Recombinant AAV2-mediated ß-globin expression in human fetal hematopoietic cells from the aborted fetuses with ß-thalassemia major.

Genetic correction of autologous hematopoietic stem cells has been proposed as an attractive treatment method for ß-thalassemia. Our previous study has shown that recombinant adeno-associated virus 2 (rAAV2) efficiently transduces human fetal liver hematopoietic cells, and mediates the expression of the human ß-globin gene in vivo. In this study, we investigated whether rAAV2 could also mediate the expression of normal ß-globin gene in human hematopoietic cells from ß-thalassemia patients. Human hematopoietic cells were isolated from aborted ß-thalassemia major fetuses, transduced with rAAV2-ß-globin, and then transplanted into nude mice. We found that rAAV2-ß-globin transduced human fetal hematopoietic cells, as determined by allele-specific PCR analysis. Furthermore, ß-globin transgene expression was detected in human hematopoietic cells up to 70 days post-transplantation in the recipient mice. High-pressure liquid chromatography analysis showed that human ß-globin expression levels increased significantly compared with control, as indicated by a 1.2-2.8-fold increase in the ratio of ß/α-globin chain. These novel data demonstrate that rAAV2 can transduce and mediate the normal ß-globin gene expression in fetal hematopoietic cells from ß-thalassemia patients. Our findings further support the potential use of rAAV-based gene therapy in the treatment of human ß-thalassemia.

[Role of sphingosine 1-phosphate receptor signaling in hematopoietic stem/progenitor cell transmigration].

OBJECTIVE: To determine the role of sphingosine 1-phosphate receptor (S1PRs ) signaling in CD34+ hematopoietic stem/progenitor cell transmigration. METHODS: CD34(+) cells were separated by Ficoll density gradient centrifugation and incubated in DMEM medium with 10% fetal calf serum. The cells were pretreated by FTY720, with or without pertussis toxin (PTX) and antiCXCR4 mAb in the medium, followed by addition of 100 ng/ml SDF-1 into the lower chamber of a Costar 24-well transwell. The migrated cells were counted using FACS and the migrating rates were determined. The expressions of sphingosine 1-phosphate receptors were analyzed in CD34(+) cells before and after the transmigration by reverse transcriptase- polymerase chain reaction (RT-PCR). Cord blood CD34(+) cells were treated with or without FTY720 (10(+) mol/L), and the expressions of CD49d (VLA-4), CD11a (LFA-1), and CD62L (L-selectin) were analyzed at 1, 8, and 16 h after the treatment. RESULTS: While FTY720 did not affect spontaneous migration, a substantial increase of SDF-1-induced transmigration was observed in the presence of FTY720 (15.26 2.14 to 28.64 2.37). The FTY720-enhanced transmigration was completely blocked by addition of PTX or antiCXCR4 mAb. S1p1-5 was expressed in fresh isolated cord blood CD34(+) cells. The migrating cells stimulated by FTY720 and SDF-1 only expressed S1P1, S1P3, and S1P4. The expressions of CD49d, CD11a and CD62L on CD34(+) cells treated with FTY720 remained unchanged at the selected time points as compared with the control. CONCLUSIONS: S1PRs are involved the transmigration of CD34(+) cells. The activation of S1PRs results in increased chemotactic response of CD34(+) to SDF-1. These effects are mediated through CXCR4 and PTX-sensitive Gi proteins. Only the CD34(+) cells expressing the specific receptors can rapidly transmigrate. The activation of the S1PRs does not affect the expressions of the adhesion molecules on cord blood CD34(+) cells.

[GFP gene transfection of dendritic cells mediated by recombinated adeno-associated virus].

OBJECTIVE: To investigate the transfection condition of Type 2 recombinant adeno-associated virus ( rAAV2) in human dendritic cells(DCs) which were induced from the bone marrow CD34+ hematopoietic stem /progenitor cells. METHODS: CD34+ hematopoietic stem /progenitor cells were purified from the bone marrow mononuclear cells by immunomagnetic beads, and the cells were cultured with IL-4 and GM-CSF and maturated by TNF-alpha on the 5th day. The rAAV2 /GFP was transfected into the induced cells at different time.The DCs were identified by electronic microscope. The expression of GFP was evaluated by flow cytometry and fluorescence microscope. RESULTS: The DCs were induced successfully. The typical morphologic characteristics of DCs were observed under the light microscope and transmission electronic microscope, and the typical phenotypes of DCs could be detected by flow cytometry. The expression rate of GFP gene on the 3rd day, the 5th day and after adding TNF-alpha was 0.45%, 13.54%, and 0.25%, respectively. CONCLUSION: DCs can be induced from the human bone marrow CD34+ hematopoietic stem /progenitor cells, and infected with the rAAV2 /GFP successfully. The longer the induction time of DCs, the higher the transfection efficiency of DC. The transfection efficiency of immature DC is higher than that of mature DC.

[Effects of granulocyte-macrophage colony stimulating factor on growth of murine bone marrow endothelial cells].

The purpose of this study was to investigate the effects of granulocyte-macrophage colony stimulating factor (GM-CSF) on the growth of mouse bone marrow endothelial cells. Endothelial cell culture medium (Endo-M) was used to culture murine bone marrow endothelial cells. Endothelial cell colonies were counted under microscope by Wright-Giemsa staining. The effect of different concentration of GM-CSF on the proliferation of bone marrow endothelial cells was observed by the formation of endothelial cell colonies, MTT and flow cytometry. The results indicated that the endothelial specific marker vWF was expressed by the colony cells, GM-CSF promoted the proliferation of bone marrow endothelial cell colonies and MTT confirmed the effect of GM-CSF on promoting the proliferation of bone marrow endothelial cells. The result of detecting cell cycle showed that the rate of cells entering into S phase was 9.3% in GM-CSF added group and the rate of cells entering into S phase was 2.1% in control. There was no significant difference in cell growth curve between the first passage and fourth passage. It is concluded that GM-CSF can promote the proliferation of bone marrow endothelial cells, the proliferation potential of bone marrow endothelial cells between the first and fourth passage no significantly changes.

[Effects of endothelial cells from cord blood on the amplification of human early hematopoietic cells from cord blood in vitro].

OBJECTIVE: To observe the effects of endothelial cells from umbilical cord blood (UCB) on the amplification of human early hematopoietic cells from UCB in vitro. METHODS: Endothelial cells from UCB were cultured by the optimized medium of endothelial cells. There were 2 experiment groups: cytokines group (SCF+IL-3+IL-6+GM-CSF, CKs group) and noncontact group (endothelial cell layer with CKs without contacting the CD34+ cells group). CD34+ cells from UCB were isolated by MiniMACS. After the cells in the CKs group and the noncontact group were cultured for 7 days, the amplifying folds of early hematopoietic cells were assayed. RESULTS: Early hematopoietic cells from UCB were expanded in the CKs group or the noncontact group. The amplifying folds of the noncontact group on early hematopoietic cells were significantly more than those of the CKs group. CONCLUSION: The amplification effect of the noncontact group on early hematopoietic cells is superior to that of the CKs group.

Effect of human thrombopoietin-modified bone marrow mesenchymal stem cells mediated by recombinant adeno-associated virus on megakaryocytopoiesis.

Recently, the research of recombinant thrombopoietin (TPO) and its subsequent use in treating thrombocytopenia following radiation therapy and chemotherapy have become more important in clinics. Our study was to determine the feasibility of recombinant adeno-associated virus (rAAV)-mediated TPO gene transfer into bone marrow-derived mesenchymal stem cells (MSCs) and to evaluate the conditioned medium (CM) obtained from TPO-transduced human (h) hMSCs for promoting the process of megakaryocytopoiesis. We constructed recombinant adeno-associated viruses expressing TPO successfully, and TPO mRNA and protein were both strongly expressed in TPO-transduced hMSCs. There was no decrease in green fluorescent protein (GFP) fluorescence expression of the transduced cells with continuous passaged culturing in vitro. The CM of TPO-transduced hMSCs has been shown to enhance the number of CD41(+) cells and megakaryocytic progenitors (colony-forming unit-megakaryocyte) significantly as compared to the nontransduced control. In this study, a novel safe and efficient method of promoting the megakaryocytopoiesis was established following the TPO-transduced hMSCs. These results provide a basis for the future studies on hematopoietic regulation by hMSCs transfected with TPO.

[Transduction efficiency of recombinant adeno-associated virus 2 in human bone marrow CD34+ hematopoietic stem/progenitor cells and mesenchyme stem cells].

OBJECTIVE: To investigate the transduction efficiency of recombinant adeno-associated virus 2 ( rAAV2) in human bone marrow CD34+ hematopoietic stem/progenitor cells and mesenchyme stem cells. METHODS: The rAAV2 containing green fluorescent protein genes (rAAV2/GFP) were constructed, packaged and purified. CD34+ hematopoietic stem/progenitor cells and mesenchyme stem cells were infected with the rAAV2/GFP. After transduction for 48 hours, the expression of GFP was detected under fluorescence microscope. Furthermore, the transduction efficiency of AAV transduced CD34+ with hydroxyurea (HU) pretreatment and that of untreated were compared. RESULTS: GFP genes were expressed in 5.3% +/- 1.7% CD34+ cells. After pretreatment with HU, the expression of the GFP gene in CD34+ cells increased to 13.2% +/- 2.8%, and 23% +/- 3.6% mesenchyme stem cells expressed the GFP gene. Conclusion The transduction efficiency of mesenchyme stem cells is higher than that of CD34+ hematopoietic stem/progenitor cells. HU pretreatment can obviously increase the transduction efficiency of CD34+ hematopoietic stem/progenitor cells.

[Promoting effects of serum-free murine bone marrow endothelial cell conditioned medium on the growth of bone marrow endothelial cells].

To study the effects of serum-free murine bone marrow endothelial cell conditioned medium (mBMEC-CM) on the growth of bone marrow endothelial cells, mBMEC-CM was collected and ultrafiltrated by Centriprep-10. The retentate of mBMEC-CM [molecular weight (MW)>10 kDa] and the filtrate of mBMEC-CM (MW<10 kDa) were obtained. The effect of bone marrow conditioned media, their components and exogenous cytokines on the formation of endothelial cell colonies were observed. The effect of bone marrow conditioned media, their components and exogenous cytokines on the proliferation of murine bone marrow endothelial cells were determined by [(3)H]-thymidine incorporation. The method of hybridizing to the Atlas cDNA array was used to determine the expression of cytokine mRNAs in bone marrow endothelial cells. The results obtained are as follows: vWF was expressed in bone marrow endothelial cells. The original mBMEC-CM and MW>10 kDa component of mBMEC-CM promoted the proliferation of bone marrow endothelial cell colonies and increased [(3)H]-thymidine incorporation of bone marrow endothelial cells. The MW<10 kDa component did not affect the production of endothelial cell colonies and did not increase [(3)H]-thymidine incorporation of endothelial cells. Six cytokines (IL-6, IL-11, SCF, GM-CSF, VEGF, bFGF) promoted the proliferation of bone marrow endothelial cell colonies. VEGF, bFGF and SCF increased [(3)H]-thymidine incorporation of bone marrow endothelial cells. According to the results of the Atlas cDNA array, GM-CSF,TGF-beta,BMP-2, bFGF, SCF, endothelin-2, thymosin beta10, MSP-1, connective tissue GF, PDGF-A chain, MIP-2 alpha, PlGF, neutrophil activating protein ENA-78, INF-gamma, IL-1, IL-6, IL-13, IL-11, inhibin-alpha mRNAs were expressed in endothelial cells. These results suggest that murine bone marrow endothelial cell conditioned medium promotes the proliferation of bone marrow endothelial cells.

[Effects of bone marrow fibroblasts conditioned media on the expansion of megakaryocytes in vitro].

OBJECTIVE: To investigate the effect of bone marrow fibroblasts conditioned media on the expansion of megakaryocytes in vitro. METHODS: Serum-free bone marrow fibroblasts conditioned medium (BMF-CM) was prepared and collected. The effect of BMF-CM or BMF-CM combined with IL-11 on the expansion of mature megakaryocytes and the effect of BMF-CM on the expansion of megakaryocytic progenitors were observed. RT-PCR was applied to determine the expression of NF-E2 mRNA in human megakaryoblastic leukemia cell line--Meg-0l. RESULTS: BMF-CM promoted the expansion of mature megakaryocytes. The effect of BMF-CM combined with IL-11 on the expansion of mature megakaryocytes was further enhanced. BMF-CM promoted the expansion of megakaryocytic progenitors. NF-E2 mRNA expression in megakaryocytic cell lines Meg-0l was enhanced when cultures were exposed to 30% BMF-CM for 4 hours, compared with control cultures exposed to IMDM for 4 hours. CONCLUSION: BMF-CM can promote the expansion of megakaryocytes in vitro.

[Anti-leukemia effect of sophora flavescens combined with the low molecular weight natural tumor suppressor of the human fetal liver and its mechanism].

OBJECTIVE: To explore the effect of sophora flavescens (SF) combined low molecular weight natural tumor suppressor(LMW-NTS) on normal human bone marrow CFU-GM and acute myelogenous leukemia cells and its mechanism. METHODS: CFU-GM colony culture, cell culture, Wrigh-Giemsa stain, 3H-TdR incorporation test, agarose gel electrophoresis of DNA fragment and RT-PCR were employed in this research. RESULTS: The combination of SF and LMW-NTS greatly inhibited the proliferation of acute meylogenous leukemia cells, induced their apoptosis, and down-regulated the c-Myc expression as compared with SF or LMW-NTS alone. CONCLUSION: The anti-leukemia effect of the combination of SF and LMW-NTS is superior to SF or LMW-NTS alone.

[Effects of murine bone marrow endothelial cell conditioned medium on the growth of yolk sac hematopoietic stem cells and progenitors].

OBJECTIVE: To investigate the effects of murine bone marrow endothelial cell conditioned medium (mBMEC-CM) on the growth of yolk sac hematopoietic progenitors. METHODS: The serum-free mBMEC-CM was obtained from subcultures of murine endothelial cell line derived from bone marrow which was established in our laboratory. The murine yolk sacs were harvested on day 8.5 postcoitus (pc) and incubated with 0.1% collagenase in 10% fetal calf serum at 37 degrees C for 40 minutes. Yolk sac cells were incubated in tissue culture dishes at 37 degrees C for 1 hour. Nonadherent cells were collected for semisolid culture assay of granulocyte-macrophage colony forming unit (CFU-GM) and high proliferative potential-colony forming cell (HPP-CFC) after being cultured in DMEM with 10% mBMEC-CM and 10% FBS for 24 hours. The number of CFU-GM and HPP-CFC was counted at day 7 and day 14 respectively. RESULTS: The growth of CFU-GM and HPP-CFC was supported by mBMEC-CM with GM-CSF. mBMEC-CM could induce the proliferation and differentiation of yolk sac hematopoietic stem cells and progenitors in liquid culture system. The percentages of CFU-GM and HPP-CFC compared with the 0 hour control were (119.5 +/- 5.7)% and (130.8 +/- 9.8)% respectively after 24 hours liquid culture (P < 0.05). The expansion effects of mBMEC-CM on CFU-GM and HPP-CFC were enhanced by compounded with flt3 ligand (FL) and thrombopoietin (TPO). The percentages of CFU-GM and HPP-CFC compared with the 0 hour control were (132.0 +/- 6.2)% and (176.9 +/- 12.8)% respectively after 24 hours liquid culture (P < 0.01). CONCLUSION: Murine bone marrow endothelial cell conditioned medium could support the growth and proliferation of yolk sac hematopoitic stem cells and progenitors, and this promoting effect was further enhanced by addition of FL and TPO.