OP9 bone marrow stroma cells differentiate into megakaryocytes and platelets.

Platelets are essential for hemostatic plug formation and thrombosis. The mechanisms of megakaryocyte (MK) differentiation and subsequent platelet production from stem cells remain only partially understood. The manufacture of megakaryocytes (MKs) and platelets from cell sources including hematopoietic stem cells and pluripotent stem cells have been highlighted for studying the platelet production mechanisms as well as for the development of new strategies for platelet transfusion. The mouse bone marrow stroma cell line OP9 has been widely used as feeder cells for the differentiation of stem cells into MK lineages. OP9 cells are reported to be pre-adipocytes. We previously reported that 3T3-L1 pre-adipocytes differentiated into MKs and platelets. In the present study, we examined whether OP9 cells differentiate into MKs and platelets using MK lineage induction (MKLI) medium previously established to generate MKs and platelets from hematopoietic stem cells, embryonic stem cells, and pre-adipocytes. OP9 cells cultured in MKLI medium had megakaryocytic features, i.e., positivity for surface markers CD41 and CD42b, polyploidy, and distinct morphology. The OP9-derived platelets had functional characteristics, providing the first evidence for the differentiation of OP9 cells into MKs and platelets. We then analyzed gene expressions of critical factors that regulate megakaryopoiesis and thrombopoiesis. The gene expressions of p45NF-E2, FOG, Fli1, GATA2, RUNX1, thrombopoietin, and c-mpl were observed during the MK differentiation. Among the observed transcription factors of MK lineages, p45NF-E2 expression was increased during differentiation. We further studied MK and platelet generation using p45NF-E2-overexpressing OP9 cells. OP9 cells transfected with p45NF-E2 had enhanced production of MKs and platelets. Our findings revealed that OP9 cells differentiated into MKs and platelets in vitro. OP9 cells have critical factors for megakaryopoiesis and thrombopoiesis, which might be involved in a mechanism of this differentiation. p45NF-E2 might also play important roles in the differentiation of OP9 cells into MK lineages cells.

Induction of functional platelets from mouse and human fibroblasts by p45NF-E2/Maf.

Determinant factors leading from stem cells to megakaryocytes (MKs) and subsequently platelets have yet to be identified. We now report that a combination of nuclear factor erythroid-derived 2 p45 unit (p45NF-E2), Maf G, and Maf K can convert mouse fibroblast 3T3 cells and adult human dermal fibroblasts into MKs. To screen MK-inducing factors, gene expressions were compared between 3T3 cells that do not differentiate into MKs and 3T3-L1 cells known to differentiate into MKs. 3T3 cells transfected with candidate factors were cultured in a defined MK lineage induction medium. Among the tested factors, transfection with p45NF-E2/MafG/MafK lead to the highest frequency of CD41-positive cells. Adult human dermal fibroblasts transfected with these genes were cultured in MK lineage induction medium. Cultured cells had megakaryocytic features, including surface markers, ploidy, and morphology. More than 90% of MK-sized cells expressed CD41, designated induced MK (iMK). Infusion of these iMK cells into immunodeficient mice led to a time-dependent appearance of CD41-positive, platelet-sized particles. Blood samples from iMK-infused into thrombocytopenic immunodeficient mice were perfused on a collagen-coated chip, and human CD41-positive platelets were incorporated into thrombi on the chip, demonstrating their functionality. These findings demonstrate that a combination of p45NF-E2, Maf G, and Maf K is a key determinant of both megakaryopoiesis and thrombopoiesis.

Long-term follow-up of reduced-intensity allogeneic hematopoietic stem cell transplantation for refractory or relapsed follicular lymphoma.

Although allogeneic hematopoietic stem cell transplantation (HSCT) is considered the only curative treatment for refractory or relapsed follicular lymphoma (FL), transplant-related mortality (TRM) greatly interferes with the success. A variety of reduced-intensity conditionings (RICs) have been used to reduce TRM, but an optimal conditioning for FL has not been fully established. We retrospectively evaluated the outcome of allogeneic HSCT for FL with RIC consisting of fludarabine and melphalan. Nineteen adult patients with relapsed or refractory FL were conditioned with fludarabine (125 mg/m2) and melphalan (140 mg/m2), and received grafts from an HLA-identical sibling (n = 6) or an unrelated donor (n = 13). For the prophylaxis of graft-versus-host disease (GVHD), cyclosporine A or tacrolimus with short-term methotrexate was given. There were no early deaths before engraftment, and all patients achieved engraftment. Three patients died of extensive-type chronic GVHD (n = 2) or bacterial infection (n = 1) without disease progression. With a median follow-up period of 75.2 months (range: 33.3­111.9 months), 16 patients were alive without disease progression. Both the 5-year overall and progression-free survival rates were 84.2% (95% CI: 67.7­100%). These results strongly suggest that allogeneic HSCT with RIC using fludarabine and melphalan could be a promising treatment choice for refractory or relapsed FL.

Drug interaction between voriconazole and tacrolimus and its association with the bioavailability of oral voriconazole in recipients of allogeneic hematopoietic stem cell transplantation.

In a previous study, we noted wide inter-individual variability in drug interactions between voriconazole and tacrolimus, but that analysis did not take into account the routes of administration. In the present study, we analyzed interactions between these two drugs when both agents were administered orally after allogeneic hematopoietic stem cell transplantation (HSCT); the effect of plasma voriconazole levels on the magnitude of the drug interaction was also examined. Twenty-five allogeneic HSCT recipients were evaluated. Trough concentrations of tacrolimus were measured prior to, and periodically for 7-10 days after, initiating voriconazole (400 mg/day) to determine the concentration/dose (C/D) ratio of tacrolimus. The median C/D ratio of tacrolimus increased significantly from 172.8 (range 28.6-1110.7) to 537.5 (range 127.8-1933.3) (ng/mL)/(mg/kg) (P < 0.01) following initiation of voriconazole; the median increase was 138.8 % (range -32.0 to 685.7 %). The plasma concentration of voriconazole did not correlate with the increase of the tacrolimus C/D ratio (ρ = 0.16, P = 0.44). These results indicate that oral voriconazole has a significant drug interaction with oral tacrolimus with a wide inter-individual variability, which cannot be explained by the bioavailability of voriconazole. Other possible mechanisms should be explored in future studies.

Effect of early posttransplantation tacrolimus concentration on the development of acute graft-versus-host disease after allogeneic hematopoietic stem cell transplantation from unrelated donors.

Only limited data are available regarding the relationship between blood concentration of tacrolimus and its efficacy in preventing acute graft-versus-host disease (aGVHD). We retrospectively evaluated the effects of the whole blood concentration of tacrolimus, which was measured by an automated microparticle enzyme immunoassay, early after allogeneic hematopoietic stem cell transplantation (HSCT) upon the development of aGVHD. Sixty patients, who underwent allogeneic HSCT from serologically human-leukocyte antigen-matched unrelated donors and received continuous infusion of tacrolimus with short-term methotrexate for GVHD prophylaxis, were included in this study. The target range of the blood concentration of tacrolimus was set at 10 to 20 ng/mL, and the level was maintained within this range in all patients. However, the mean blood concentration of tacrolimus during the third week after HSCT was significantly associated with the grades of aGVHD (17.3 ± 2.1 in patients with grades 0-I vs 15.9 ± 2.8 in II-IV and 14.8 ± 2.1 in III-IV; P < .05 and <.01, respectively). Multivariate analysis also demonstrated that higher age (≥35) of donor (odds ratio [OR] = 4.28) and lower mean blood concentrations of tacrolimus during the second (OR = 0.75; 95% confidence interval [CI]: 0.58-0.98) and third weeks (OR = 0.76; 95% CI: 0.58-0.98) after HSCT were significant risk factors for grades II-IV aGVHD (P < .05). We conclude that the early posttransplantation blood concentration of tacrolimus had a significant impact on the development of moderate-to-severe aGVHD after allogeneic HSCT from an unrelated donor.