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Successful islet transplantation (ITx) is not only dependent on the number of islets, but also their quality, including viability, metabolic activity, and function. Islet quality decreases during cultivation after the isolation procedure. To overcome this obstacle, we established the practice of islet and mesenchymal stem cells (MSCs) coculture. This coculture condition improved the ATP (adenosine-5'-triphosphate)/ADP (adenosine-5'-diphosphate) ratio and insulin secretory function in vitro. It is believed that the enhancement of islet quality in islet-MSCs cocultures may be caused by the secretion of active agents by MSCs. Herein we have shown that interleukin-6 (IL-6), vascular endothelial growth factor-A (VEGF-A), hepatocyte growth factor (HGF), and transforming growth factor-beta (TGF-beta) were significantly increased as measured by enzyme-linked immunosorbent assay (ELISA) in MSCs-cultured medium, factors that have been shown to improve the survival, function, and angiogenesis/revascularization of islets. These results indicated that the quality of human islets was enhanced by trophic molecules secreted by MSCs, which influence the intracellular islet ATP content and insulin secretory function.

Background There is an increasing need for suitable animal models for the study of the human immune system and disease. The purpose of this study was to develop a practical in vivo model of human immune cell repopulation using ex vivo expanded human fetal liver-derived CD34+ hematopoietic stem cells and subrenally coimplanted fetal liver/thymus tissues. Methods Freshly isolated fetal liver-derived CD34+ hematopoietic stem cells were frozen until injected and ex vivo expanded with various cytokines for 7 days. After fetal liver/thymus tissues were subrenally coimplanted into preirradiated Rag2−/−γc−/− mice, frozen and ex vivo expanded CD34+ cells were injected intravenously. The peripheral blood of the mice was monitored for the detection of human cell engraftment using flow cytometry. Then we confirmed human T-cell function by in vitro function assays. Results After fetal liver/thymus tissues were coimplanted into the irradiated Rag2−/−γc−/− mice, with frozen and ex vivo expanded CD34+ hematopoietic stem cells, human cell engraftments were determined using hCD45 and multilineage markers. The cultured cells with the cytokine combination of stem cell factor, thrombopoietin, Flk2/Flk3 ligand (FL), and interleukin-3 showed stable and long-term engraftment compared to other combinations. The ex vivo expanded human fetal liver-derived CD34+ hematopoietic stem cells, under our culture conditions, accomplished a large volume of expanded cells that were sustained, demonstrating self-renewal of the evaluated markers, which may have indicated long- term repopulation activity. Conclusion The results of this study demonstrated a practical mouse model of expanded human immune cells especially T cells in Rag2−/−γc−/− mice.

We investigated whether epigenetic modification agents can convert naïve T cells to regulatory T cells (T(regs)) which are responsible for limiting immune responses and maintaining self-tolerance. We treated splenic CD4(+)/CD25(-) naïve T cells from BALB/c mice with the DNA-methyltransferase inhibitor 5-aza-2'-deoxycytidine (5AzaD) or the histone protein deacetylase (HDAC) inhibitor Trichostatin A (TSA), and measured the effects on the expression of FOXP3, which encodes a transcription factor (FOXP3) that regulates T(reg) development. FOXP3 expression in naïve T cells was increased by 5AzaD or TSA treatment, administered 72 hours after T-cell receptor (TCR) stimulation with anti-CD3 plus anti-CD28. The T(regs) induced by 5AzaD or TSA expressed greater amounts of the FOXP3 protein than the control and the natural T(regs). The analysis of T(reg)-associated markers also showed T(reg) phenotypes (CD25(+)/CTLA4(+)/GITR(+)/CD127(-)). Finally, the induced T(reg) population also displayed T-cell suppression. These data suggested that epigenetic modification agents can induce FOXP3 expression, promoting the conversion of naïve T cells to T(regs).

Background. Due to ethical issues, in vivo studies of the human immune system have been difficult. Thus, small-animal xenotransplantation models have been employed, although they scarcely sustain a human immune response. In this study, we compared human cell repopulation tendencies and functionality in Rag2 -/- γc -/- mice following various ex vivo expanded human hematopoietic stem cells (HSCs). Methods. Human umbilical cord blood (UCB) CD34 + cells were cultured for 7 days with a cytokine combination of stem cell factor, Flk2/Flt3 ligand, and thrombopoietin, with absence or presence of rhIL-3, then transplanted into Rag2 -/- γc -/- mice. Reconstituted human lymphocytes were analyzed based on the expression of CD45 as well as CD3, CD19, and CD56 in peripheral blood (PB) until 16 weeks after transplantation. BrdU assay and functional analysis of reconstituted human lymphocytes used PHA- or rhIL-2-stimulated splenocytes and bone marrow cells from recipient mice. Results. The percentage of human CD45 dim cells, not CD45 bright cells, in PB of mice transplanted with cultured HSCs with rhIL-3 was much higher than in the group without rhIL-3 (approximately 2.5-fold at week 10 posttransplantation). The humanized mice showed systemic repopulation with a comprehensive array of human lymphohematopoietic cells, including T, B, natural killer (NK) cells, and even dendritic cells. However, the expression level was also dim. The number of CD3 + T cells and CD56 + NK cells was especially increased in the presence of rhIL-3. In addition, after in vitro restimulation proliferation assays and NK activity of interferon-y secretion showed greater effects in the presence of rhIL-3. Conclusion. These data suggested that the development of a diverse repopulation of human lymphocytes was possible in Rag2 -/- γc -/- mice after transplantation of cultured UCB CD34 + HSCs with interleukin-3.

Background Solid organ xenotransplantation is a potential solution to current organ shortages in allotransplantation. We performed four heart transplantations from alpha1, 3-galactosyltransferase gene-knockout (GT-KO) pigs to cynomolgus monkeys and monitored immunological parameters before and after transplantation. Methods After blood typing of the cynomolgus monkeys, we assessed the binding activity of immunoglobulin G (IgG) and IgM of monkey serum and serum toxicity toward porcine peripheral blood mononuclear cells (PBMCs) using flow cytometry. Immunosuppressive protocols consisted of anti-thymocyte globulin (25 mg/kg), rituximab (20 mg/kg), anti-CD154mAb (20 mg/kg), cobra venom factor (0.05 mg/kg), tacrolimus, and steroid. Cynomolgus monkeys with A or AB blood type with the lowest antibody binding and serum toxicity activity on porcine PBMCs were selected as recipients. Results Absolute numbers of CD3 + T cells, CD20 + B cells, and CD3 + CD95 + memory T cells in the peripheral blood were suppressed upto 24 days after transplantation. Interferon gamma production of T cells in response to porcine antigens were also significantly suppressed. Heart xenografts from GT-KO pigs survived for upto 24 days without pathologic evidence of rejection. Conclusion We successfully performed 4 heart xenotransplantations using GT-KO pigs. We overcame hyperacute rejection by using GT-KO pigs, and all of the heart xenografts from the GT-KO pigs survived between 11 and 24 days without pathologic evidence of rejection, disseminated intravascular coagulation, or consumptive coagulopathy; however, we need to optimize protocols for immune modulation and postoperative care to attain long-term survival of solid organ xenografts.