A hematopoietic tissue, bone marrow, is located inside the bone. The functional interaction between skeletal and hematopoietic systems has been revealed recently. The roles of osteoblasts as a component of the hematopoietic microenvironment have been intensively studied, which is now an important research field. In contrast, the functions of hematopoietic cells as regulators of osteoblasts have also been elucidated gradually. This review describes how innate immune cells such as macrophages and neutrophils take part in bone metabolism and also how the deviation of inter-organ communication between skeletal and hematopoietic systems is importantly associated with clinical hematology.

Myeloproliferative neoplasms are clonal hematopoietic stem cell disorders characterized by the proliferation of cells of one or more of the myeloid lineages. The three major subgroups of MPN, namely PV, ET, and PMF, have been shown to exhibit the same gene mutations such as JAK2, MPL, and CALR. As JAK2 mutant mice developed three type of MPNs as PV, ET, or PMF, and CALR mutant mice developed ET, both JAK2 and CALR mutants played a driver role in MPN. The frequencies of hematopoietic stem cells (HSCs) in bone marrow (BM) increased in mice with JAK2 or CALR mutation compared to WT mice, however, HSCs with JAK2 mutation or CALR mutation exhibited a reduced growth advantage relative to wild-type (WT) HSCs in competitive transplantation assay, suggesting that JAK2 mutation or CALR mutation is insuffi cient for MPN onset. In addition to the above driver mutations, mutations in epigenetic regulators have been reported in MPN patients. Among them, TET2 mutation was observed in 10-20% of MPN patients, and frequently associated with JAK2 mutation. Cooperation between mutant JAK2 and mutant TET2 compensated for impaired HSC functioning by JAK2 mutation, and developed MPNs in the serial transplantation assay.

We previously identifi ed very primitive CD34-negative (CD34^-) severe combined immunodefi ciency (SCID)-repopulating cells (SRCs) in human cord blood (CB), using the intra-bone marrow injection technique. However, it is diffi cult to clarify the characteristics of these CD34^- SRCs because of their low incidence (1/25,000). Therefore, we developed a highresolution purifi cation method for CD34^- SRCs, using 18 Lineage (18Lin)-specifi c antibodies and CD133 antibody. Our results showed that CD34⁺ and CD34^- SRCs were enriched to approximately 1/100 and 1/140 in 18Lin^-CD34⁺CD133⁺ and 18Lin^-CD34^-CD133⁺ fractions, respectively. To further elucidate the characteristics of human CD34^+/- hematopoietic stem cells (HSCs), we aimed to identify additional positive markers for more effi cient purifi cation of CB-derived CD34^+/- SRCs. Subsequently, we elucidated that glycosylphosphatidylinositol-anchored protein GPI-80 was expressed on the portion of CB-derived 18Lin^-CD34⁺CD38^- and 18Lin^-CD34^- fractions. GPI-80 was fi rst identifi ed as a surface antigen on neutrophils; it is associated with the CD11b/CD18 (Mac-1) complex and regulates adherence and migration of neutrophils. We next performed limiting dilution analyses, which revealed that the frequencies of SRCs in the 18LinCD34⁺CD38^-GPI-80^+/- and 18Lin^-CD34^-GPI-80^+/- fractions were 1/21, 1/35 and 1/28, 1/874, respectively. These results clearly show that GPI-80 is a useful marker for the effi cient purifi cation of human CB-derived CD34^+/- SRCs (HSCs).

The hematological malignancy is very rare, however it is as diverse as those in adults. Based on these characteristics, multicenter clinical trial is necessary to provide high quality diagnosis and treatment to patients. We have performed

diagnosis and classifi cation by fl ow cytometry (FCM) method. As a result, it became applicable not only to the diagnosis of each hematopoietic tumor case but also to the characteristics of the malignancy, the prognosis prediction, the target treatment, and it became possible to more clearly characterize the hematopoietic tumor. In this paper, we analyzed 652

cases diagnosed in Osaka University from August 2011 to March 2017. The ALL subtype was detected at the same frequency as previously reported. We were able to detect CRLF 2, NG 2 as part of the research. DNA index was also examined, and ploidy could be well detected. FCM detects the protein expressed on the surface of various cells, not only examines the number and ratio, it is also possible to study the characteristic and function of the expressed protein on the cells. The procedure is simple and can be diagnosed and quantifi ed quickly. Even in the genome era, the FCM, which can search for the type and amount of protein expressed on tumor or normal cells at once, is still an excellent method, and it is necessary to provide important knowledge.

Immunophenotypic analysis of hematopoietic tumors by fl ow cytometry (FCM) is an important technique in diagnosing hematologic malignancies and evaluating therapeutic effectiveness. By improving the accuracy and stability of flow cytometer and analysis software over the years, it has become possible to acquire highly reliable data at voluntary facilities. However, FCM is not mature; it is not a complete technology. The development of devices and fl uorescent dyes that enable multicolor analysis is still ongoing. Therefore, clinical fl ow cytometry is a typical Laboratory developed test (LDT). From this, it is necessary to defi ne appropriate strategies in order to obtain equivalent results among labs, and standard guidelines with appropriate validation are required. In 2013 the International Council for Standardization in Haematology (ICSH) and the International Clinical Cytometry Society (ICCS) reported comprehensive guidelines for LDTs using FCM, including from analysis to post-analysis report, at the request of the FDA. In standardization of hematopoietic tumor analysis using FCM, one of the most dynamic approaches is the guideline proposed by EuroFlow, a division of European Scientifi c Foundation for Laboratory Hemato Oncology (ESLHO). A number of proposals have been reported, including the 2006 Bethesda International Consensus Panel, the recommended panel based on WP 10 of European Leukemia Net (ELN), and the Harmonemia project to obtain the same data with various fl ow cytometers. In this paper, we introduce this information and consider the direction of standardization of FCM.