Elevated expression of DNMT1 is associated with increased expansion and proliferation of hematopoietic stem cells co-cultured with human MSCs

BACKGROUND: Mesenchymal stem cells (MSCs) play an important role in hematopoietic stem cell (HSC) maintenance, proliferation, and apoptosis. DNA methyltransferase 1 (DNMT1) is considered an essential factor in the maintenance of HSCs in mammalian cells. Therefore, this study was conducted to evaluate the mRNA expression level of DNMT1 during cord blood (CB)-HSC ex vivo expansion with MSCs. METHODS: Ex vivo cultures of CB-HSCs were performed in three culture conditions for 7 days: cytokines, cytokines with MSCs, and only MSCs. Total and viable cell numbers were counted after 5 and 7 days using trypan blue stain, and the stem cell percentage was then evaluated by flow cytometry. Moreover, in vitro colony-forming unit assay was carried out to detect clonogenic potential of HSCs at days 0 and 7 using MethoCult H4434. Finally, DNMT1 mRNA expression level was evaluated by real-time polymerase chain reaction. RESULTS: Maximum CB-CD34⁺ cell expansion was observed on day 7 in all the three cultures. After 7 days, ex vivo expansion of CB-CD34⁺ cells indicated a significant decrease in DNMT1 expression in the cytokine cultures, whereas in the two co-culture conditions DNMT1 expression was increased. A significant difference between the number of CD34⁺ and CD34⁻ cells in the cytokine co-culture system was observed. CONCLUSION: These data indicated that an elevated expression of DNMT1 is associated with increased expansion and proliferation of HSCs co-cultured with human MSCs. Hence, DNMT1 may be a potential factor in the maintenance of expanded HSCs co-cultured with human MSCs.

Ex vivo Expansion of Human Natural Killer Cells from Blood Retained in a Disposable Platelet Apheresis Set / 대한수혈학회지

BACKGROUND: Natural killer cells expanded from human peripheral blood (PB) have been used in cancer immunotherapy research. Although most research teams have access to human PB, it is necessary to find a source of blood that can be easily obtained. We have tested the possibility of using blood retained in a disposable platelet apheresis set as an alternative source, with special interest in expansion of NK cells for use in cancer immunotherapy research. METHODS: For expansion of NK cells, peripheral blood mononuclear cells (PBMCs) were isolated from an MCS+ platelet apheresis kit (Haemonetics, Braintree, USA) and PB from the same donor (n=7) and co-cultured with 100-Gy gamma ray-irradiated K562 cells expressing the 4-1BB ligand and membrane-bound IL-15 for three weeks in RPMI1640 medium in the presence of IL-2 and IL-15. Cytotoxicity was measured using WST-1 at 1:1, 2:1, and 4:1 effector-to-target (E:T) ratios for a period of four hours. RESULTS: Mean rate of expansion of NK cells was 1,097-fold and their purity was 94.4% from blood retained in a disposable platelet apheresis set; mean rate of expansion of NK cells was 953-fold and their purity was 92.0% from PB after a period of three weeks. No differences in cytotoxicity against K562, 697, Raji, and RPMI8226 were observed between NK cells expanded from two blood sources. CONCLUSION: Blood retained in a disposable platelet apheresis set is a useful and convenient source for expansion of NK cells for use in cancer immunotherapy research.

The Cross-talk between ROS and p38MAPKα in the Ex Vivo Expanded Human Umbilical Cord Blood CD133+ Cells / 华中科技大学学报（医学）（英德文版）

This study investigated the correlation between and compared the effects of reactive oxygen species (ROS) and p38 mitogen-activated protein kinase a (p38MAPKα) in the ex vivo expanded umbilical cord blood (hUCB) CD133+ cells.hUCB CD133+ cells were cultured in the hematopoietic stem cells (HSCs) culture medium with N-acetylcysteine (NAC,an anti-oxidant),p38MAPKα-specific inhibitor (SB203580) or their combination.The levels of ROS and expression of phosphorylated p38MAPKα (p-p38) in CD133+ cells were flow cytometrically detected.The efficacy of ex vivo expansion was evaluated by the density of CD 133+ cell sub-group colony-forming cells (CFC) and cobblestone area-forming cells (CAFC) assay.Our results showed decreased ROS levels in NAC,SB203580,and their combination treatment groups were almost 37％,48％,and 85％,respectively.Furthermore,SB203580 abrogated the activation of p38MAPKα more obviously than NAC.Moreover,the CD133+ cells in SB203580 treatment group had a 21.93±1.36-fold increase,and 14.50±1.19-fold increase in NAC treatment group,but only 10.13±0.57-fold increase in control group.In addition,SB203580 treatment led a higher level increase in the number of CFU and CAFC than NAC did.These findings suggested that,in expanded CD133+ cells,ROS activates p38MAPKα,which,in turn,induces ROS production,and p38MAPKα might be the most suitable regulator in ROS- p38MAPKα pathway for the promotion ofHSCs ex vivo expansion.

Proliferation of CD4(+)CD25(high+)Foxp3(+) regulatory T lymphocytes in ex vivo expanded ascitic fluid from primary and recurrent ovarian carcinoma / 부인종양

OBJECTIVE: Regulatory T lymphocytes evoke the immune tolerance by suppressing and inactivating cytotoxic T lymphocytes. The objective of this study was to compare the proportion of regulatory T lymphocytes, precisely defined as CD4(+)CD25(high+)Foxp3(+) T lymphocytes, in primary and recurrent ovarian carcinoma before and after ex vivo expansion of ascites with interleukin-2 (IL-2). METHODS: Ascitic fluid samples were obtained from 26 patients with ovarian carcinoma. Lymphocytes were isolated from ascites and cell markers were analyzed by flow cytometry using anti-CD3/CD4/CD8/CD16/CD56/CD25 and anti-Foxp3 antibodies. Lymphocytes were incubated for 2 to 3 weeks and expanded ex vivo by IL-2 stimulation and their phenotypes were analyzed by flow cytometry. RESULTS: Following ex vivo expansion, ascitic fluid lymphocytes increased by a greater extent in the recurrent group than in the primary group. The proportion of ex vivo-expanded lymphocytes changed as follows; CD4(+) T lymphocytes increased, CD8(+) T lymphocytes decreased, and the proportion of CD3(-)CD16(+)56(+) NK cells was unchanged. The proportion of CD4(+)CD25(high+)Foxp3(+) regulatory T lymphocytes in CD4(+) T lymphocytes increased after ex vivo expansion in both groups, but to a greater degree in the recurrent group. CONCLUSION: This study showed that regulatory T lymphocytes, neither cytotoxic T lymphocytes nor NK cells, were extensively increased after ex vivo expansion, especially in recurrent ovarian carcinoma. These results may provide information that helps to guide the future development of adoptive immunotherapy against ovarian carcinoma.

In vitro Expansion of Natural Regulatory T Lymphocytes Useful for Cell Therapy in Allotransplantation

Immunotherapy with regulatory T lymphocytes is considered to be an attractive new therapeutic modality to prevent allograft rejection. The success of this new therapy is critically dependent on the preparation of highly effective and enough number of regulatory T cells. Here, we tried to establish a proper strategy for the ex vivo expansion of regulatory T cells and evaluated their characteristics. CD4+CD25h+CD62L+ T cells were isolated from the recipient mice and weekly stimulated with various stimuli in the presence of IL-2. The most efficient protocol for the expansion of regulatory T cells maintaining Foxp3 expression and regulatory activity was the three cycles stimulation with donor bone marrow-derived dendritic cells (BM-DCs) which yielded around 400 fold expansion of regulatory T cells. The in vitro-expanded regulatory T cells expressing lymph node homing receptors on their cell surface, were composed of polyclonal population, and did not acquire the ability to produce effector cytokines. Importantly, these expanded regulatory T cells induced a modest prolongation of skin allograft survival when combined with transient T cell depletion in recipient mice. These data indicate that our protocol could be used to obtain an effective population of natural regulatory T cells available for the regulatory T cell therapy to prevent allograft rejection.

Clinical Grade of Gerneration of Dendritic Cells for Immunotherapy / 华中科技大学学报（医学）（英德文版）

In order to develop a protocol for clinical grade generation of dendritic cells (DCs) for cancer immumotherapy, aphereses were performed with the continuous flow cell separator and materials were derived from 10 leukemia patients that had achieved complete remission. Peripheral blood monocytes were cultured in vitro with GM-CSF, IL-4 for 6 days, then TNF-α (the TNF-α group) or TNF-α, IL-1β, IL-6, PGE2 (the cytokine mixture group) were added to promote maturation. Cell number was counted by hematology analyzer, and phenotype study (CD1a, CD14, CD83) was carried out by flow cytometry, and the function of DCs was examined by mixed lymphocyte reaction. The results showed that (0.70±0.13)×107/mL (the TNF-α group) and (0.79±0.04)×107/mL (the cytokine mixture group) DCs were generated respectively in peripheral blood obtained by leucapheresis. The phenotypes were as follows: CD1a+ (74.65±4.45)%, CD83+(39.50±4.16)%, CD14+(2.90±1.76)% in TNF-α group, and CD1a+ (81.86±5.87)%, CD83+ (81.65±6.36)%, CD14+ (2.46±1.68)% in the cytokine mixture group. It was concluded that leucapheresis may be a feasible way to provide large number of peripheral blood monocytes for DC generation, and combined administration of TNF-α, IL-1β,IL-6, and PGE2 may greatly promote maturity.

The Effect of Leptin on the Proliferation of the Endothelial Progenitor Cells from Peripheral Blood / 대한수혈학회지

BACKGROUND: Leptin has been found to be involved in the regulation of hematopoiesis processes and angiogenesis. Therefore, we investigated the effect of leptin in the proliferation and angiogenesis of peripheral blood (PB)-derived endothelial progenitor cells (EPCs). METHODS: Mononuclear cells were isolated from PB of healthy male volunteers and were cultured in endothelial cell growth medium-2 (EGM-2). After 6 days of culture, cells were treated with 50 ng/mL vascular endothelial growth factor (VEGF) and/or with various concentrations of leptin (10 ng/mL, 100 ng/mL, 1microgram/mL, and 10 microgram/mL) and were further cultured for one week. Proliferation of EPCs was examined by an assay measuring the uptake of 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbo cyanine-labeled acetylated LDL (Dil-ac-LDL) and tubule formation on a matrigel. The control group of cells was not treated with VEGF and/or leptin. RESULTS: The number of Dil-ac-LDL labeled-EPCs, tubule formation on matrigel and the number of cells present along tubules were significantly increased in the leptin-treated groups of cells as compared to the control group or VEGF treated group of cells (P<.05). The effect was synergistically increased in the group of cells co-treated with leptin and VEGF. The number of EPCs was increased in a leptin dose-dependent manner that was maximal at a concentration of 1microgram/mL leptin. CONCLUSION: This study shows that leptin increased in vitro proliferation and angiogenesis of EPCs derived from peripheral blood.

Immunological evaluation of ex vivo expanded umbilical cord blood T lymphocytes / 부인종양

OBJECTIVE: The objectives of this study were, first to characterize the immunological differences of the T lymphocytes of umbilical cord blood (UCB) compared to those of adult; second to expand the T lymphocytes in ex vivo condition of a short term culture, and to assess the immunological function of the expanded T lymphocytes. METHODS: The immunophenotypic study of 40 UCB and 10 adult peripheral blood (PB) was performed. The fresh UCB mononuclear cells (MNCs) were isolated. The nonadherent MNC fractions were then cultured with the anti-CD3 antibody with or without Loranthus yadoriki (10 microgram/ml). The MNCs were cultured in the anti-CD3 antibody-coated flasks for 4 days, and then transferred to the non-coated flasks, which were added IL-2 175 U/ml and cultured for another 10 days. Proliferative ability of UCB T lymphocytes, cell surface markers, and cytotoxicity assays of the expanded T lymphocytes were performed. RESULTS: Cell surface markers of the UCB T lymphocytes were different from those of adult T cells. The UCB T lymphocytes cultured with the anti-CD3 antibody 100 ng/ml showed a significant increase in the proliferative ability (p<0.05). After culture in the anti-CD3 antibody coated flask with IL-2, expression of the activated T lymphocytes were increased significantly. The cultured cells exhibited substantial killing activity on the SK-OV-3 target cells compared to the fresh UCB lymphocytes. CONCLUSION: The ex vivo combination of the anti-CD3 antibody and IL-2 significantly enhanced proliferation, activation, and maturation of the UCB T lymphocytes. Moreover cytotoxic potential of expanded UCB T cells was observed.

Inverse Tendency between Ex Vivo Expansion Potential of Hematopoietic Progenitors and Time to Engraftment after Hematopoietic Stem Cell Transplantation / 대한진단검사의학회지

BACKGROUND: The CD34+ cell dose and infused number of committed progenitor cells in transplantation are important factors in hematologic engraftment. However, the relationship between expansion potential of progenitor cells and hematologic engraftment remains controversial. We evaluated whether expansion potential of progenitor cells is a predictive factor of post-transplantation hematologic engraftment. METHODS: Mononuclear cells isolated from mobilized peripheral blood and bone marrow were cultured with cytokine cocktail for 7 days. Progenitor cells and committed progenitors were analyzed using stem cell markers (CD34 and CD133) and lineage specific markers. Hematologic engraftment was defined as neutrophil counts over 500/microliter and platelet counts over 20,000/microliter without transfusion. Acute and chronic graft-versus-host disease (GVHD) were investigated. RESULTS: There was inverse tendency between the number and fold expansion of progenitor cells or committed (granulocytic or megakaryocytic) progenitors and time to engraftment. Especially, fold expansion of CD34(+)/CD33(+) cells was significantly correlated with time to neutrophil engraftment in bone marrow transplantation (r=-0.56, P=0.04). The infused number and fold expansion of lymphoid progenitors were not related to the occurrence of acute or chronic GVHD. CONCLUSIONS: We could not prove that expansion potential of progenitor cells and committed progenitor cells is correlated to hematologic engraftment although there is a correlation between CD34(+)/ CD33(+) cells and time to neutrophil engraftment. But, a further study on the value of expansion potential is required because there is an inverse tendency.

Characterization of regulatory T cells in ex vivo expansion of human umbilical cord blood / 대한산부인과학회지

OBJECTIVE: Regulatory T cells, which expressing CD4 and CD25, have a crucial role in suppressing immune systems to self-antigens and preventing autoimmune diseases. This study aims to evaluate the role of regulatory T cells in maternal tolerance to the fetus by comparing the proportion of regulatory T cells in peripheral blood of pregnant and non-pregnant women with those in umbilical cord blood. Also we analyze the changes of proportions of regulatory T cells of umbilical cord blood according to ex vivo expansion. METHODS: An immunophenotypic study on 10 peripheral blood of pregnant and non-pregnant women and 10 cord blood was performed by means of FACSort flow cytometry using anti-CD4, anti-CD25 and anti-CD152 antibodies. Fresh cord blood mononuclear cells (MNCs) were isolated by Ficoll-Hypaque density centrifugation. The MNCs were cultured in anti-CD3 Ab-coated flasks for 4 days. The cells were then transferred to non-coated flasks with IL-2 175 U/mL and were cultured for another 17 days. The expression of CD4, CD25, CD152 and FoxP3 PCR were analyzed in accordance with days in culture. We also performed FoxP3 PCR in isolated CD25+ and CD25- T cells using MACSTM kit. RESULTS: Umbilical cord blood had a higher proportion of CD4+CD25++ regulatory T cells than adults, and term-pregnant women had a lower proportion than non-pregnant women (p<0.05). After ex vivo expansion in anti-CD3 Ab coated flask with IL-2, we observed a significantly increased expression of CD4, CD25, CD152 at 4th day after culture and decrease thereafter. The result was the same as the expression of FoxP3 PCR. CONCLUSION: Umbilical cord blood contained a high proportion of CD4+CD25++ regulatory T cells and regulatory T cells increased on culture day 4 and declined thereafter. Umbilical cord blood may serve as a readily available source of regulatory T cells for immunotherapy.

Effect of Angiotensin Ⅱ on Cord Blood CD34+ Cells Expansion in vitro / 华中科技大学学报（医学）（英德文版）

In order to investigate the influence of angiotensin Ⅱ on hematopoietic system, CD34+cells in cord blood were purified, and the effects of angiotensin Ⅱ in combination with various cytokines on their growth and differentiation were studied by cell culture in vitro. It was found that angiotensin Ⅱ in suspending medium could stimulate both BFU-E and CFU-GM expansion. The number of BFU-E and CFU-GM was increased with the increases of angiotensin Ⅱ concentrations during a certain range. In addition, the expansion fold of CFU-GM was increased from 2.3±0.8 times to 7.8±2.3 times when angiotensin Ⅱ was added in the presence of SCF+G-CSF+GM-CSF+IL-3 cytokines mixture. Similarly, the expansion fold of BFU-E was increased from 3.1 ±1.8 times to 9.2±2.3 times with angiotensin Ⅱ in the presence of SCF+EPO+TPO+IL-3. In the semi-solid medium, angiotensin Ⅱ could stimulate CFU-GM expansion but had no effect on the growth of BFU-E. In conclusion, angiotensin Ⅱ had some stimulating effects on cord blood hematopoietic progenitors expansion in vitro in the presence of other cytokines.

Ex vivo Expansion and Clonal Maintenance of CD34+ Selected Cells from Cord Blood and Peripheral Blood / 소아과

PURPOSE: Because of the unavailability of marrow transplantation, umbilical cord blood (CB) is increasingly being used. We evaluated the potential of ex vivo expansion and clonality in CD34+ cells separated from cord blood source and mobilized peripheral blood (PB) in a serum-free media. METHODS: The CD34+ cells, selected from CB and mobilized PB, were expanded with hematopoietic growth factors. They were then cultured for burst-forming units of erythrocytes (BFU-E), colony-forming units of granulocytes and monocytes (CFU-GM) and colony-forming units of megakaryocytes (CFU-Mk) at culture days 0, day 4, day 7, and day 14 with various growth factors. RESULTS: The CB-selected CD34+ cells showed significantly higher total cell expansion than those from the PB at day 7 (2 fold increase than PB). The CB-selected CD34+ cells produced more BFU-E colonies than did the PB on culture at days 7 and at day 14. Also, the CB-selected CD34+ cells produced more CFU-Mk colonies than did the PB on culture at day 4 and at day 7. CONCLUSION: The ex vivo expansion of the CB cells may be promising in producing total cellular expansion, CFU-Mk and BFU-E compared with PB for 7 to 14 days. The growth factors combination including megakaryocyte growth and development, flt3-ligand and interleukin-3 showed more expansion in the view of total cells and clonal maintenance compared with less combination.

Ex vivo expansion of umbilical cord blood-derived T-lymphocyte with autologous cord blood plasma / 대한산부인과학회지

OBJECTIVE: The objective of this study was to establish a clinically applicable culture system by investigating the use of autologous cord blood plasma (ACBP) instead of fetal bovine serum (FBS) for the ex vivo expansion of umbilical cord blood (UCB) T-lymphocytes. METHODS: Fresh UCB mononuclear cell (MNC) fractions were isolated by Ficoll-Hypaque density centrifugation. The nonadherent MNC fractions were then cultured with the anti-CD3 antibody 5 microgram/mL plus IL-2 175 U/mL in the presence of 10% FBS, 10% ACBP or homologous cord blood plasma (HCBP). On day 8, proliferation rate, cell surface markers, cytotoxic assay of UCB T-lymphocytes according to the medium supplemented with FBS, ACBP or HCBP were evaluated. RESULTS: Proliferation studies demonstrated a significant increase in the proliferative ability of UCB T-lymphocytes incubated in anti- CD3 and IL-2 irrespective of the medium supplemented with FBS or ACBP. In the FBS supplemented medium, expressions of the activated T-lymphocytes were increased significantly after culture: CD3+CD8+, CD3+CD25+, CD3+CD38+, and CD45RO+ (p<0.05). Also in the ACBP supplemented medium, expressions of the activated T-lymphocytes were increased significantly after culture: CD3+ CD8+, CD3+CD25+, and CD45RO+ (p<0.05). In the HCBP supplemented medium, expressions of the activated T-lymphocytes were increased significantly after culture as in the ACBP: CD3+CD8+, CD3+CD25+, and CD45RO+ (p<0.05). Of the activated T-lymphocytes, increase of cytotoxic CD3+CD8+ cells increased significantly in the ACBP and HCBP groups compared to FBS group (p<0.05). CONCLUSION: These findings support the feasibility of ex vivo expansion of umbilical cord blood T-lymphocytes in the medium supplemented with autologous cord blood plasma, instead of fetal bovine serum, for future adoptive cellular immunotherapy.

Preparation and Characterization of SDF-1alpha Recombinant Adenovirus for ex vivo Expansion of Hematopoietic Stem Cells

Stromal cell-derived factor 1 (SDF-1/CXCL12) is a multifunctional cytokine implicated in normal hematopoiesis. We previously reported that SDF-1alpha enhanced the survival of hematopoietic stem and progenitor cells in synergy with other cytokine such as GM-CSF, steel factor, or thrombopoietin. As adult stem cells are very rare, many investigators are trying to expand hematopoietic stem/progenitor cells in vitro. In this study, we constructed an adenoviral vector and produced high titer of recombinant adenoviruses directing robust expression of SDF-1alpha determined by ELISA. We also produced control empty adenoviruses and recombinant LacZ adenoviruses. In order to check the feasibility of SDF-1alpha in ex vivo expansion system, we compared HUVEC cells tranduced by a SDF-1alpha recombinant virus with HUVEC cells transduced by a LacZ recombinant virus in supporting activity of hematopoietic cells, and found that expression of SDF-1alpha in HUVEC cells increased viable blood cell population obtained from the same number of CD34+ cells. The SDF-1alpha recombinant adenovirus seems to be useful for future application in hematopoiesis studies.

The Expression of Fas antigen and Bax and Apoptosis in Ex Vivo Expanded Hematopoietic Progenitor Cells / 대한혈액학회지

BACKGROUND: During ex vivo expansion of cord blood (CB) CD34+ cells, differentiation of the expanded cells happened and hematopoietic potential of the progenitor cells decreased. In this study, we evaluate the effect of the expression of Fas antigen, Bcl-2, and Bax on CD34+ or AC133+ hematopoietic progenitor cells during ex vivo expansion. METHODS: CD34+ and AC133+ cells isolated from human CB were cultured in serum free medium supplemented with several cytokines for 7 days. After expansion culture, we re isolated CD34+ and AC133+ cells and compared the numbers of granulocyte-macrophage colony-forming units (CFU-GM) and granulocyte, erythrocyte, monocyte, and macrophage colony-forming units (CFU-GEMM), and expression of Fas antigen, Bcl-2, and Bax with unexpanded cells. RESULTS: CFU-GM was expanded 23.94 fold in CD34+ cells and 15.22 fold in AC133+ cells at day 7 of culture but CFU-GEMM was not expanded. The expression of Fas antigen and Bax was 7.44% and 2.75%, respectively, in fresh isolated CD34+ cells and increased to 19.71 % and 33.67%, respectively, in expanded CD34+ cells at day 7 culture, but Bcl-2 was not changed. In case of AC133+ cells, the expression of Fas antigen and Bax were also increased from 5.87% and 6.19% to 24.85% and 22.83%, respectively, and Bcl-2 was slightly decreased. Apoptosis was not changed in CD34+ cells and AC133+ cells during ex vivo expansion. CONCLUSION: These results indicate that the nature of expansion was similar between CD34+ and AC133+ cells, and expression of Fas antigen and Bax increased on CD34+ and AC133+ cells during ex vivo expansion. Selection of the expanded progenitor cells without apoptosis may be useful for the hematopoietic stem cell transplantation.

Ex vivo Expansion of Hematopoietic Progenitor Cells in Co-culture of Cord Blood CD34+Cells with Human Umbilical Vein Endothelial Cells / 대한혈액학회지

BACKGROUND: We examined an ex vivo expansion system for cord blood (CB) hematopoietic progenitor cells, which is based upon a co-culture of CD34+cells with human umbilical endothelial cells (HUVECs) in the presence of stromal cell-derived factor-1 (SDF-1) and hematopoietic growth factors. METHODS: Cord blood CD34+cells were either incubated a liquid suspension culture or co-cultured on HUVEC monolayers with hematopoietic growth factors in the presence or absence of SDF-1. After 7~14 days of culture, cells were harvested and analyzed for fold-increase in nucleated cells, CD34+ cells, and colony-forming cells (CFCs) and apoptosis. RESULTS: Seven-day suspension culture of CD34+ cells in the presence of a cytokine combination consisting of throbmopoietin, flk-2 ligand, and kit-ligand (TFK) led to a 43-fold increase of nucleated cells, a 19-fold increase of CD34+ cells, and 14-fold increase of CFCs, respectively. The addition of SDF-1 to TFK slightly further increased this expansion. A co-culture of CD34+ cells with HUVECs significantly enhanced the expansion of both CD34+ cells and CFCs compared with a liquid suspension culture. This was further increased by the addition of SDF-1. A co-culture of CD34+ cells on HUVECs transfected with null-adenoviral vector led to a better fold increase of haemtopoietic progenitor cells compared with a culture with non-transfected HUVECs. Adding SDF-1 to the co-culture diminished the annexn V-positive cells both in the supernatant and adherent cell layers. CONCLUSION: A co-culture of cord blood cells with HUVECs in the presence of hematopoietic growth factors and SDF-1 could be a new model for the efficient expansion of hematopoietic progenitors.

Ex vivo Expansion of Hematopoietic Cells and Amifostine Effects / 대한혈액학회지

BACKGROUND: The possibility of cord blood transplantation in adults was limited by the amount of cord blood that could be collected. Cord blood transplantation after ex vivo expansion with cytokines have already been tried in adults. Amifostine is a phosphorylated aminothiol that affords broad cytoprotection from the myelosuppressive effects of antineoplastic agents. The purposes of this study were to investigate expansion of progenitor and myeloid cells after ex vivo culture of mononuclear cells (MNCs) in umbilical cord blood with growth factor and characterize hematopoietic activities of amifostine. METHODS: MNCs were cultured and ex vivo expanded into myeloid progenitors by using hematopoietic growth factors (IL-1beta, IL-3, IL-6, G-CSF, GM-CSF, SCF, EPO) which are known to stimulate differentiation and proliferation of myeloid progenitors. MNCs exposed to the appropriate amount of amifostine for 15 min were cultured in semisolid media and harvested at 24h intervals, and then apoptosis was assessed by propidium iodide staining. RESULTS: Myeloid colonies were successfully produced from MNCs. Maximal expansion was obtained with the combination of IL-3+SCF+G-CSF+GM-CSF. SCF was thought to be the most important growth factor for expansion of myeloid progenitor. Pretreatment with amifostine for 15 min stimulated formation of hematopoietic colonies at clinically relevant concentrations ranging from 1 to 100 micrometer. Increase in colony number compare to control were comparable after pretreatment with amifostine (10micrometer), and CFU-GEMM and BFU-E were highly responsive. Further enhancement of colony was not observed after prolonging the duration of pre- incubation exposure to 1, 8 and 24 hours. Amifostine enhanced IL-1 and IL-3 induced formation of CFU-GEMM and BFU-E. Incubation of MNCs with amifostine in suspension culture increased recovery of secondary colonies. Treatment with amifostine retarded cell loss and apoptosis, and promoted cell survival at 24, 48 and 72 hours in cytokine-deficient medium. CONCLUSION: Cord blood MNCs can be successfully expanded into myeloid progenitors by using hematopoietic growth factors. This investigation extend the previously recognized hematologic effects of amifostine, and indicate that in addition to its cytoprotective properties, amifostine is a stimulant of hematopoietic progenitor growth.

Expansion and Differentiation of Dendritic Cells in Clinical Scale from Human Cord CD34+ Progenitor Cells

Dendritic cells are the most potent antigen presenting cells and appear to be the only cell type capable of limiting a primary T-cell dependent immune response. Recently, progress in the understanding of DCs biology has been relatively fast and systems using CD34+ stem cells stimulated with GM-CSF and tumor necrosis factor-alpha(TNF-alpha have been described. These systems have been further modified by others to increase the diversity and the yield. Indeed, several studies have shown distinct clinical responses after vaccination with tumor antigen- loaded, autologous DC. Despite this progress, the total number of DC available for immunotherapy remains limited. In vitro human DC can be generated from human CD34+ bone marrow and peripheral blood progenitor cells after culture with different cytokine combinations or from peripheral blood CD14+ monocytes when grow in the presence of GM-CSF and IL-4. Here, we have explored another source of DC precursors, human CD34+ cord blood cells, which in contrast to monocytic precursors, expand when cultured in the presence of GM-CSF and TNF-alpha The CD34+ cells were purified using MACS and expanded in culture with cytokine mixtures (SCF, Flt-3 TPO, IL-3, and IL-6). The CD34+ cells (4.0 +/-1.8 x105) isolated from cord blood cultured for 1, 2, 3, and 4 weeks resulted in a mean increase of total cell number of 41.5 +/-26.2 x105 (10-fold), 143.8 +/-78.9 x105 (36 fold), 197.5 +/-145.5 x105 (49-fold), 241.5 +/-167.4 x105 (60-fold), respectively. The precursor cells progressively lose most of the CD34 expression in culture and are over 95% positive for CD38 and low expression for CD3/CD19 indicating that all precursors are from myeloid origin. The percentage of CD14 positive precursors was significantly increased according to the expansion duration. The CD1a expression of expanded DC precursors was all negative (0.15-0.57%). The CD1a expression, which were in immature DCs, was high (28-78%), and CD40, CD80, CD11c and HLA-DR was positive after expanded precursor DCs were cultured for 1 weeks using GM-CSF and IL-4. The immature DC derived from all precursor culture conditions were negative for CD83. TNF-alphaactivated DCs derived from the four precursor culture condition according to the day of culture were used as stimulator cells in allogenenic MLR. When the total DC population was used, the expanded DCs for 2 weeks induced a slightly but reproducibly stronger MLR than those for 4 weeks. In this study, we show the sequential culture method after expansion is particularly appropriate for immunotherapeutical approaches, because relatively large numbers of DC can from cord blood be generated to overcome the limitation of cell count, which are needed for repetitive vaccination.

Stem Cells Expressing Homing Receptors Could be Expanded From Cryopreserved and Unselected Cord Blood

We assessed the cytokine combinations that are best for ex vivo expansion of cord blood (CB) and the increment for cell numbers of nucleated cells, as well as stem cells expressing homing receptors, by an ex vivo expansion of cryopreserved and unselected CB. Frozen leukocyte concentrates (LC) from CB were thawed and cultured at a concentration of 1x10(5)/mL in media supplemented with a combination of SCF (20 ng/mL)+TPO (50 ng/mL)+FL (50 ng/mL)+/-IL-6 (20 ng/mL)+/-G-CSF (20 ng/mL). After culturing for 14 days, the expansion folds of cell numbers were as follows: TNC 22.3+/-7.8~26.3+/-4.9, CFU-GM 4.7+/-5.1~11.7+/-2.6, CD34+CD38- cell 214.0+/-251.9~464.1+/-566.1, CD34+CXCR4+ cell 4384.5+/-1664.7~7087.2+/-4669.3, CD34+VLA4+ cell 1444.3+/-1264.0~2074.9+/-1537.0, CD34+VLA5+ cell 86.2+/-50.9~ 113.2+/-57.1. These results revealed that the number of stem cells expressing homing receptors could be increased by an ex vivo expansion of cryopreserved and unselected CB using 3 cytokines (SCF, TPO, FL) only. Further in vivo studies regarding the engraftment after expansion of the nucleated cells, as well as the stem cells expressing homing receptors will be required.

Megakaryocytic Differentiation of Human Cord Blood CD34+Cells During ex vivo Expansion / 대한혈액학회지

BACKGROUND: Thrombopoietin (TPO) has been currently used for ex vivo expansion of hematopoietic progenitor cells. Previously, we have reported that TPO induces a characteristic pattern of apoptosis during ex vivo expansion of cord blood (CB) CD34+cells. In the present study, we have investigated on the relationship between the TPO-induced apoptosis and megakaryocytic differentiation. METHODS: CD34+cells, purified from human CBs, were expanded in serum-free conditions stimulated with TPO. Multidimensional flow cytometry and TUNEL assay as well as electron microscopy were applied for analysis of apoptosis. Asociation of megakaryocytic differentiation and apoptosis was studied by FACS-sorting and immunocytochemistry. Clonogenic potential was studied by CFU-MK assay. RESULTS: The TPO-induced apoptotic cells appeared in CD61+fractions. Immunocytochemical analysis of the FACS-sorted fractions showed that the apoptosis-associated CD44low fraction expressed CD61. Clonogenic assay revealed 7.4+-0.50-fold increase of total CFU-MKs during the initial 9 days. Thereafter, the number of CFU-MKs decreased, which was parallel with the increase of apoptosis. When the MK colonies were subdivided according to size, the proportion of large colonies progressively decreased, while that of medium and small colonies increased. In particular from day 6, small colonies became predominant. CONCLUSION: These results suggested that the MK progenitors matured as they were expanded during ex vivo expansion with TPO, and then proceeded to apoptosis.