Pharmacokinetics of immunosuppressive agents during hemoperfusion in a sheep model.

Introduction: Hemoadsorption shows promising signals in organ preservation and post lung transplantation. However, its potential impact on the pharmacokinetics of immunosuppressant drugs (ID) is still unknown. Methods: In this interventional study, CytoSorb® hemoperfusion was tested in healthy sheep (n = 5) against a sham extracorporeal circuit (n = 3). Seven different ID (tacrolimus (TAC), cyclosporin A (CYA), mycophenolate mofetil (MMF), everolimus (EVER), basiliximab (BAS), methylprednisolone (MP) and prednisolone (PRED)) were administered in clinically relevant doses and combinations. Their levels were measured repeatedly in blood samples from the extracorporeal circulation over 6 h following administration. Population pharmacokinetic modeling analysis (NONMEM® 7.5) was performed. Results: Negligible clearance was observed for PRED and BAS. For all other substances, a saturable adsorption sub-model with linear decrease of the adsorption effect over the adsorbed amount best described the measured concentrations. The maximum absolute adsorbed amounts (95% CI) for TAC, CYA, MMF, EVER, and MP were 0.040 (0.028-0.053), 1.15 (0.39-1.91), 4.17 (2.00-6.35), 0.0163 (0.007-0.026), and 53.4 mg (20.9-85.9), respectively, indicating an adsorption of less than 5% of the daily administered dosages for all investigated substances. Discussion: In this large animal model, CytoSorb® hemoperfusion appears to have a limited effect on the clearance of tested ID.

Generation of the human erythroblast-derived iPSC line UBTi001-A.

We performed reprogramming of human erythroblasts derived from CD34+ hematopoietic stem / progenitor cells of a healthy donor. CD34+ cells were differentiated in-vitro into a pure population of CD36+ erythroblasts and nucleofected with four episomal plasmids expressing SOX2, OCT3/4, KLF4, LIN28, L-MYC and TP53-shRNA. The established iPSC line showed normal karyotype. Pluripotency was confirmed by expression of pluripotency markers and in-vitro differentiation into tissues of all three germ layers. The UBTi001-A iPSC line might provide an attractive source for developmental research on human hematopoiesis and erythropoiesis.

Membrane Properties of Human Induced Pluripotent Stem Cell-Derived Cultured Red Blood Cells.

Cultured red blood cells from human induced pluripotent stem cells (cRBC_iPSCs) are a promising source for future concepts in transfusion medicine. Before cRBC_iPSCs will have entrance into clinical or laboratory use, their functional properties and safety have to be carefully validated. Due to the limitations of established culture systems, such studies are still missing. Improved erythropoiesis in a recently established culture system, closer simulating the physiological niche, enabled us to conduct functional characterization of enucleated cRBC_iPSCs with a focus on membrane properties. Morphology and maturation stage of cRBC_iPSCs were closer to native reticulocytes (nRETs) than to native red blood cells (nRBCs). Whereas osmotic resistance of cRBC_iPSCs was similar to nRETs, their deformability was slightly impaired. Since no obvious alterations in membrane morphology, lipid composition, and major membrane associated protein patterns were observed, reduced deformability might be caused by a more primitive nature of cRBC_iPSCs comparable to human embryonic- or fetal liver erythropoiesis. Blood group phenotyping of cRBC_iPSCs further confirmed the potency of cRBC_iPSCs as a prospective device in pre-transfusional routine diagnostics. Therefore, RBC membrane analyses obtained in this study underscore the overall prospects of cRBC_iPSCs for their future application in the field of transfusion medicine.

Relaxin and Erythropoietin Significantly Reduce Uterine Tissue Damage during Experimental Ischemia-Reperfusion Injury.

Successful uterus transplantation, a potential treatment method for women suffering from absolute uterine infertility, is negatively affected by ischemia-reperfusion injury (IRI). The aim of this study is to investigate the protective effect of relaxin (RLX) or/and erythropoietin (EPO) on experimental uterus IRI. Eighty rats, randomly assigned into eight groups (n = 10/group), were pretreated with either saline, 5 µg/kg human relaxin-2, 4000 IU/kg recombinant human erythropoietin or their combination. Ischemia was achieved by clamping the aorta and ovarian arteries for 60 min, following 120 min of reperfusion and tissue sampling. For sham animals, clamping was omitted during surgery. There were no differences in tissue histological score, malondialdehyde (MDA) and superoxide dismutase (SOD) levels, myeloperoxidase (MPO) and TUNEL-positive cell count between all sham-operated rats. Pretreatment with RLX preserved normal tissue morphology, reduced MDA levels, MPO and TUNEL-positive cell count, preserved SOD activity and upregulated NICD and HES1 gene expression when compared to the control group. Pretreatment with EPO reduced MDA levels. In conclusion, pretreatment with RLX, EPO or a combination of both EPO and RLX significantly alleviates uterine tissue damage caused by IRI.

Enhanced Ex Vivo Generation of Erythroid Cells from Human Induced Pluripotent Stem Cells in a Simplified Cell Culture System with Low Cytokine Support.

Red blood cell (RBC) differentiation from human induced pluripotent stem cells (hiPSCs) offers great potential for developmental studies and innovative therapies. However, ex vivo erythropoiesis from hiPSCs is currently limited by low efficiency and unphysiological conditions of common culture systems. Especially, the absence of a physiological niche may impair cell growth and lineage-specific differentiation. We here describe a simplified, xeno- and feeder-free culture system for prolonged RBC generation that uses low numbers of supporting cytokines [stem cell factor (SCF), erythropoietin (EPO), and interleukin 3 (IL-3)] and is based on the intermediate development of a "hematopoietic cell forming complex (HCFC)." From this HCFC, CD43+ hematopoietic cells (purity >95%) were continuously released into the supernatant and could be collected repeatedly over a period of 6 weeks for further erythroid differentiation. The released cells were mainly CD34+/CD45+ progenitors with high erythroid colony-forming potential and CD36+ erythroid precursors. A total of 1.5 × 107 cells could be harvested from the supernatant of one six-well plate, showing 100- to 1000-fold amplification during subsequent homogeneous differentiation into GPA+ erythroid cells. Mean enucleation rates near 40% (up to 60%) further confirmed the potency of the system. These benefits may be explained by the generation of a niche within the HCFC that mimics the spatiotemporal signaling of the physiological microenvironment in which erythropoiesis occurs. Compared to other protocols, this method provides lower complexity, less cytokine and medium consumption, higher cellular output, and better enucleation. In addition, slight modifications in cytokine addition shift the system toward continuous generation of granulocytes and macrophages.