Expansion of CD34~+ cells from human umbilical cord blood by FL and/or TPO gene transfected human marrow stromal cell lines

To elucidate the effect of gene transfected marrow stromal cell on expansion of human cord blood CD34+ cells, a culture system was established in which FL and TPO genes were transfected into human stromal cell line HFCL. To establish gene transfected stromal cells co-culture system, cord blood CD34+ cells were purified by using a magnetic beads sorting system. The number of all cells and the number of CD34+ cells and CFC (CFU-GM and BFU-E) were counted in different culture systems. The results showed that in all 8 culture systems, SCF+IL-3+HFT manifested the most potent combination, with the number of total nucleated cells increasing by (893.3±52.1)-fold, total progenitor cells (CFC) by (74.5±5.2)-fold and CD34+ cells by 15.7-fold. Maximal expansions of CFC and CD34+ cells were observed at the end of the second week of culture. Within 14 days of culture, (78.1±5.5)-fold and (57.0±19.7)-fold increases in CFU-GM and BFU-E were obtained. Moreover, generation of LTC-IC from amplified CD34+ cells within 2     

Ex vivo differentiation of natural killer cells from human umbilical cord blood CD34+ progenitor cells

Abstract

Natural Killer (NK)-cells are peripheral blood lymphocytes that represent an important arm of the innate immune system. NK-cells play a critical role in the immune surveillance against tumors and virally infected cells in a major histocompatibiliy complex (MHC)-unrestricted fashion. We have explored such capacities of NK-cells after differentiation from hematopoietic stem and progenitor cells derived from human umbilical cord blood. Several culture conditions have been established supporting proliferation and subsequent differentiation of these cells in terms of receptor expression and specific lysis depending on the growth conditions in the presence and absence of supportive stromal feeders. We show that acquisition of Killer Immunoglobulin Receptor (KIR) as well as NK Cytotoxicity Receptor expressions is independent of culture condition whereas absence of stromal feeders did not support acquisition of CD94/NKG2A expression. Such KIR-positive/NKG2A-negative cells generated under different culture conditions showed strong and specific cytolytic activity which could have impact on further immunotherapeutic strategies.     

Support of hMSCs transduced with TPO/FL genes to expansion of umbilical cord CD34+ cells in indirect co-culture

A novel indirect co-culture system was established to support ex vivo expansion of hematopoietic progenitors in umbilical cord blood (UCB) by using thrombopoietin (TPO)/Flt-3 ligand (FL)-transduced human-marrow-derived mesenchymal stem cells (tfhMSCs) as a feeder. UCB CD34⁺ cells were isolated and cultured by using five culture systems in serum-containing or serum-free medium. Suitable aliquots of cultured cells were taken to monitor cell production, clonogenic activity, and long-term culture-initiating culture (LTC-IC) output. Finally, the severe-combined immunodeficient mouse (SCID) repopulating cell (SRC) assay was performed to confirm the ability of the indirect co-cultured cells from the tfhMSCs system to reconstitute long-term hematopoiesis. Results showed significant differences in the number of total nucleated cells (TNCs) among the culture systems with respect to serum-containing medium or serum-free medium during 14-day culture. In addition, on day 14, the outputs of CD34⁺ cells, the colony-forming units (CFUs) in culture, and the CFUs in mixed colonies containing erythroid and myeloid cells and megakaryocytes in the tfhMSC indirect co-culture system were significantly enhanced. The LTC-IC assay demonstrated that the tfhMSCs indirect co-culture system had the strongest activity. The SCID-SRC assay confirmed the extensive ability of the expanded cells from the tfhMSCs indirect co-culture systems to reconstitute long-term hematopoiesis. Furthermore, polymerase chain reaction analysis demonstrated the presence of human hematopoietic cells in the bone marrow and peripheral blood cells of non-obese diabetic/SCID mice. Thus, hMSCs transduced with TPO/FL, in combination with additive cytokines, can effectively expand hematopoietic progenitors from UCB in vitro. The tfhMSC indirect co-culture system may therefore be a suitable system for ex vivo manipulation of primitive progenitor cells under non-contact culture conditions.This work was supported by the Zhejiang Scientific Foundation (no. 2003C23015).     

Co-culture of human CD34+ cells with mesenchymal stem cells increases the survival of CD34+ cells against the 5-aza-deoxycytidine- or trichostatin A-induced cell death

It has been suggested that epigenetic regulation plays an important role in maintaining the stemness and lineage differentiation of hematopoietic stem cells (HSCs), 5-aza-deoxycytidine (aza-D) and Trichostatin A (TSA) being candidate additives for HSC ex vivo expansion. Although they have potent activity to maintain the stemness, they can also cause serious cell death. This study examined the effects of mesenchymal stem cells (MSCs) on the maintenance of CD34+ cells driven by aza-D and TSA in culture with the combined cytokines of thrombopoietin, flt-3 ligand, stem cell factor, interleukin-3, and interleukin-6. In cultures without MSCs, although aza-D and TSA retained the CD34 frequency 4 to 8 times more than in the cytokines alone, a large portion of cells underwent apoptotic cell death. Consequently, CD34+ cell expansion could not be achieved in any condition without MSCs. In cultures with MSCs, the total cell number was higher in aza-D or TSA than in any conditions in the cultures without MSCs. The CD34 frequency was also similar to the level in the cultures in aza-D or TSA without the MSCs. These results suggest that a co-culture of CD34+ cells with the MSCs might not simply deliver the proliferation signals but also stemness and survival signals, and overlap the action of epigenetic regulators.     

S-亚硝基谷胱甘肽诱导脐带血CD34+细胞来源的巨核细胞产生血小板

为了探讨一氧化氮供体S-亚硝基谷胱甘肽（S—nitrosoglutathione,GSNO）对脐带血CD34^＋细胞分化来源的巨三核细胞产生血小板的可能作用,我们采用免疫磁珠法从8例健康产妇足月顺产的胎儿脐带血中分选CD34^＋细胞,并在含血小板生成素（thrombopoietin,TPO,50ng／ml）、白细胞介素-3（IL-3,10ng／ml）、干细胞因子（stem cell factor,SCF,50ng／ml）和重组人粒-巨噬细胞刺激因子（rHu GM—CSF,20ng／ml）的无血清培养基中培养14d。随后,用免疫磁珠法分选CD61^＋细胞。CD61^＋细胞在含有（实验组）和缺乏（对照组）GSNO（20mg／ml）的无血清培养基[含TPO（50ng／ml）、IL-3（10ng／ml）、SCF（50ng／ml）]中培养不同时间（30min、2h）。采用流式细胞仪检测培养体系中的血小板数;电子显微镜观察巨核细胞的形态学;倒置显微镜和流式细胞仪观察凝血酶诱导的血小板凝集;ELISA方法检测巨核细胞中cGMP的含量。结果显示,与对照组比较,实验组血小板数量明显增加（P〈0．05）;电子显微镜下可见巨核细胞有明显伪足形成和突出;凝血酶诱导后在倒置显微镜和流式细胞仪上均可观察到血小板凝集现象;GSNO作用后巨核细胞中的cGMP明显升高（P〈0．05）。这些结果提示,GSNO可以促进脐带血CD34^＋细胞来源的巨核细胞产生具有一定功能的血小板,其产生的机制可能部分与cGMP途径有关。     

Role of macrophage colony-stimulating factor in the differentiation and expansion of monocytes and dendritic cells from CD34+ progenitor cells

The present study focused on whether it is possible to expand monocytic cells from CD34⁺ progenitor cells by using macrophage colony-stimulating factor (M-CSF) in the absence and presence of mast cell growth factor (MGF) and IL-6. It was demonstrated that CD34⁺ cells differentiate without expansion to functional mature monocytic cells in the presence of M-CSF or combinations of M-CSF plus IL-6 and MGF. A different response pattern was observed for the number of clonogenic cells. The addition of IL-6 or both IL-6 and MGF to M-CSF containing cultures resulted in significant higher numbers of colony-forming unit-macrophage (CFU-M) as tested in clonogenic and³H-thymidine assays. Furthermore, M-CSF plus both IL-6 and MGF appeared to be the most potent combination to preserve the monocytic precursor in cell suspension culture assays. These results indicate that IL-6 and MGF in conjunction with M-CSF affect CD34⁺ cells especially at precursor level without distinct effect on the more mature stages. Secondly we studied whether M-CSF is only critical for the monocytic lineage or also affects dendritic cell (DC) development. Indeed, we were able to culture CD83⁺ DC from CD34⁺ progenitor cells in the presence of M-CSF in conjunction with TNF-α, IL-4, and MGF although their absolute number is almost threefold lower than the number of CD83⁺ cells yielded from GM-CSF plus TNF-α, IL-4, and MGF stimulated CD34⁺ cells.     

FLT3配基在人骨髓基质细胞系中的基因转移与表达

目的：研究逆转录病毒介导的FL在骨髓基质细胞系HFCL中的表达。方法：采用脂质体法将重组质粒pLF-SN/HFCL和空载体pLXSN/HFCL转染包装细胞PA317,G418筛选抗性克隆,用抗性克隆上清液感染HFCL。RT－PCR和基因组DNA－PCR检测外源基因mRNA水平的表达及染色体的整合,小鼠CFU－GM集落法检测FL生物学活性。结果：在mRNA水平上有FL的表达,染色体基因组中整合有标记neo基因和FL基因。活性测试结果显示转染的骨髓基质细胞分泌FL。结论：提示骨髓基质细胞可作为基因治疗的靶细胞。     

Stimulation of immune suppressive CD34+ cells from normal bone marrow by Lewis lung carcinoma tumors

 Progressive growth of metastatic Lewis lung carcinoma (LLC-LN7) tumors is associated with increased levels of bone-marrow-derived CD34⁺ cells having natural suppressor (NS) activity toward T cells. The present studies determined whether tumor-derived products are responsible for this induction of NS activity. Culturing normal bone marrow cells with LLC-LN7-conditioned medium (LLC-CM) or with recombinant granulocyte/macrophage-colony-stimulating factor (GM-CSF) resulted in the appearance of NS activity. The development of NS activity coincided with a prominent increase in the levels of CD34⁺ cells. That the CD34⁺ cells were responsible for the NS activity of the bone marrow cultures containing LLC-CM was shown by the loss of NS activity when CD34⁺ cells were depleted. The stimulation of CD34⁺ NS cells by LLC-CM was attributed to tumor production of GM-CSF, since neutralization of GM-CSF within the LLC-CM reduced its capacity to increase CD34⁺ cell levels. Studies also showed that the induction of CD34⁺ NS cells by LLC-CM and GM-CSF could be overcome by including in the cultures an inducer of myeloid differentiation, 1α,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃]. These results demonstrate that the mechanism by which the LLC-LN7 tumors stimulate increased levels of CD34⁺ NS cells from normal bone marrow is by their production of GM-CSF and that this can be blocked with the myeloid differentiation inducer 1,25(OH)₂D₃. Received: 8 December 1997 / Accepted: 27 February 1998     

Red blood cells derived from peripheral blood and bone marrow CD34+ human haematopoietic stem cells are permissive to Plasmodium parasites infection

The production of fully functional human red cells in vitro from haematopoietic stem cells (hHSCs) has been successfully achieved. Recently, the use of hHSCs from cord blood represented a major improvement to develop the continuous culture system for Plasmodium vivax. Here, we demonstrated that CD34+hHSCs from peripheral blood and bone marrow can be expanded and differentiated to reticulocytes using a novel stromal cell. Moreover, these reticulocytes and mature red blood cells express surface markers for entrance of malaria parasites contain adult haemoglobin and are also permissive to invasion by P. vivax and Plasmodium falciparum parasites.     

Effect of dimethyl sulfoxide on post-thaw viability assessment of CD45+ and CD34+ cells of umbilical cord blood and mobilized peripheral blood

BACKGROUND: The effect of dimethyl sulfoxide (Me2SO) on enumeration of post-thaw CD45+ and CD34+ cells of umbilical cord blood (HPC-C) and mobilized peripheral blood (HPC-A) has not been systematically studied. METHODS: Cells from leukapheresis products from multiple myeloma patients and umbilical cord blood cells were suspended in 1, 2, 5, or 10% Me2SO for 20 min at 22 degrees C. Cells suspended in Me2SO were then immediately assessed or assessed following removal of Me2SO. In other samples, cells were suspended in 10% Me2SO, cooled slowly to -60 degrees C, stored at -150 degrees C for 48 h, then thawed. The thawed cells in 10% Me2SO were diluted to 1, 2, 5, or 10% Me2SO, held for 20 min at 22 degrees C and then immediately assessed or assessed after the removal of Me2SO. CD34+ cell viability was determined using a single platform flow cytometric absolute CD34+ cell count technique incorporating 7-AAD. RESULTS: The results indicate that after cryopreservation neither recovery of CD34+ cells nor viability of CD45+ and CD34+ cells from both post-thaw HPC-A and HPC-C were a function of the concentration of Me2SO. Without cryopreservation, when Me2SO is present recovery and viability of HPC-C CD34+ cells exposed to 10% Me2SO but not CD45+ cells were significantly decreased. Removing Me2SO by centrifugation significantly decreased the viability and recovery of CD34+ cells in both HPC-A and HPC-C before and after cryopreservation. DISCUSSION: To reflect the actual number of CD45+ cells and CD34+ cells infused into a patient, these results indicate that removal of Me2SO for assessment of CD34+ cell viability should only be performed if the HPC are infused after washing to remove Me2SO.     

Role of the plasma membrane ROS-generating NADPH oxidase in CD34+ progenitor cells preservation by hypoxia

Hypoxia favored the preservation of progenitor characteristics of hematopoietic stem and progenitor cells (HSPCs) in bone marrow. This work aimed at studying the role of reactive oxygen species (ROS)-generating NADPH oxidase system regulated by hypoxia in ex vivo cultures of cord blood CD34⁺ cells. The results showed that NADPH oxidase activity and ROS generation were reduced in hypoxia with respect to normal oxygen tension. Meanwhile the ROS generation was found to be inhibited by diphenyleneiodonium (the NADPH oxidase inhibitor), or N-acetylcysteine (the ROS scavenger). Accordingly NADPH oxidase mRNA and p67 protein levels decreased in hypoxia. The analysis of progenitor characteristics, including the proportion of cultured cells expressing the HSPCs marker CD34⁺CD38⁻, colony production ability of the colony-forming cells (CFCs), and the re-expansion capability of the cultured CD34⁺ cells, showed that either 5% pO₂ or reduced ROS favored preserving the characteristics of CD34⁺ progenitors, and promoted the expansion of CD34⁺CD38⁻ cells as well. The above results demonstrated that hypoxia effectively maintained biological characteristics of CD34⁺ cells through keeping lower intracellular ROS levels by regulating NADPH oxidase.     

N-糖基化在肿瘤细胞逃逸CD8+T细胞中的意义和机制

肿瘤在发生发展过程中会产生异常糖基化结构,改变细胞功能,使其能逃逸机体细胞的免疫识别和免疫攻击,实现免疫逃逸。细胞膜表面蛋白的N-糖基化参与调控肿瘤细胞的增殖、迁移、侵袭、干细胞特性等。近年来,一系列研究进一步证实,肿瘤细胞免疫检查点分子的N-糖基化,以及识别N-糖基化的动物凝集素在肿瘤免疫逃逸CD8⁺T细胞中发挥了重要作用,如PD-L1的N-糖基化能增强其自身蛋白质的稳定性,促进与其受体PD-1的相互作用,并且靶向PD-1、PD-L1蛋白N-糖基化的抗体,能够有效抑制肿瘤免疫逃逸和肿瘤发展。     

Partition behavior of CD133+ stem cells from human umbilical cord blood in aqueous two‐phase systems: In route to establish novel stem cell primary recovery strategies

Aqueous two‐phase systems (ATPS) represent a promising strategy for the recovery of CD133⁺ stem cells. This particular type of stem cells has great potential for research and clinical applications. Traditional [polyethylene glycol (PEG), dextran (DEX), and ficoll] and novel (Ucon) polymer–polymer ATPS were exploited to study the partitioning behavior of CD133⁺ stem cells and contaminants from human umbilical cord blood (HUCB). The aim of the study was to select conditions under which the product of interest and the contaminants concentrate in opposite phases. To accomplish this, three independent samples were tested: (1) enriched CD133⁺ sample, (2) whole HUCB (contaminants), and (3) complex sample (CD133⁺ stem cells and contaminants). The objective of this research was to evaluate the partition behavior of CD133⁺ in ATPS in route to establish the basis for the development of a novel and scalable purification bioprocess. In conclusion, the partitioning behavior of CD133⁺ stem cells and contaminants from complex samples was as follows: 59% of CD133⁺ stem cells fractionated to the top phase when employing ficoll 400,000–DEX 70,000 or 100% to the bottom phase with Ucon‐DEX 75,000 and PEG 8,000‐DEX 500,000 ATPS. In average, 35% of the contaminants partitioned to the top phase of the ficoll 400,000‐DEX 70,000 ATPS, 99% to the dextran rich phase of the Ucon‐DEX 75,000 systems and 97% to the bottom phase of the PEG 8,000‐DEX 500,000. Cell viability was at least 98% after ATPS recovery. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:700–707, 2014     

Recombinant human (rh) stem cell factor and rhIL-4 stimulate differentiation and proliferation of CD3+ cells from umbilical cord blood and CD3+ cells enhance FcepsilonR1 expression on fetal liver-derived mast cells in the presence of rhIL-4

We previously reported that rhIL-4 induced apoptosis and rhIL-6 mediated protection of human mast cells derived from cord blood mononuclear cells. Based on the result, we attempted to obtain the phenotypes and differentiation of CD3+ cells from cord blood by investigating their cell surface markers in the presence of rhSCF plus rhIL-4. The effect of co-cultured CD3+ cells on fetal liver mast cells (FLMCs) was also determined. Phenotypes from cord blood-derived cells were analyzed by flow cytometry and cell numbers were determined. Fetal liver mast cells were cultured with cord blood-derived cells (mainly CD3+) in the presence of rhSCF and/or rhIL-4 and were analyzed to determine cell number and expression of Kit+ and FcepsilonR1. The percentage of CD3+ cells from cord blood-derived cells on day 0 was about 41 +/- 13.5%, following monocytes and granulocytes. CD3+ cells increased in number (1.5-fold) and purity (90%), whereas other cell types did not survive. More than 60% of CD3+ cells from cord blood at day 0 were CD4(-)CD8-. These double-negative cells dramatically decreased by 1 week of culture, while CD4+CD8+ cells increased in number and purity through 3 weeks of culture, and then decreased as greater numbers of single-positive T cells emerged. We also found that FcepsilonR expression on FLMC increased in the presence of rhIL-4, but was not affected by the T cells that developed from cord blood mononuclear cells. The results indicate that IL-4, a Th2 type cytokine, together with rhSCF, can induce T cell proliferations, differentiation, and maturation from cord blood progenitor cells.