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.     

Radiation sensitivity of megakaryocyte colony-forming cells in human placental and umbilical cord blood

The in vitro radiation sensitivity of CFU-Meg isolated from human placental and umbilical cord blood was evaluated in plasma clot cultures stimulated by recombinant human cytokines, including thrombopoietin, the FLT3 ligand (FLT3LG), interleukin-3, interleukin-11 and stem cell factor. The CD34(+) cells were irradiated with X rays at a dose rate of 73 cGy/ min. The megakaryocyte colonies were identified by using an FITC-conjugated antibody to glycoprotein IIbIIIa and were classified into two groups based on colony size: large colonies (immature CFU-Meg) and small colonies (mature CFU-Meg). Treatment with thrombopoietin alone or in combination with FLT3LG and/or interleukin-11 gave exponential radiation survival curves (D(0) for immature CFU-Meg = 56-77 cGy, D(0) for mature CFU-Meg = 86 cGy-1.12 Gy), while marked shoulders were observed on the survival curves for colonies supported by the combination of thrombopoietin, interleukin-3 and stem cell factor (D(0) for immature CFU-Meg = 89- 98 cGy; D(0) for mature CFU-Meg = 1. 25-1.31 Gy). Our results showed that the immature CFU-Meg were more radiosensitive than the mature CFU-Meg and that the combination of cytokines, including thrombopoietin, interleukin-3 and stem cell factor, affected the radiation sensitivity of CFU-Meg to the same extent as with thrombopoietin alone or in combination with FLT3LG and/or interleukin-11.     

Regeneration of megakaryocytopoiesis and thrombopoiesis in vitro from X-irradiated human hematopoietic stem cells

In the present study, we investigated whether X-irradiated hematopoietic stem cells can be induced to undergo megakaryocytopoiesis and thrombopoiesis in vitro using cytokine combinations that have been demonstrated to be effective for conferring increased survival on irradiated human CD34(+) megakaryocytic progenitor cells (colony-forming unit megakaryocytes; CFU-Meg), such as thrombopoietin (TPO), interleukin 3 (IL3), stem cell factor and FLT3 ligand. Culture of nonirradiated CD34(+) cells in serum-free medium supplemented with multiple cytokine combinations led to an approximately 200- to 600-fold increase in the total cell numbers by day 14 of culture. In contrast, the growth of X-irradiated cells was observed to be one-sixth to one-tenth that of the nonirradiated cultures. Similarly, total megakaryocytes were increased by 50- to 130-fold, while culture of X-irradiated cells yielded one-fourth to one-eighth of the control numbers. At this time, CD41(+) particles, which appeared to be platelets, were produced in the medium harvested from nonirradiated and irradiated cultures. Although radiation suppressed cell growth and megakaryocytopoiesis, there were no significant differences in thrombopoiesis between the two types of culture. These results suggest that X-irradiated CD34(+) cells can be induced to undergo nearly normal terminal maturation through megakaryocytopoiesis and thrombopoiesis by stimulation with appropriate cytokine combinations.     

Different radiosensitive megakaryocytic progenitor cells exist in steady-state human peripheral blood

CD34 antigen is a novel marker for human hematopoietic stem/progenitor cells. In the present study, two cell fractions, CD34low and CD34high, were prepared from steady-state human peripheral blood on the basis of CD34 antigen expression. The colony-forming unit megakaryocytes (CFU-Meg) contained in each cell fraction were compared for X-radiation sensitivity and cytokine action. The content of CD34+CD45+ cells in the CD34low and CD34high cell fractions was 74.8% and 88.8%, respectively, and the frequency of thrombopoietin (TPO)-supported CFU-Meg in the CD34low cell fraction was 1.9 times higher than that in CD34high. The CFU-Meg in CD34high were more radiosensitive than those in CD34low, indicating that steady-state human peripheral blood contains different types of CFU-Meg. However, no significant differences were observed between cell fractions in the radiation survival curves of CFU-Meg stimulated by TPO plus cytokines except granulocyte colony-stimulating factor (G-CSF). TPO plus interleukin 3 was the optimal combination for survival of both types of CFU-Meg after X irradiation. The present study also demonstrated that TPO plus G-CSF is able to increase the survival of irradiated CD34low CFU-Meg. These results suggest that two megakaryocytic progenitor populations with different radiosensitivity and cytokine responses are found in steady-state human peripheral blood.     

Severe damage of human megakaryocytopoiesis and thrombopoiesis by heavy-ion beam radiation

Heavy ions have a unique efficacy for tumor control in radiotherapy. To clarify the effects of heavy-ion beams on hematopoietic stem/progenitor cells, the effects of carbon-ion beams on megakaryocytopoiesis and thrombopoiesis in CD34(+) cells derived from human placental and umbilical cord blood were investigated. The cells were exposed to carbon-ion beams (LET = 50 keV/microm) and then were treated with thrombopoietin (TPO) alone or TPO plus other cytokines. Megakaryocytic progenitor cells, such as megakaryocyte colony-forming units (CFU-Meg), were far more sensitive to carbon-ion beams than to X rays, and no restoration of carbon-ion beam-irradiated CFU-Meg by treatment with any cytokine combination was observed. However, total cell expansion in liquid culture was not different after either carbon-ion beam or X irradiation of CD34(+) cells. The activation of gamma-H2AX, a marker of DNA double strand-breaks (DSBs), was promoted by the cytokine treatment in X-irradiated CD34(+) cells but not in carbon-ion-irradiated cells. These results showed that carbon-ion beams inflicted severe damage on megakaryocytopoiesis and thrombopoiesis and that a better combination of cytokines and other agents may be needed to stimulate the recovery of hematopoietic cells and repair this damage.     

A new role of thrombopoietin enhancing ex vivo expansion of endothelial precursor cells derived from AC133-positive cells

We previously reported that CD31(bright) cells, which were sorted from cultured AC133(+) cells of adult peripheral blood cells, differentiated more efficiently into endothelial cells than CD31(+) cells or CD31(-) cells, suggesting that CD31(bright) cells may be endothelial precursor cells. In this study, we found that CD31(bright) cells have a strong ability to release cytokines. The mixture of vascular endothelial growth factor (VEGF), thrombopoietin (TPO), and stem cell factor stimulated ex vivo expansion of the total cell number from cultured AC133(+) cells of adult peripheral blood cells and cord blood cells, resulting in incrementation of the adhesion cells, in which endothelial nitric oxide synthase and kinase insert domain-containing receptor were positive. Moreover, the mixture of VEGF and TPO increased the CD31(bright) cell population when compared with VEGF alone or the mixture of VEGF and stem cell factor. These data suggest that TPO is an important growth factor that can promote endothelial precursor cells expansion ex vivo.     

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.     

Placental/umbilical cord blood-derived mesenchymal stem cell-like stromal cells support hematopoietic recovery of X-irradiated human CD34+ cells

AimsThe potential of human mesenchymal stem cell-like stroma prepared from placental/umbilical cord blood for hematopoietic regeneration by X-irradiated hematopoietic stem cells is herein assessed.Main methodsPlacental/umbilical cord blood-derived mesenchymal stem cell-like stromal cells were applied to a regenerative ex vivo expansion of X-irradiated human CD34⁺ cells in a serum-free liquid culture supplemented with a combination of interleukine-3 plus stem cell factor plus thrombopoietin.Key findingsThe total number of cells and of lineage-committed myeloid hematopoietic progenitor cells generated in the co-culture of both non-irradiated and X-irradiated cells with stromal cells was significantly higher than those in the stroma-free culture. In addition, the number of CD34⁺ cells and CD34⁺/CD38^? cells, immature hematopoietic stem/progenitor cells also increased more than the stroma-free culture. The stromal cells produced various types of cytokines, although there was little difference between the co-cultures of non-irradiated and X-irradiated cells with stromal cells. Furthermore, when X-irradiated cells came in contact with stromal cells for 16 h before cytokine stimulation, a similar degree of hematopoiesis was observed, thus suggesting the critical role of cell-to-cell interaction.SignificanceThe present results showed the potential efficacy of human mesenchymal stem cell-like stroma for hematopoietic regeneration from irradiated hematopoietic stem/progenitor cells.     

低氧对CD34^＋造血干／祖细胞增殖分化及其对细胞因子依赖性的影响

To elucidate the effects of hypoxia on the proliferation and differentiation of CD34(+) hematopoietic stem/progenitor cells and their response to cytokines, the cells were isolated from umbilical cord blood by using a high-gradient magnetic cell sorting system (MACS). Mononuclear cells (MNC) and CD34(+) cells were incubated in severe hypoxia (1% oxygen) culture system, and the colony forming cells and antigen expression were studied by colony forming assays and FACS analysis. The results showed that incubation in severe hypoxia increased the number of erythroid bursts (BFU-E) (324.8+/-41.4/10(4) cells) generated from CD34(+) cells (191.2+/-34.5/10(4) cells in the control group, P<0.01). Severe hypoxia also enhanced the maintenance and cloning efficiency of BFU-E in a liquid culture system without growth factors, the number of BFU-E (152.4+/-22.6/10(4)cells) was much bigger than that in the control group (74.2+/-9.3/10(4) cells, P<0.01). In cultures incubated in hypoxia, the percentage of CD34(+) cells was significantly higher (2.5+/-1.2-fold, P<0.05) than in those incubated in air. BFU-E cloning efficiency of MNC was not significantly affected by hypoxia. The above results show that hypoxia enhances the maintenance of erythroid progenitor cells generated from CD34(+) hematopoietic stem/progenitor cells, no matter growth factors are present or not. These positive effects of hypoxia did not occur for the other progenitors.     

IL-9 enhances the growth of human mast cell progenitors under stimulation with stem cell factor

We examined the effects of IL-9 on human mast cell development from CD34(+) cord blood (CB) and peripheral blood cells in serum-deprived cultures. IL-9 apparently enhanced cell production under stimulation with stem cell factor (SCF) from CD34(+) CB cells. A great majority of the cultured cells grown with SCF + IL-9 became positive for tryptase at 4 wk. In methylcellulose cultures of CD34(+) CB cells, IL-9 increased both the number and size of mast cell colonies grown with SCF. Furthermore, SCF + IL-9 caused an exclusive expansion of mast cell colony-forming cells in a 2-wk liquid culture of CD34(+) CB cells, at a level markedly greater than for SCF alone. Clonal cell cultures and RT-PCR analysis showed that the targets of SCF + IL-9 were the CD34(+)CD38(+) CB cells rather than the CD34(+)CD38(-) CB cells. IL-9 neither augmented the SCF-dependent generation of progeny nor supported the survival of 6-wk-cultured mast cells. Moreover, there was no difference in the appearance of tryptase(+) cells and histamine content in the cultured cells between SCF and SCF + IL-9. The addition of IL-9 increased numbers of mast cell colonies grown with SCF from CD34(+) peripheral blood cells in children with or without asthma. It is of interest that mast cell progenitors of asthmatic patients responded to SCF + IL-9 to a greater extent than those of normal controls. Taken together, IL-9 appears to act as a potent enhancer for the SCF-dependent growth of mast cell progenitors in humans, particularly asthmatic patients.     

Cytokine-augmented culture of haematopoietic progenitor cells in a novel three-dimensional cell growth matrix

Studies aimed at the in vitro expansion of haematopoietic progenitor cells (HPCs) have suffered from the conflict of increasing cell numbers while maintaining long-term repopulating ability. We have developed a long-term bone marrow bioreactor culture system resembling the marrow-microenvironment that cultures HPCs in an inert, three-dimensional, porous biomatrix termed Cellfoam. Previous studies have shown that the short-term culture of CD34(+)cells in Cellfoam improved the maintenance and multipotency of haematopoietic stem cells compared to cells cultured on plastic dishes. In this study, we examined the effects of low concentrations of cytokines including stem cell factor (SCF), IL-3, and Flk-2/Flt-3 ligand, on the maintenance, preservation and multipotency of CD34(+) cells cultured for 3 or 6 weeks in Cellfoam. Analysis of cell yields using flow cytometry showed that in SCF and Flk-2/Flt-3 ligand-supplemented cultures as well as cytokine-free cultures, a higher number of CD45(+)34(+) and CD45(+)34(+)38(-) cells is observed in Cellfoam cultures as compared to plastic cultures. The function of cultured cells was evaluated in colony-forming assays. The data demonstrate that Cellfoam cultures supplemented with SCF and Flk-2/Flt-3 ligand resulted in a higher output of colony activity compared to plastic cultures. Analysis of CAFC (29 days) activity also demonstrated that primitive progenitors were maintained to a greater extent in Cellfoam cultures containing either no cytokines or low concentrations of early-acting cytokines. These data suggest that culture of HPCs in three-dimensional bioreactors such as Cellfoam for extended periods may benefit from the addition of low levels of early-acting cytokines, including SCF and Flk-2/Flt-3 ligand, resulting in high yields of cells that are enriched for multipotent haematopoietic progenitors. These findings demonstrate that a three-dimensional matrix promotes the survival of primitive HPCs in culture and may modulate the in vitro effects of cytokines.     

Ex vivo expansions of megakaryocytopoiesis from placental and umbilical cord blood CD34(+) cells in serum-free culture supplemented with proteoglycans extracted from the nasal cartilage of salmon heads and the nasal septum cartilage of whale

As a possible approach to the treatment of thrombopocytopenia, the ex vivo expansion of megakaryocytic progenitor cells may be a useful tool to accelerate platelet recovery in vivo. Our objective was to assess the promoting effect of proteoglycans in a serum-free culture condition using human cord blood CD34(+) cells. Highly purified proteoglycan (PG) extracted from the nasal cartilage of salmon heads and the nasal septum cartilage of a whale were applied to the ex vivo expansion of megakaryocytopoiesis and thrombopoiesis from placental and umbilical cord blood CD34(+) cells in serum-free cultures stimulated with a combination of thrombopoietin (TPO) and interleukin-3 (IL-3). Each PG (0.5 and 5 mug) was applied to the culture with three different concentrations of TPO (50, 5 and 0.5 ng/ml) and IL-3 (100, 10 and 1 ng/ml). Both of the PGs showed no promoting effects on the mononuclear cell proliferation rate in any of the cultures. However, the whale-PG promoted the generation of megakaryocytic progenitor cells and megakaryocytes in the culture with a lower dose of cytokines, respectively. In addition, whale-PG led to a significant increase in CD42a(+) particles which seemed to be platelets. While the salmon-PG failed to promote such production in almost all of the cultures. Although whale-PG is an attractive molecule for the ex vivo expansion of human megakaryocytopoiesis, its action may depend on the glycosaminoglycans sulfation pattern and the ability of the binding affinity and the kinetics to interact with the cytokines and hematopoietic stem/progenitor cells.     

The comparison of different protocols for expansion of umbilical-cord blood hematopoietic stem cells

Hematopoiesis is maintained by the activity of multipotent stem cells, which have the dual capacity to self-renew and to differentiate into all of the blood cell lineages. The major challenge of stem cells based regenerative therapy is to expand ex vivo the primitive compartment to increase transplantable stem cells number. The present study was designed to evaluate several culture systems for in vitro maintenance of umbilical cord blood stem cells. The influences of different growth conditions such as stromal feeder layer, cytokines supplement and placental conditioned medium (PCM) have been evaluated over a relatively short period of time on CD34(+) cell expansion and maintenance of clonogenic progenitors. When cells were expanded on feeder layer in the presence of added cytokines and PCM on average a 2.96-fold increase of CD34(+)CD71(-) and a 3.13-fold increase of CD34(+)HLA-DR(-) was observed. The total number of colony forming cells (35 +/- 2.65) indicated also that the yield of clonogenic progenitors obtained with a combination of all factors was two folds higher than each of these factors alone and ten time above control (3.67 +/- 2.52). In conclusion, the results of our study clearly show that the ex vivo expansion of hematopoietic progenitor cells obtained from human umbilical cord blood is dependent on controlled experimental conditions, which might be helpful when designing culture systems for clinical applications.     

Differential regulation of human blood dendritic cell subsets by IFNs

Based on the relative expression of CD11c and CD1a, we previously identified subsets of dendritic cells (DCs) or DC precursors in human peripheral blood. A CD1a(+)/CD11c(+) population (CD11c(+) DCs), also called myeloid DCs, is an immediate precursor of Langerhans cells, whereas a CD1a(-)/CD11c(-) population (CD11c(-) DCs), sometimes called lymphoid DCs but better known as plasmacytoid DCs, is composed of type I IFN (IFN-alpha beta)-producing cells. Here, we investigate the effects of IFN-alpha beta and IFN-gamma as well as other cytokines on CD11c(+) and CD11c(-) DC subsets, directly isolated from the peripheral blood, instead of in vitro-generated DCs. IFN-gamma and IFN-alpha, rather than GM-CSF, were the most potent cytokines for enhancing the maturation of CD11c(+) DCs. Incubation of CD11c(+) DCs with IFN-gamma also resulted in increased IL-12 production, and this IL-12 allowed DCs to increase Th1 responses by alloreactive T cells. In contrast, IFN-alpha did not induce IL-12 but, rather, augmented IL-10 production. IFN-alpha-primed matured CD11c(+) DCs induced IL-10-producing regulatory T cells; however, this process was independent of the DC-derived IL-10. On the other hand, IFN-alpha by itself neither matured CD11c(-) DCs nor altered the polarization of responding T cells, although this cytokine was a potent survival factor for CD11c(-) DCs. Unlike IFN-alpha, IL-3 was a potent survival factor and induced the maturation of CD11c(-) DCs. The IL-3-primed CD11c(-) DCs activated T cells to produce IL-10, IFN-gamma, and IL-4. Thus, CD11c(+) and CD11c(-) DC subsets play distinct roles in the cytokine network, especially their responses to IFNs.     

An increase in circulating mast cell colony-forming cells in asthma

We compared a potential to generate mast cells among various sources of CD34(+) peripheral blood (PB) cells in the presence of stem cell factor (SCF) with or without thrombopoietin (TPO), using a serum-deprived liquid culture system. From the time course of relative numbers of tryptase-positive and chymase-positive cells in the cultured cells grown by CD34(+) PB cells of nonasthmatic healthy individuals treated with G-CSF, TPO appears to potentiate the SCF-dependent growth of mast cells without influencing the differentiation into mast cell lineage. CD34(+) PB cells from asthmatic patients in a stable condition generated significantly more mast cells under stimulation with SCF alone or SCF+TPO at 6 wk of culture than did steady-state CD34(+) PB cells of normal controls. Single-cell culture studies showed a substantial difference in the number of SCF-responsive or SCF+TPO-responsive mast cell progenitors in CD34(+) PB cells between the two groups. In the presence of TPO, CD34(+) PB cells from asthmatic children could respond to a suboptimal concentration of SCF to a greater extent, compared with the values obtained by those of normal controls. Six-week cultured mast cells of asthmatic subjects had maturation properties (intracellular histamine content and tryptase/chymase enzymatic activities) similar to those derived from mobilized CD34(+) PB cells of nonasthmatic subjects. An increase in a potential of circulating hemopoietic progenitors to differentiate into mast cell lineage may contribute to the recruitment of mast cells toward sites of asthmatic mucosal inflammation.     

Improved survival, vascular differentiation and wound healing potential of stem cells co-cultured with endothelial cells

In this study, we developed a methodology to improve the survival, vascular differentiation and regenerative potential of umbilical cord blood (UCB)-derived hematopoietic stem cells (CD34(+) cells), by co-culturing the stem cells in a 3D fibrin gel with CD34(+)-derived endothelial cells (ECs). ECs differentiated from CD34(+) cells appear to have superior angiogenic properties to fully differentiated ECs, such as human umbilical vein endothelial cells (HUVECs). Our results indicate that the pro-survival effect of CD34(+)-derived ECs on CD34(+) cells is mediated, at least in part, by bioactive factors released from ECs. This effect likely involves the secretion of novel cytokines, including interleukin-17 (IL-17) and interleukin-10 (IL-10), and the activation of the ERK 1/2 pathway in CD34(+) cells. We also show that the endothelial differentiation of CD34(+) cells in co-culture with CD34(+)-derived ECs is mediated by a combination of soluble and insoluble factors. The regenerative potential of this co-culture system was demonstrated in a chronic wound diabetic animal model. The co-transplantation of CD34(+) cells with CD34(+)-derived ECs improved the wound healing relatively to controls, by decreasing the inflammatory reaction and increasing the neovascularization of the wound.     

Human lymphoid and myeloid cell development in NOD/LtSz-scid IL2R gamma null mice engrafted with mobilized human hemopoietic stem cells

Ethical considerations constrain the in vivo study of human hemopoietic stem cells (HSC). To overcome this limitation, small animal models of human HSC engraftment have been used. We report the development and characterization of a new genetic stock of IL-2R common gamma-chain deficient NOD/LtSz-scid (NOD-scid IL2Rgamma(null)) mice and document their ability to support human mobilized blood HSC engraftment and multilineage differentiation. NOD-scid IL2Rgamma(null) mice are deficient in mature lymphocytes and NK cells, survive beyond 16 mo of age, and even after sublethal irradiation resist lymphoma development. Engraftment of NOD-scid IL2Rgamma(null) mice with human HSC generate 6-fold higher percentages of human CD45(+) cells in host bone marrow than with similarly treated NOD-scid mice. These human cells include B cells, NK cells, myeloid cells, plasmacytoid dendritic cells, and HSC. Spleens from engrafted NOD-scid IL2Rgamma(null) mice contain human Ig(+) B cells and lower numbers of human CD3(+) T cells. Coadministration of human Fc-IL7 fusion protein results in high percentages of human CD4(+)CD8(+) thymocytes as well human CD4(+)CD8(-) and CD4(-)CD8(+) peripheral blood and splenic T cells. De novo human T cell development in NOD-scid IL2Rgamma(null) mice was validated by 1) high levels of TCR excision circles, 2) complex TCRbeta repertoire diversity, and 3) proliferative responses to PHA and streptococcal superantigen, streptococcal pyrogenic exotoxin. Thus, NOD-scid IL2Rgamma(null) mice engrafted with human mobilized blood stem cells provide a new in vivo long-lived model of robust multilineage human HSC engraftment.