Application of proteoglycan extracted from the nasal cartilage of salmon heads for ex vivo expansion of hematopoietic progenitor cells derived from human umbilical cord blood

Highly purified proteoglycan (PG) extracted from the nasal cartilage of salmon heads was applied to the ex vivo expansion of hematopoietic progenitor cells prepared from human umbilical cord blood in serum-free cultures supplemented with the combination of early-acting cytokines, thrombopoietin (TPO), interleukin-3 (IL-3) and stem cell factor (SCF). PG showed no promoting effects on the cell proliferation rate; however, they promoted the generation of progenitor cells for granulocyte-macrophages, erythrocytes and/or megakaryocytes in culture with TPO alone or SCF plus TPO. However, no promoting effect was observed in a combination of IL-3 plus SCF, which showed the highest cell proliferation rate. PG failed to promote the generation of mixed colony-forming units (i.e. the relatively immature cells in hematopoiesis). These results suggest that PG acts on the relatively mature stem/progenitor cells, and may function as a regulatory factor in the differentiation pathway of hematopoiesis.     

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 differentiation of umbilical cord blood progenitor cells in the presence of placental conditioned medium

Hematopoetic stem cells (HSC) are the progenitors for the lympho-hematopoietic system, with long lifespan and high proliferation potential. Transplantation of HSC from bone marrow or peripheral blood represents a standard therapy in severe hematological conditions. A possible alternative source of HSC is the umbilical cord blood, prepared by various separation procedures followed by expansion in cultures supplemented with hematopoietic growth factors. In order to check the effects of placental conditioned medium (PCM) from placental cells culture upon viability of HSC, we added plasma, PCM, dimetil sulfoxyde or hemin in HSC cultures. Flow cytometry or direct scoring of solid cultures using CD45+, CD34+, CD71+ and CD14+ fluorescent-labeled monoclonal antibodies evaluated the effects upon cell proliferation and colony forming ability of HSC cultures, versus controls. PCM produced the highest proliferation, followed by plasma, DMSO and hemin. PCM improved the survival time and maintained a higher proportion of immature cells. PCM stimulates the differentiation towards myeloid lineage progenitor cells (>90% being CD45+), increasing the percentage of CD14+, granulocites /monocytes precursors. It is highly suggestive that PCM contains growth factors or cytokines, which regulate the development of HSC. Characterization of these factors is in progress.     

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.     

Improved immunomagnetic enrichment of CD34(+) cells from umbilical cord blood using the CliniMACS cell separation system

Background aimsCD34+ enrichment from cord blood units (CBU) is used increasingly in clinical applications involving ex vivo expansion. The CliniMACS instrument from Miltenyi Biotec is a current good manufacturing practice (cGMP) immunomagnetic selection system primarily designed for processing larger numbers of cells: a standard tubing set (TS) can process a maximum of 60 billion cells, while the larger capacity tubing set (LS) will handle 120 billion cells. In comparison, most CBU contain only 1–2 billion cells, raising a question regarding the optimal tubing set for CBU CD34+ enrichment. We compared CD34+ cell recovery and overall viability after CliniMACS processing of fresh CBU with either TS or LS.MethodsForty-six freshly collected CBU (≤ 36 h) were processed for CD34+ enrichment; 22 consecutive units were selected using TS and a subsequent 24 processed with LS. Cell counts and immunophenotyping were performed pre- and post-selection to assess total nucleated cells (TNC), viability and CD34+ cell content.ResultsTwo-sample t-tests of mean CD34+ recovery and viability revealed significant differences in favor of LS (CD34+ recovery, LS = 56%, TS = 45%, P = 0.003; viability, LS = 74%, TS = 59%, P = 0.011). Stepwise linear regression, considering pre-processing unit age, viability, TNC and CD34+ purity, demonstrated statistically significant correlations only with the tubing set used and age of unit.ConclusionsFor CD34+ enrichment from fresh CBU, LS provided higher post-selection viability and more efficient recovery. In this case, a lower maximum TNC specification of TS was not predictive of better performance. The same may hold for smaller scale enrichment of other cell types with the CliniMACS instrument.     

Pattern of cytokine expression by rat liver epithelial cells supporting long-term culture of human CD34(+)umbilical cord blood cells

Fetal liver is the main site of haematopoiesis during mid-gestation. The adult liver still provides a favourable environment for extramedullary haematopoiesis. Nevertheless, regulation of liver haematopoiesis by cell-cell contacts or by cytokines remains poorly understood. Recently, we have shown that rat liver epithelial cells (RLECs) support long-term survival and multilineage differentiation of adult human CD34(+)and CD34(+)/CD38(-)haematopoietic cells obtained from granulocyte-colony stimulating factor mobilized peripheral blood and from bone marrow respectively. In addition, the importance of physical proximity between haematopoietic cells and RLECs was clearly demonstrated. Here, our findings give evidence that RLECs belonging to the epithelial but non-parenchymal liver compartment also sustain the long-term production of progenitors from human CD34(+)umbilical cord blood cells. Moreover, to better analyse the regulation of haematopoiesis in this RLEC coculture model, we have investigated the cytokine expression by RLECs alone and by RLECs coming from coculture with CD34(+)cells from umbilical cord blood. We demonstrated that a broad spectrum of cytokines acting at different stages of haematopoiesis is produced by RLECs. Interestingly, an upregulation of leukemia inhibitory factor expression by RLECs in presence of CD34(+)haematopoietic cells was observed. These data suggest an important role of cell-cell interactions in the regulation of haematopoiesis.     

Protective effects of thrombopoietin and stem cell factor on X-irradiated CD34+ megakaryocytic progenitor cells from human placental and umbilical cord blood

In previous studies we characterized the radiosensitivity of CFU-megakaryocytes from human placental and umbilical cord blood and the effects of various early-acting cytokines. We found that the maximal clonal growth of CFU-megakaryocytes in vitro and maximal protection against X-ray damage were supported by a combination of thrombopoietin and stem cell factor. However, the mechanism by which the two cytokines exert a synergistic effect remained unclear, so we extended these studies to investigate the radioprotective action of synergistic thrombopoietin and stem cell factor on the survival of X-irradiated CD34(+) CFU-megakaryocytes. A combination of thrombopoietin and stem cell factor led to activation of mitogen-activated protein kinase and extracellular signal-regulated protein kinase and to suppression of caspase 3 in X-irradiated CD34(+) cells. When PD98059 and various synthetic substrates-specific inhibitors of these proteins-were used, the combination had less effect on the clonal growth of X-irradiated CD34(+) CFU-megakaryocytes. However, the addition of wortmannin, a specific inhibitor of the phosphatidylinositol-3 kinase pathway, did not alter the synergistic action of thrombopoietin plus stem cell factor. We suggest that part of this synergistic effect can be explained by activation of mitogen-activated protein kinase and extracellular signal-regulated protein kinase and by suppression of the caspase cascade.     

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.     

Individual and synergistic cytokine effects controlling the expansion of cord blood CD34(+) cells and megakaryocyte progenitors in culture

Background aimsExpansion of hematopoietic progenitors ex vivo is currently investigated as a means of reducing cytopenia following stem cell transplantation. The principal objective of this study was to develop a new cytokine cocktail that would maximize the expansion of megakaryocyte (Mk) progenitors that could be used to reduce periods of thrombocytopenia.MethodsWe measured the individual and synergistic effects of six cytokines [stem cell factor (SCF), FLT-3 ligand (FL), interleukin (IL)-3, IL-6, IL-9 and IL-11] commonly used to expand cord blood (CB) CD34⁺ cells on the expansion of CB Mk progenitors and major myeloid populations by factorial design.ResultsThese results revealed an elaborate array of cytokine individual effects complemented by a large number of synergistic and antagonistic interaction effects. Notably, strong interactions with SCF were observed with most cytokines and its concentration level was the most influential factor for the expansion and differentiation kinetics of CB CD34⁺ cells. A response surface methodology was then applied to optimize the concentrations of the selected cytokines. The newly developed cocktail composed of SCF, thrombopoietin (TPO) and FL increased the expansion of Mk progenitors and maintained efficient expansion of clonogenic progenitors and CD34⁺ cells. CB cells expanded with the new cocktail were shown to provide good short- and long-term human platelet recovery and lymphomyeloid reconstitution in NOD/SCID mice.ConclusionsCollectively, these results define a complex cytokine network that regulates the growth and differentiation of immature and committed hematopoietic cells in culture, and confirm that cytokine interactions have major influences on the fate of hematopoietic cells.     

CD133 immunomagnetic separation: effectiveness of the method for CD133(+) isolation from umbilical cord blood

Background aimsUmbilical cord blood (UCB) is a rich source of stem cells, the characterization and isolation of which requires specific stem cell markers and reliable and reproducible protocols.MethodsWe assessed CD133 isolation in 39 UCB samples, using a commercial immunomagnetic cell-sorting protocol, and, because of its non-reproducibility, we applied optimized protocols in an effort to improve it. These included extra-labeling of the selected CD133⁺ subpopulation and indirect labeling using anti-phycoerythrin (PE) microbeads, goat anti-mouse IgG microbeads or a combination of both. The CD34 isolation was used as a control.ResultsThe mononuclear cell fraction expressed 0.53 ± 0.06% CD133. The corresponding value for CD34 was 1.64 ± 0.15%. Following the manufacturer's instructions, the CD34 isolation resulted in a population expressing 93 ± 1.25% CD34 while, after the corresponding process, CD133⁺ expression ranged from 10% to 85% (median 60%). The optimized isolation protocols did not result in improved CD133⁺ yield. The variation in the purity of the CD133 population cannot be attributed to the different clones of CD133 used, because they do not cross-block, while other factors such as glycosylation, which could possibly interfere, do not apply in normal hematopoietic stem cells (HSC).ConclusionsCD34 isolation by the immunomagnetic method results in highly pure CD34⁺ population, while the efficient and reproducible yield of a pure CD133⁺ population is not feasible. Therefore quantification of the positive cells should follow each isolation procedure in order to confirm the number of CD133⁺ cells.     

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.     

Cryopreservation of umbilical cord blood: 1. Osmotically inactive volume,hydraulic conductivity and permeability of CD34(+) cells to dimethyl sulphoxide

Umbilical cord blood (UCB) is an accepted treatment for the reconstitution of bone marrow function following myeloablative treatment predominantly in children and juveniles. Current cryopreservation protocols use methods established for bone marrow and peripheral blood progenitors cells that have largely been developed empirically. Such protocols can result in losses of up to 50% of the nucleated cell population: losses unacceptable for cord blood. The design of optimal cryopreservation regimes requires the development of addition and elution protocols for the chosen cryoprotectant; protocols that minimise damaging osmotic transients. The biophysical parameters necessary to model the addition and elution of dimethyl sulphoxide to and from cord blood CD34(+) cells have been established. An electronic particle counting method was used to establish the volumetric response of CD34(+) cells to changes in osmolality of the suspending medium. The non-osmotic volume of the cell was 0.27 of the cells isotonic volume. The permeation kinetics of CD34(+) cells to water and dimethyl sulphoxide were investigated at two temperatures, +1.5 and +20 degrees C. Values for the hydraulic conductivity were 3.2 x 10(-8) and 2.8 x 10(-7)cm/atm/s, respectively. Values for the permeability of dimethyl sulphoxide at these temperatures were 4.2 x 10(-7) and 7.4 x 10(-6)cm/s, respectively. Clonogenic assays indicated that the ability of CD34(+) cells to grow and differentiate was significantly impaired outside the limits 0.6-4x isotonic. Based on the Boyle van't Hoff plot, the tolerable limits for cell volume excursion were therefore 45-140% of isotonic volume. The addition and elution of cryoprotectant was modelled using a two-parameter model. Current protocols for the addition of cryoprotectant based on exposure at +4 degrees C would require additional time for complete equilibration of the cryoprotectant. During the elution phase current protocols are likely to cause CD34(+) cells to exceed tolerable limits. The addition of a short holding period during elution reduces the likelihood of this occurring.     

Optimisation of transfection conditions of CD34+ hematopoietic cells derived from human umbilical cord blood

Human umbilical cord blood is frequently used as a source of transplantable hematopoietic cells and more recently as a target of gene therapy - a new approach for treatment of various disorders. The aim of our study was optimisation of the transfection conditions of cord blood-derived CD34(+) hematopoietic cells. Mononuclear cells fraction was isolated from cord blood samples by density gradient centrifugation. Subsequently, CD34(+) hematopoietic cells were separated on immunomagnetic MiniMACS columns. Pure population of CD34(+) cells was incubated in a serum free medium supplemented with thrombopoietin, stem cell factor and Flt-3 ligand for 48 h and then transfected with plasmid DNA carrying the enhanced version of green fluorescent protein (EGFP) as a reporter gene. We studied the influence of various pulse settings and DNA concentrations on the transfection efficiency, measured by flow cytometry as the fluorescence of target cells due to the expression of EGFP. The optimal settings were as follows: 4 mm cuvette, 1600 microF, 550 V/cm, and 10 microg of DNA per 500 microl. With these settings we obtained a high transfection frequency (41.2%) without a marked decrease of cell viability. An increase of the pulse capacitance and/or of DNA concentration resulted in a greater electroporation efficiency, but also in a decrease of cell viability. In conclusion, the results described here allow one to recommend electroporation as an efficient method of gene delivery into CD34(+) hematopoietic cells derived from human umbilical cord blood.     

Co-culture of cord blood CD34(+) cells with human BM mesenchymal stromal cells enhances short-term engraftment of cord blood cells in NOD/SCID mice

BACKGROUND: The major challenge for cord blood transplantation (CBT) is higher rates of delayed and failed engraftment. In an attempt to broaden the application of CBT to more candidates, ex vivo expansion of hematopoietic stem/progenitor cells in CB is a major area of investigation. The purpose of this study was to employ human BM mesenchymal stromal cells (hBM-MSC) as the feeding-layer to expand CB cells ex vivo. METHODS: In this study, hBM-MSC were isolated and characterized by morphologic, mmunophenotypic and RT-PCR analysis. The hBM-MSC at passage 3 were employed as the feeding-layer to expand CB CD34(+) cells in vivo in the presence of thrombopoietin, flt3/flk2 ligand, stem cell factor and G-CSF. The repopulating capacity of the ex vivo-expanded CB cells was also evaluated in a NOD/SCID mice transplant experiment. RESULTS: After 1 or 2 weeks of in vitro expansion, hBM-MSC supported more increasing folds of CB in total nucleated cells, CD34(+) cells and colony-forming units (CFU) compared with CB without hBM-MSC. Furthermore, although NOD/SCID mice transplanted with CB cells expanded only in the presence of cytokines showed a higher percentage of human cell engraftment in BM than those with unexpanded CB CD34(+) cells, expanded CB cells co-cultured with hBM-MSC were revealed to enhance short-term engraftment further in recipient mice. DISCUSSION: Our study suggests that hBM-MSC enhance in vitro expansion of CB CD34(+) cells and short-term engraftment of expanded CB cells in NOD/SCID mice, which may be valuable in a clinical setting.     

A comparison of CFU-GM, BFU-E and endothelial progenitor cells using ex vivo expansion of selected cord blood CD133(+) and CD34(+) cells

BACKGROUND: CD133 is a newly developed hematopoietic stem cell marker but little is known about its function. Whether CD133(+) cell selection provides any advantage over CD34(+) selection for hematopoietic stem cell isolation and transplantation is unclear. The present study compared colony formation and endothelial cell differentiation of these two cell types from umbilical cord blood (UCB). METHODS: Mononuclear cells from the same UCB samples were used for both CD133(+) and CD34(+) cell selection. Cells with 97.1% purity were incubated in semi-solid culture medium containing stem cell growth factor (SCGF) and G-CSF or erythropoietin (EPO). Purified cells were also cultured in M199 containing vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and insulin-like growth factor-1 (IGF-1). RESULTS: CD34(+) and CD133(+) cells produced similar numbers of CFU-GM colonies (median 43.25 and 30.5, respectively; P>0.2). However, a greater than four-fold difference in BFU-E colony formation was observed from CD34(+) cells compared with CD133(+) cells (median 35 and 8, respectively; P<0.04). CD34(+) cells gave rise to endothelial-like cells when stimulated with VEGF, bFGF and IGF-1. CD133(+) cells were unable produce this cell type under the same conditions. DISCUSSION: CD133(+) cells produced smaller BFU-E colonies and were unable to differentiate into mature endothelial cells. CD34(+) cells contained endothelial progenitors that could differentiate into mature cells of this lineage. Based on these data, it appears that CD133 offers no distinct advantage over CD34 as a selective marker for immunoaffinity-based isolation of hematopoietic stem cells and endothelial progenitor cells.     

Hematopoietic reconstitution of CD34+ cells derived from short-term cultured cord blood mononuclear cells

Ex vivo expansion of hematopoietic stem cells (HSCs) is very important for clinical applications of cord blood (CB). With the aim to find proper culture duration for ex vivo expansion, mononuclear cells (MNC) was applied as starting culture cells to expand HSCs and the repopulating potential of seven-day and fourteen-day cultured CD34⁺ cells were compared. The average expansion of total cells and CD34⁺ cells cultured for 7 days were higher than those cultured for 14 days. The results of phenotypic analysis of fresh and cultured cells showed that the percentage of CD3⁺ cells declined and the percentage of CD33⁺ cells increased during culture. The engraftment levels of fourteen-day cultured CD34⁺ cells were higher than those of fresh and seven-day cultured CD34⁺ cells. Fourteen-day cultured CD34⁺ cells also showed better multilineage reconstitution ability than fresh and seven-day cultured CD34⁺ cells. The results of the present study demonstrated that prolonged culture could preserve the hematopoietic reconstitution ability of ex vivo cultured CB cells and improve the engraftment level in NOD/SCID mice.