Ex vivo expansion of neutrophil precursor cells from mobilized peripheral blood cells: similar results in cancer patients and normal donors

BACKGROUND: Infusion of ex vivo differentiated myeloid progenitors may reduce or abrogate severe neutropenia following mobilized peripheral blood transplantation. We compared the ex vivo expansion of myeloid progenitor cells starting from cancer patients (CP) and from normal donors (ND) and evaluated the influence of the CD34(+) cell mobilization on the capacities of cells to be expanded. METHODS: The ex vivo-expanded cells were evaluated for their phenotype, the presence of primary and secondary granules and their functional capacities (oxidative burst activity and phagocytosis). RESULTS: We did not observe significant differences between ND and CP for the total leukocyte and CD34(+) cell expansions nor for the myeloid progenitor production. In CP as well as in ND, the expanded cells were functionally competent. DISCUSSION: This suggests that the capacities of CD34(+) cells to proliferate and differentiate ex vivo are not impaired by prior chemotherapy and/or disease status. On the other hand, we did not observe any significant correlation between the number of mobilized CD34(+) cells before apheresis and the cell expansion. In conclusion, the ex vivo expansion of CP and ND cells is comparable and achievable even with a low CD34(+) cell number in mobilized peripheral blood.     

Ex vivo expansion of umbilical cord blood

The efficacy of cord blood (CB) transplantation is limited by the low cell dose available. Low cell doses at transplant are correlated with delayed engraftment, prolonged neutropenia and thrombocytopenia and elevated risk of graft failure. To potentially improve the efficacy of CB transplantation, approaches have been taken to increase the cell dose available. One approach is the transplantation of multiple cord units, another the use of ex vivo expansion. Evidence for a functional and phenotypic heterogeneity exists within the HSC population and one concern associated with ex vivo expansion is that the expansion of lower 'quality' hematopoietic progenitor cells (HPC) occurs at the expense of higher 'quality' HPC, thereby impacting the reserve of the graft. There is evidence that this is a valid concern while other evidence suggests that higher quality HPC are preserved and not exhausted. Currently, ex vivo expansion processes include: (1) liquid expansion: CD34+ or CD133+ cells are selected and cultured in medium containing factors targeting the proliferation and self-renewal of primitive hematopoietic progenitors; (2) co-culture expansion: unmanipulated CB cells are cultured with stromal components of the hematopoietic microenvironment, specifically mesenchymal stem cells (MSC), in medium containing growth factors; and (3) continuous perfusion: CB HPC are cultured with growth factors in 'bioreactors' rather than in static cultures. These approaches are discussed. Ultimately, the goal of ex vivo expansion is to increase the available dose of the CB cells responsible for successful engraftment, thereby reducing the time to engraftment and reducing the risk of graft failure.     

Ex vivo expansion of cord blood mononuclear cells on mesenchymal stem cells

BACKGROUND: Cord blood (CB) cells are being used increasingly as a source of hematopoietic cells to support high dose chemotherapy. However, CB units contain low numbers of cells, including CD34+ cells, and thus their use is associated with significant delays in engraftment of neutrophils and platelets. Exvivo expansion of CB has been proposed to increase the numbers of cells available. We and others have reported the requirement of CD34 selection for optimal expansion of CB products'; however, the selection of frozen CB products in clinical trials results in significant loss of CD34+ cells, with a median recovery of 50, but less than 40% recovery in more than one-third of products. In the present studies we evaluated the potential of mesenchymal stem cells (MSC) to support ex vivo expansion of unselected CB products. METHODS: Mononuclear cells (MNC) from CB products were isolated and cultured on preformed MSC layers in T150 flasks containing 50 mL Stemline II media plus hematopoietic growth factors. Various culture conditions were compared for optimal expansion of the CB MNC. RESULTS: Ex vivo expansion of CB MNC on MSC resulted in 10- to 20-fold expansion of total nucleated cells, seven- to 18-fold expansion of committed progenitor cells, two- to five-fold expansion of primitive progenitor cells and 16- to 37-fold expansion of CD34+ cells. DISCUSSION: These studies demonstrated significant expansion of CB products without CD34 cell selection using culture conditions that are clinically applicable. Our current focus is to initiate clinical trials to evaluate the in vivo potential of CB cells expanded with these conditions.     

Ex vivo expansion of hematopoietic stem cells derived from umbilical cord blood in rotating wall vessel

Expansion of umbilical cord blood mononuclear cells (UCB MNCs) was carried out in a rotating wall vessel (RWV) bioreactor and tissue culture flasks (T-flasks) in serum-containing medium supplemented with relatively low doses of purified recombinant human cytokines (5.33 ng/ml IL-3, 16 ng/ml SCF, 3.33 ng/ml G-CSF, 2.13 ng/ml GM-CSF, 7.47 ng/ml FL and 7.47 ng/ml TPO) for 8 days. The cell density, pH and osmolality of the culture medium in the two culture systems were measured every 24h. Flow cytometric assay for CD34+ cells was carried out at 0, 144 and 197 h and methylcellulose colony assays were performed at 0, 72, 144 and 197 h. The pH and osmolality of the medium in the two culture systems were maintained in the proper ranges for hematopoietic stem cells (HSCs) and progenitors culture. The RWV bioreactor, combined with a cell-dilution feeding protocol, was efficient to expand UCB MNCs. At the end of 200 h culture, the total cell number was multiplied by 435.5+/-87.6 times, and CD34+ cells 32.7+/-15.6 times, and colony-forming units of granulocyte-macrophage (CFU-GM) 21.7+/-4.9 times. While in T-flasks, however, total cells density changed mildly, CD34+ cells and CFU-GM decreased in number. It is demonstrated that the RWV bioreactor can provide a better environment for UCB MNCs expansion, enhance the contact between HSCs and accessory cells and make the utilization of cytokines more effective than T-flask.     

Isolation and expansion of oligodendrocyte progenitor cells from cryopreserved human umbilical cord blood

Background aimsOligodendrocyte precursor cells (OPC) hold promise as a cellular therapy for demyelinating diseases. The feasibility of using OPC-based therapies in humans depends upon a reliable, readily available source. We have previously described the isolation, expansion and characterization of oligodendrocyte-like cells from fresh human umbilical cord blood (UCB). We now describe the isolation and expansion of OPC from thawed, cryopreserved UCBMethodsWe thawed cryopreserved UCB units employing a standard clinical protocol, then isolated and plated mononuclear cells under previously established culture conditions. All OPC cultures were trypsinized at 21 days, counted, then characterized by flow cytometry after fixation, permeablization and labeling with the following antibodies: anti-oligodendrocyte marker 4 (O4), anti-oligodendrocyte marker 1 (O1) and anti-myelin basic protein (MBP). OPC were also placed in co-culture with shiverer mouse neuronal cells then stained in situ for beta tubulin III (BT3) and MBP as a functional assay of myelination.ResultsThe average OPC yield per cryopreserved UCB unit was 64% of that seen with fresh UCB. On flow cytometric analysis, 74% of thawed UCB units yielded cells with an O4-expression level of at least 20% of total events, compared with 95% of fresh UCB units. We observed myelination of shiverer neurons in our functional assay, which could be used as a potency assay for release of OPC cells in phase I human clinical trials.ConclusionsOur results demonstrate that OPC can be derived reliably from thawed, cryopreserved UCB units, and support the feasibility of using these cells in human clinical trials.     

Ex vivo expansion, adipogenesis and neurogenesis of cryopreserved human bone marrow mesenchymal stem cells

This study aimed to investigate the potentials of ex vivo expansion and pluridifferentiation of cryopreserved adult human bone marrow mesenchymal stem cells (hMSCs) into adipocytes and neurocytes. Cryopreserved hMSCs were resuscitated and cultured for 15 passages, and then induced to adipocytes and neurocytes with corresponding induction medium. The induced cells were observed for morphological properties and expression of triglyceride or neuron-specific enolase and nestin was detected. The result showed that the resuscitated cells cultured in induction medium consisting of dexamethasone, 3-isobutyl-1-methylxanthine, indomethacin and insulin-like growth factor I (IGF-I) showed adipogenesis, and lipid vacuole accumulation was detectable after 21days. The resuscitated hMSCs were also induced into neurocytes and expressed nestin and neuron-specific enolase (NSE), which are special surface markers associated with neural cells at different stages. This study suggested that resuscitated hMSCs should still be a population of pluripotential cells and should be accessible for establishing an abundant hMSC reservoir for further experiment and treatment of various clinical diseases.     

Ex vivo expansion and clonality of CD34+ selected cells from bone marrow and cord blood in a serum-free media

Although umbilical cord blood is increasingly being used in allogeneic marrow transplantation, delayed platelet engraftment is often a concern for cord blood transplant recipients. We evaluated the potential of ex vivo expansion and clonality in CD34+ cells separated from a bone marrow source, and cord blood, in a serum-free Media. The CD34+ cells, selected from bone marrow (BM) and umbilical cord blood (CB), 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 days 0, 4, 7, and 14 under the combination of growth factors, with cell counts. The cytokines included the recombinant human megakaryocyte growth and development (100 ng/ml), interleukin-3 (10 ng/ml), stem cell factor (100 ng/ml), flt-3 ligand (50 ng/ml) and interleukin-11 (200 ng/ml). The CB-selected CD34+ cells showed significantly higher total cell expansion than those from the BM at day 7 (3.0 fold increase than BM), day 14 (2.4 fold), and day 17 (2.6 fold). The colony count of the BFU-E/CFU-E per CD34+ cell at day 0 was 0.14 +/- 0.023 in the CB, which was significantly higher than 0.071 +/- 0.015 in the BM. The CB-selected CD34+ cells produced more BFU-E colonies than the BM on culture days 4, 7, and 14. The BFU-E colonies from the CB cells increased markedly on culture days 4 and 7, with a 4-fold increase at day 14. The colony count of the CFU-Mk per CD34+ cell at day 0 was 0.047 +/- 0.011 in the CB-selected CD34+ cells cultures, which was higher than the 0.026 +/- 0.014 in the BM. The CB-selected CD34+ cells produced more CFU-Mk colonies than the BM on culture days 4, 7 and 14. In conclusion, the ex vivo expansion of the CB cells may be very promising in producing total cellular expansion, CFU-Mk and BFU-E compared with BM, especially at day 7. The ex vivo expansion of the CB may have rationale in making an ex vivo culture for 7 to 14 d.     

Ex vivo expansion of human umbilical cord blood hematopoietic progenitor cells in a novel three-dimensional culture system

A novel three-dimensional culture system for the ex vivo expansion of human umbilical cord blood (CB) hematopietic progenitor cells (HPCs) was developed by growing CB mononuclear cells on highly porous CultiSpher G microspheres coated with human bone marrow stromal cells in stirred flasks in the presence of supplemented cytokines. After 12 days, the number of total viable cells, colony-forming units in culture (CFU-C) and CD34⁺ cells present in the cultures reflected average increases of 7.7, 23.3 and 9.6-fold, respectively, and marked hematopoietic islands were formed on the surface of CultiSpher G.     

Mesenchymal stem cells from cryopreserved human umbilical cord blood

Umbilical cord blood (UCB) is well known to be a rich source of hematopoietic stem cells with practical and ethical advantages, but the presence of mesenchymal stem cells (MSCs) in UCB has been disputed and it remains to be validated. In this study, we examined the ability of cryopreserved UCB harvests to produce cells with characteristics of MSCs. We were able to obtain homogeneous plastic adherent cells from the mononuclear cell fractions of cryopreserved UCB using our culture conditions. These adherent cell populations exhibited fibroblast-like morphology and typical mesenchymal-like immunophenotypes (CD73+, CD105+, and CD166+, etc.). These cells presented the self-renewal capacity and the mesenchymal cell-lineage potential to form bone, fat, and cartilage. Moreover, they expressed mRNAs of multi-lineage genes including SDF-1, NeuroD, and VEGF-R1, suggesting that the obtained cells had the multi-differentiation capacity as bone marrow-derived MSCs. These results indicate that cryopreserved human UCB fractions can be used as an alternative source of MSCs for experimental and therapeutic applications.     

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.     

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

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.     

Ex vivo expanded umbilical cord blood T cells maintain naive phenotype and TCR diversity

BACKGROUND: Umbilical cord blood (CB) is a promising source of hematopoietic stem cells for allogeneic transplantation. However, delayed engraftment and impaired immune reconstitution remain major limitations. Enrichment of donor grafts with CB T cells expanded ex vivo might facilitate improved T-cell immune reconstitution post-transplant. We hypothesized that CB T cells could be expanded using paramagnetic microbeads covalently linked to anti-CD3 and anti-CD28 Ab. METHODS: CB units were divided into three fractions: (1) cells cultured without beads, (2) cells cultured with beads and (3) cells cultured with beads following CD3+ magnetic enrichment. All fractions were cultured for 14 days in the presence of IL-2 (200 IU/mL). RESULTS: A mean 100-fold expansion (range 49-154) of total nucleated cells was observed in the CD3+ magnetically enriched fraction. Following expansion, CB T cells retained a naive and/or central memory phenotype and contained a polyclonal TCR diversity demonstrated by spectratyping. DISCUSSION: Our data provide evidence that naive and diverse CB T cells may be expanded ex vivo and warrant additional studies in the setting of human CB transplantation.     

脐血造血细胞体外保存及扩增的研究

研究了脐血造血细胞在4℃冰箱保存过程中单核细胞活力、造血性能的变化以及细胞因子种类、组合、不同培养基添加成分对造血细胞扩增的影响。研究表明脐血在4℃冰箱保存应不超过3d;细胞因子组合SCF IL-6 FL TPO扩增CFU—C的效果最佳;培养基体系中添加脐血混合血浆对扩增CFU—C作用明显,脐血混合血浆的最佳浓度为25％。     

Isolation and expansion of high yield of pure mesenchymal stromal cells from fresh and cryopreserved placental tissues

The injection of placental stromal cells isolated from fetal human tissues (f-hPSC) was reported to indirectly induce tissue regeneration in different animal models. A procedure of f-hPSC isolation from fragments of both selected fresh or cryopreserved bulk placental neonate tissues is proposed, based on their high migratory potential,. The fragments of the desired fetal placental tissues are adhered to a culture dish by traces of diluted fibrin and covered with culture medium. Spontaneous migration of pure f-hPSC from the tissue fragments to the cell culture dishes is followed by their rapid expansion by numerous passages. The isolated f-hPSC express typical mesenchymal surface antigens, including CD29, CD105, CD166 and CD146, with negative expression of white blood cell lineage and endothelial cells markers. Optimal yields of f-hPSC cultures can also be obtained from tissue samples cryopreserved in medium composed of 10% dimethyl sulfoxide (M₂SO) and 50% fetal calf serum. Slightly better yields are obtained with media supplemented with 1% human albumin. Medium with 5% M₂SO and/or 0.25 mg/ml PEG yielded inferior results. The f-hPSC from fresh or cryopreserved tissues express similar cell markers and growth kinetics. The proposed isolation protocol may also be applied for high yield isolation of stromal cells from fresh and cryopreserved tissue of other organs.     

低温冻存对脐血树突状细胞生物特性的影响

目的 :观察低温冻存对脐血来源的树突状细胞 (DCs)培养生物特性的影响 ,为DCs的应用提供冻存方法。方法 :设未冻存脐血细胞 (UFCB)经增殖培养后产生的DCs(UFDCs)为对照组 ,来源于经低温冻存的脐血 (CPCB)的DCs和经低温冻存的DCs(CPDCs)为实验组 ,比较实验组与对照组DCs生物特性 ,包括细胞增殖量 ,不染色和染色的细胞形态 ,台盼蓝拒染回收率 (TBR) ;细胞表面抗原 ;混合淋巴细胞培养剌激指数 (SI)、抗毒 杀灭效应处理率(ER)。结果 :CPCB与UFCB比 ,形成DCs的时间迟 ,细胞增殖量、树突状细胞量、CD1a、CD83、HLA DR表达、SI、ER均低。CPDCs与UFDCs比 ,形成贴壁树突状细胞迟 ,细胞增殖量、树突状细胞量、CD1a、CD83、HLA DR表达、SI、ER均低。CPCB的TBR为 95.8% ,CPDCs的TBR为 88.7%。结论 :脐血细胞和经增殖培养的脐血DCs ,经低温冻存后复温培养 ,DCs的生物特性有一定的损伤 ,但基本功能仍比较完整 ,回收率均 >85%。