Multicenter study on in vitro characterization of dendritic cells.

BACKGROUND: There is growing interest in the use of in vitro-expanded dendritic cells (DC) in cancer immunotherapy as cellular-based vaccines. However, the methods used for in vitro preparation vary widely between institutions. Therefore, a strong need exists for standardization, characterization and quality control (QC) of such vaccines. A first prospective multicenter pilot study was performed to investigate basic QC parameters of frozen/thawed DC. The study design was focused on comparison of test results for cell counts, immunophenotyping and cell viability. METHODS: CD14+ monocytes were isolated from three healthy volunteers. The cells were expanded in vitro, matured and cryopreserved using a standardized protocol in one laboratory. The aliquots of cryopreserved DC and a panel of reagents were shipped to eight laboratories worldwide. The objective was to compare the results of non-functional QC assays between sites by testing identical DC vaccines and using a pre-defined test protocol. RESULTS: Measurements of nucleated cell (NC) content of thawed DC vaccines with different types of hematology analyzers (HA) gave similar results for the majority of sites. Immunophenotyping using identical clones of monoclonal antibodies for the detection of surface antigens (i.e. CD1a, CD14, CD16, CD83, CD86 and HLA-DR) provided mostly comparable results between laboratories with an acceptable level of variation. In contrast, highly different results between study sites were generated for measuring the viability of thawed DC by flow cytometry using 7-amino-actinomycin D (7-AAD) dye exclusion. DISCUSSION: In characterizing frozen/thawed DC vaccines, NC counts generated by HA yielded similar results between different laboratories. Furthermore, immunophenotyping of DC vaccines can be standardized between centers, i.e. by using identical reagents. Because of highly variable results between laboratories, 7-AAD viability testing of thawed DC needs to be studied further to identify potential causes for the observed variability.

Multi-laboratory evaluation of procedures for reducing the volume of cord blood: influence on cell recoveries.

BACKGROUND: Various procedures can be used to isolate stem and progenitor cells from cord blood. This study evaluated the hydroxyethyl starch sedimentation (HES) with two centrifugation steps, and the top and bottom (T&B) isolation of buffy coat following a single centrifugation, and two filter systems for processing cord blood, one developed by Asahi Kasei Medical (filter A) and the second by Terumo (filter B). METHODS: Each of seven laboratories was randomly assigned the evaluation of either the HES or T&B method and one of the filter methods (n=8 cord blood units, per laboratory, for each method). The leukocyte-containing fraction with the stem/progenitor cells was recovered from the filters by reverse flushing. Utilizing the routine traditional processing and testing procedures of each laboratory, in vitro parameters were determined, with samples obtained after collection, after processing and after freezing/thawing. The results were expressed as the percentage recovery of viable cells in processed vs. collected samples (performance 1; PF1) and in thawed vs. processed samples (performance 2; PF2). The composite results obtained by the seven laboratories were summarized. RESULTS: The median PF1 percentage recovery of total nucleated cells (TNC) was comparable with both traditional methods (HES 79%, T&B 86%) and statistically reduced with both filtration procedures (filter A 58%, filter B 61%). Mononuclear cell (MNC) PF1 recovery was highest statistically with the T&B method (91%) and reduced on using filter A (77%) and filter B (70%) and the HES method (72%). CD34+ cell recovery was judged to be essentially comparable with the four methods, although the range of unit recoveries differed. The percentage recovery of TNC and MNC in PF1 was influenced by the volume of the collected cord blood, especially with use of the filtration procedures. This correlated with TNC content. A greater percentage of red cells and platelets was removed during processing with both filter methods. The time to process cord blood preparations with filter A was significantly shorter than the other methods. Processing with the HES method took the longest time. The recoveries for TNC, MNC and CD34+ cells in PF2 did not appear to be influenced by the specific processing procedure. DISCUSSION: These data indicate that filters that capture stem and progenitor cells may be an appropriate methodology for processing cord blood collected for banking.

High sensitivity of megakaryocytic progenitor cells contained in placental/umbilical cord blood to the stresses during cryopreservation.

In placental/umbilical cord blood (PCB) banking and PCB transplantation (PCBT), long-term cryopreservation of hematopoietic stem and progenitor cells is a unique requirement as compared to that for bone marrow transplantation and cytokine-mobilized peripheral blood transplantation. A long period of severe thrombocytopenia is a problem in many patients after PCBT. The object of this study was to define whether megakaryocytic progenitor cells (CFU-Meg), which produce platelets, are more sensitive to cryopreservation than the other myeloid progenitor cells in PCB. The leukocyte concentrates (LCs) obtained from clinical PCB banks were cryopreserved, and progenitor cell recoveries were determined by differential count of colony-forming cells (CFCs). The LCs were exposed to stresses which cells face during freezing, thawing, and washing out cryoprotectants. Most of the myeloid progenitor cells contained in the LCs showed good survival when cryopreserved at slow cooling rates, although cellular injury was observed at higher cooling rates and higher osmolalities. In contrast, the recovery rate of CFU-Meg was significantly lower than other progenitor cells, indicating a higher sensitivity to the various stresses they were exposed to during cryopreservation. Thrombocytopenia observed in patients receiving PCBT may be explained, at least in part, by the disappearance of CFU-Meg during cryopreservation.

Bcl-2, Bcl-xL and c-FLIP(L) potentially regulate the susceptibility of human peripheral blood monocyte-derived dendritic cells to cell death at different developmental stages.

We examined the susceptibility of human monocyte-derived dendritic cells (DCs) to spontaneous and CD95-mediated cell death at different developmental stages. Time course experiments revealed that the susceptibility of mature dendritic cells (mDCs) to spontaneous cell death was significantly lower than that of immature dendritic cells (iDCs) in a long-term culture under cytokine-free conditions, and the treatment with GM-CSF rescued these cells from spontaneous cell death at the late culture period. iDCs and mDCs expressed similar levels of CD95 whereas both cell types were relatively resistant to CD95-mediated cell death. Antigen (Ag)-specific and nonspecific cognate interaction with T cells failed to cause cell death of iDCs and mDCs. iDCs constitutively expressed transcripts and intracellular products of Bcl-2 and Bcl-xL, but not cellular FLICE-inhibitory protein(long (c-FLIP(L)), while the increased expressions of Bcl-2, Bcl-xL and c-FLIP(L) were observed in mDCs. These results suggest that the selective expressions of Bcl-2, Bcl-xL and c-FLIP(L) may be involved in the difference in the susceptibility to cell death between iDCs and mDCs.

Transient hematopoietic stem cell rescue using umbilical cord blood for a lethally irradiated nuclear accident victim.

We performed stem cell rescue and allogeneic skin transplantation on a lethally neutron-irradiated nuclear accident victim. HLA-DRB1 mismatched unrelated umbilical cord blood cells (2.08 x 10(7)/kg recipient body weight) were transplanted to an 8-10 Gy equivalent neutron-irradiated patient because of a lack of a suitable bone marrow or peripheral blood donor. Pre-transplant conditioning consisted of anti-thymocyte gamma-globulin alone, and GVHD prophylaxis was a combination of cyclosporine (CYA) and methylprednisolone (mPSL). Granulocyte colony-stimulating factor (G-CSF), erythropoietin (EPO), and thrombopoietin (TPO) were concurrently administered after transplantation. The absolute neutrophil count reached 0.5 x 10(9)/l on day 15, the reticulocyte count rose above 1% on day 23, and the platelet count was over 50 x 10(9)/l on day 27, respectively. Cytogenetic studies of blood and marrow showed donor/recipient mixed chimerism. Rapid autologous hematopoietic recovery was recognized after withdrawal of CYA and mPSL. Repeated pathological examinations of the skin revealed no evidence of acute GVHD. Eighty-two days after the irradiation, skin transplantation was performed to treat radiation burns. Almost 90% of the transplanted skin engrafted. Immunological examination after autologous hematopoietic recovery revealed an almost normal T cell count. However, immune functions were severely impaired. The patient died from infectious complication 210 days after the accident.

Enhancement of migratory and aggregate activities of human peripheral blood monocyte-derived dendritic cells by stimulation with RANTES.

We examined the effects of various chemokines on the functional activation of granulocyte-macrophage colony-stimulating factor (GM-CSF) plus interleukin-4 (IL-4)-generated human peripheral blood monocyte-derived immature dendritic cells (iDC). Stimulation of iDC with regulated on activation normal T cell expressed and secreted (RANTES) resulted in the promotion of their chemotactic migratory capacity in response to RANTES when compared with that of unstimulated cells. TNF-alpha induced a homotypic aggregated cluster formation of iDC in a dose-dependent manner, whereas the combination of TNF-alpha and RANTES exhibited more potent induction. IDC stimulated with RANTES were more efficient than unstimulated iDC in the production of endogenous RANTES. Treatment of iDC with the combination of TNF-alpha and RANTES was just little effective for the enhancement of allogeneic T-cell stimulatory capacity as compared with that of TNF-alpha treated iDC. These results suggest that endogenous secretions of RANTES from iDC stimulated with RANTES be potentially involved in RANTES-induced changes of properties with respect to morphology and function.

[Effects of glycosaminoglycans on the in vitro colony formation of CD34+ megakaryocytic progenitor cells in human placental/umbilical cord blood].

The in vitro effect of various glycosaminoglycans (GAGs) on the clonal growth of CD34+ megakaryocytic progenitor cells (CFU-Megs) isolated from human placental/umbilical cord blood (CB) was evaluated in human plasma containing semisolid culture stimulated by recombinant human thrombopoietin (TPO). The GAGs, including hyaluronic acid from human umbilical cords (HA-h), pig skins (HA-p) and rooster combs (HA-r), or keratan sulfate (KS), various chondroitin sulfates (CS-A, B, C, D, E), and heparan sulfate (HS), were tested. Each GAG alone did not affect the clonal growth of CFU-Meg. In the presence of TPO, adding of HA-p or HS (100 micrograms/ml) resulted in an approximately 1.3-fold increase, in the total number of colonies, due to an increase in large megakaryocyte colonies. In contrast, CS-E led to a marked decrease in CFU-Meg growth. At the end of the culture, the total number of cells increased 3.0-fold of the initial value of the control, but adding HA-p or HS showed an approximately 9.1-fold or 18.3-fold increase. Similarly, the total number of CFU-Meg detected in the harvested cells increased to 4.8-fold of the initial value, while, an approximately 18.3-fold or 38.8-fold increase was observed in the culture containing HA-p or HS, respectively. Flow cytometric analysis of the harvested cells showed no significant difference in the expression of surface antigens and DNA ploidy distribution of megakaryocytes between the control and GAG treatments. These results suggest that HA-p and HS promote the proliferation of immature CB CD34+ CFU-Meg in the presence of TPO.

Repopulating activities of human cord blood cells separated by a stem cell collection filter in NOD/SCID mice: a comparative study of filter method and HES method.

BACKGROUND: Volume reduction and removal of RBCs are essential for cost-efficient cord blood (CB) banking. It has previously been shown that a newly developed device, a stem cell-collection filter (SCCF), can reduce the CB volume and remove RBCs efficiently, giving high recovery rates for CD34+ cells, colony-forming cells, and long-term culture-initiating cells with short operation time. The aim of this study was to compare the quality of CB cells separated by SCCF and HES by analyzing repopulation in NOD/SCID mice. STUDY DESIGN AND METHODS: A total of 1 x 10(6) or 5 x 10(6) nucleated cells derived from SCCF- or HES-separated, cryopreserved, thawed, and washed CB were transplanted into NOD/SCID mice. Eight weeks after transplantation, bone marrow cells of the recipient mice were examined by flow cytometry and hematopoietic progenitor assay for the engraftment of human cells. RESULTS: Mice given human CB cells, separated by SCCF, showed degrees of engraftment similar to those in mice given HES-separated CB cells. There was no significant difference in the lymphohematopoietic reconstitution pattern in the two groups of mice. CONCLUSION: SCCF processing does not appear to reduce the number of repopulating cells in NOD/SCID mice or alter the number of HPCs. It is now shown that these cells can be captured by SCCF and removed, and that they will engraft.

Stem cell collection filter system for human placental/umbilical cord blood processing.

BACKGROUND AND OBJECTIVES: The hydroxyethyl starch method and the Top & Bottom method have been used worldwide for the volume reduction of human placental/umbilical cord blood (PCB) units. To simplify the preparation of nucleated cell (NC) concentrates, we developed a new filter device--the stem cell collection filter system (SCF SYSTEM)--which can collect mononuclear cells (MNC) at a high recovery rate. MATERIALS AND METHODS: The SCF SYSTEM consisted of a filter and two bags. Multilayered polyethylene terephthalate non-wovens, coated with a hydrophilic polymer, were used as filter media. PCB units were filtered by gravity (n = 12). Red blood cells, platelets and plasma were drained into the drain bag, and the NC trapped on the filter media was collected in the recovery bag by reverse washing. Recovered cell fractions were evaluated. RESULTS: The volume of cell concentrate recovered was 27.4 +/- 2.2 ml (mean +/- SD, n = 12). The whole time required for processing was less than 30 min, and handling was very simple. The viability of recovered NC was 97.8 +/- 3.2%. The recovery of lymphocytes, monocytes and granulocytes was 79.5 +/- 16.9%, 79.8 +/- 20.4% and 39.0 +/- 19.5%, respectively. The recovery rate of granulocytes was significantly lower than that of monocytes and lymphocytes (P < or = 0.0001). The recovery rates of CD3+ cells, CD19+ cells and CD56+ cells were almost the same as that of MNC. The recovery rates of CD34+ cells, total colony-forming cells and long-term culture-initiating cells were 81.7 +/- 27.0% (n = 11), 80.8 +/- 27.7% (n = 12) and 75.0 +/- 18.4% (n = 2), respectively. CONCLUSION: The new filter system was shown to be efficient for PCB processing, encompassing a very simple handling procedure with a good recovery of haematopoietic progenitor cells. Hence, the SCF SYSTEM is potentially useful for the volume reduction of PCB units for cord blood banking.

Expansion of megakaryocyte progenitors from cryopreserved leukocyte concentrates of human placental and umbilical cord blood in short-term liquid culture.

Long-term severe thrombocytopenia following human placental and umbilical cord blood (CB) transplantation is a significant clinical problem. We studied the ex vivo expansion of megakaryocytic progenitor cells (CFU-Meg) from cryopreserved/thawed leukocyte concentrates (LC) of CB prepared by the Tokyo Cord Blood Bank protocol. The LC cells were cultured in serum-free culture medium supplemented with a combination of early-acting cytokines including thrombopoietin (TPO), flt3-ligand (FL), and stem cell factor (SCF). Combination of TPO plus FL, TPO plus SCF, and all of these cytokines together resulted in 8.9-, 7.7-, and 8.4-fold increases in CFU-Meg, respectively, by Day 5 of culture. Our results showed that this simple expansion strategy has the potential for expanding CFU-Meg from cryopreserved/thawed LC cells from CB.

Effect of murine kidney extracts on the proliferation of hematopoietic progenitor cells in human umbilical cord blood.

We examined the effect of murine kidney extract (MKE) on the clonal growth of highly purified CD34+ hematopoietic progenitor cells from human umbilical cord blood. MKE did not affect the total number of colonies of erythroid burst-forming units (BFU-E), granulocyte-macrophage colony-forming units (CFU-GM) or granulocyte-erythroid-macrophage-megakaryocyte colony-forming units (CFU-Mix/CFU-GEMM) in a methylcellulose culture with exogenous recombinant human granulocyte colony-stimulating factor, granulocytemacrophage colony-stimulating factor, interleukin-3, stem cell factor and erythropoietin. MKE significantly increased the proportion of BFU-E- or CFU-Mix-derived colonies, and suppressed the formation CFU-GM-derived colonies depending on the MKE dose. However, because of an increase in small megakaryocyte colonies derived from mature CFU-Meg MKE increased by approximately 40% the growth of megakaryocyte colony-forming units (CFU-Meg) in plasma clot culture stimulated by recombinant human thrombopoietin. Also MKE promoted an increase in hyperploid megakaryocytes, suggesting that the active factor(s) in MKE acts on the mature CFU-Meg and promotes the maturation of megakaryocytes. Gel-filtration high performance liquid chromatography of MKE showed that the promoting factor(s) in MKE was approximately 45 kDa. These results indicate that the factor(s) detected in MKE influence human hematopoiesis in vitro, especially thrombopoiesis.

Dysfunctional regulation of the development of monocyte-derived dendritic cells in cancer patients.

Dendritic cells (DCs) are highly effective antigen (Ag)-presenting cells (APCs) that are required for the initiation of the immune response. DCs derived from cancer patients have been shown to be defective in several phenotypic and functional properties. However, little is known about the capacity of monocytes derived from cancer patients to differentiate into DCs. Herein, we examined the differentiation of monocyte-derived DCs in cancer patients. Flow cytometric analysis revealed that monocytes derived from cancer patients cultured with granulocyte-macrophage colony-stimulating factor (GM-CSF) plus interleukin-4 (IL-4) exhibited lower levels of CD11c, CD40, CD86, and HLA-DR expression as compared with those of monocyte-derived DCs from healthy volunteers. Furthermore, the capacities of DCs derived from cancer patients' monocytes to stimulate allogeneic T cell responses and to migrate in response to regulated-on-activation normal T cells expressed and secreted (RANTES) were impaired in comparison with those of monocyte-derived DCs from healthy volunteers. However, the two cell types had similar pinocytotic capacities for fluorescein isothiocyanate labeled-dextran (FITC-DX) and lucifer yellow (LY). These results suggest that monocytes from cancer patients may be defective in the capacity to develop into DCs.

IL-12 responsiveness and expression of IL-12 receptor in human peripheral blood monocyte-derived dendritic cells.

We analyzed the expression of IL-12Rbeta1 and IL-12Rbeta2 and the role of IL-12 in the activation of monocyte-derived dendritic cells (DCs) via IL-12Rbeta1-mediated signaling events. Flow cytometric analysis revealed that IL-12Rbeta1 was expressed in T cells, Con A blasts, and monocyte-derived DCs, but not in monocytes, while its transcript was detected in all of these cell types. Transcriptional expression of IL-12Rbeta2 was observed in T cells, Con A blasts, and monocyte-derived DCs, but not monocytes. The ligation of DCs as well as Con A blasts by IL-12 induced the production of GM-CSF, IL-1beta, IL-6, TNF-alpha, and IFN-gamma at the transcription levels. Furthermore, stimulation of DCs with IL-12 induced IL-12p40 transcript, but not IL-12p35 transcript, whereas this stimulation caused the expressions of both transcripts in Con A blasts. Stimulation of DCs with IL-12 caused a tyrosine phosphorylation of several intracellular proteins, and the pattern of these events were distinct from those of IL-12-stimulated Con A blasts. IL-12 also induced tyrosine phosphorylation of IL-12Rbeta1 as well as recruitment of several tyrosine-phosphorylated proteins to IL-12Rbeta1 in DCs and Con A blasts. Receptor engagement of DCs as well as Con A blasts by IL-12 resulted in activation of Janus kinase 2 and Tyk2 kinases and Stat3 and Stat4 transcription factors and the association of these proteins to IL-12Rbeta1. Stimulation with IL-12 caused a tyrosine phosphorylation and enzymatic activity of a family of mitogen-activated protein kinases, p38mapk. These results suggest that IL-12 acts directly on DCs to induce their functional activation via IL-12Rbeta1-mediated signaling events.

Autocrine activation-induced cell death of T cells by human peripheral blood monocyte-derived CD4+ dendritic cells.

Mature T cells activated by antigen (Ag)-presenting cells are subject to various downmodulatory processes designed to maintain T cell homeostasis. Here we describe experiments in which mature T cells were subjected to apoptosis following stimulation with CD4(+) dendritic cells (DCs) during Ag presentation. The proliferative response of allogeneic T cells was increased by DCs at stimulator to responder (S/R) ratios ranging from 10(-3) to 1, whereas this response was decreased at S/R ratios ranging from 2 to 10. Allogeneic T cells stimulated with DCs at an S/R ratio of 5 underwent apoptosis, whereas this event was not observed in allogeneic T cells stimulated with DCs at an S/R ratio of 0.5. Stimulation of T cells with DCs at an S/R ratio of 5 induced a higher level of expression of CD95 ligand (CD95L) than stimulation of T cells cultured with DCs at an S/R ratio of 0.5, whereas similar levels of expression of CD28 and CD154 were observed in both cells. The abortive proliferation of mature T cells stimulated with DCs was prevented by blocking the CD95-CD95L system. Our results suggest that the CD4(+) DCs play counterregulatory roles in dictating T cell responses during Ag presentation.

TGF-beta 1 reciprocally controls chemotaxis of human peripheral blood monocyte-derived dendritic cells via chemokine receptors.

We examined the effect of TGF-beta 1 on the chemotactic migratory ability of human monocyte-derived dendritic cells (DCs). Treatment of immature DCs with TGF-beta 1 resulted in increased expressions of CCR-1, CCR-3, CCR-5, CCR-6, and CXC chemokine receptor-4 (CXCR-4), which were concomitant with enhanced chemotactic migratory responses to their ligands, RANTES (for CCR-1, CCR-3, and CCR-5), macrophage-inflammatory protein-3 alpha (MIP-3 alpha) (for CCR-6), or stromal cell-derived growth factor-1 alpha (for CXCR-4). Ligation by TNF-alpha resulted in down-modulation of cell surface expressions of CCR-1, CCR-3, CCR-5, CCR-6, and CXCR-4, and the chemotaxis for RANTES, MIP-3 alpha, and stromal cell-derived growth factor-1 alpha, whereas this stimulation up-regulated the expression of CCR-7 and the chemotactic ability for MIP-3beta. Stimulation of mature DCs with TGF-beta 1 also enhanced TNF-alpha-induced down-regulation of the expressions of CCR-1, CCR-3, CCR-5, CCR-6, and CXCR-4, and chemotaxis to their respective ligands, while this stimulation suppressed TNF-alpha-induced expression of CCR-7 and chemotactic migratory ability to MIP-3 beta. Our findings suggest that TGF-beta 1 reversibly regulates chemotaxis of DCs via regulation of chemokine receptor expression.

Radiation sensitivity of megakaryocyte colony-forming cells in human placental and umbilical cord blood.

The in vitro radiation sensitivity of CFU-Meg isolated from human placental and umbilical cord blood was evaluated in plasma clot cultures stimulated by recombinant human cytokines, including thrombopoietin, the FLT3 ligand (FLT3LG), interleukin-3, interleukin-11 and stem cell factor. The CD34(+) cells were irradiated with X rays at a dose rate of 73 cGy/ min. The megakaryocyte colonies were identified by using an FITC-conjugated antibody to glycoprotein IIbIIIa and were classified into two groups based on colony size: large colonies (immature CFU-Meg) and small colonies (mature CFU-Meg). Treatment with thrombopoietin alone or in combination with FLT3LG and/or interleukin-11 gave exponential radiation survival curves (D(0) for immature CFU-Meg = 56-77 cGy, D(0) for mature CFU-Meg = 86 cGy-1.12 Gy), while marked shoulders were observed on the survival curves for colonies supported by the combination of thrombopoietin, interleukin-3 and stem cell factor (D(0) for immature CFU-Meg = 89- 98 cGy; D(0) for mature CFU-Meg = 1. 25-1.31 Gy). Our results showed that the immature CFU-Meg were more radiosensitive than the mature CFU-Meg and that the combination of cytokines, including thrombopoietin, interleukin-3 and stem cell factor, affected the radiation sensitivity of CFU-Meg to the same extent as with thrombopoietin alone or in combination with FLT3LG and/or interleukin-11.

In vitro generation of dendritic cells derived from cryopreserved CD34+ cells mobilized into peripheral blood in lymphoma patients.

BACKGROUND: Dendritic cells (DC) are APC that initiate primary T-cell dependent immune responses. They have been shown to be generated from CD34+ cells in BM, placental/umbilical cord blood (CB), and G-CSF mobilized peripheral blood CD34+ cells (PBSC). In recent clinical studies, DC were used as a vaccine for cancer patients and showed induction of their antitumor effects. Cryopreservation of CD34+ cells is important to extend the availability of cellular therapy with DC. However, little is known about the effect of cryopreservation on the functional maturation of DC. METHODS: PBSC harvested from lymphoma patients mobilized with G-CSF and undergoing leukapheresis were cryopreserved at -135 degrees C for 3 days. Freshly isolated or cryopreserved PBSC were cultured with GM-CSF/SCF/tumor necrosis factor-alpha (TNF-alpha). After 14 days of culture, DC were harvested, washed, and used for phenotypical and functional analysis. RESULTS: Cryopreserved PBSC, as well as freshly-isolated PBSC cultured for 14 days, gave rise to CD1a+ /CD4+ /CD11c+ /CD14low+ /CD25( -)/CD40+ / CD45RO+/CD80+/CD83+/CD86+/HLA-DR+ cells with dendritic morphology. DC derived from cryopreserved PBSC mobilized with G-CSF showed a similar endocytic capacity and chemotactic migratory capacities when compared with DC derived from freshly-isolated G-CSF mobilized PBSC. These DC also exhibited similar capacities in the primary allogeneic T-cell response. DISCUSSION: These results indicate that cryopreserved G-CSF mobilized PBSC cultured with GM-CSF/SCF/TNF-alpha gave rise to DC that were morphologically, phenotypically and functionally similar to DC derived from fresh G-CSF mobilized PBSC. The observation indicates the clinical usefulness of cryopreserved CD34+ cells from lymphoma patients.

[Current status of cord blood banks in the world].

Since the first successful transplantation of placental/umbilical cord blood (PCB) in 1988 at St. Louis Hospital in Paris, the effort has been made to establish large scale cord blood banks for allogeneic transplantation around the world. Recent reports from New York Blood Center which stocks 9000 units and shipped 700 units to US and other countries indicates that PCB transplantation is as effective as bone marrow transplantation. There are many cord blood banks in the world now and the number of PCB units stocked in the world are increasing rapidly. The methods for collection and processing of PCB, and test items for screening for viral infections, etc. differ greatly among banks. Therefore, the quality of PCB units stocked worldwide has become an important issue and some major banks are trying to introduce GMP or ISO system for the quality assurance of PCB. In this chapter, we introduce the activities of major cord blood banks in the world. Guidelines or regulation for hematopoietic stem cell processing including PCB has been enacted in some countries though the situation of support from their governments differ. International registry for PCB transplantation have been developed by EUROCORD. The international network of cord blood banks, NETCORD, has been established recently with major banks in Europe, United States and Japan to ensure the rapid supply of safe and efficient PCB units to the patients throughout the world. Obtaining information of international activities of cord blood banks in the world and cooperation with them should bring the National Cord Blood Bank in Japan into concordance with a developing world standards.

Immunological reconstitution after cord blood transplantation for an adult patient.

We transplanted 4.1x10(7) unrelated umbilical cord blood cells into a 27-year-old patient suffering from transformed acute myelocytic leukemia. The thawing method was the same as described by Rubinstein et al (Proc. Natl. Acad. Sci. USA 1995; 92: 10119-10122). ANC reached over 500x10(9)/l on day 19, and the patient was free from RBC and platelet transfusion on days 26 and 38, respectively. Cytogenetic and molecular analysis after transplant revealed complete chimerism. The CD3+CD4+ lymphocyte count became greater than 100x10(9)/l at 5 weeks after transplantation. The CD3+CD8+ count became greater than 500x10(9)/l at 7 weeks and thereafter progressively increased in spite of administration of CYA. This immunological reconstitution pattern after umbilical cord blood transplantation was different from that after bone marrow transplantation, and resistance of CD3+CD8+ lymphocytes to CYA was the distinguishing characteristic. The rapid hematological recovery and immunological reconstitution may be attributed to the high dose of transfused nucleated cells and umbilical cord blood transplantation may become a promising method for treatment for such cases.