A wide variety of RNA molecules are now known to have important or essential biological functions in cells. Some function simply through complementary base pairing, while others through forming a variety of threedimensional shapes. RNA aptamers are the best example of the latter functional RNAs, which are experimentally selected from a random pool of RNA sequences and have high affinity and specificity to the target of interest. Their unique and potent characteristics have greatly promoted the research and development of RNA aptamers for medical and analytical applications. Notably, the first aptamer-based drug, called Macugen (VEGF antagonist), was approved by the U.S. FDA in 2004 far ahead of any RNAi therapeutics.

Nucleic acid aptamers are ligands that specifically bind to a target molecule, such as a wide variety of small molecules, proteins, viruses, and cells, obtained by an in vitro selection method using nucleic acid libraries with a random sequence. The potential of aptamers that bind to proteins and inhibit their activities has been realized in the form of aptamer-based diagnostic and pharmaceutical products. However, the limited 4-base components (A, G, C, and T/U) of nucleic acids restrict the further development of aptamers with increased functionality. To address this problem, researchers have been developing artificial, extra base pairs, unnatural base pairs, that work with the natural A-T and G-C base pairs in replication and transcription. Unnatural base pairs could expand the genetic alphabet and enable the site-specific incorporation of extra components into DNA and RNA. This report describes a novel RNA aptamer creation by using unnatural base pair systems.

Hematopoiesis is a dynamic and strictly regulated process orchestrated by self-renewing hematopoietic stem cells (HSCs) and the supporting microenvironment. However, the exact mechanisms in which individual human HSCs involved to sustain hematopoietic homeostasis remain to be clarified. To understand how the long-term repopulating cell (LTRC) activity of individual human HSCs and the hematopoietic hierarchy are maintained in the BM microenvironment, we traced the repopulating dynamics of human HSC. Our study presented several lines of evidence regarding the in vivo dynamics of human hematopoiesis. We demonstrated that human LTRCs existed in a rare population of CD34^＋CD38^－cells that localized to the stem cell niches and maintained their stem cell activities while being in a quiescent state. We further demonstrated that human mesenchymal stem cells differentiated into key components of the niche and maintained LTRC activity by closely interacting with quiescent human LTRCs, resulting in the increased number of LTRCs.

To elucidate the cellular and molecular mechanisms underlying normal and malignant hematopoiesis, it is critical to isolate the stem cell compartment as well as downstream lineage-restricted progenitor populations. Recent advances in the fluorescence-activated cell sorting (FACS) technology have greatly contributed to the progress of stem/progenitor isolation, providing the multicolor platform for simultaneous analyses of cell-surface antigens and/or specific gene expression profiles. In murine system, the almost ultimate purification of hematopoietic stem cell (HSC) has been achieved, demonstrating a long-term hematopoietic reconstitution by the single HSC transplantation. A variety of lineage-committed progenitors are also isolatable at the diverse checkpoints in the hierarchy of normal murine hematopoiesis. In human system, the emergence of xenotransplant models utilizing immunodeficient mice helped the identification of hematopoietic stem and progenitor populations, demonstrating that the expression patterns of surface antigens as well as the developmental pathways are considerably different between human and mouse. Furthermore, the verification of the concept of cancer stem cell is in progress. In this review, we summarize the phenotypic and functional properties of murine and human hematopoietic stem/progenitor cells and also discuss the recent topics of leukemic stem cells.

The Japanese Committee for Clinical Laboratory Standards, Area Committee on Hematology, and Subcommittee on Flow Cytometry (the so-called JCCLS FCM-Working Group) has published three guideline documents for flow cytometry:, (1)“Guidelines for Performing Surface Antigen Analysis on Peripheral Lymphocytes using Flow Cytometry”(JCCLS H1-A V2.0), (2)“Guidelines for Performing Surface Antigen Analysis on Hematopoietic Malignant Cells”(JCCLS H2-P V1.0), and (3)“Guidelines for CD34⁺ Cell Determination by Flow Cytometry”(JCCLS H3-P V1.0). The first guideline has already been approved by JCCLS, and the Working Group is currently seeking public comments on the proposed CD34 guideline document. Common subjects among these flow cytometry tests, i.e., optimizing instrument settings, work procedures after data acquisition and analysis, maintaining instruments, and managing reagents, are only described in JCCLS H1-A V2.0, the earliest published guideline document. The latter two guideline documents contain subjects related to each application. These guidelines are useful in accurate clinical diagnosis by flow cytometry, particularly for assuring accuracy and precision of flow cytometry in clinical laboratories as well as minimizing interlaboratory imprecision in external Quality Control programs.

Research has shown that high temperatures can damage and kill cancer cells, usually with minimal injury to normal tissue cells. However, the precise mechanism of hyperthermia-induced cell death is not yet fully under stood. CCD32SK diploid normal human fibroblasts were transiently transfected with short interfering RNA (siRNA) specific for human p53 (CCD/p53i) and negative siRNA (CCD/NEGi) for 24h. After p53 siRNA transfection (final concentration: 100 nM), CCD32SK cells were heated at 45°C for 0.5h. After the 45°C hyperthermia treatment, CCD/NEGi cells exhibited normal nuclear shapes, and almost all cells were dead at 120h. In contrast, CCD/p53i cells showed a marked increase in abnormal nuclear shapes after hyperthermia. While CCD/NEGi cells were arrested in G1 and G2 after heat shock, significant numbers of mitot ic cells with multiple poles appeared in CDD/p53i cells. Subsequently, cells with multiple micronuclei increased in number as tumor cells, with time, and after heat shock. We then investigated the induction of centrosome amplification by p53 siRNA transfection after hyperther mia. There was a small increase in the frequency of centrosome amplification in CCD/p53i cells (5.0%) without hyperthermia treatment. In contrast, at 48 h after hyperthermia treatment, CCD/p53i cells exhibited pronounced centrosome amplification (45%). In the present study, we found that siRNA-mediated silencing of p53 in normal human fibroblasts, and,together, with DNA damage by hyperthermia, centrosome amplification and mitotic catastrophe is efficiently induced. However, these phenomena are not induced by either siRNA-mediated silencing of p53 or hyper thermia alone.

Flow cytometric techniques are used widely in studies of cell death, and particularly in the identification of apoptotic cells. Darzynkiewicz reviewed methods of detecting apoptosis by the analysis of DNA fragmentation. Mitotic catastrophe is an important mechanism of inducing cell death in cancer cells by radiation and hyperthermia, which damage DNA. This process is facilitated by defects in the G1 and G2 checkpoints of the cell cycle, which allow the cells to enter mitosis with DNA damage. DNA fragmentation and micronuclei have also been observed in mitotic catastrophe, and a sub-G1 peak is therefore detected. To elucidate the effects of radiation on cell death, we examined the cell cycle in KK47 bladder cancer cells following irradiation. Twenty-four hours after 5-Gy irradiation, irradiated cells were arrested in G2 phase. Laser scanning cytometry performed 48 h after irradiation revealed the following two pathways : 1) catastrophic cell death ; or 2) failure to undergo cytokinesis, resulting in polyploidy. Nuclear fragmentation and micronuclei were observed in the accumulating sub-G1 population 48 h after irradiation. The tumor suppressor protein p53 is a critical regulator of the G1/S and G2 checkpoints. We found that short interfering RNA-mediated silencing of p53 in normal human fibroblasts, together with DNA damage by irradia tion, efficiently induced mitotic catastrophe. In this experiment, too, nuclear fragmentation and micronuclei were observed in the accumulating sub-G1 population. In conclusion, when a sub-G1 peak is observed by cytometry, not only apoptosis, but also mitotic cell death,may be present.