Multilineage-differentiating stress-enduring (Muse) cells are distinct stem cells in mesenchymal cell populations with the capacity to self-renew, to differentiate into cells representative of all three germ layers from a single cell, and to repair damaged tissues by spontaneous differentiation into tissue-specific cells without forming teratomas. We describe step-by-step procedures for isolating and evaluating these cells. Muse cells are also a practical cell source for human induced pluripotent stem (iPS) cells with markedly high generation efficiency. They can be collected as cells that are double positive for stage-specific embryonic antigen-3 (SSEA-3) and CD105 from commercially available mesenchymal cells, such as adult human bone marrow stromal cells and dermal fibroblasts, or from fresh adult human bone marrow samples. Under both spontaneous and induced differentiation conditions, they show triploblastic differentiation. It takes 4-6 h to collect and 2 weeks to confirm the differentiation and self-renewal capacity of Muse cells.

Lecithin:cholesterol acyltransferase (LCAT) and cholesteryl ester transfer protein (CETP) are responsible for the esterification of cell-derived cholesterol and for the transfer of newly synthesized cholesteryl esters (CE) from HDL to apoB-containing lipoproteins in human plasma. LCAT and CETP are also crucial factors in HDL remodeling, a process by which HDL particles with a high capacity for cell cholesterol uptake are generated in plasma. In the present study, cholesterol esterification and transfer were evaluated in 60 patients with isolated hypercholesterolemia (HC, n = 20) and isolated (HTG, n = 20) or mixed hypertriglyceridemia (MHTG, n = 20) and in 20 normolipidemic healthy individuals (NL). Cholesterol esterification rate (CER) and net CE transfer rate (CETR) were measured in whole plasma. LCAT and CETP concentrations were determined by specific immunoassays. HDL remodeling was analyzed by monitoring changes in HDL particle size distribution during incubation of whole plasma at 37 degrees C. Mean CER and CETR were 48% and 73% higher, respectively, in hypertriglyceridemic (HTG + MHTG) versus normotriglyceridemic individuals. HDL remodeling was also significantly accelerated in plasma from hypertriglyceridemic patients. Strong positive correlations were found in the total sample between plasma and VLDL triglyceride levels and CER (r = .722 and r = .642, respectively), CETR (r = .510 and r = .491, respectively), and HDL remodeling (r = .625 and r = .620, respectively). No differences in plasma LCAT and CETP concentrations were found among the various groups except for a tendency toward higher CETP levels in hypercholesterolemic patients (+51% in MHTG and +20% in HC) versus control subjects (NL). By stepwise regression analysis, VLDL triglyceride level was the sole significant predictor of CER and CETR and contributed significantly together with baseline HDL particle distribution to HDL remodeling. These results indicate that plasma triglyceride level is a major factor in the regulation of cholesterol esterification/transfer and HDL remodeling in human plasma, whereas LCAT/CETP concentrations play a minor role in the modulation of reverse cholesterol transport.

Heterologous immunologic memory has been considered a potent barrier to tolerance induction in primates. Induction of such tolerance for a previously transplanted organ may be more difficult, because specific memory cells can be induced and activated by a transplanted organ. In the current study, we attempted to induce tolerance to a previously transplanted kidney allograft in nonhuman primates. The conditioning regimen consisted of low dose total body irradiation, thymic irradiation, antithymocyte globulin, and anti-CD154 antibody followed by a brief course of a calcineurin inhibitor. This regimen had been shown to induce mixed chimerism and allograft tolerance when kidney transplantation (KTx) and donor bone marrow transplantation (DBMT) were simultaneously performed. However, the same regimen failed to induce mixed chimerism when delayed DBMT was performed after KTx. We found that significant levels of memory T cells remained after conditioning, despite effective depletion of naïve T cells. By adding humanized anti-CD8 monoclonal antibody (cM-T807), CD8 memory T cells were effectively depleted and these recipients successfully achieved mixed chimerism and tolerance. The current studies provide 'proof of principle' that the mixed chimerism approach can induce renal allograft tolerance, even late after organ transplantation if memory T-cell function is adequately controlled.

Transplant tolerance, defined as indefinite allograft survival without immunosuppression, has been regularly achieved in laboratory mice but not in nonhuman primates or humans. In contrast to laboratory mice, primates regularly have high frequencies of alloreactive memory T cells (TMEMs) before transplantation. These TMEMs are poorly sensitive to conventional immunosuppression and costimulation blockade, and the presence of donor-reactive TMEMs in primates may account for their resistance to transplant tolerance protocols that have proven consistently effective in mice. We measured the frequencies of anti-donor TMEMs before and after transplantation in a series of rejecting and tolerant monkeys that underwent nonmyeloablative conditioning, short-term immunosuppression, and combined allogeneic kidney/cell transplantation. Transplants were acutely rejected in all the monkeys with high numbers of donor-specific TMEMs before transplantation. In contrast, long-term survival was observed in the recipients harboring lower frequencies of anti-donor TMEMs before transplantation. Similar amounts of TMEM homeostatic expansion were recorded in all transplanted monkeys upon hematopoietic reconstitution; however, only the tolerant monkeys had no expansion or activation of donor-reactive TMEMs after transplantation. These results indicate that the presence of high frequencies of host donor-reactive TMEMs before transplantation impairs tolerance induction to kidney allografts in this nonhuman primate model. Indeed, recipients harboring a low anamnestic reactivity to their donor before transplantation were successfully rendered tolerant via infusion of donor cells and short-term immunosuppression. This suggests that selection of allogeneic donors with low memory responses in recipients may be essential to successful transplant tolerance induction in patients.