ES cells are a high potential source for use in regenerative medicine because of their high growth capacity and differentiation-competence. Endoderm cells compose respiratory, digestive and urological organs such as thyroid, lung, liver, pancreas, intestine, prostrate, bladder. In-vitro produced Hepatocytes and pancreatic beta cells from ES cells are expected to use for the treatment of liver injury and diabetes mellitus. Our strategy is to monitor intermediate stages of in-vivo endoderm development. Increasing of intermediate stage cells growth and differentiation efficiency can improve the therapeutic cell differentiation efficiency. A higher function is expected using a differentiation system according to in vivo development. We have developed the endoderm specific induction and monitoring system to separate the Goosecoid+ Sox17+ definitive endoderm stem cells with cell surface markers CXCR4 and E-cadherin. Moreover, purified definitive endoderm stem cells can dif ferentiate into various visceral organ cells including hepatocytes. Cancer is a heterogeneous disease charac terized by tumor composing of various cell types and difference among individuals, which make it difficult to treat. Our established cell-monitoring system is so useful to identify the specific cells in heterogeneous cell population from ES cells that it can be applied as a method for analysis of heterogeneous tumor cell population in the cancer.
Mouse hematopoietic stem cells (HSCs) are the best-studied stem cells because functional assays for mouse HSCs were established earliest and purification techniques for mouse HSCs have progressed furthest. Here we describe our current protocols for the purification of CD34-/lowc-Kit+Sca-1+lineage marker- (CD34-KSL) cells, the HSC population making up approximately 0.005% of bone marrow cells in adult C57BL/6 mice. Purified HSCs have been characterized at cellular and molecular levels. Since clonal analysis is essential for the study of self-renewal and lineage commitment in HSCs. We also introduce our immunostaining procedures for small numbers of HSCs, which are useful for signal transduction analysis.
Toll-like receptors (TLRs) play essential roles in the innate immune system, which recognize pathogen-asso ciated molecular patterns and initiate the acquired immune responses. Previous studies revealed that expres sion levels of TLR2 mRNA and protein on monocytes from sepsis patients are significantly up-regulated com pared to those from healthy subjects. In those studies, flow cytometric analysis was used to examine the mod ulation of TLR2 protein levels. Generally, flow-cytometer is a high precision tool to quantify the expression level of an antigen on cell sur face. However, it is hard to compare the measurements assayed at different time-points, because the inter assay variations are inevitable by the fluctuating factors, such as cytometerʼs performance and other assay conditions. To perform a longitudinal follow-up study for TLR2 protein levels on monocytes from the same patients, we developed a new quantitative analysis system for TLR2. For the first step, we created a set of standard beads which bore 4 differing amounts of recombinant TLR2 protein on their surface. On every assay, mean fluores cence intensity (MFI) of these standard beads was assessed by flow-cytometer to draw an analytical curve. Simultaneously, TLR2 levels on monocytes from the subjects were assayed, and the MFI values were convert ed to the numbers of anti-TLR2 monoclonal antibody binding sites with the analytical curve. By using this method, we followed-up TLR2 modulation of patients with bacterial infections. Through this follow-up study, some characteristics of TLR2 modulation on monocyte were exposed, which provide critical information about clinical conditions and medical treatment. Consequently, in clinical our developed TLR2 quantitative assay system is likely to provide more reliable measurement than conventional methods. Our methodology may be applicable to quantify not only TLR2 level but also other useful cell-surface markers, which are worth carrying out time-interval assay during clinical courses.
Cord blood transplantation (CBT) has become a standard therapeutic option in recent years. Compared with bone marrow transplantation (BMT), CBT has some advantages; rapid availability and low risk of severe acute graft-versus host disease (GVHD). However, CBT in adults is limited by graft cell dose. Multiple cord blood transplant (MCBT) in which two or more umbilical cord blood units are given is reported to enhance the engraftment compared with single unit CBT. However, little is known about the mechanism of enhanced engraftment. To elucidate the early engraftment kinetics of two units CBT, we developed the flow cytometry -based method of chimerism analysis using anti-HLA monoclonals.
The determination of the number of CD34 positive cells is essential for evaluating the availability of hematopoietic stem cells in autologous and homologous peripheral stem cell transplantations and for optimizing the timing of collecting a sufficient number of peripheral stem cells. Recently the CD34 Count Kit has been marketed by DAKO to flow-cytometrically determine the absolute number of CD34 positive cells by the single platform method, following the International Society for Cellular Therapy (ISCT) guidelines for stem cell analysis in human peripheral blood or its fractions. We assessed the performance of this kit using peripheral blood and simultaneously compared it with the Stem-kit, which is made by Beckman-Coulter Inc. on the same principle.
There was no essential difference between the two kits in their performance including cellular staining as well as time required for the whole procedure, which was completed within 30 minutes. The absolute number of CD34 cells collected from peripheral blood ranged from 4 to 161/μl, and the number obtained by the CD34Count Kit (Y) agreed well with that obtained by the Stem Kit (X) with a regression of y = 1.019 + 1.5 and a coefficient of determination (R2) of 0.9984. Similarly, the white cell counts obtained by the two kits agreed well with each other in the range of 3,900 to 70,000 /μl with a regression of y=0.966 x -240.6 and an R2 of 0.9984. However, when the two kits were compared using bone mallow cells, forward scatter versus side scatter cytograms of the CD34 Count Kit allowed many non-specifically stained cells such as debris and platelets to enter the stem cell region, occasionally giving falsely high counts of CD34 positive cells and leukocytes. This difference is due to the effect of the solution used to suspend internal standard particles in the Stem Kit, enhancing forward scatter of CD34 positive cells and thus discriminating them from non-specifically stained debris.
We performed DNA-ploidy analysis by flow cytometry (FCM) in patients with malignant lymphoma (ML). The subjects consisted of 732 patients who were suspected of having ML and underwent immunological examination by FCM and DNA-ploidy analysis between June 1997 and June 2007. Biopsy specimens or body cavity fluid samples were used for analysis. The amount of DNA was measured via dissolves with cytoplasmic membrane used the nonionic detergent TritonX-100 and staining with propidium iodide. The incidence of DNA-aneuploidy and DNA Index distribution were examined according to disease types. As a result, DNA-aneuploidy was not observed in reactive lesions but was detected in 35.1% of patients with B-ML and 25.3% of those with T/NK-ML. Thus, tumor proliferation could be detected by DNA-ploidy analysis in about 30% of patients with ML. The DNA Index was widely distributed from 0.83 to 2.34. However, the index was present near diploidy (1.00 ± 0.24) in 75% of the patients and near tetraploidy (2.00 ± 0.24) in 20%. Since index concentration in areas near the G0/G1 and G2/M phases (95%) was observed, overlapping with their peaks may reduce the detection rate. Therefore, the selection of a high-resolution method and avoidance of the influences of aggregating cells are important.
Models for immunodeficient mice reconstituted with a functional human immune system would be a valuable tool for the in vivo study of human immune response, notably the field of cancer biology, allergy, autoimmunity,human-specific viral infections such as HIV, and transplantation immunology. A model of SCID mice transplanted with human peripheral blood lymphocytes (hu-PBL-SCID) by a single i.p. injection was developed in the late 1980s. Reconstitution is characterized by a low rate of success, low and transient levels of chimerism in early models, but recent advances revealed that the abolishment of NK cell activity in NOD/SCID mice by antiNK antibody treatment or intercross with NK defective knock out mice results in a high degree of engraftment. Both human T and B lymphocytes were successfully developed after intra-splenic transplantation of cord blood mononuclear cells (CBMC) in novel immunodeficent mice (NOD/SCID/Jak3 deficient mice). Multicolor flowcy-tometric analysis revealed that both CD4＋and CD8＋Naive T lymphocytes (CD45RA＋CD62L＋) differentiated into the Central memory (CD45RA－CD62L＋) and Effector memory (CD45RA－CD62L－) phenotype in the mice. Naive B lymphocytes (CD19+IgD+ CD38+) also differentiated into Germinal center type(CD19＋IgD－CD38＋＋) and Memory(CD19＋IgD－CD38＋～－) phenotype. These results indicate that this system (NOD/SCID/Jak3 defi- cient mice intra-splenically xenografted with human CBMC) will be an excellent model for studies of human immuno-reaction against infectious diseases and vaccine development.
A new concept for the genome structure of mammalian cells is introduced with a model (A fractal structure model) constructed based on observations that have been reported. Briefly, the DNA structure is a self-similar fractal with a unit of opposite-handed twin circles. The DNA in human diploid cells is constructed with six hierarchies whose sizes are 325, 324, 323, 322, 321, and 320 with a unit of 200 DNA base pairs, corresponding to a genome, a chromosome, a chromosome band, a replicon, a rosette loop (a gene), and a nucleosome, respectively. This model may solve the questions why the band number of chromosomes decreases in M phase, why R-bands are replicated earlier than G-bands, why the telomere repeat is required for cells, and why the homologous chromosome can cynapsize in meiosis.
A new model for the genome structure of mammalian polyploid cells is introduced. It was constructed using the fractal structure model that was described in the preceding review. When cell division is inhibited and DNA syn thesis progresses, replicated DNA will be stacked keeping the initial structure of opposite-handed twin-circles. When inhibitors are removed, the polyploidized cells may return to the initial ploidy, because the stacked DNA loops have not been linked. When the stacked DNA twin-loops are linked with a proper configuration, the cells may become polyploid cells that can proliferate. There is a distinct difference in the genome structure between polyploidized and polyploid cells. The ploidy of polyploid cells is not stable, because the homologous chromo somes are arrayed mirror-symmetrically. The polyploid cells can return abruptly to a diploid state and repossess the point symmetry, or they gradually decrease the DNA level and become stable hypoploid cells whose DNA structure deviates from mirror symmetry. Experimental results will be introduced to verify the prediction.