Stem cells responsible for tissue maintenance and repair are found in a number of organs. Hepatic stem cells assumed to play a key role in liver development and regeneration remain to be well characterized. By combining single-cell-based assays with fluorescence activated cell sorting (FACS), we prospectively identified hepatic stem cells with multilineage differentiation potential and self-renewing capability. These cells could be clonally propagated in culture, where they continuously produced hepatocytes and cholangiocytes as descendants while maintaining primitive stem cells. When cells that expanded in vitro were transplanted into recipient animals, they morphologically and functionally differentiated into hepatocytes and cholangiocytes, with reconstitution of hepatocyte and bile duct structures. Furthermore, these cells differentiated into pancreatic ductal and acinar cells or intestinal epithelial cells when transplanted into pancreas or duodenal wall. Flowcytometric sorting enabled to identify and purify pluripotent hepatic stem cells reside in the developing liver and allowed to clear a further potential for differentiation. Our data demonstrate that manipulation of hepatic stem cells may provide new insight into therapies for diseases of the digestive system.

- mesenchymal interactions (EMI) play an essential role in normal organogenesis during development in many tissues, including the hair follicle. The hair follicle is a suitable model organ for studying EMI because of its distinct structure and the existence of a functional assay system. The hair follicle is composed of several follicular epithelium layers and hair specific mesenchymal cells, called dermal papilla (DP) cells. DP cells are difficult to isolate from hair follicles since they are enclosed by the follicular epithelium. We utilized the mesenchymal specific enhancer element from the gene for one of fibroblast producing proteoglycans, named versican, to express the green fluorescent protein (GFP) reporter gene in DP cells of transgenic mice, and we have successfully isolated a large number of DP cells from neonatal skin cells by high-speed cell sorting. These isolated GFP-positive DP cells had a hair inductive ability in a grafting assay in vivo. Cultured DP cells lost versican expression and GFP fluorescence as they were passaged over time and they also lost their hair inductivity. These results indicate that this model is useful to isolate pure DP cells, and that it is also useful to test hair inductivity with GFP as an indicator. This model should lead to a better understanding of the signaling cascade that occurs during EMI. High speed cell sorting is sufficient and useful to collect a large number of cells within a short time while maintaining high yield and viability.

The green fluorescent protein (GFP) produces the fluorescence similar to that of fluorescein isothiocyanate,within living cells. Using flowcytometry, we can analyze and sort the cells expressing GFP fluorescence to accomplish the cellular and molecular biological experiments. We made the transgenic mice, and the knockin mice, in which the expression of the GFP protein was driven by the transcriptional activity of the GATA-2 gene regulatory region. The GATA-2 is a transcription factor essential to the hematopoiesis, but its roles are not clarified yet. These mice produced the fluorescence in the hematopoietic cells, recapitulating the endogenous expression of the GATA-2 gene. The GFP expressing cells in the fetal liver had the same surface marker profile to that of hematopoietic stem/progenitor cells. We found that the GFP was also expressed in the aorta and adjacent tissues, where the production of adult-type hematopoietic stem/progenitor cells occurred. These cells had the morphology of the mesenchymal cells and expressed no CD45 hematopoietic cell antigens, but produced abundant hematopoietic cells after culture, indicating that the GATA-2 was expressed in the early precursors of the hematopoietic cells. Further, we used the GFP to identify the cells infected by retrovirus, and revealed the inhibitory role of GATA-2 expression to produce hematopoietic cells from these precursor mesenchymal cells. Thus, the GFP techniques with flowcytometry, combined with the developmental biotechnology, are now the strong tool to reveal the physiological molecular mechanisms in the individual cells forming mammalian tissues.

Embryonic stem (ES) cell lines are derived from the inner cell mass of blastocysts and possess a totipotency which is equivalent with that of epiblast cells. A part of the totipotency of ES cells can be revealed in vitro by using various methods like embryoid body formation in suspension cultures as well as stromal cell-dependent induction of differentiation. By combining with fluorescence activated cell sorting (FACS), in vitro differentiation of ES cells provides not only a therapeutic means to isolate cells intended for transplantation but also an experimental tool to study developmental process of a given cell lineage. This review summarizes a culture system of murine ES cells in which lateral mesoderm cells and their descendants such as endothelial cells and hematopoietic progenitors are sequentially isolated by FACS. Especially, ES cell clones that revealed activity of the Gata1 and Gata2 genes by expressing a GFP transgene as a marker enabled us to examine expression and function of these genes in the development of hematopoietic cell lineages. Expression of the Gata2 gene was already detected in a fraction of the earliest lateral mesoderm cells developed in a culture of the ES cells. The Gata2-expressing mesoderm cells purified by FACS exclusively contained primitive erythroid colony-forming cells (CFU-EryP). Expression of the Gata1 gene in mesoderm cells became detectable later than that of the Gata2 gene. CFU-EryP was highly enriched in the Gata1-expressing mesoderm cell fraction. Mesoderm cells which did not express the Gata1 gene contained high frequency of endothelial cell progenitors. Endothelial cells derived from these progenitors possessed a potential to give rise to definitive hematopoietic cells. The results suggested that the primitive hematopoietic cell lineage diverges directly from lateral mesoderm whereas the definitive hematopoietic cell lineage originates in the endothelium.

To date, the appropriate condition for the collection and storage of samples in flowcytometorical analysis has not been studied. In this study, flowcytometorical analysis was performed using different tubes (EDTA,heparin, RPMI with heparin) at different points (within 6 hours or at 24hours) from sample collection, and the influence by changes of storage conditions on the results of flowcytometorical analysis was examined. When the analysis was performed within 6hours, more than 85% of cells were viable, and there was no change in fluorescence intensity or positive rate in all tubes. However, the percentage of dead cells was increased in the samples kept in the EDTA tube for 24 hours. Furthermore, a couple of samples showed the changes of fluorescence intensity and scatter of the cytogram after 24 hours regardless of the kinds of tubes. These results suggest that a careful review of the results is necessary for an appropriate report of flowcytometorical analysis, especially when samples left for long time after collection are applied.

Flow cytometric analysis is indespensable for identifying immunophenotypically hematopoietic cells. In such analysis, it is the most important issue how to choose flow-cytometrically target cells, namely gating. To differentiate target cells from the other cells, three gating approaches are generally known; morphological gating derived from the SSC/FSC cytogram, specific gating of the Leuko-gate(BD) using CD45 and CD14 measurement, and two-color gating using two CD antibodies relevant to target cells. In this paper, we reviewed our experiences of the problematic issues and relevance of gating in flow cytometric analysis as a routine hematological test.

Flow cytometric analysis of blood cells is an important technique for the differential diagnosis of leukemias. With regard to the analysis of leukemia cells, the combined use of intracellular antigens or nuclear DNA content with other routine cellular surface markers is quite useful for the analysis of immunophenotyping, and enables the differentiation of leukemia cells from the contaminating non-malignant cells, particularly in the analysis of the minimal residual diseases (MRD). We established a flow cytometric method for the simultaneous detection of cell surface antigens, intracellular antigens and relative DNA content. The reagent consists of phosphate-buffered saline containing 0.5% paraformaldehyde and 0.5% saponin (PFS) for fixation and permeabilization of cells. The method permits rapid cell processing, preservation of cell surface antigens, and sensitive staining of intracellular antigens. The method also exhibits very low coefficients of variation in the determination of nuclear DNA content. Use of this flow cytometric method substantially facilitates the diagnosis and detection of leukemia cells.

Escapee phenomenon in flow cytometric analysis (FCM) of lymphocyte subsets is defined as a variable number of lymphocytes escaping from the conventional gate of lymphocytes to those of other hematological cells by correlated analysis of forward and side scatters. Although this phenomenon is well recognized and is an artifactual problem in FCM, little information is available about its mechanism. Recently, we examined a new mechanism of escapee phenomenon in FCM of lymphocytes, and this issue was reviewed. Using dot plots of forward scatter versus fluorescent intensity of anti-CD8 antibody labeled with FITC, CD8+ cells escaping from the conventional lymphocyte gate were separated to two populations: one was CD8+ escaping cells that were the size of granulocytes (type I escapee), and the other was CD8+ escaping cells that were the size of monocytes (type II escapee). Type I escapee also expressed CD13, and the fluorescent intensity of CD8 on these cells was equal to that of CD8 on non-escapee cells, which were within the conventional lymphocyte gate. Type I escapee appeared to contain a CD8+ lymphocyte and a granulocyte. Type II escapee expressed neither CD33 nor CD14, and the fluorescent intensity of CD8 on these cells was two times the intensity of CD8 on non-escapee cells. Type II escapee appeared to contain two CD8+ lymphocytes. Type I escapee disappeared by treatment with unlabeled anti-CD32 antibody, which can block FcRII, before staining with FITClabeled anti-CD8 antibody. Type I escapee was also reduced by treatment with anti-CD16 antibody, which can block FcRIII. Type II escapee was reduced by treatment with anti-CD32 or anti-CD16 antibodies. These results showed that escapee phenomenon of peripheral CD8+ lymphocytes in FCM occurred through Fc receptors. Thus, we propose the following model of the escapee phenomenon; peripheral CD8+ lymphocytes bind to other cells through the binding between Fc receptors on other cells and anti-CD8 antibody recognizing CD8+ lymphocytes, resulting in a shift from the conventional lymphocyte gate to the gate of granulocytes or monocytes in FCM.

To apply UV-excited fluorochromes to a BD LSR benchtop flow cytomerty system, we examined fixation and staining conditions using UV-excitable dyes such as Hoechst 33342, DAPI and Indo-1. The BD LSR is available as a two-laser system featuring an argon laser (488 nm) and an UV-laser (325 nm). Flow cytometric analysis of DNA content with DAPI without RNase-treatment exhibited lower CV value of G₀/G₁ phase peak than that with PI. Analysis of human tumor cells and mouse lymphocytes for simultaneous staining of DNA and cell surface markers such as CD98 revealed that fixation with 2-4% paraformaldhyde followed by permeabilization with 0.1% saponin give the best result. Flow cytometric analysis of DNA content in intact cells treated with Hoechst 33342 displayed a CV value of G₀/G₁ phase peak comparable to fixed cells. Thus, we established simultaneous analysis of DNA content and multiple surface markers by using UV-excited fluorochromes and BD LSR cytometer. Next, we used BD LSR to measure intracellular Ca²⁺ concentration in murine thymocytes that had been loaded with the Ca²⁺-sensitive, UV-exitable dye Indo-1. We could demonstrate the elevation of intracellular Ca²⁺ after CD3-stimulation characteristically in CD4⁺CD8⁺ and CD4^-CD8^- subsets. Thus,the combined application of UV-excited dye, Indo-1 and BD LSR enable us to simultaneously analyze intracellular Ca²⁺ and various cell surface markers.

The DNA ploidy and DNA indices (DI) of 414 colorectal cancer patients treated in Tottori University Hospital between 1976 and 1994 were analyzed. The incidence of patients with hepatic recurrence was found to be significantly higher in those with aneuploid tumors and a DI above 1.5 than in those with diploid tumors and a DI below 1.4 or in those with diploid tumors and a DI equal to 1.0. A total of 110 colorectal cancer patients was randomly divided into two groups as a prophylactic portal infusion chemotherapy (PPIC) group and control I (a peripheral venous chemotherapy group) to confirm the effectiveness of PPIC as adjuvant therapy for prevention of hepatic recurrence. 5-FU (3mg/kg/day) was administered for two postoperative weeks and mitomycin C (10mg/body) was administered at day 0, day 7, day 14, via respective route. The incidence of hepatic recurrence in the patients with cancer of DI above 1.5 was significantly lower in the PPIC group compared to control group I (8% vs. 38%). Next, we examined that, which is superior as adjuvant chemotherapy via hepatic artery or via peripheral vein, using the same anticancer drugs and dosages mentioned above. There was no difference in recurrence rate between hepatic arterial infusion chemotherapy (HAIC) and peripheral vein group (control II) (22% vs. 23%), even in the patients with DI above 1.5 (13% vs. 17%). These findings suggest that colorectal cancer with DI above 1.5 may have a strong tendency to metastasize to the liver, and that PPIC may be more effective for prevention of hepatic recurrence compared to HAIC. We are processing the next regimen of PPIC using continuous infusion of 5FU (150mg/m²/day) and cisplatin (3mg/m²/day) for 3 weeks to confirm the effectiveness of new PPIC in the patients with cancer of DI above 1.5 as a high risk group of hepatic recurrence. Preliminary data showed the better disease free 5-year survival in new PIPC compared to control III(without infusion chemotherapy) (88% vs. 64%), and the reduced hepatic recurrence (no cases in PPIC and 4 cases in control). The new PIPC for the patients with DI above 1.5 was effective for prevention of hepatic recurrence.

Although the pathways of cell growth and apoptosis have been extremely investigated, it seems still unclear how cell growth inhibition and apoptosis are controlled or switched over. Cancer cells enter the cell cycle by being stimulated by a growth factor, such as epidermal growth factor (EGF) and insulin-like growth factor (IGF). Recently, vascular endothelial growth factor (VEGF) known as an inducer of angiogenesis is focused on,because of its function as growth factor. In fact, all human lung cancer cell lines we maintain secrete VEGF,and VEGF is similarly suspected to function as a growth factor. On the other hand, anticancer chemotherapeutic agents induce cell growth inhibition or apoptosis. DNAdamaging anticancer agents stimulate wild type p53 production, and p53 has a key role in the control of G1/S check point and then decides the outcome of cells to apoptosis or cell growth inhibition. Thus, p53 is very important to assess the efficiency of chemotherapeutic agents. DNA damage reaches apoptosis through two pathways, mitochondrial pathway initiated by Bcl-2 family and death receptor pathway stimulated by TNFreceptor superfamily activation. However, we found that all human lung cancer cell lines we maintained expressed Fas, a member of TNF-receptor superfamily. Fas was localized in the cytoplasm in exponentially growing cells and in the membrane in confluent cells. Interestingly, Fas levels in confluent cells were significantly correlated with their doubling times (r = 0.757, p = 0.0088). Moreover, growth factor stimulation such as EGF, IGF, and VEGF induced Fas internalization. From these results we suppose that Fas may function as a cell growth inhibitor as well as a death receptor just like p53.

The study of cytokines has been of great interest to researchers in many scientific disciplines,including Immunology and Cell Biology. Multiple stimulatory and inhibitory cytokines and their receptors form the basis for a sophisticated cellular communication network. Because of the complexity of the cytokine network, the simultaneous measurement of multiple cytokines in individual biological samples is desirable.^1,2）Herein, we describe the BD Cytometric Bead Array (CBA) System and its application to specifically and reproducibly measure several distinct cytokine proteins within small volume samples.