Effects of adriamycin, 4-hydroperoxycyclophosphamide and ASTA Z 7557 (INN mafosfamide) on the release of IL-2 and IL-1 in vitro

Adriamycin (ADR), 4-hydroperoxycyclophosphamide (4-OOHCYP) and the new cyclophosphamide derivative AZ 7557 (AZ) were tested for their effects on the release of the Interleukins IL-2 and IL-1 from rat spleen cells and peritoneal exudate macrophages respectively. ADR was found to enhance IL-2 release and less effectively IL-1 release. 4-OOHCYP and AZ were found to inhibit IL-2 release but had no effect on IL-1 production. It is suggested that the modulation of IL-2 levels by these drugs is unlikely to be mediated by their effects on IL-1.     

Phenotypic characterization of murine primitive hematopoietic progenitor cells isolated on basis of aldehyde dehydrogenase activity

There are several different technical approaches to the isolation of hematopoietic stem cells (HSCs) with long-term repopulating ability, but these have problems in terms of yield, complexity, or cell viability. Simpler strategies for HSC isolation are needed. We have enriched primitive hematopoietic progenitors from murine bone marrow of mice from different genetic backgrounds by lineage depletion followed by selection of cells with high aldehyde dehydrogenase activity using the Aldefluor reagent (BD Biosciences, Oxford, U.K.). Lin- ALDH(bright) cells comprised 26.8 +/- 1.0% of the total Lin- population of C57BL6 mice, and 23.5 +/- 1.0% of the Lin- population of BALB/c mice expressed certain cell-surface markers typical of primitive hematopoietic progenitors. In vitro hematopoietic progenitor function was substantially higher in the Lin- ALDH(bright) population compared with the Lin- ALDH(low) cells. These cells have higher telomerase activity and the lowest percentage of cells in S phase. These data strongly suggest that progenitor enrichment from Lin- cells on the basis of ALDH is a valid method whose simplicity of application makes it advantageous over conventional separations.     

TGF-β1和/或TNF-α反义PS-ODNS对造血干/祖细胞体外扩增的作用

目的：研究TGF-β1和／或TNF—α反义硫代寡核苷酸(PS—ODNS)对造血干／祖细胞体外扩增的调节作用。方法：采用免疫磁珠分离技术从新鲜脐带血中分离CD34^ 细胞，用淋巴细胞分离液从骨髓血中分离单个核细胞，应用液体培养及造血祖细胞集落分析检测TGF-β1和／或TNF—α反义PS—ODNS对CD34^ 细胞数及多向性造血祖细胞(CFU—mix)、粒-单祖细胞(CFU—GM)、红系祖细胞(BHU—E和CFU—E)集落数的影响。结果：培养体系中加入TGF-β1反义PS—ODNS后CD34^ 细胞数、CFU—mix、CFU—GM、BFU—E和CFU—E集落数分别是对照组(仅加细胞因子组)的4、2．6、2．7、1．8、2．1倍；加入TNF—α反义PS—ODNS后CD34^ 细胞数、CFU—mix、CFU—GM、BHU—E和CFU—E集落数分别是对照组的4、2．9、2．6、1．7、1．8倍；同时加入TGF-β1和TNF—α反义PS—ODNS后CD34^ 细胞数、CFU—mix、CFU—GM、BHU—E和CFU—E集落数分别是对照组的5．3、2．1、2．7、1．9、1．8倍。结论：采用反义PS—ODNS阻断内源性TGF-β1和TNF—α是造血干／祖细胞体外扩增的有效措施之一。     

The Rho kinase inhibitor fasudil augments the number of functional endothelial progenitor cells in ex vivo cultures

Rho kinase (ROCK) has been implicated in the regulation of vascular tone, endothelial dysfunction, inflammation and remodeling. Endothelial progenitor cells (EPC) have been proven to have the efficacy of therapeutic neovascularization in ischemia. However, the scarcity of EPCs limits cell therapy. Using an in vitro EPC culture assay, Y27632 was found to increase the number of adherent EPCs. In this study, we investigated the effect of fasudil, another ROCK inhibitor being used in the clinic, on EPC number and examined whether EPCs expanded by fasudil are functional in vitro and in vivo. In ex vivo cultures of EPCs, fasudil effectively increased the number of ac-LDL/UEA-1 positive cells as well as adherent cells, in contrast to H89, a less selective ROCK inhibitor. Fasudil also increased EPC numbers in culture up to 10 μM, in a dose-dependent manner. When EPCs expanded with fasudil were examined for the migratory activity toward stromal cell-derived factor-1 and vascular endothelial growth factor, these cells retained functional properties in migration, albeit with some decrease. Fasudil-cultured EPCs labeled with PKH26 showed an activity similar to non-treated EPCs for cellular adhesion into an endothelial cell (EC) monolayer and incorporation into capillary-like structures formed by ECs. Finally, when EPCs cultured with fasudil (106 cells/mouse) were injected into ischemic limbs, these cells showed a blood flow recovery at a level comparable to non-treated control EPCs and increased neovascularization. Therefore, these data suggest that the ROCK inhibitor fasudil can provide a beneficial effect in the treatment of ischemic diseases by increasing EPC numbers.     

Effect of the surface density of nanosegments immobilized on culture dishes on ex vivo expansion of hematopoietic stem and progenitor cells from umbilical cord blood

Umbilical cord blood (UCB) is an attractive source of hematopoietic stem and progenitor cells for hematopoietic stem cell (HSC) transplantation. However, the low number of HSCs obtainable from a single donor of UCB limits direct transplantation of UCB to the treatment of pediatric patients. In this study, we investigated the ex vivo expansion of HSCs cultured on biomaterials grafted with several nanosegments, i.e. polyamine, fibronectin, RGDS, and CS1 (EILDVPST), at several surface densities. No direct correlation was found between fold expansion of HSCs and physical parameters of the culture dishes, i.e. surface roughness and water contact angle of the culture dishes. However, a small amount of grafted amino groups, less than 0.8 residual μmol cm(-2), on the dishes was effective for the ex vivo expansion of HSCs. A high amount of grafted amino groups hindered the ex vivo expansion of HSCs on the dishes. HSCs cultured on dishes with a high concentration of CS1 (2.40 residual μmol cm(-2)) showed greater expansion of HSCs and more pluripotent colony-forming units (i.e. colony-forming unit-granulocyte, erythroid, macrophage, and megakaryocyte (CFU-GEMM)) than those on fibronectin-grafted and polyamine-grafted dishes. These data suggest that the specific interaction between HSCs and CS1 helps to maintain the pluripotency of HSCs during the ex vivo expansion of HSCs.     

细胞因子短期扩增对造血干/祖细胞黏附和迁移能力的影响

目的：探讨干细胞因子（SCF）+白细胞介素-6（IL-6）短期扩增对CD34+造血干/祖细胞黏附和迁移能力的影响。方法:用密度剃度离心的方法分离脐血CD34+细胞，经SCF和IL-6孵育48 h，用CCK-8方法检测CD34+细胞增殖能力；用流式细胞仪检测处理前后的CD49d（VLA-4）、CD11a（LFA-1）、CD62L（L-selectin）及CD184（CXCR4）的表达。用纤连蛋白（FN）包被96孔板，检测经或未经因子扩增的CD34+细胞的黏附能力。扩增的CD34+细胞悬浮于transwell培养板的上层，下层添加基质细胞衍生因子（SDF-1），流式细胞仪检测迁移细胞数，计算迁移率。结果:经SCF+IL-6处理48h后CD34+细胞扩增近3倍；表达CD49d、CD11a、CD62L及CD184的CD34+细胞的百分数分别由原来的26.34%±5.37%、17.63%±4.57%、46.38%±6.61%和9.58%±1.56%增加到65.67%±8.72%、56.67%±6.34%、84.76%±9.57%和19.32%±3.64%（P<0.01）。扩增后的CD34+细胞对FN的黏附能力及在SDF-1诱导下的迁移作用都显著增强（P<0.01）。结论:SCF+IL-6短期扩增CD34+ 造血干/祖细胞显著增加细胞的黏附能力，增加SDF-1诱导的迁移作用，可能是SCF+IL-6促进归巢的主要机制之一。     

黄芪多糖对小鼠骨髓及外周血造血干细胞的增殖及动员作用

探讨APS-P对正常或化疗后小鼠的骨髓、脾及外周血造血干细胞的促增殖及外周动员作用。给正常或经环磷酰胺化疗后的C57BL／6小鼠注射APS-P，然后取骨髓细胞、外周血细胞及脾细胞，并用荧光抗体PerCP-Sca-1，APE-c-kit及PE-Lineage(CD3，CD4，CD8，Trll9，B220 CDllb，Gr-1)进行染色标记，用流式细胞仪检测小鼠造血干细胞(Lin c-kit^ Sca-1^ )数量变化。注射APS-P能使正常及化疗小鼠骨髓、脾及外周血中的造血干细胞有不同程度的增加。这一作用可能是由于APS-P促进骨髓造血干细胞的增殖，而在外周血及脾中的增加则可能是由于APS-P对骨髓造血干细胞的外周动员作用。     

Effect of the Aldehyde Dehydrogenase Inhibitor, Cyanamide, on the Ex Vivo Sensitivity of Murine Multipotent and Committed Hematopoietic Progenitor Cells to Mafosfamide (Asta Z 7557)

The ex vivo sensitivity of murine multipotent (CFU-GEMM) and committed (CFU-Mk, CFU-GM, BFU-E and CFU-E) hematopoietic progenitor cells to mafosfamide was quantified with and without concurrent exposure to cyanamide, an inhibitor of aldehyde dehydrogenase activity. In the absence of cyanamide, CFU-GEMM, CFU-Mk and CFU-GM were approximately equisensitive to mafosfamide while the erythroid progenitors were more sensitive to the drug. Cyanamide potentiated the cytotoxicity of mafosfamide toward CFU-GEMM and CFU-Mk, but not toward CFU-GM, BFU-E and CFU-E. Cellular aldehyde dehydrogenases are known to catalyze the oxidation of 4-hydroxycyclophos-phamide/aldophosphamide, the major intermediate in cyclophosphamide and mafosfamide activation, to the relatively nontoxic acid, carboxyphosphamide. Thus, our findings indicate that 1) murine CFU-GEMM contain the relevant aldehyde dehydrogenase activity, and 2) the relevant aldehyde dehydrogenase activity is retained upon differentiation to progenitors committed to the megakaryocytoid lineage, but lost upon differentiation to progenitors committed to the granulocytoid/monocytoid and erythroid lineages. The relative insensitivity of CFU-GM to mafosfamide is apparently due to a cellular determinant that influences their sensitivity to all cross-Unking agents since CFU-GM were found to be relatively insensitive to non-oxazaphosphorine cross-linking agents as well.     

Effect of the Aldehyde Dehydrogenase Inhibitor,Cyanamide, on the Ex Vivo Sensitivity of Murine Multipotent and Committed Hematopoietic Progenitor Cells to Mafosfamide (Asta Z 7557)

Abstract

The ex vivo sensitivity of murine multipotent (CFU-GEMM) and committed (CFU-Mk, CFU-GM, BFU-E and CFU-E) hematopoietic progenitor cells to mafosfamide was quantified with and without concurrent exposure to cyanamide, an inhibitor of aldehyde dehydrogenase activity. In the absence of cyanamide, CFU-GEMM, CFU-Mk and CFU-GM were approximately equisensitive to mafosfamide while the erythroid progenitors were more sensitive to the drug. Cyanamide potentiated the cytotoxicity of mafosfamide toward CFU-GEMM and CFU-Mk, but not toward CFU-GM, BFU-E and CFU-E. Cellular aldehyde dehydrogenases are known to catalyze the oxidation of 4-hydroxycyclophos-phamide/aldophosphamide, the major intermediate in cyclophosphamide and mafosfamide activation, to the relatively nontoxic acid, carboxyphosphamide. Thus, our findings indicate that 1) murine CFU-GEMM contain the relevant aldehyde dehydrogenase activity, and 2) the relevant aldehyde dehydrogenase activity is retained upon differentiation to progenitors committed to the megakaryocytoid lineage, but lost upon differentiation to progenitors committed to the granulocytoid/monocytoid and erythroid lineages. The relative insensitivity of CFU-GM to mafosfamide is apparently due to a cellular determinant that influences their sensitivity to all cross-Unking agents since CFU-GM were found to be relatively insensitive to non-oxazaphosphorine cross-linking agents as well.     

脐血CD34+细胞体外定向诱导分化为T淋巴细胞的实验研究

目的：建立利用人造血干／祖细胞体外定向诱导分化为T淋巴细胞的方法，为研究T细胞生物学特性及细胞免疫提供技术平台。方法：MACS方法分离人脐带CD34^ 细胞接种到人胎儿胸腺基质单层细胞上，IMDM液体培养基含20％人AB血清并加入FL、IL-12、IL-7和IL-2细胞因子组合，于培养7、14、21、28、35、42天取非贴壁细胞利用流式细胞仪对细胞表型进行检测，并进行细胞形态学分析。结果：2周后，CD4^ CD8^ 非成熟T淋巴细胞占细胞总数的0．3％-13．3％，4-5周CD4^ CD8^ T淋巴细胞达到高峰占16．6％-26．5％，且CD3^ CD4^ CD8^ 和CD3^ CD4^-CD8^ T淋巴细胞逐渐增多，6周后达26．5％～64．9％和11．6％-38．9％。培养成熟的T淋巴细胞经PHA IL-2刺激后瑞氏染色鉴定可见大原始淋巴细胞存在。结论：利用人脐血CD34^ 在体外人胎儿胸腺基质单层细胞上加FL、IL-12、IL-7和IL-2细胞因子组合条件下，可诱导分化出T淋巴细胞，并且培养的T细胞对有丝分裂素刺激有增殖反应。     

Effects of mannosylated glycopolymers on specific interaction to bone marrow hematopoietic and progenitor cells derived from murine species

Poly[N-pvinylbenzyl-O-D-galactopyranosyl-(1-4)-D-glucoamide], poly[N-pvinylbenzyl-O-D-glucopyranosyl-(1-4)-D-glucoamide], and poly[N-p-vinylbenzyl-O-mannopyranosyl-(1-4)-D-gluconamide] (referred to as PVLA, PVMA, and PV-Man) are polystyrene derivatives that contain galactose, glucose, and mannose moieties, which interact with hematopoietic cells (HCs). To clarify the specific interaction between the glucopolymers and hematopoietic cells, glycopolymers labeled with fluorescent isothiocyanate (FITC) were used to follow the specific interaction, which was visualized by confocal laser microscopy. We found that PV-Man binds strongly to HCs, probably because of a specific interaction mediated by specific receptors present on the cell membrane, while some cytotoxicity when was observed when PV-Man interacted with the cell membrane. The fluorescence intensity between PV-Man and HCs was up to four-fold (0.14 +/- 0.04) that of PVMA and PVLA with hematopoietic HCs (0.033 +/- 0.01). Moreover, cellular fluorescence increased significantly with increasing incubation time and increasing polymer concentration. Using hematopoietic lineage-specific antibodies, cells were stained and analyzed by flow cytometry to confirm which HCs showed specific binding with glycopolymers, especially hematopoietic stem cells and progenitor cells (c-kit+), B-lymphocyte progenitor cells (B220+), monocyte cells (CD11b+), and erythrocytes (Ter119+).     

腺病毒载体介导的人IL-24基因对HL-60白血病细胞体外培养的影响

目的 构建人IL-24的腺病毒载体Ad-IL24,观察Ad-IL24对HL-60细胞体外培养的影响.方法 以pcDNA3.0-IL24重组质粒为模板PCR扩增IL-24基因,酶切并连接到带有绿色荧光蛋白(GFP)标记的pAdTrack-CMV质粒上,Pme Ⅰ线性化重组质粒pAdTrack-CMV-IL24,并与腺病毒质粒pAdEasy-1共转化BJ5183细菌,获得重组腺病毒载体pAdEasy-1-pTrack-CMV-IL24,经Pac Ⅰ线性化后转染QBI-293A细胞,收获重组病毒Ad-IL24.将它感染HL-60细胞,通过激光扫描共聚焦显微镜(LSCM)、MTT和流式细胞术(FCM)法检测IL-24对HL-60细胞生长的影响,免疫细胞化学分析凋亡因子的改变,ELISA检测重组病毒对HL-60细胞分泌γ干扰素(IFN-γ)和肿瘤坏死因子-α(TNF-α)的影响.结果 成功构建重组腺病毒载体pAdEasy-1-pTrack-CMV-IL24并获得高滴度的重组腺病毒Ad-IL24,各种检测方法表明IL-24基因能抑制HL-60细胞生长,诱导凋亡,IL-24还能下调HL-60细胞Bcl-2的表达并刺激它分泌二级细胞因子IFN-γ和TNF-α.结论 IL-24能抑制HL-60细胞生长、诱导凋亡,它可通过下调HL-60细胞Bcl-2的表达并刺激其分泌具有抗肿瘤活性的二级细胞因子来发挥效应.     

Effect of interleukin 10 on the hematopoietic progenitor cells from patients with aplastic anemia.

The overproduction of cytokines with inhibitory effects on hematopoiesis is considered to play a role in the pathogenesis of aplastic anemia. While interleukin 10 (IL-10) is a cytokine production inhibitory factor, the possibility of immunosuppressive therapy using IL-10 for aplastic anemia has not been explored. In this study, therefore, we examined the effect of IL-10 on progenitor cells obtained from seven patients with severe aplastic anemia. Our study indicated that IL-10 dramatically enhanced the erythroid colony formation in a dose-dependent manner in two of the seven cases examined. When we examined the concentration of cytokines in the culture supernatants of unstimulated bone marrow cells, the spontaneous production of interferon-gamma (IFN-gamma) was observed in one of these two cases, and this production was completely inhibited by addition of IL-10. These findings suggested that IL-10 enhanced the erythroid colony formation by inhibiting the pathological production of IFN-gamma in this case. This study provides an experimental support for the clinical application of IL-10 in some patients with aplastic anemia.     

Expression of cell surface determinant(s) on murine myeloma stem cells and hematopoietic stem cells

Murine B cell lymphomas and myelomas were examined for the expression of a determinant previously found exclusively on normal pluripotent stem cells colony-forming unit-spleen (CFU-s). This determinant(s), which is defined by a rabbit antimouse brain antiserum (R alpha MB), is present on the tumor stem cell population of some but not all B cell neoplasms examined. The determinant is not detected on tumor cells of the macrophage or T cell lineage. Absorption of the activity in R alpha MB with myeloma cells, concomitantly removed reactivity with the normal stem cell, CFU-s, and the myeloma stem cell, plasmacytoma CFU-s. Sorting analysis further showed that the antigen was diminished within a positive tumor population as cells acquired the capacity to secrete immunoglobulin. These studies suggest that this normal stem cell-associated antigen may also be an early differentiation antigen for the B cell lineage, and is expressed on some stem cells of B cell tumors.     

Effect of tresperimus on ex vivo expansion of CD34+CD38(-)-enriched cord blood cells

Tresperimus, an analogue of 15-deoxyspergualine (15-DSG), has been found, in rodents, to induce a potent state of tolerance after organ and bone marrow allografts. In a previous study, we have reported that tresperimus at the optimal concentration of 0.5 microgram/ml supports the clonogenic potential of human cord blood CD34+ cells. Dose dependent inhibition of clonogenesis was also observed with complete reversibility following drug withdrawal. In this study, we tested the effect of 0.5 microgram tresperimus/ml on ex vivo expansion of primitive human cord blood CD34+CD38- cells. Our findings revealed that the total number of expanded cells was decreased in the presence of tresperimus. However, the multipotential and erythroid colonies were significantly increased in the presence of tresperimus compared with control cultures done without the test drug. Progenitor cell morphology was comparable in both test and control cultures. The telomerase activity was consistently lower in tresperimus-treated hematopoietic progenitors than in control cultures. These results suggest that tresperimus preserves primitive CD34+CD38- cells in a state of high potentiality while limiting the total number of their differentiated progeny. Bearing in mind that the test drug supports the clonogenic potential of CD34+ cells, the overall findings emphasize the importance of assessing the effect of tresperimus on in vivo long-term hematopoiesis which could predict the potential clinical use of tresperimus in the prevention of graft-versus-host disease in recipients of allogeneic bone marrow.     

Effect of hyaluronidase immobilized using electron-beam synthesis nanotechnology on sensitivity of progenitor cells to regulatory factors

In vitro experiments demonstrated increased colony-forming capacity of erythroid, granulomonocytic, and mesenchymal progenitors of the bone marrow and parenchymal progenitor elements of the liver after treatment with immobilized hyaluronidase. Increased sensitivity of these progenitor cells to erythropoietin, granulocyte colony-stimulating factor, fibroblast growth factor, and stem cell factor, respectively, was demonstrated. Immobilized hyaluronidase enhanced the formation of tissue-specific hepatic CFU against the background of reduced yield of stromal precursors in liver tissue culture containing insulin.     

The impact of progenitor enrichment,serum, and cytokines on the ex vivo expansion of mobilized peripheral blood stem cells: a controlled trial

The aim of this study was to verify, and possibly improve, culture conditions to expand human mobilized peripheral blood stem cells (PBSCs). We investigated the role of three parameters: A) the culture medium (serum-free versus serum-dependent); B) the initial cell population (Ficoll-separated mononucleated cells versus CD34(+)-selected cells), and C) the low concentration of recombinant cytokines, flt3 ligand, and thrombopoietin in association with a basic cocktail of stem cell factor, interleukin (IL)-6, IL-3, GM-CSF, and erythropoietin. Eighteen leukapheresis samples were monitored in static culture for 15 days. The expansion potential was assessed at day 10 and 15 by total nuclear cells, colony-forming-units (CFUs) (burst-forming units-erythroid [BFU-E], colony-forming units-granulocyte-macrophage [CFU-GM], and colony-forming units-granulocyte-erythroid-macrophage-megakaryocyte [CFU-GEMM]), and flow cytometry immunophenotyping (CD34(+)/CD38(-), CD38(+), CD33(+), CD41(+), GlyA(+) progenitor cells). The results, evaluated by multivariate analysis of variance, emphasize that some variables affected the outcome of stem and progenitor cell expansion. CD34(+) enrichment increased expansion of total nuclear cells, number of CD38(+) and CD33(+) late precursors, and number of the CFU-GM compartment. Interestingly, however, quantitative expansion of GlyA(+) and the early progenitor cells (CD34(+)/CD38(-), CFU-GEMM, BFU-E) are favored by the use of unselected mononucleated cells. Regarding the role of serum, no significant difference was observed except for expansion of total nuclear cells, CFU-GM, and BFU-E. Cytokine combinations, in particular the use of flt3 ligand, stimulated expansion of almost all the cellular subsets, reaching a statistical significance for total nuclear cells and CFU-GM. Our study indicates that progenitor and late precursor multilineage cell compartments of mobilized PBSCs may be significantly expanded in short-term cultures by well-defined experimental conditions. Furthermore, these data might be useful when evaluating ex vivo expansion of hematopoietic cells for clinical purposes.     

The influence of extracellular matrix derived from skeletal muscle tissue on the proliferation and differentiation of myogenic progenitor cells ex vivo

Skeletal muscle relies upon regeneration to maintain homeostasis and repair injury. This process involves the recruitment of the tissue's resident stem cell, the muscle progenitor cell, and a subsequent proliferative response by newly generated myoblasts, which must then align and fuse to generate new muscle fibers. During regeneration, cells rely on environmental input for direction. Extracellular matrix (ECM) represents a crucial component of a cell's microenvironment that aids in guiding muscle regeneration. We hypothesized that ECM extracted from skeletal muscle would provide muscle progenitor cells and myoblasts with an ideal substrate for growth and differentiation ex vivo. To test this hypothesis, we developed a method to extract ECM from the large thigh muscles of adult rats and present it to cells as a surface coating. Myogenic cells cultured on ECM extract experienced enhanced proliferation and differentiation relative to standard growth surfaces. As the methodology can be applied to any size muscle, these results demonstrate that bioactive ECM can be readily obtained from skeletal muscle and used to develop biomaterials that enhance muscle regeneration. Furthermore, the model system demonstrated here can be applied to the study of interactions between the ECM of a particular tissue and a cell population of interest.