Shin-ichi Mizuno

Reciprocal Activation of GATA-1 and PU.1 Marks Initial Specification of Hematopoietic Stem Cells into Myeloerythroid and Myelolymphoid Lineages

A hierarchical hematopoietic development with myeloid versus lymphoid bifurcation has been proposed downstream of the multipotent progenitor (MPP) stage, based on prospective isolation of progenitors capable of generating only myeloerythroid cells (common myeloid progenitor, CMP) or only lymphocytes (common lymphoid progenitor, CLP). By utilizing GATA-1 and PU.1 transcription factor reporters, here we identified progenitor populations that are precursors for either CMPs or CLPs. Two independent populations expressing either GATA-1 or PU.1 resided within the CD34(+)Sca-1(+)c-Kit(+) MPP fraction. The GATA-1(+) MPP displayed potent myeloerythroid potential without giving rise to lymphocytes, whereas the PU.1(+) MPP showed granulocyte/monocyte/lymphoid-restricted progenitor activity without megakaryocyte/erythroid differentiation. Furthermore, GATA-1(+) and PU.1(+) MPPs possessed huge expansion potential and differentiated into the original CMPs and CLPs, respectively. Thus, the reciprocal activation of GATA-1 and PU.1 primarily organizes the hematopoietic lineage fate decision to form the earliest hematopoietic branchpoint that comprises isolatable myeloerythroid and myelolymphoid progenitor populations.

Involvement of interferon-γ and macrophage colony-stimulating factor in pathogenesis of haemophagocytic lymphohistiocytosis in adults

Summary We investigated the role of monocyte/macrophage‐activating cytokines in pathogenesis of haemophagocytic lymphohistiocytosis (HLH) in 21 adult patients. Sera from patients with active HLH contained extremely high levels of macrophage colony‐stimulating factor (M‐CSF) and of interferon‐γ (IFN‐γ). These levels returned to almost normal during remission. Neither interleukin‐4 nor granulocyte/macrophage colony‐stimulating factor could be detected. Active HLH sera also contained high concentrations of inflammatory monokines, such as interleukin‐6 (IL‐6) and tumour necrosis factor‐α (TNF‐α). Serum concentrations of soluble CD8 and soluble interleukin‐2 receptor were extremely high during active HLH, and returned to virtually normal levels during remission. Circulating CD2+ T‐cells obtained from patients with active HLH spontaneously secreted M‐CSF and IFN‐γin vitro, whereas circulating monocytes did not produce detectable levels of both M‐CSF and IFN‐γ, but produced high levels of IL‐6 and TNF‐α. These findings suggest that IFN‐γ and M‐CSF at least partly from T‐cells, such as CD8+ T‐cells, might contribute to activation of monocytes or histiocytes, resulting in the up‐regulated monokine production and haemophago‐cytosis in HLH.

Identification of eosinophil lineage–committed progenitors in the murine bone marrow

Eosinophil lineage–committed progenitors (EoPs) are phenotypically isolatable in the steady-state murine bone marrow. Purified granulocyte/monocyte progenitors (GMPs) gave rise to eosinophils as well as neutrophils and monocytes at the single cell level. Within the short-term culture of GMPs, the eosinophil potential was found exclusively in cells activating the transgenic reporter for GATA-1, a transcription factor capable of instructing eosinophil lineage commitment. These GATA-1–activating cells possessed an IL-5Rα+CD34+c-Kitlo phenotype. Normal bone marrow cells also contained IL-5Rα+CD34+c-Kitlo EoPs that gave rise exclusively to eosinophils. EoPs significantly increased in number in response to helminth infection, suggesting that the EoP stage is physiologically involved in eosinophil production in vivo. EoPs expressed eosinophil-related genes, such as the eosinophil peroxidase and the major basic protein, but did not express basophil/mast cell–related mast cell proteases. The enforced retroviral expression of IL-5Rα in GMPs did not enhance the frequency of eosinophil lineage read-outs, whereas IL-5Rα+ GMPs displayed normal neutrophil/monocyte differentiation in the presence of IL-5 alone. Thus, IL-5Rα might be expressed specifically at the EoP stage as a result of commitment into the eosinophil lineage. The newly identified EoPs could be the cellular target in the treatment of a variety of disorders mediated by eosinophils.

Cyclin A Is Redundant in Fibroblasts but Essential in Hematopoietic and Embryonic Stem Cells

Cyclins are regulatory subunits of cyclin-dependent kinases. Cyclin A, the first cyclin ever cloned, is thought to be an essential component of the cell-cycle engine. Mammalian cells encode two A-type cyclins, testis-specific cyclin A1 and ubiquitously expressed cyclin A2. Here, we tested the requirement for cyclin A function using conditional knockout mice lacking both A-type cyclins. We found that acute ablation of cyclin A in fibroblasts did not affect cell proliferation, but led to prolonged expression of another cyclin, cyclin E, across the cell cycle. However, combined ablation of all A- and E-type cyclins extinguished cell division. In contrast, cyclin A function was essential for cell-cycle progression of hematopoietic and embryonic stem cells. Expression of cyclin A is particularly high in these compartments, which might render stem cells dependent on cyclin A, whereas in fibroblasts cyclins A and E play redundant roles in cell proliferation.

FLT3-ITD up-regulates MCL-1 to promote survival of stem cells in acute myeloid leukemia via FLT3-ITD–specific STAT5 activation

Myeloid cell leukemia-1 (MCL-1) is an essential survival factor for hematopoiesis. In humans, hematopoietic stem cells (HSCs) express MCL-1 at the highest level in response to FMS-like tyrosine kinase-3 (FLT3) signaling. We here show that this FLT3-dependent stem cell maintenance system also plays a critical role in survival of leukemic stem cells (LSCs) in acute myeloid leukemia (AML). The CD34(+)CD38(-) LSC fraction expresses high levels of FLT3 as well as MCL-1, even compared with normal HSCs. Treatment with FLT3 ligand induced further MCL-1 up-regulation in LSCs in all AML cases tested. Interestingly, the group of samples expressing the highest levels of MCL-1 constituted AML with FLT3-internal tandem duplications (ITD). In FLT3-ITD AML cell lines, cells expressed a high level of MCL-1, and an inhibition of MCL-1 induced their apoptotic cell death. A tyrosine kinase inhibitor suppressed MCL-1 expression, and induced apoptosis that was reversed by the enforced MCL-1 expression. Finally, transduction of FLT3-ITD into HSCs strongly activated MCL-1 expression through its signal transducer and activator of transcription 5 (STAT5)-docking domains. This effect was completely abrogated when STAT5 activation was blocked. Thus, the acquisition of FLT3-ITD ensures LSC survival by up-regulating MCL-1 via constitutive STAT5 activation that is independent of wild-type FLT3 signaling.

Cytomegalovirus (CMV) antigenaemia for rapid diagnosis and monitoring of CMV‐associated disease after bone marrow transplantation

A technique for the rapid detection of cytomegalovirus (CMV) antigen‐positive blood leucocytes (CMV antigenaemia) was evaluated in 15 marrow transplant patients as a means of diagnosis and for monitoring CMV‐associated disease. CMV antigenaemia was determined by direct immunoperoxidase staining of leucocytes with a peroxidase‐labelled monoclonal antibody, HRP‐C7, which binds an immediate‐early antigen of human CMV.

Increased incidence of cytomegalovirus (CMV) infection and CMV-associated disease after allogeneic bone marrow transplantation from unrelated donors

Cytomegalovirus (CMV) infection and CMV-associated disease were monitored using the CMV antigenemia assay in 72 patients who received allogeneic bone marrow transplantation (BMT), and their incidences were compared between related and unrelated donor transplant patients. The incidence of CMV infection after BMT was significantly higher in patients who received transplants from HLA-matched unrelated donors than from HLA-matched sibling donors (87% vs 53%, P < 0.05). cmv-associated disease developed in 73% of unrelated and in 14% of sibling donor transplant patients (P < 0.01). the peak levels of cmv antigenemia were significantly higher in unrelated donors than in sibling donor transplant patients (16 vs 1 CMV antigen-positive cells per 50 000 WBCs, P < 0.01). the median number of cmv antigen-positive cells on first detection was also significantly higher in unrelated donor transplant patients (15 vs 1, P < 0.01). the detection of cmv antigen-positive cells preceded the development of cmv-associated disease in 18% of unrelated donor transplant patients, suggesting a lower predictive value of cmv antigenemia for subsequent cmv- associated disease in unrelated donor bmt. careful monitoring and further studies are needed for the early diagnosis and prevention of cmv-associated disease in unrelated donor bmt.

The effects of a simplified method for cryopreservation and thawing procedures on peripheral blood stem cells

A simplified method for cryopreservation at −80°C of peripheral blood stem cells (PBSC) has been increasingly used for autologous PBSC transplantation in Japan. Although this method, using 6% hydroxyethyl starch (HES) and 5% dimethyl sulfoxide (DMSO) as a cryoprotectant without rate-controlled freezing, has several advantages over the conventional method using 10% DMSO with rate-controlled freezing, little is known about effects of long-term cryopreservation for years and thawing process on hematopoietic progenitors. We examined the recovery rates of BFU-E and CFU-GM in sample tubes cryopreserved by the simplified method under various conditions as follows: (1) long-term storage for 1–5 years; (2) DMSO exposure for 1 h after rapid thawing; and (3) thawing at a lower temperature other than 37°C. In our study, we found that the recovery rates of BFU-E and CFU-GM were not affected by the length of cryopreservation period; they remained at more than 70% on average for 16–61 months. In our hands, a 1-h exposure to DMSO after rapid thawing was not toxic for hematopoietic progenitors. Furthermore, there was no significant difference in the recovery rates of BFU-E and CFU-GM between thawing at 37°C and 20°C. These observations indicate that PBSC cryopreserved for at least 5 years by the simplified method can be used clinically without losing hematopoietic activity, and suggest that hematopoietic activity of the thawed PBSC may be unaffected when PBSC are infused slowly within 60 min or even when PBSC are thawed gradually at room temperature.

Quantitative analysis of AML1/ETO transcripts in peripheral blood stem cell harvests from patients with t(8;21) acute myelogenous leukaemia.

Summary Peripheral blood stem cells (PBSC) have been used increasingly for haemopoietic reconstitution after marrow‐ablative chemotherapy in patients with acute leukaemia because of the possibility that there is a lower risk of leukaemic contamination. We have developed a titration assay using a competitive reverse transcriptase polymerase chain reaction (RT‐PCR) which is able to estimate the number of AML1/ETO transcripts so that minimal residual disease (MRD) can be monitored quantitatively in patients with t(8;21) acute myelogenous leukaemia (AML). Using a qualitative RT‐PCR method, AML1/ETO transcripts could be detected in all samples from 15 first PBSC harvests and 11 second PBSC harvests obtained from 15 patients with t(8;21) AML. With our competitive RT‐PCR assay, the number of AML1/ETO transcripts was found to be lower in the second PBSC harvest than that in the first in every individual. Furthermore, MRD in PBSC harvests was less than that in the corresponding bone marrow obtained on the day of PBSC collection in the individual patients studied. In 10 patients who received autologous blood stem cell transplantation (ABSCT), we could not find a relationship between the number of AML1/ETO transcripts in the infused PBSC harvests and the clinical outcome after ABSCT. The present study clearly indicates that although PBSC harvests collected after consolidation chemotherapy are contaminated by leukaemic cells, the degree of leukaemic contamination may decrease as chemotherapy is repeated. The mobilization of PBSC by repeated chemotherapy may provide an advantageous source of haemopoietic stem cells for ABSCT.

Aberrant expression of HLA-G antigen in interferon γ-stimulated acute myelogenous leukaemia

We have analysed the expression of HLA‐G in 40 leukaemia samples of various subtypes [seven cases of acute lymphoblastic leukaemia (ALL), 28 cases of acute myelogenous leukaemia (AML), three cases of chronic myelogenous leukaemia (CML) and two cases of chronic lymphocytic leukaemia (CLL)] by flow cytometry using HLA‐G‐specific monoclonal antibody. No leukaemia samples expressed HLA‐G without incubation with interferon (IFN)‐γ. However, six out of 28 (21%) AML samples expressed HLA‐G upon incubation with IFN‐γ. These six samples derived from one out of seven M2, two out of eight M4 and three out of five M5. The results indicated that AML cells, especially myelomonocytic leukaemia samples, are capable of expressing the HLA‐G molecule.