiation and functional restoration of the BAMG when administered with VEGF in neurogenic bladder. Our results indicate that NGF may be a useful cytokine for enhancing the regeneration of a functional bladder following acellular matrix grafting in a neurogenic rat model.     

Efficacy of dynamic CT perfusion imaging in conjunction with three dimensional CT angiography for the evaluation of acute ischemic stroke

BACKGROUND AND PURPOSE: Through the use of a high-speed spiral CT scanner (GEMedical HiSpeedZX/i), CT/P/A technique, where conventional CT, CT perfusion imaging (CTP) and CT angiography (CTA) are consecutively performed, can now be performed with an imaging time of 90 seconds and a total contrast medium volume of 100 ml. A prospective clinical study was performed to ascertain the effectiveness of CT/P/A in diagnosing acute ischemic strokes. METHODS: 29 consecutive patients of Teraoka Memorial Hospital suspected of suffering from the occlusion or constriction of cerebral arteries and who underwent CT/P/A within 3 hours from the onset served as subjects. The sensitivity, specificity, or Odds ratio of CTP and CTA in detecting lesions that caused cerebral infarction was calculated. RESULTS: CTP detected a hypoperfusion area with a sensitivity, specificity, and Odds ratio of 80%, 64%, and 7.2. The sensitivity in lobar infarcts, white matter infarcts, basal ganglia infarcts, and brainstem infarcts was 100%, 100%, 100%, 0% (p = 0.0022). The sensitivity and Odds ratio of CT/P/A in cerebral infarcts differed according to the diameter of the infarcts. That with infarcts of 10 mm or more was 91%, 20. That with infarcts smaller than 10 mm was 50%, 2. CTA detected arterial lesions that caused cerebral ischemic attack with a sensitivity of 94% and specificity of 90%. The examination time for CT/P/A was 18 minutes, total radiation time being 90 seconds. CONCLUSIONS: Although CT/P/A was ineffective for the diagnosis of brainstem infarcts and lesions smaller than 10 mm, CT/P/A was useful in detecting moderate-sized hypoperfusion areas and arterial lesions three-dimensionally before an infarct is completed.