Human embryonic stem (ES) cells can be maintained as undifferentiated cells in culture without apparent limits. They also have the capacity to differentiate into all cell types. Therefore, they are expected to be a new cell source in the regenerative medicine. We have recently established a novel method inducing human ES cells to differentiate into blood cells in vitro in coculture with stromal cells derived from murine embryonic hematopoietic tissues. However, there remain a number of problems to be solved for the clinical application of human ES cell-derived blood cells.

With significant attention paid to the field of tissue-specific stem cells, the identification of stem cell-specific markers and stem cell niche is of considerable importance. Recently, we showed that high expression of integrin β3 is an attribute of both rabbit corneal epithelial stem cells and mouse hematopoietic stem cells (HSCs). Additionally, we mainly utilized HSCs to elucidate the relationship between tissue-specific stem cells and integrin β3, and demonstrated that the expression of integrin β3 (CD61) is correlated with properties of HSCs. Moreover, our results strongly indicate that the integrin β3 subunit on HSCs is associated with integrin α3v (CD51), but not integrin αIIb (CD41). These results suggest that integrin β3 subunit is available as a common surface marker of tissue-specific stem cells. In addition, induction of integrin β3 signaling on HSCs contributes to the maintenance of HSC activity during ex vivo culture, which is dependent on the presence of thrombopoietin (TPO). Thus, our finding demonstrates that integrin β3 is essential for cytokine-dependent maintenance of stem cell activity. Thus, we believe that the further study for integrin β3 on tissue-specific stem cells provides a breakthrough that permits further characterization of the stem cell niche.

We have commercialized the world's first damage-less cell sorter (PERFLOW® Sort: http://www.furukawa.co. jp/bio/english/) for analyzing and sorting living cells, especially human induced pluripotential stem (iPS) cells and human embryonic stem (ES) cells. We have succeeded in generating monoclonal human iPS cells from single cells with high survival of 66%. Different from the conventional sorting methods using ultrasonic wave, charge and high pressure, which bring physical damage to cells, PERFLOW Sort adopts high-performed micro-flow control and fixed optical fiber alignment to monitor flow speed of target cells precisely, as guarantees living cells to be sorted with less physical damage. On the other hand, PERFLOW Sort adopts transmitted light signals instead of conventional forward scatter to achieve high-sensitivity analysis.

The disposable microfluidic chip based compact flow cytometer, FISHMAN-R, has many advantages. For the purpose of relief from the user's load of the measurement setup, the FISHMAN-R adopts a useful system that the fluorescence compensation is executed after measurements. We reacted human peripheral blood mononuclear cells with the fluorescence-labeled antibodies, and measured those samples in the various instrument settings including the powers of the lasers and the sensitivities of the detectors of the FISHMAN-R. These results showed that the data from different settings brought about same outcomes for the antigen expression after the compensation. One advantage by using the microfluidic chip is that the requirement of cell samples is only a small amount. Our data indicated that human peripheral blood 10μL was used in the analysis of blood cell markers and the quantities of antibodies was suppressed. The other advantage is that it is possible to collect samples after measurements by the FISHMAN-R. In experiments with stimulation of human lymphocytes, there was no difference between responses of control and collected cells. It demonstrated that collected cells after measurement were available for further experiments.

During solid tumor development, invasion of tumor cells in peripheral blood occur known as circulating tumor cells (CTC). Although mechanisms of invasive and metastatic behaviors of CTC are not fully understood, CTC becomes an important prognostic marker for cancer patients, which provides information of the therapy and the follow-up. We have challenged the enumeration of CTC by flow cytometry (FCM). In this article, we focus on our recent development of intact CTC enumeration and analysis procedure (iCeap), which enables to enumerate CTC keeping their cellular integrity. The iCeap is designed to perform a flow cytometric CTC enumeration and any downstream analysis after the measurement. Two applications with iCeap are shown; Live/Dead discrimination of CTC and cultivation of spiked cancer cell line. A prospective usefulness of CTC qualification in addition to quantification is discussed.

Development of organism is a dynamic but coordinated process that involves cell proliferation, migration and changes in cell function. Molecular biology and genome science promoted this realm of biomedical science by elucidating various common rules. Fluorescence live imaging has made it possible to quantitatively analyze multicellular process in 4 dimensions, thus providing coherent understandings of distinct levels of description form molecular levels to tissue, organ and organism. Application of this approach has given further insight and comprehension of dynamic process, not mere description of molecular hierarchy, into developmental biology and medicine. In this review paper, we, through introducing current topics in the body patterning, overview how the live imaging of fluorescent proteins has shed new lights on developmental biology.

Objective: In order to improve the diagnostic accuracy of urothelial tumors, a technology of molecular cytogenetic analysis was applied to bladder washings. Study design: Multicolor - FISH (fluorescence in situ hybridization, UroVysion^® ) was applied to specimens prepared by the liquid-based cytology (LBC) method for 305 bladder washings. FISH data was compared with cytological findings. Results: The cytological examination classified tumors into three categories; negative, equivocal, and malignant for 34.1%, 30.2%, and 35.7% of 305 specimens, respectively. UroVysion using three centromere probes for chromosomes 3, 7, and 17, and a locus specific probe for 9p21 harboring p16^INK4a allowed diagnosis as follows. When more than 10% of epithelial cells show numerical aberrations of signal spots at least in one of 3 CEPs or when more than 7% of epithelial cells have less than 2 spots of 9p21, the tumor is classified as urothelial carcinoma. Furthermore, the tumor in which the 'variant fraction size' of CEPs is less than 15% is categorized as low-grade carcinoma. The tumor in which the 'variant fraction size' of CEPs is more than 15% is categorized as high-grade carcinoma. Negative, low-grade carcinoma, and high-grade carcinoma accounted for 35.4%, 27.5%, and 37.1% of 305 cases, respectively. In this series, 69.5% of cytogenetically negative tumors were cytologically diagnosed as negative, and 80.5% of high-grade carcinomas were cytologically diagnosed as positive. Conclusion: The multicolor-FISH analysis allows a diagnosis of urothelial tumors.

Since Wolf found platelet dust or platelet vesicle which was a bleb from activated platelet in 1960s, it has become to be known well that this vesicle has coagulant activity and recently is termed as microparticles. It is well known that microparticles are generated from many kinds of activated and/or apoptotic cells and suggested that micoparticles increase in diabetes mellitus, metabolic syndrome and coronary syndrome. In the present study, we measured the number of platelet-derived microparticles (PDMP) and that of endothelium-derived microparticles (EDMP) from healthy subjects (n=29) and patient subjects (n=20) who received percutaneous coronary intervention operation by using of flow cytometer (FCM) with FITC labeled CD41 monoclonal antibody or CD144 polyclonal antiserum for the purpose of proposing standardization of PDMP and EDMP determination. PDMP gate was set to include particles of >0.5μm and <1.0μm in forward scatter (FS) and EDMP gate was set to include particles of >0.5μm and <2.0μm in FS. FITC positive events in the gate of FS-FL1 scatter were counted by using of FCM. PDMP were found even in the healthy subjects (113±78events/μl) but numerous PDMP were found in the patient subjects (490±660events/μl). EDMP were rare in the healthy subjects (1.5±0.9events/μl) but substantial number of EDMP were found in the patient subjects (10.0±6.0events/μl). Events detected by isotype control staining were very fewer than PDMP and EDMP. These data suggest that our gate setting is convincing for measuring PDMP and EDMP by using of FCM and we propose the present gate setting for standardization of PDMP and EDMP determination.