HMG-CoA reductase inhibitor augments the serum total cholesterol-lowering effect of human adipose tissue-derived multilineage progenitor cells in hyperlipidemic homozygous Watanabe rabbits.

Familial hypercholesterolemia (FH) is an autosomal codominant disease characterized by high concentrations of proatherogenic lipoproteins secondary to deficiency in low-density lipoprotein (LDL) receptor. We reported recently the use of in situ stem cell therapy of human adipose tissue-derived multilineage progenitor cells (hADMPCs) in lowering serum total cholesterol in the homozygous Watanabe heritable hyperlipidemic (WHHL) rabbits, an animal model of homozygous FH. Here we demonstrate that pravastatin, an HMG-CoA reductase inhibitor, augmented the cholesterol-lowering effect of transplanted hADMPCs and enhanced LDL clearance in homozygous WHHL rabbit. The results suggest the potential beneficial effects of in situ stem cell therapy in concert with appropriately selected pharmaceutical agents, in regenerative medicine.

Transplantation of human adipose tissue-derived multilineage progenitor cells reduces serum cholesterol in hyperlipidemic Watanabe rabbits.

Familial hypercholesterolemia (FH) is an autosomal codominant disease characterized by high concentrations of proatherogenic lipoproteins and premature atherosclerosis secondary to low-density lipoprotein (LDL) receptor deficiency. We examined a novel cell therapy strategy for the treatment of FH in the Watanabe heritable hyperlipidemic (WHHL) rabbit, an animal model for homozygous FH. We delivered human adipose tissue-derived multilineage progenitor cells (hADMPCs) via portal vein and followed by immunosuppressive regimen to avoid xenogenic rejection. Transplantation of hADMPCs resulted in significant reductions in total cholesterol, and the reductions were observed within 4 weeks and maintained for 12 weeks. (125)I-LDL turnover study showed that the rate of LDL clearance was significantly higher in the WHHL rabbits with transplanted hADMPCs than those without transplanted. After transplantation hADMPCs were localized in the portal triad, subsequently integrated into the hepatic parenchyma. The integrated cells expressed human albumin, human alpha-1-antitrypsin, human Factor IX, human LDL receptors, and human bile salt export pump, indicating that the transplanted hADMPCs resided, survived, and showed hepatocytic differentiation in vivo and lowered serum cholesterol in the WHHL rabbits. These results suggested that hADMPC transplantation could correct the metabolic defects and be a novel therapy for inherited liver diseases.

Properties of hepatocyte-like cell clusters from human adipose tissue-derived mesenchymal stem cells.

There are only a few reports that describe the hepatocytic differentiation potential of human adipose tissue-derived mesenchymal stem cells (hADMSCs) and no reports that describe the in vivo functions of hepatocyte-like cells differentiated from somatic stem cells including hADMSCs. In this study, we established a new method for generation of functional hepatocyte-like cell clusters using floating culture method and induced functional hepatocyte-like cell clusters, which functioned effectively not only in vitro but also in vivo. The generated hepatocyte-like cell clusters were characterized by gene expression analysis, functional assays, and transplantation into non-obese diabetic severe combined immunodeficiency (NOD-SCID) mouse with chronic liver injury. The generated hepatocyte-like cell clusters expressed various genes normally found on mature hepatocytes. The cell clusters exhibited functional characteristics of hepatocytes: they expressed albumin, secreted urea, had cytochrome P450 activity, could take up low-density lipoprotein, and stored glycogen. Transplantation of these cell clusters into NOD-SCID mouse with chronic liver injury resulted in a significant improvement of serum albumin and total bilirubin levels. In summary, we established a new protocol for efficient induction of hADMSCs into functional hepatocyte-like cell clusters.

Transdifferentiation of human adipose tissue-derived stromal cells into insulin-producing clusters.

Type 1 diabetes mellitus is caused by autoimmune destruction of insulin-producing beta cells. The major obstacle to transplantation of insulin-producing cells to cure the disease is the limited source of these cells. To overcome this problem, we describe here a multistep protocol for generation of insulin-producing islet-like clusters from human adipose tissue-derived stromal cells (ADSCs). Analysis using reverse transcription polymerase chain reaction detected enhanced expression of various pancreatic genes during the differentiation of ADSCs. Immunofluorescence analysis revealed functional similarities between cells derived from ADSCs and pancreatic islet cells, i.e., the presence of insulin- and C-peptide-coexpressing cells in the clusters and glucagon expression on the cell surface. The glucose challenge tests revealed the production of insulin, and such production was regulated via physiological signaling pathways. Our insulin-producing cells derived from ADSCs could be potentially used for cell therapy of type 1 diabetes mellitus.

Creation of a rich subcutaneous vascular network with implanted adipose tissue-derived stromal cells and adipose tissue enhances subcutaneous grafting of islets in diabetic mice.

Subcutaneous tissue was proposed as an optimal transplant site for islets in treatment for type I diabetes mellitus. However, vascular networks in subcutaneous tissue are too poor in their natural state to allow survive and function of the transplanted graft. This study examined whether subcutaneous implantation of adipose tissue-derived stromal cells (ADSCs) combined with minced adipose tissue could form vascular-rich beds suitable to support islet transplantation. ADSCs were isolated from male C57BL/6J mouse inguinal subcutaneous adipose tissue. ADSCs and minced adipose tissue were implanted syngeneically in subcutaneous tissue of the back of recipient mice. Four weeks later, vascularization in the implanted subcutaneous tissue was evaluated, and islets were transplanted onto the vascularized pockets. Mice that received ADSCs mixed with minced adipose tissue showed a richly vascularized pocket of tissue with significantly higher capillary density than in mice implanted with either ADSCs or minced adipose tissue only. All recipient mice of the combination ADSCs and minced adipose tissue group showed correction in blood glucose level within a week after islet transplantation and maintained normoglycemia for over 8 weeks. These mice became hyperglycemic again after removal of the subcutaneous grafts. This novel method will expand the indications for islet transplant therapy and potential clinical application of cell-based therapy.

Localized giant inflammatory polyposis of the ileocecum associated with Crohn's disease: report of a case.

Although inflammatory polyposis is one of the common complications in patients with inflammatory bowel disease, it is rare that each poly grows up to more than 1.5 cm. We describe a case of localized giant inflammatory polyposis of the ileocecum associated with Crohn's disease. A 40-year-old man who had been followed for 28 years because of Crohn's disease was hospitalized for right lower abdominal pain after meals. Barium enema and colonoscopy showed numerous worm-like polyps in the ascending colon which grew up to the hepatic flexure of the colon from the ileocecum, causing an obstruction of the ileocecal orifice. Since histology of a biopsy specimen taken from the giant polyps showed no dysplasia, he was diagnosed with ileus due to the localized giant inflammatory polyposis. A laparoscopically assisted ileocecal resection was performed. The resected specimen showed that the giant polyps grew up into the ileocecum. Histological examination revealed inflammatory polyposis without neoplasm. Generally, conservative treatment is indicated for localized giant inflammatory polyposis because this lesion is regarded as benign. However, occasionally serious complications arise, requiring surgical treatment.

Graft Duodenal Perforation due to Internal Hernia after Simultaneous Pancreas-Kidney Transplantation: Report of a Case.

Although complications including graft thrombosis, graft pancreatitis, and rejection have been well documented after pancreas transplantation, the occurrence of graft duodenal perforation is uncommon. In this article, we report a case of graft duodenal perforation due to internal hernia after simultaneous pancreas-kidney transplantation (SPK). A patient with type I diabetes mellitus and diabetic nephropathy had undergone SPK from a cadaveric donor. One year later, she was admitted to our hospital for severe lower abdominal pain with preshock status. She was immediately examined by abdominal computed tomography and both peripancreas graft fluid accumulation and severe dilatation of the ileum were detected. On emergency operation, two punched holes located at the graft duodenal side near the suture line and an obstruction of herniated bowel behind the graft pancreas were detected. These holes were repaired and the internal hernia was reduced. However, a control of the intraabdominal infection was very difficult despite intensive treatment with antibiotics and additional abdominal drainage. Finally, a graft pancreatectomy was unavoidably required. When complications, including symptomatic intraabdominal infection, require re-laparotomy after pancreas transplantation, the therapeutic focus should be switched from salvaging the graft to the preservation of life.

Development of beta-cells in the native pancreas after pancreas allo-transplantation in the Spontaneously Diabetic Torii rat.

BACKGROUND: We previously demonstrated the development of beta-cells in the native pancreas after syngeneic pancreas transplantation (PTx) in a model of type 2 diabetes, namely the Spontaneously Diabetic Torii (SDT; RT1 a) rat. In this study, we evaluated the effect of fully allogeneic PTx (allo-PTx) under immunosuppression on the native pancreases in the recipients. MATERIALS AND METHODS: Diabetic 25-week-old SDT rats were divided into two groups: untreated controls and PTx-treated recipients. Dark Agouti (RT1 a) pancreases were then transplanted into the SDT rats. FK506 was administered daily postoperatively. Each group was examined for 15 weeks. RESULTS: Control SDT rats showed a disappearance of the pancreatic and duodenal homeobox-1 (PDX-1) expression of the pancreases with the development of diabetes. In addition, the islets were gradually replaced by fibrosis, thus resulting in a marked decrease in the beta-cell mass at 40 weeks of age. On the other hand, in PTx recipients, islet-like cell clusters were found in the native pancreases. The beta-cell mass significantly increased in the native pancreases in the recipients at 10 and 15 weeks posttransplantation in comparison to the age-matched controls. Moreover, we observed the re-expression of PDX-1 in the islet-like cell clusters. Interestingly, insulin and glucagon double-positive stained cells in the mesenchyme and insulin single-positive cells in the ductal epithelium were also observed. CONCLUSIONS: Our results indicated that the benefits of avoiding glucose toxicity by allo-PTx under immunosuppression could therefore induce the PDX-1 expression in the native pancreases, thus potentially resulting in the development of beta-cells in type 2 diabetic recipients.

Pancreas transplantation using type I and type II spontaneously diabetic rats--our experimental experience.

Pancreas transplantation (PTx) is the only therapy that can cure type 1 diabetes mellitus. With the recent advance of surgical procedures and immunosuppression, the outcome of PTx has become better than it used to be before, but some problems still remain. It is rather difficult to induce tolerance and to reverse rejection once it occurred because pancreas graft itself has a strong immunogenicity. Another important issue is regarding the recurrence of autoimmune disease in the pancreatic graft, therefore, some animal models are necessary to delineate and regulate those immune responses specific for PTx. Recently, PTx is also clinically applicable for type 2 diabetic patients with end-stage renal disease. It has been shown that insulin resistance was improved by PTx in type 2 diabetic recipients. In the current study, we have introduced some useful type 1 and type 2 diabetic models mainly based on our experimental experiences.

Effect of tandem forms of DAF(CD55) on complement-mediated xenogeneic cell lysis.

BACKGROUND: It is difficult to produce a transgenic animal with high expression of decay-accelerating factor (CD55: DAF) or other molecules. The purpose of this study was to assess the effect of tandem forms of DAF on a xenogeneic cell membrane against human complement. METHODS: cDNAs of the delta-Short Consensus Repeat (SCR) 1-DAF, the double-DAF, the triple-DAF, and the tetra-DAF with a FLAG-tag were established. Chinese hamster ovary (CHO) cell lines and a pig endothelial cell (PEC) line expressing these molecules were established. The amelioration of complement-mediated lysis by the transfectant molecules on these cells was examined. The CHO cell transfectants were also incubated with normal human serum, and the amount of C3 deposited was determined by FACS analysis. RESULTS: Stable CHO cells and PEC transfectants, in which each molecule was clearly expressed, and Western blots showed that each band corresponded to the expected molecular weight. The extent of amelioration of complement-mediated lysis by these four molecules was then examined. A clear tendency was found, as follows: The higher the tandem number of DAF, the greater was the effect on cytotoxicity. Additional experiments focusing on triple-DAF and tetra-DAF did not indicate any significant difference in complement-mediated lysis. Consistent with the complement-regulatory ability, the inhibitory effect of the deposition of C3 fragments by these molecules was closely related to the degree of amelioration. CONCLUSION: These data indicate that tandem DAF, especially a triple-DAF, is a very effective form for protecting against complement activation.

A study of the xenoantigenicity of neonatal porcine islet-like cell clusters (NPCC) and the efficiency of adenovirus-mediated DAF (CD55) expression.

BACKGROUND: The pig pancreas is considered to be the most suitable source of islets for xenotransplantation in patients with type I diabetes. The objective of this study was to assess the antigenicity of neonatal porcine islet-like cell clusters (NPCC), including the Galalpha1-3Galbeta1-4GlcNAc-R (alpha-Gal) and Hanganutziu-Deicher (H-D) antigens, and the pathway involved in human complement activation. The efficiency of expression of human decay-accelerating factor (DAF: CD55) on NPCC by adenoviral transduction was also examined, and the functional capacity of DAF was also estimated. METHODS: The deposition of human natural antibodies, immunoglobulin (Ig)G and IgM, and the expression of alpha-Gal and H-D antigens on NPCC were investigated by FACS analysis. The downregulation in the antigenicity to human natural antibodies, including the alpha-Gal and H-D antigens on NPCC by treatment with tunicamycin, PDMP and neuraminidase were also examined. In addition, complement-mediated islet lysis was examined using factor D-deficient and C1-deficient sera. An adenovirus encoding DAF under the control of the cytomegalovirus promoter, Ad.pCMV-DAF, was then constructed, and used for transducing NPCC. The amelioration of complement-dependent cytotoxicity of the NPCC by the transduced DAF was assessed as an in vitro hyperacute rejection model of a pig to human xenograft. RESULTS: The NPCC clearly expressed the alpha-Gal epitope, and the human natural antibodies, IgG and IgM, and the anti-H-D antibody also reacted with the NPCC. Treatment of NPCC with tunicamycin led to a drastic reduction in the extent of deposition of IgG, indicating the importance of N-linked sugars on the islets, presumably related to alpha-Gal expression on N-linked sugars. Neuraminidase treatment indicated the presence of, not only the H-D antigen, but also other sialic acid antigens which reacted with the human natural antibody, especially IgG. The complement deposition of factor B on NPCC was clear, and the alternative pathway-mediated NPCC killing accounted for approximately 30% of that by the total complement pathway. On the other hand, approximately 90% of the NPCC could be transduced to express DAF by the adenovector, Ad.pCMV-DAF. The expressed DAF showed an approximately 50-62% suppression in complement-dependent NPCC lysis. CONCLUSION: The origin of the antigenicity of NPCC is mainly N-linked sugars including alpha-Gal and sialic acid antigens, and NPCC expressed the transduced molecule in high efficiency by the adenovector.