Kayoko Matsumoto

Factors affecting the volume of umbilical cord blood collections

Objective. Our purpose was to find factors that may help increase the number of the HSC ( CD34+) collected from umbilical cord blood for transplantation.

Differences Between Peripheral Blood and Cord Blood in the Kinetics of Lineage‐Restricted Hematopoietic Cells: Implications for Delayed Platelet Recovery Following Cord Blood Transplantation

Cord blood (CB) cells are a useful source of hematopoietic cells for transplantation. The hematopoietic activities of CB cells are different from those of bone marrow and peripheral blood (PB) cells. Platelet recovery is significantly slower after transplantation with CB cells than with cells from other sources. However, the cellular mechanisms underlying these differences have not been elucidated. We compared the surface marker expression profiles of PB and CB hematopoietic cells. We focused on two surface markers of hematopoietic cell immaturity, i.e., CD34 and AC133. In addition to differences in surface marker expression, the PB and CB cells showed nonidentical differentiation pathways from AC133+CD34+ (immature) hematopoietic cells to terminally differentiated cells. The majority of the AC133+CD34+ PB cells initially lost AC133 expression and eventually became AC133−CD34− cells. In contrast, the AC133+CD34+ CB cells did not go through the intermediate AC133−CD34+ stage and lost both markers simultaneously. Meanwhile, the vast majority of megakaryocyte progenitors were of the AC133−CD34+ phenotype. We conclude that the delayed recovery of platelets after CB transplantation is due to both subpopulation distribution and the process of differentiation from AC133+CD34+ cells.

Spontaneous and rapid reexpression of functional CXCR4 by human steady-state peripheral blood CD34+ cells

Although only 5% of steady-state peripheral blood (PB) CD34+ cells were found to express chemokine receptor CXCR4, 45% of the cells became CXCR4+ after incubation at 37°C for 4 hours. In contrast, there were no remarkable differences between PB CD34+ cells before and after the 37°C incubation in their expression of selectin ligand, VLA-4, and VLA-5 or in their affinity for VCAM-1 or fibronectin.This increase in CXCR4 expression level was inhibited by the addition of brefeldin A, actinomycin D, or cycloheximide.When PB CD34+ cells with CXCR4 expression levels enhanced by a 4-hour preincubation at 37°C or bone marrow (BM) CD34+ cells were exposed overnight to stromal cell-derived factor 1 (SDF-1), the expression levels of CXCR4 were greatly reduced, and when SDF-1 was removed, CXCR4 levels were thereafter up-regulated.The reexpressed CXCR4 was able to elicit integrin-dependent migration of hematopoietic progenitor cells.There was no difference in the severe combined immunodeficient mouse repopulating cell activity between PB CD34+ cells with and cells without a 37°C preincubation.

Down-regulation of ASY/Nogo transcription associated with progression of adult T-cell leukemia/lymphoma

Adult T‐cell leukemia/lymphoma (ATLL) is an aggressive form of human leukemia/lymphoma. Although this disease is initiated by infection with human T‐lymphotropic virus type 1 (HTLV‐1), many HTLV‐1 carriers survive for a long period without aggressive illness, suggesting that other factors may play roles in the progression of ATLL to an aggressive state. However, the mechanism involved in this progression still remains unclear. Previously, we have reported that ASY/Nogo mRNA was markedly down‐regulated in human small‐cell lung carcinomas, whereas it was expressed in normal tissues and other lung carcinomas, such as adenocarcinoma and squamous cell carcinoma. To understand whether or not ASY/Nogo gene is involved in the progression of ATLL, we examined the expression of ASY/Nogo mRNA in smoldering, chronic and aggressive ATLL, and found that the expression level of ASY/Nogo mRNA was markedly reduced in clinically aggressive ATLL. HTLV‐1 Tax expression was not affected by the down‐regulation of ASY/Nogo mRNA. These results indicate that the ASY/Nogo gene may act as a suppressor against ATLL progression, independent of Tax expression.

Multiple ETS Family Proteins Regulate PF4 Gene Expression by Binding to the Same ETS Binding Site

In previous studies on the mechanism underlying megakaryocyte-specific gene expression, several ETS motifs were found in each megakaryocyte-specific gene promoter. Although these studies suggested that several ETS family proteins regulate megakaryocyte-specific gene expression, only a few ETS family proteins have been identified. Platelet factor 4 (PF4) is a megakaryocyte-specific gene and its promoter includes multiple ETS motifs. We had previously shown that ETS-1 binds to an ETS motif in the PF4 promoter. However, the functions of the other ETS motifs are still unclear. The goal of this study was to investigate a novel functional ETS motif in the PF4 promoter and identify proteins binding to the motif. In electrophoretic mobility shift assays and a chromatin immunoprecipitation assay, FLI-1, ELF-1, and GABP bound to the −51 ETS site. Expression of FLI-1, ELF-1, and GABP activated the PF4 promoter in HepG2 cells. Mutation of a −51 ETS site attenuated FLI-1-, ELF-1-, and GABP-mediated transactivation of the promoter. siRNA analysis demonstrated that FLI-1, ELF-1, and GABP regulate PF4 gene expression in HEL cells. Among these three proteins, only FLI-1 synergistically activated the promoter with GATA-1. In addition, only FLI-1 expression was increased during megakaryocytic differentiation. Finally, the importance of the −51 ETS site for the activation of the PF4 promoter during physiological megakaryocytic differentiation was confirmed by a novel reporter gene assay using in vitro ES cell differentiation system. Together, these data suggest that FLI-1, ELF-1, and GABP regulate PF4 gene expression through the −51 ETS site in megakaryocytes and implicate the differentiation stage-specific regulation of PF4 gene expression by multiple ETS factors.

HVJ (Sendai Virus) Stimulates Release of Interferon from Leukocytes Used Once for Interferon Production

Leukocytes, subjected once to interferon (IFN) induction by HVJ (Sendai virus), were studied for their capability to produce IFN after a second similar stimulus. Substantial amounts of IFN (about 30 000 IU/ml) were recovered. Experiments using cycloheximide or actinomycin D and kinetic studies showed that this IFN originated mainly in IFN which resided within the cell as a result of the first induction and was released after the second stimulation. Increasing amounts of HVJ used for the second stimulus resulted in proportionally increased yields of IFN, reaching a plateau at the same dose of HVJ (1000 HAU/ml) as that which gave optimal yields after the first stimulation. Evidence is presented that the capacity of HVJ to trigger the production of a second IFN harvest is closely associated with its infectivity.

Expression and Affinity of Homing-Related Molecules on Steady-State Adult and Neonate Human PB CD34+ Cells and Their SRC Activity

Although the vast majority of hematopoietic progenitor cells (HPCs) reside within the bone marrow (BM), a small number of HPCs also continuously circulate in the peripheral blood (PB). The examination of the fate of blood-borne HPCs in parabiotic mice, which are surgically conjoined and share a common circulation, recently revealed that steady-state PB HPCs play a physiological role in, at least, the functional re-engraftment of unconditioned BM. To assess the possibility that human HPCs have a similar function, in this study we examined the expression level and affinity of the homing-related molecules, as well as the SCID mouse reconstituting cell (SRC) activity of human PB CD34+ cells, and compared adults with neonates.There was no remarkable difference between adults and neonates in the expression of E- and/or P-selectin ligands by PB CD34+ cells or in these cells’ affinity to VCAM-1. In contrast, the expression level of CXCR4 on PB CD34+ cells was much lower in adults than in neonates. Adult cells also showed a much lower SRC activity than neonates.These results suggest that human PB HPCs may contribute to steady-state hematopoiesis in the BM of neonates to some extent, but not so much in adults.