In the present paper, we described disadvantages and advantages and future state of cord blood transplantation. One of most important disadvantages is a limited number of nucleated cells obtained from cord blood for transplantation. In order to overcome with the limited number of the cells, ex vivo expansion is considered to be essential for cord blood cell transplantation to adult patients. We also should develop the techniques of antigen presenting cells expanded ex vivo such as dendric cells pulsed with tumor specific antigen. Furthermore, we should create and supply unique cells from cord blood using genetic manipulation for the treatment of hematological disorders such as red cells without any red-cell antigen those can be transfused the patient with any blood type.

An IMI channel on automated hematology analyzer SE-9000 was used for detection of stem cells in cord blood in comparison with other standard methods such as flow cytometry analysis and progenitor assays. IMI count in cord blood samples correlated with the numbers of CD34+ cells, CFU-GM and BFU-E. Also the cell count in HPC area showed a correlation with CD34+ cells number, but to lesser extent than IMI count, and there was no significant correlation with the numbers of progenitor cells. Cord blood contains higher frequency of immature subclasses of progenitor cells, and it might explain the difference in the result of HPC module between cord blood and peripheral blood stem cell sample. The results demonstrated a usefulness of the measurement of IMI-positive cells to estimate stem cell content in cord blood samples as a screening test for cell banking.

We describe 3 different ways to determine the number of hematopoietic stem or progenitor cells in peripheral blood and harvest juice for peripheral blood stem cell transplantation. Flow cytometric analysis of CD34 positive cells gives us relatively quick and reliable results, although it's quite expensive. Automated hematology analyzer, SE-9000, exhibits real time appearance of stem cell population in a least expensive way. Colony formation in vitro such as CFU-GM is a direct way to detect hematopoietic committed cells, however this is time-consuming. We have to choose proper ways for each institute.

To date blood transfusion is considered to be one of tissue or organ transplantation. In the hematopoietic stem cell transplantation, donors of bone marrow, peripheral blood stem cells (PBSC) or cord blood should be tested to keep safety of donors and patients. Donors of PBSC, cord blood and bone marrow should be selected on the results of HLA class I and class II. Furthermore, donors of bone marrow should be tested for general anesthesia in order to keep safety of donors. In the present paper, we described the lists of tests for patients and donors of blood, PBSC, cord blood and bone marrow before and after stem cell transplantation.

Allogeneic peripheral blood stem cell transplantation (PBSCT) has been increasingly used as an alternative to allogeneic bone marrow transplantation. Allo-PBSCT can provide rapid engraftment of neutrophils and platelets. Although the recipients of allogeneic PBSCT are infused 10-fold T cells compared with BMT, there is no evidence for a significant difference between PBSCT and BMT with regard to incidence and severity of acute graft-versus-host disease (GVHD). On the other hand, several reports have indicated a high risk for developing chronic GVHD after allogeneic PBSCT as opposed to BMT.

Allogeneic peripheral blood stem cell transplantation (allo-PBSCT) has been increasingly used as an alternative to allogeneic bone marrow transplantation (allo-BMT). Medication of granulocyte colony stimulating factor (G-CSF) and apheresis are well tolerated by donors and supply adequate numbers of stem cells for the engraftment. Patients engraft sooner using PBSCT compared to allo-BMT. Allo-PBSCT is a safe alternative to allo-BMT and has distinct advantages for donors and patients. Faster engraftment results in fewer transfusion, shorter hospitalization, and decreased cost. However future research to determine if long-term side effects from G-CSF will negatively affect donors is essential. Data regarding durability of hematopoiesis and incidence for graft versus host disease warrant further analysis.

Relapse due to either residual host disease or reinfused tumor cells remains the principal cause of treatment failure after autologous stem cell transplantation. It is very attractive to remove contaminated tumor cells from autologous grafts prior to transplant and many purging techniques including hyperthermia we developed, have been reported on this subject. However, there are only limited data suggesting that purging autografts have any favorable effect on relapses or disease-free survival. Drug-treated purging and CD34 positive cell selections that remove substantial numbers of T cells or destroy stem cells may have adverse effects such as delayed hematopoietic or T cell recovery. Therefore, there is a critical need for large, well-designed trials to re-evaluate ex vivo and in vivo purging effects on relapses and disease-free survival after autologous stem cell transplantation.

Peripheral blood stem cells can be stored by the following 3 methods: liquid storage, non-rate controlled freezing and rate controlled freezing. Methods of processing of these cells including thawing, ex vivo purging and infusion are described in detail.

Recently, mobilized peripheral blood stem cells (PBSC) are increasingly used as an alternative to bone marrow for engrafting procedures. Chemotherapy followed by recombinant human granulocyte-colony stimulating factor (rhG-CSF) or rhG-CSF alone are the most commonly used PBSC mobilization schedules. Current timing of apheresis has now been yet decided on time by flowcytometric analysis of CD34 positivity of peripheral blood whole white cell count. Because apheresis procedure has been established quite well, PBSC harvest procedure becomes fast, safe and stable.

Umbilical cord blood has provides an alternative source to bone marrow for transplantation in children and some adults. However it has been thought to be necessary to expand hematopoietic stem cells in cord blood for adult-size transplantation. Flow-cytometric analysis revealed that most immature hematopoietic progenitors express gp130 but not interleukin-6 receptor (IL-6R). We established an ex vivo expansion system of human hematopoietic stem/progenitor cells using soluble IL-6R (sIL-6R). A combination of sIL-6R/IL-6 complex and SCF expanded CFU-Mix approximately 60-fold in both serum-containing and serum-free cultures by day 14. Addition of anti-gp130 mAbs and anti-IL-6R mAb to the above cultures dose-dependently inhibited the expansion of progenitors, suggesting that gp130 signalling initiated by the sIL-6R/IL-6 complex is important for significant expansion of human hematopoietic stem/progenitor cells. Addition of thrombopoetin and/or Flk2/Flt3 ligand (FL) to culture with sIL-6R/IL-6/SCF augmented the expansion of not only hematopoietic progenitors assayable in clonal culture but also hematopoietic stem cells estimated by NOD/SCID mice. Recent evidence shows that hematopoietic stem cells first occur and expand in aorta-gonad-mesonephros(AGM) region at 10 to 11 days post coitum(dpc) in murine, which suggests that AGM region at this stage provides a microenvironment suitable for development for hematopoietic stem cells. We reported here on a novel stromal cell line derived from the AGM region at day 10.5 dpc, which supported for 6 weeks generation of human multipotential hematopoietic progenitors from cord blood CD34+38- primitive hematopoietic cells in a co-culture system. This cell line is expected to elucidate molecular mechanisms regulating early hematopoiesis, and pave the way for developing strategies for ex vivo expansion of human transplantable hematopoietic stem cells.

Current reports from Europe and USA show that unrelated cord blood transplantation (CBT) gives good results in treating with children with malignancies and other disorders. However, there are not enough patients in the adults to perform a comparison with other stem cell transplantations. In unrelated CBT, HLA disparity is not a limiting factor and the incidence and the severity of acute and chronic GVHD is lower, even in the state of HLA 2 or 3 antigen-mismatched transplant, than in other stem cell transplantation, but the number of cells infused is important. At this stage, unrelated CBT is an optional source of stem cells in adults patients with malignancies who lack HLA compatible donors in bone marrow donor registries. However, the choice of cord blood units containing large number of cells and good disease status at transplantation might improve results.

Quality control (QC) of cryopreserved cord blood (CB) cells is very important for the safety of transplantation. In this article, the cell processing of CB cells, the laboratory tests for CB specimens, and the system of QC in CB bank is discussed.

In order to collect the umbilical cord blood for the separation of peripheral blood stem cells, obstetrician have to get the document of informed consent from the pregnant women for the umbilical cord blood donation during pregnancy. After the fetal delivery, umbilical cord blood is easier collected from the umbilical vein of the placenta in utero than that of the delivered placenta. After the sterilization of umbilical cord, the special bag attached with tube and needle is more useful to collect the blood disinfectively and abundantly than the syringe. Blood stem cells should be separated as soon as possible after collection, but umbilical blood may be stored in the refrigerator until 24 hours after the collection, if the immediate separation is impossible. There is the positive correlation between the collected blood volume or the preserved hours until the separating management and the separated stem cells. Hearing on the health of the child should be followed until 6 months after delivery.

The era of stem cell transplantation is now moving from PBSCT to umbilical cord blood transplantation. In compensate demerits of those therapies, another new way would be found for the next generation. For instance, utilization of more immature stem cells with multi potential hematopoietic activity, combined transplantation of functional lymphocytes like natural killer cells for prevention of GVHD, effective introduction of HSV-tk gene, standardization for cell processing, and establishment of international cooperative center for cell processing. We will be needed to consider the genome therapy in the next century.

A hematopoietic stem cell is considered to be one of the ideal targets for gene therapy, and there is expectation that gene therapy will be established based on the technology of hematopoietic stem cell transplantation. However, in recent clinical trials of stem cell gene therapy for monogenic diseases, significant clinical improvement has not been reported. One of the main obstacles is the low efficiency of gene transfer into hematopoietic stem cells. Many investigators have been trying to improve the transduction efficiency to the clinically applicable level. Another approach to solve this problem is to develop the method for selective expansion of transduced hematopoietic stem cells in vivo. We are currently developing novel regulatory genes (selective amplifier genes) for stem cell gene therapy.

A 35-year-old male had advanced nonseminomatous germ cell tumor (stage IIIC, embryonal cell carcinoma) which proved refractory to conventional PVB combined chemotherapy. He was then treated with an ultra high-dose chemotherapy consisting of carboplatin (1.5 g/m2) and etoposide (1.3 g/m2), followed by the transplantation of peripheral blood stem cells (PBSCT) with a total of 1.9 x 10(5)/kg granulocyte colony-forming cells (CFU-GM). Because he developed lung metastasis, escalated doses of carboplatin (2.0 g/m2), and etoposide (1.8 g/m2) combined with cyclophosphamide (7.0 g/m2) were given with peripheral blood stem cell transplant of 3.2 x 10(5)/kg CFU-GM. He has remained free of any recurrence without maintenance therapy.

A 65-year-old woman was admitted to our hospital with arthritis and agranulocytosis. She had given a diagnosis of acute myelogenous leukemia (FAB classification M1) a year ago and treated with 3 cycles of cytarabine and anthracycline or etoposide for 4 months, achieving complete remission state. Her bone marrow aspirate revealed normocellularity with normal karyotype (46, XX [20]) without apparent dysplastic feature at this time. She received autologous peripheral blood stem cell transplantation subsequently after conditioning regimen consisted of granulocyte colony-stimulating factor, busulfan, Ara-C and etoposide. Three months later, she started to manifest low grade fever, polyarthralgia and agranulocytosis and she admitted to our hospital after nine months. Bone marrow aspirate revealed marked hypocellularity with dysplastic features in three series of hematopoietic cells. Arthritis was dramatically improved after administration of prednisolone, but low granulocyte count continued. Bone marrow aspirate revealed karyotypic abnormality with monosomy 7 and we diagnosed her as myelodysplastic syndrome. Chemotherapy-induced myelodysplasia has been reported so far. This case would represent secondary myelodysplastic syndrome after chemotherapy. We could not clarify the etiology for polyarthritis but could be one of the paraneoplastic syndrome. We should note subsequent occurrence of myelodysplasia when planning treatment schedule.