We performed an intravenous cyclosporine (CsA) drip infusion method for 3 hours and C3 monitoring in pediatric hematopoietic stem cell transplantation and examined the internal change and monitoring method of CsA. A total of six cases comprised five cord blood transplantations and one related allogeneic bone marrow transplantation. We started CsA 1.5 mg/kg at the day before transplantation by intravenous drip infusion (twice a day) for three hours. We controlled the dose so that the optimal peak value of C3 reached 800-1000 ng/ml. We recognized the C3 peak occurred three hours after initiation of infusion, and the blood CsA concentration was restored to the baseline value (C0) to (C12) 12 hours after that. We found a strong correlation between the C3 value (r = 0.90), and AUC(0-12). Two patients had grade II acute graft-versus-host disease (GVHD), but one needed no treatment, and the other recovered with short-term dosage of prednisolone. Apart from these instances, no serious complication occurred. In pediatric hematopoietic stem cell transplantation, it seems that regulation of the appropriate blood CsA concentration is enabled by using C3 monitoring at around 3 hours after commencing the intravenous drip infusion method for CsA.

TRALI is a rare but serious complication associated with transfusion, and known to occur following infusion of all types of plasma-containing blood products. However, only one adult case of TRALI after allogenic marrow graft has been reported. In this study, we present a pediatric case possibly associated with allogenic marrow infusion. A 10-yr-old girl was referred to our hospital for the treatment of acute myeloid leukemia. She underwent allogenic bone marrow transplantation from her HLA-2-loci-mismatched mother. During conditioning, she suffered from bacterial sepsis, but it had improved with antibiotics until day 0 of transplantation. Two h after starting the marrow infusion, she developed severe hypoxia. We discontinued the infusion and started steroids, which improved her respiratory condition. However, she developed respiratory failure again after resuming infusion of the graft. Despite intensive care with mechanical ventilation, the patient died of endotoxin shock five days after transplantation. Although we could not identify the antibody which might have been involved in the respiratory distress, the clear temporal relationship between marrow infusion and respiratory distress suggested that similar acute lung injury to TRALI might have occurred following allogenic marrow infusion in the present case.

The temporal pattern of embryonic, fetal, and adult globin expression in the alpha (zeta --> alpha) and beta (epsilon --> gamma and gamma --> beta) clusters were quantitatively analyzed at the transcriptional and translational levels in erythrocytes induced from primate embryonic stem cells in vitro. When vascular endothelial growth factor receptor-2(high) CD34(+) cells were harvested and reseeded onto OP9 stromal cells, two-wave erythropoiesis occurred sequentially. Immunostaining and real-time reverse transcription-polymerase chain reaction analyses of floating mature erythrocytes revealed that globin switches occurred in parallel with the erythropoietic transition. Colony-forming assays showed replacement of primitive clonogenic progenitor cells with definitive cells during culturing. A decline in embryonic zeta- and epsilon-globin expression at the translational level occurred in individual definitive erythroid progenitors. Expression of beta-globin in individual definitive erythroid progenitors was upregulated in the presence of OP9 stromal cells. Thus, this system reproduces early hematopoietic development in vitro and can serve as a model for analyzing the mechanisms of the globin switch in humans.

The mechanism of commencement of hematopoiesis in blood islands of the yolk sac and the aorta-gonad-mesonephros (AGM) region during primate embryogenesis remains elusive. We previously showed the development of both primitive and definitive hematopoiesis when cynomolgus monkey embryonic stem cells were co-cultured with OP9 stromal cells. In this study, we examined the hematopoietic potential of endothelial cells developing in our coculture system and demonstrated that VE-cadherin+CD45− endothelial cells derived from embryonic stem cells were able to generate primitive and definitive hematopoietic cells sequentially, as revealed by immunostaining of floating erythrocytes and colony-forming assay in cultures. All floating erythrocytes which emerged initially expressed ε- and ζ-globins, while β-globin expression was hardly detected. The percentage of floating erythrocytes positive for β-globin gradually increased thereafter, and almost all erythrocytes were positive by day 40. Meanwhile, expression of ε- and ζ-globins declined gradually. Clonal analysis revealed that single bipotential cells for hematopoietic and endothelial lineages were included in this endothelial cell population. Hemogenic activity of endothelial cells was observed exclusively in the α4-integrin+ subpopulation. RT-PCR data showed that Runx1, a transcriptional factor associated with definitive hematopoiesis, was expressed in the hemogenic α4-integrin+ subpopulation, but not the non-hemogenic α4-integrin− subpopulation among embryonic stem cell-derived endothelial cells. The kinetics of this hemogenic subpopulation was similar to that of hemogenic endothelial cells previously reported in the yolk sac and the AGM region in vivo, in that they emerged only for a limited time. On the other hand, VE-cadherin−CD45−α4-integrin+ cells gave rise to more primitive erythrocytes than VE-cadherin+CD45−α4-integrin+ cells, but hardly contributed to definitive hematopoiesis. These results indicate that VE-cadherin+CD45−α4-integrin+ endothelial cells generate primitive and definitive hematopoietic cells sequentially, while VE-cadherin−CD45−α4-integrin+ cells are primary sources for primitive hematopoiesis. It seems that precursors of primitive and definitive erythropoiesis arise simultaneously but that the definitive precursors require a period of maturation before they differentiated into blood cells. We suggest that a subset of endothelial cells is involved in primitive as well as definitive hematopoiesis during primate embryogenesis, and that α4-integrin marks the hemogenic subpopulation in primates.

The close developmental association between hematopoietic and endothelial cells suggests that both lineage cells share a common precursor, the hemangioblast. The vascular endothelial growth factor (VEGF)-A system has been proven to have roles in the embryonic development of hemangioblast in mouse by the use of embryos or embryonic stem (ES) cells deprived of the genes encoding its ligand or receptors (VEGFR-1 and VEGFR-2). On the other hand, there have been only a few reports on the hemangioblast development during primate (human and monkey) embryogenesis.

Globin switching of erythrocytes during primate (human and monkey) hematopoietic development has been most thoroughly investigated not only as a model of tissue- and temporally specific transcriptional control but also as a tool for drug discovery against hemoglobinopathies. However, the regulatory mechanisms of globin switching in primates remains to be unresolved in primates mainly due to lack of available model system to date which reproduces the process of hematopoiesis to reflect accurately in vivo development. Recently primate ES cell lines were established, which are expected to serve as an experimental model for tissue growth and development, along with an efficacy and toxicity screening system for new drugs and a cell source for regeneration therapy. Among them, we previously demonstrated that the transition from primitive into definitive erythropoiesis was induced from primate ES cells by coculture with OP9 stromal cells. We also demonstrated that the VEGFR-2high CD34+ cells, emerging onto OP9 stromal layer after initial 6-day differentiation, contain the hemogenic progenitors.